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Serotonergic and non-serotonergic projections from the raphe nuclei to the piriform cortex in the rat: a cholera toxin B subunit (CTb) and 5-HT immunohistochemical study
Abstract
Retrograde axonal transport of the cholera toxin B subunit (CTb) was combined with 5-HT immunohistochemistry to determine the origin of the serotonergic innervation of the piriform cortex (PC) in the rat. After iontophoretic CTb injections in the PC, a substantial number of retrogradely labeled cells were found in the middle and medio-ventral part of the dorsal raphe nucleus (RD). A few retrogradely labeled cells were also observed in the median raphe nucleus (MnR) and the B9 serotonergic cell groups. Following CTb and 5-HT immunohistochemistry on the same sections, double-labeled cells were observed in the RD, MnR and B9 groups. In the RD, 30% of CTb stained cells were immunoreactive to 5-HT. After colchicine or nialamide (a monoamine oxidase inhibitor) pretreatment the percentage of these double-labeled cells reached 70%. These results indicate that both 5-HT and non-5-HT neurons in the RD innervate the PC and that the percentage of double-labeled cells is influenced by drug pretreatment. To determine the terminal fields of the RD efferent fibers in the PC, injections of the anterograde tracer PHA-L were also performed. Analysis of the fiber distribution in the PC further revealed some medio-lateral and antero-posterior differences.
... These studies revealed that DRN neurons project widely throughout the neuraxis, innervating most of the structures in the forebrain, midbrain, and brainstem. The organization of DRN projections to cerebral cortex (Kosofsky and Molliver, 1987), nucleus submedius (Matsuzaki et al., 1993), substantia nigra (Corvaja et al., 1993), piriform cortex (Datiche et al., 1995), hypothalamic paraventricular nucleus (Larsen et al., 1996), suprachiasmatic nucleus (Meyer-Bernstein and Morin, 1996;Hay-Schmidt et al., 2003), subcommissural organ (Mikkelsen et al., 1997), substantia innominata and nucleus basalis magnocellularis (Gasbarri et al., 1999), and central amygdaloid nucleus (Commons et al., 2003) has also been examined in detail using PHA-L tracing. ...
... with reported values ranging from 15% to >97% (O'Hearn and Molliver, 1984;Beitz et al., 1986;Ma et al., 1991;Petrov et al., 1992;Van Bockstaele et al., 1993;Datiche et al., 1995;Kirifides et al., 2001;Kim et al., 2004). It is known that the supramammillary nucleus (Kiss et al., 2002) and the inferior colliculus (Schwarz and Schwarz, 1992) are targeted by projections arising from glutamatergic DRN cells. ...
... These findings suggest that the collateralized component should be regarded as a representative sample of the serotonergic and non-serotonergic projections to the two sites. These estimates are within the considerable range of reported proportions (15% to >97%) of extrinsically-projecting DRN neurons which are 5-HT-positive (Beitz et al., 1986;Ma et al., 1991;Petrov et al., 1992;Van Bockstaele et al., 1993;Datiche et al., 1995;Kirifides et al., 2001). Although it is not known which transmitter substance(s) are present in the nonserotonergic DRN cells projecting to the vestibular nuclei and CeA, there are several possible candidates. ...
Preclinical and clinical evidence indicates that the serotonergic system regulates processing in the vestibular nuclei and in pathways linking balance function with emotional responses and affect. Previous studies conducted in this laboratory demonstrated that the serotonergic innervation of the vestibular nuclei is derived largely from the dorsal raphe nucleus (DRN), and revealed that the DRN also sends a nonserotonergic projection to the vestibular nuclei. The purpose of these experiments was to characterize the organization of the serotonergic and nonserotonergic components of the DRN projection to the vestibular nuclei. In Chapter 3, we describe retrograde tracing experiments that examined whether DRN cells send collateralized projections to the vestibular nuclei and central amygdaloid nucleus (CeA), regions involved in the clinical linkage between disorders of balance control and anxiety, and concluded that a subset of the serotonergic and nonserotonergic projections to the vestibular nuclei also project to CeA.Chapter 4 describes experiments with the anterograde tracer biotinylated dextran amine (BDA) that identified the terminal distribution of DRN projections within the vestibular nuclei. This study revealed that DRN projections descending in the ventricular plexus and the medial longitudinal fasciculus terminate within distinct vestibular terminal fields. In Chapter 5, BDA was used in combination with the serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) to selectively anterogradely trace nonserotonergic DRN projections to the vestibular nuclei. These experiments demonstrated that nonserotonergic DRN projections descend exclusively within the ventricular plexus and terminate primarily within the periventricular aspect of the vestibular nuclei.The purpose of the experiments in Chapter 6 was to map the distribution of serotonergic DRN terminals within the vestibular nuclei; 5,7-DHT was injected directly into DRN and silver staining was used to visualize the resulting pattern of terminal degeneration. It appears that projections from serotonergic DRN neurons terminate within both medial and lateral regions of the vestibular nuclei.Based on these findings, we conclude that major differences exist in the course of descent and termination patterns of serotonergic and nonserotonergic DRN projections to the vestibular nuclei, indicating that serotonergic and nonserotonergic cells give rise to distinct DRN projection systems that may selectively modulate processing within specific functional domains of the vestibular nuclei.
... Retrograde tract tracing studies have repeatedly found that only a subset of raphe cells projecting to forebrain structures contain serotonin. These nonserotonergic cells have been reported to project to such areas as the HPC (Aznar et al., 2004;Montone et al., 1988), septum (Aznar et al., 2004;Montone et al., 1988), cortex (Datiche et al., 1995;Koh et al., 1991;O'Hearn and Molliver, 1984), suprachiasmatic nucleus (Hay-Schmidt et al., 2003;Leander et al., 1998;Meyer-Bernstein and Morin, 1996), medial preoptic nucleus (Leanza et al., 1995), lateral geniculate nucleus (Villar et al., 1988), medial vestibular nucleus (Halberstadt and Balaban, 2007), and striatum (Steindler et al., 1983). Although some studies have reported that cells containing dopamine (Trulson et al., 1985) and GABA (Stamp and Semba, 1995) and various neuropeptides such as substance P, corticotropin-releasing factor (Valentino and Commons, 2005), galanin, somatostatin, and neurotensin (Araneda et al., 1999;Melander et al., 1986;Morin and Beaudet, 1998) can be found in the midbrain raphe nuclei, no study to date has conclusively identified the neurochemical phenotype of any nonserotonergic projection neurons. ...
... It has long been appreciated that as much as half of raphe projections to the HPC and MS (Aznar et al., 2004;Datiche et al., 1995;Montone et al., 1988) as well as other forebrain structures (Datiche et al., 1995;Hay-Schmidt et al., 2003;Koh et al., 1991;Leander et al., 1998;Leanza et al., 1995;Meyer-Bernstein and Morin, 1996;O'Hearn and Molliver, 1984;Steindler et al., 1983;Villar et al., 1988) are not serotonergic. This is the first study to tie nonserotonergic raphe neurons projecting to the forebrain to a particular cell phenotype, namely VGLUT3. ...
... It has long been appreciated that as much as half of raphe projections to the HPC and MS (Aznar et al., 2004;Datiche et al., 1995;Montone et al., 1988) as well as other forebrain structures (Datiche et al., 1995;Hay-Schmidt et al., 2003;Koh et al., 1991;Leander et al., 1998;Leanza et al., 1995;Meyer-Bernstein and Morin, 1996;O'Hearn and Molliver, 1984;Steindler et al., 1983;Villar et al., 1988) are not serotonergic. This is the first study to tie nonserotonergic raphe neurons projecting to the forebrain to a particular cell phenotype, namely VGLUT3. ...
The brainstem raphe nuclei are typically assigned a role in serotonergic brain function. However, numerous studies have reported that a large proportion of raphe projection cells are nonserotonergic. The identity of these projection cells is unknown. Recent studies have reported that the vesicular glutamate transporter VGLUT3 is found in both serotonergic and nonserotonergic neurons in both the median raphe (MR) and dorsal raphe (DR) nuclei. We injected the retrograde tracer cholera toxin subunit B into either the dorsal hippocampus or the medial septum (MS) and used triple labeled immunofluorescence to determine if nonserotonergic raphe cells projecting to these structures contained VGLUT3. Consistent with previous studies, only about half of retrogradely labeled MR neurons projecting to the hippocampus contained serotonin, whereas a majority of the retrogradely labeled nonserotonergic cells contained VGLUT3. Similar patterns were observed for MR cells projecting to the MS. About half of retrogradely labeled nonserotonergic neurons in the DR contained VGLUT3. Additionally, a large number of retrogradely labeled cells in the caudal linear and interpeduncular nuclei projecting to the MS were found to contain VGLUT3. These data suggest the enigmatic nonserotonergic projection from the MR to forebrain regions may be glutamatergic. In addition, these results demonstrate a dissociation between glutamatergic and serotonergic MR afferent inputs to the MS and hippocampus suggesting divergent and/or complementary roles of these pathways in modulating cellular activity within the septohippocampal network.
... In addition to the olfactory bulb, the raphe nuclei also project heavily to the piriform cortex (De Olmos and Heimer 1980; Datiche et al. 1995). The fiber distribution appears heavier in the anterior portion of the PC as compared with the posterior. ...
... The fiber distribution appears heavier in the anterior portion of the PC as compared with the posterior. As with NE, there also appears to be some laminar specificity as 5HT fibers are the densest in layers I and III (Datiche et al. 1995). Both 5HT1 and 5HT2 receptors are present in the PC and are mainly distributed in the deeper layers (Pompeiano et al. 1992Pompeiano et al. , 1994). ...
The mammalian olfactory system is well established for its remarkable capability of undergoing experience-dependent plasticity. Although this process involves changes at multiple stages throughout the central olfactory pathway, even the early stages of processing, such as the olfactory bulb and piriform cortex, can display a high degree of plasticity. As in other sensory systems, this plasticity can be controlled by centrifugal inputs from brain regions known to be involved in attention and learning processes. Specifically, both the bulb and cortex receive heavy inputs from cholinergic, noradrenergic, and serotonergic modulatory systems. These neuromodulators are shown to have profound effects on both odor processing and odor memory by acting on both inhibitory local interneurons and output neurons in both regions.
... Neurons projecting to the medial prefrontal cortex are clustered in the medial DRN (Van Bockstaele et al., 1993). The anterior thalamic nucleus receives a serotonergic projection from the ventromedial and ventrolateral part of the ipsilateral DRN (Gonzalo-Ruiz et al., 1995), whereas the piriform cortex is innervated by neurons in the ventromedial DRN (Datiche et al., 1995). Rat raphe neurons innervating the ventricular system appear to be distributed in the intermediate dorsomedial DRN (Simpson et al., 1998). ...
... Interestingly, in rats, DRN neurons projecting to the visual cortex are clustered in the ventromedial/interfascicular region, but are con-fined, almost exclusively, to the rostral two-thirds of the DRN (Waterhouse et al., 1986(Waterhouse et al., , 1993. The population of rat DRN neurons projecting to the piriform cortex is restricted to the ventromedial part of the DRN as well (Datiche et al., 1995) and appears to include the interfascicular region. The caudal interfascicular/ventromedial DRN may be anatomically different from the rostral ventromedial DRN. ...
Recent evidence suggests that the dorsal raphe nucleus (DRN) of the brainstem is a collection of neuronal clusters having different neurochemical characteristics and efferent projection patterns. To gain further insight into the neuroanatomic organization of the DRN, neuronal populations projecting to the superior colliculus (SC) were mapped in a highly visual rodent, the Mongolian gerbil (Meriones unguiculatus). Retrograde tracers Fluoro-Gold (FG) or cholera toxin subunit-B (CTB) were injected into the superficial layers of the SC, and serotonin (5-hydroxytryptamine, 5-HT) -positive cells were identified by using immunocytochemistry in the FG-injected animals. Based on its projections to the SC, the DRN was divided into five rostrocaudal levels. In the rostral and middle levels of the DRN, virtually all FG-filled cells occurred in the lateral DRN, and 36-55% of 5-HT-immunoreactive (5-HT-ir) cells were also double-labeled with FG. Caudally, FG-filled cells occurred in the lateral, ventromedial, and interfascicular DRN; and 44, 12, and 31% of 5-HT-ir cells, respectively, were also FG-filled. The dorsomedial DRN contained only a small proportion of FG-filled cells at its most caudal level and was completely devoid of FG-filled cells more rostrally. The CTB-injected animals showed a similar distribution of retrogradely labeled cells in the DRN. Topographically, the dorsal tegmental nucleus and the laterodorsal tegmental nucleus appeared to be closely associated with 5-HT-ir cells in the caudal DRN. These results suggest that the lateral DRN and the ventromedial/interfascicular DRN may be anatomically, morphologically, and neurochemically unique subdivisions of the gerbil DRN.
... Dysfunctions of the serotonergic system are implicated in several neurological and psychiatric disorders, including depression [15] and epilepsy [16,17]. RN 5-HT fibers densely innervate the primary olfactory cortex (aPC, anterior piriform cortex) with both the dorsal raphe nucleus (DRN) and median raphe nucleus (MRN) neurons contributing to this projection [18]. The most prominent effect of exogenously applied 5-HT in the aPC is a 5HT 1A receptor-mediated hyperpolarization of principal neurons [19] and a 5HT 2 and 5HT 3 receptor-mediated depolarization of local interneurons [20][21][22][23], which coincides with an increase in inhibitory postsynaptic potentials (IPSPs) in principal neurons [24]. ...
Serotonin (5-hydroxytriptamine, 5-HT) is an important monoaminergic neuromodulator involved in a variety of physiological and pathological functions. It has been implicated in the regulation of sensory functions at various stages of multiple modalities, but its mechanisms and functions in the olfactory system have remained elusive. Combining electrophysiology, optogenetics and pharmacology, here we show that afferent (feed-forward) pathway-evoked synaptic responses are boosted, whereas feedback responses are suppressed by presynaptic 5-HT1B receptors in the anterior piriform cortex (aPC) in vitro. Blocking 5-HT1B receptors also reduces the suppressive effects of serotonergic photostimulation of baseline firing in vivo. We suggest that by regulating the relative weights of synaptic inputs to aPC, 5-HT finely tunes sensory inputs in the olfactory cortex.
... 1,2 GM1 is ubiquitously expressed in vertebrates but is found in high relative abundance in the nervous system, including in the periphery of motor neurons at the neuro-muscular junction; for this reason CTB has been used extensively as a neuronal tracer. [3][4][5][6] It is usually detected via antibody-mediated detection using fluorescence or 3 0 -Diaminobenzidine (DAB) immunohistochemistry, [7][8][9] but direct fluorophore conjugates are also available 10,11 as well as CTB-horseradish peroxidase (HRP) conjugates for detection via peroxidase chemistry. 4,12 The ability to detect the tracer directly without the need of antibodies is advantageous. ...
Tracing of neurons plays an essential role in elucidating neural networks in the brain and spinal cord. Cholera toxin B subunit (CTB) is already widely used as a tracer although its use is limited by the need for immunohistochemical detection. A new construct incorporating non-canonical azido amino acids (azido-CTB) offers a novel way to expand the range and flexibility of this neuronal tracer. Azido-CTB can be detected rapidly in vivo following intramuscular tongue injection by ‘click’ chemistry, eliminating the need for antibodies. Cadmium selenide/zinc sulfide (CdSe/ZnS) core/shell nanoparticles were attached to azido-CTB by strain-promoted alkyne-azide cycloaddition to make a nano-conjugate. Following tongue injections the complex was detected in vivo in the brainstem by light microscopy and electron microscopy via silver enhancement. This method does not require membrane permeabilization and so ultrastructure is maintained. Azido-CTB offers new possibilities to enhance the utility of CTB as a neuronal tracer and delivery vehicle by modification using ‘click’ chemistry.
... These brain areas are suggested to support noradrenergic, dopaminergic and serotonergic innervation respectively, and play a nonnegligible function in shaping information processing and synaptic plasticity in the PC (Bouret and Sara, 2002;Fletcher and Chen, 2010;Narla et al., 2015). Consistent with the previous axon tracing studies using traditional tracers (De Olmos and Heimer, 1980;Datiche et al., 1995), we found that the APC received obviously more DR inputs than the PPC did (data not shown). Although the role that the serotonergic system plays in olfactory processing within the PC is not well known, it is possible that the serotonergic neuromodulation may be implicated in enhancing the signalto-noise ratio of odor inputs in the APC (Fletcher and Chen, 2010), because a previous electrophysiology study reported that activation of DR serotonin neurons may inhibit spontaneous activities in the APC, but not influence the odor induced response (Lottem et al., 2016). ...
The piriform cortex (PC) is a key brain area involved in both processing and coding of olfactory information. It is implicated in various brain disorders, such as epilepsy, Alzheimer’s disease, and autism. The PC consists of the anterior (APC) and posterior (PPC) parts, which are different anatomically and functionally. However, the direct input networks to specific neuronal populations within the APC and PPC remain poorly understood. Here, we mapped the whole-brain direct inputs to the two major neuronal populations, the excitatory glutamatergic principal neurons and inhibitory γ-aminobutyric acid (GABA)-ergic interneurons within the APC and PPC using the rabies virus (RV)-mediated retrograde trans-synaptic tracing system. We found that for both types of neurons, APC and PPC share some similarities in input networks, with dominant inputs originating from the olfactory region (OLF), followed by the cortical subplate (CTXsp), isocortex, cerebral nuclei (CNU), hippocampal formation (HPF) and interbrain (IB), whereas the midbrain (MB) and hindbrain (HB) were rarely labeled. However, APC and PPC also show distinct features in their input distribution patterns. For both types of neurons, the input proportion from the OLF to the APC was higher than that to the PPC; while the PPC received higher proportions of inputs from the HPF and CNU than the APC did. Overall, our results revealed the direct input networks of both excitatory and inhibitory neuronal populations of different PC subareas, providing a structural basis to analyze the diverse PC functions.
... In addition we show that serotonin transporter proteins (SERT) were highly reduced in laminated brain regions such as hippocampus, cortex, and cerebellum, which may explain the involvement of reelin in serotonergic neurons activity. The cortical neurons receive axonal projections from supralemniscal serotonergic cell group (B9) [73][74][75][76] rostral, ventral part and intrafascicular and dorsal parts of dorsal raphe nucleus [77][78][79][80][81][82] but not from lateral parts of dorsal raphe neurons, which project mainly to subcortical structures [83][84][85] and the contribution of B9 to the cortical innervation is probably much less than the 50% reduction in SERT, which explained that the remaining serotonin neurons on the midline may well have altered forebrain projections in reeler mice. [86] Mentioned that serotonergic signaling is most importantly regulated by the activity of SERT, which actually controls the concentration of active serotonin outside the cell. ...
Serotonin (5-HT) acts as both a morphogenetic factor during early embryonic development and a neuromodulator of circuit plasticity in the mature brain. Dysregulation of serotonin signaling during critical periods is involved in developmental neurological disorders, such as schizophrenia and autism. In this study we focused on the consequences of defect reelin signaling for the development of the brainstem serotonergic raphe system. We observed that reelin signaling components are expressed by serotonergic neurons during the critical period of their lateral migration. Further, we found that reelin signaling is important for the normal migration of rostral, but not caudal hindbrain raphe nuclei and that reelin deficiency results in the malformation of the paramedian raphe nucleus and the lateral wings of the dorsal raphe nuclei. Additionally, we showed that serotonergic neurons projections to laminated brain structures were severely altered. With this study, we propose that the perturbation of canonical reelin signaling interferes with the orientation of tangentially, but not radially, migrating brainstem 5-HT neurons. Our results open the window for further studies on the interaction of reelin and serotonin and the pathogenesis of neurodevelopmental disorders.
... The Pir is highly innervated by serotoninergic neurons of the raphe nucleus [35] and midbrain dopaminergic neurons [36], neurotransmitters that can modulate the activity of principal cells and interneurons [37,38]. To evaluate dopaminergic and serotoninergic function in the Pir, we measured the levels of these neurotransmitters and their metabolites by HPLC-ED. ...
Background:
Autism spectrum disorder (ASD) is characterized by impaired social interactions and repetitive patterns of behavior. Symptoms appear in early life and persist throughout adulthood. Early social stimulation can help reverse some of the symptoms, but the biological mechanisms of these therapies are unknown. By analyzing the effects of early social stimulation on ASD-related behavior in the mouse, we aimed to identify brain structures that contribute to these behaviors.
Methods:
We injected pregnant mice with 600-mg/kg valproic acid (VPA) or saline (SAL) at gestational day 12.5 and evaluated the effect of weaning their offspring in cages containing only VPA animals, only SAL animals, or mixed. We analyzed juvenile play at PD21 and performed a battery of behavioral tests in adulthood. We then used preclinical PET imaging for an unbiased analysis of the whole brain of these mice and studied the function of the piriform cortex by c-Fos immunoreactivity and HPLC.
Results:
Compared to control animals, VPA-exposed animals play less as juveniles and exhibit a lower frequency of social interaction in adulthood when reared with other VPA mice. In addition, these animals were less likely to investigate social odors in the habituation/dishabituation olfactory test. However, when VPA animals were weaned with control animals, these behavioral alterations were not observed. Interestingly, repetitive behaviors and depression-related behaviors were not affected by social enrichment. We also found that VPA animals present high levels of glucose metabolism bilaterally in the piriform cortex (Pir), a region known to be involved in social behaviors. Moreover, we found alterations in the somatosensory, motor, and insular cortices. Remarkably, these effects were mostly reversed after social stimulation. To evaluate if changes in glucose metabolism in the Pir correlated with changes in neuronal activity, we measured c-Fos immunoreactivity in the Pir and found it increased in animals prenatally exposed to VPA. We further found increased dopamine turnover in the Pir. Both alterations were largely reversed by social enrichment.
Conclusions:
We show that early social enrichment can specifically rescue social deficits in a mouse model of ASD. Our results identified the Pir as a structure affected by VPA-exposure and social enrichment, suggesting that it could be a key component of the social brain circuitry.
... For example, the rostral DRN projects to the caudate-putamen and substantia nigra, the middle DRN to the amygdala, whereas the caudal DR projects to the lateral and medial septum, ventral hippocampus, bed nucleus of the stria terminalis, locus coeruleus and hypothalamus. These subfield projections are likely involved in different behavior functions (Azmitia and Segal, 1978;Imai et al., 1986a,b;Datiche et al., 1995). In this study, we chose 5-HT neurons in the midline of vmDRN subdivisions of the DRN because in our immunohistochemistry study, this population of the neurons were most densely labeled by TPH, the marker of 5-HT neurons. ...
The serotonin (5-HT) system originating in the dorsal raphe nucleus (DRN) is implicated in various mood- and emotion-related disorders, such as anxiety, fear and stress. Abnormal activity of DRN 5-HT neurons is the key factor in the development of these disorders. Here, we describe a crucial role for the Kv7.4 potassium channel in modulating DRN 5-HT neuronal excitability. We demonstrate that Kv7.4 is selectively expressed in 5-HT neurons of the DRN. Using selective Kv7.4 opener fasudil and Kv7.4 knock-out mice, we demonstrate that Kv7.4 is a potent modulator of DRN 5-HT neuronal excitability. Furthermore, we demonstrate that the cellular redox signaling mechanism is involved in this 5-HT activation of Kv7.4. The current study suggests a new strategy for treating psychiatric disorders related to altered activity of DRN 5-HT neurons using K+ channel modulators.
... In the olfactory bulb (OB), 5-HT can affect odor processing by inhibiting glutamate release from the terminals of olfactory receptor neurons (Petzold et al., 2009) and exciting external tufted cells (Liu et al., 2012). The primary olfactory cor-tex or anterior piriform cortex (aPC) is also a significant target of DRN innervation (De Olmos and Heimer, 1980;Datiche et al., 1995). Principal neurons in aPC receive two major sources of input: (1) feedforward input from the OB via the lateral olfactory tract (LOT); and (2) feedback input comprising the recurrent collaterals of local principal neurons and top-down innervation from other brain regions (Haberly, 1998;Isaacson, 2010). ...
Unlabelled:
Serotonin (5-hydroxytriptamine; 5-HT) is implicated in a variety of brain functions including not only the regulation of mood and control of behavior but also the modulation of perception. 5-HT neurons in the dorsal raphe nucleus (DRN) often fire locked to sensory stimuli, but little is known about how 5-HT affects sensory processing, especially on this timescale. Here, we used an optogenetic approach to study the effect of 5-HT on single-unit activity in the mouse primary olfactory (anterior piriform) cortex. We show that activation of DRN 5-HT neurons rapidly inhibits the spontaneous firing of olfactory cortical neurons, acting in a divisive manner, but entirely spares sensory-driven firing. These results identify a new role for serotonergic modulation in dynamically regulating the balance between different sources of neural activity in sensory systems, suggesting a possible role for 5-HT in perceptual inference.
Significance statement:
Serotonin is implicated in a wide variety of (pato)physiological functions including perception, but its precise role has remained elusive. Here, using optogenetic tools in vivo, we show that serotonergic neuromodulation prominently inhibits the spontaneous electrical activity of neurons in the primary olfactory cortex on a rapid (<1 s) timescale but leaves sensory responses unaffected. These results identify a new role for serotonergic modulation in rapidly changing the balance between different sources of neural activity in sensory systems.
... While MR innervation was conspicuously localized in the glomerular cell layer, offering a perfect anatomical substrate for a direct control of primary olfactory inputs (Petzold et al. 2009), the DR innervation was restricted to the granule cell layer in a position to be likely implicated in the control of secondary olfactory processing. The importance of DR 5-HT in the secondary olfactory control is further supported by the dense DR innervation of secondary olfactory relays including the anterior olfactory nuclei and the piriform cortex (Datiche et al. 1995). Another interesting aspect of 5-HT innervation to the OB is the existence of a rhythmic release of 5-HT in the OB (Corthell et al. 2013). ...
Serotoninergic innervation of the central nervous system is provided by hindbrain raphe nuclei (B1–B9). The extent to which each raphe subdivision has distinct topographic organization of their projections is still unclear. We provide a comprehensive description of the main targets of the rostral serotonin (5-HT) raphe subgroups (B5–B9) in the mouse brain. Adeno-associated viruses that conditionally express GFP under the control of the 5-HT transporter promoter were used to label small groups of 5-HT neurons in the dorsal (B7d), ventral (B7v), lateral (B7l), and caudal (B6) subcomponents of the dorsal raphe (DR) nucleus as well as in the rostral and caudal parts of the median raphe (MR) nucleus (B8 and B5, respectively), and in the supralemniscal (B9) cell group. We illustrate the distinctive and largely non-overlapping projection areas of these cell groups: for instance, DR (B7) projects to basal parts of the forebrain, such as the amygdala, whereas MR (B8) is the main 5-HT source to the hippocampus, septum, and mesopontine tegmental nuclei. Distinct subsets of B7 have preferential brain targets: B7v is the main source of 5-HT for the cortex and amygdala while B7d innervates the hypothalamus. We reveal for the first time the target areas of the B9 cell group, demonstrating projections to the caudate, prefrontal cortex, substantia nigra, locus coeruleus and to the raphe cell groups. The broad topographic organization of the different raphe subnuclei is likely to underlie the different functional roles in which 5-HT has been implicated in the brain. The present mapping study could serve as the basis for genetically driven specific targeting of the different subcomponents of the mouse raphe system.
... For example, the lateral wings receive diverse afferent inputs (Peyron et al. 1998) and provide 5-HT innervation to the thalamus (De Olmos and Heimer 1980;Waterhouse et al. 1993), nucleus accumbens (Van Bockstaele et al. 1993), lateral geniculate nucleus (Pasquier and Villar 1982), and superior colliculus (Waterhouse et al. 1993). The ventromedial DRN receives more restricted afferent input (Peyron et al. 1998) and is a source of 5-HT innnervation to the cortex (Datiche et al. 1995;Waterhouse et al. 1993Waterhouse et al. , 1986, septum , hippocampus , and ventral tegmental area (Simon et al. 1979). This arrangement would allow specific stimuli to differentially alter 5-HT release in selective targets of the DRN through activation of selective CRF afferents. ...
... In the Mongolian gerbil, serotonergic neurons in the caudal ventromedial DRN and interfascicular DRN (Levels 4 and 5) project to the superior colliculus (SC); whereas at more rostral levels very few, if any, ventromedial neurons project to the SC . In rats, the ventromedial DRN also projects to the visual cortex (Waterhouse et al., 1986) and the piriform cortex (Datiche et al., 1995). In the Mongolian gerbil, neurons projecting to the primary visual cortex (V1) appear to be localized in the ventromedial subdivisions of the DRN as well (Janušonis et al., 2000). ...
Recent studies suggest that the dorsal raphe nucleus (DRN) of the brainstem contains several subdivisions that differ both anatomically and neurochemically. The present study examined whether variation of c-Fos expression across the 24-hour light-dark cycle may also be different in these subdivisions. Animals were kept on a 12:12 light-dark cycle, were perfused at seven different time points, and brain sections were processed by using c-Fos immunocytochemistry. At all coronal levels of the DRN, c-Fos expression reached a peak 1 hour after the light-dark transition (lights-off) and reached its lowest levels in the middle of the light period. In contrast to the light-dark transition, c-Fos levels did not change significantly after the dark-light transition (lights-on). One-way analysis of variance (ANOVA) revealed that the diurnal variation of c-Fos expression was highly significant in the caudal ventral DRN. Similar variation in c-Fos expression also was observed in the other DRN subdivisions, but this variation appeared to gradually diminish in the caudal-to-rostral and ventromedial-to-dorsomedial directions. Double-label immunocytochemistry revealed that, 1 hour after lights-off, only 11% of c-Fos-positive neurons in the caudal ventral DRN were serotonin (5-HT)-immunoreactive. These results suggest that DRN subdivisions may differ functionally with regard to the diurnal cycle, and that these differences may be reflected in the activity of nonserotonergic cells in the DRN. J. Comp. Neurol. 440:31–42, 2001. © 2001 Wiley-Liss, Inc.
... Raphe circuits involve direct modulation of the HPA axis as well as indirectly mediated influences orchestrated by raphe projections to other limbic structures (e.g., the hippocampus, amygdala, and medial prefrontal cortex) and brainstem areas that regulate the autonomic nervous system. MR and DR project to many of the same forebrain regions but also have distinct projections; additionally DR subfields project to different brain regions (Azmitia and Segal, 1978;Datiche et al., 1995;Imai et al., 1986a;Imai et al., 1986b;Johnson et al., 2008;Lowry, 2002;Vertes, 1991;Vertes et al., 1999;Vertes and Martin, 1988). For example, the rostral DR projects to the caudate-putamen and substantia nigra, the middle DR to the amygdala, whereas the caudal DR projects to the lateral and medial septum, ventral hippocampus, bed nucleus of the stria terminalis, locus coeruleus and hypothalamus. ...
The median (MR) and dorsal raphe (DR) nuclei contain the majority of the 5-hydroxytryptamine (5-HT, serotonin) neurons that project to limbic forebrain regions, are important in regulating homeostatic functions and are implicated in the etiology and treatment of mood disorders and schizophrenia. The primary synaptic inputs within and to the raphe are glutamatergic and GABAergic. The DR is divided into three subfields, i.e., ventromedial (vmDR), lateral wings (lwDR) and dorsomedial (dmDR). Our previous work shows that cell characteristics of 5-HT neurons and the magnitude of the 5-HT(1A) and 5-HT(1B) receptor-mediated responses in the vmDR and MR are not the same. We extend these observations to examine the electrophysiological properties across all four raphe subfields in both 5-HT and non-5-HT neurons. The neurochemical topography of glutamatergic and GABAergic cell bodies and nerve terminals were identified using immunohistochemistry and the morphology of the 5-HT neurons was measured. Although 5-HT neurons possessed similar physiological properties, important differences existed between subfields. Non-5-HT neurons were indistinguishable from 5-HT neurons. GABA neurons were distributed throughout the raphe, usually in areas devoid of 5-HT neurons. Although GABAergic synaptic innervation was dense throughout the raphe (immunohistochemical analysis of the GABA transporters GAT1 and GAT3), their distributions differed. Glutamate neurons, as defined by vGlut3 anti-bodies, were intermixed and co-localized with 5-HT neurons within all raphe subfields. Finally, the dendritic arbor of the 5-HT neurons was distinct between subfields. Previous studies regard 5-HT neurons as a homogenous population. Our data support a model of the raphe as an area composed of functionally distinct subpopulations of 5-HT and non-5-HT neurons, in part delineated by subfield. Understanding the interaction of the cell properties of the neurons in concert with their morphology, local distribution of GABA and glutamate neurons and their synaptic input, reveals a more complicated and heterogeneous raphe. These results provide an important foundation for understanding how specific subfields modulate behavior and for defining which aspects of the circuitry are altered during the etiology of psychological disorders.
... Little is known regarding the functional specificity of interfascicular neurons but these cells appear to form a distinct subpopulation. Compared to the majority of DRN neurons, this caudal area shows a specific arrangement of afferent projections (Datiche et al., 1995;Janusonis et al., 1999Janusonis et al., , 2003, unique electrophysiological properties (Rasmussen et al., 1984), and, in some rodents, a distinctive pattern of diurnal c-fos expression (Janusonis and Fite, 2001). The increased activation observed following clock-resetting nonphotic procedures and the lack of activation following physical restraint point to a potential role of these cells in non-photic phase control. ...
Some procedures for stimulating arousal in the usual daily rest period (e.g., gentle handling, novel wheel-induced running) can phase shift circadian rhythms in Syrian hamsters, while other arousal procedures are ineffective (inescapable stress, caffeine, modafinil). The dorsal and median raphe nuclei (DRN, MnR) have been implicated in clock resetting by arousal and, in rats and mice, exhibit strong regionally specific responses to inescapable stress and anxiogenic drugs. To examine a possible role for the midbrain raphe nuclei in the differential effects of arousal procedures on circadian rhythms, hamsters were aroused for 3 h in the mid-rest period by confinement to a novel running wheel, gentle handling (with minimal activity) or physical restraint (with intermittent, loud compressed air stimulation) and sacrificed immediately thereafter. Regional expression of c-fos and tryptophan hydroxylase (TrpOH) were quantified immunocytochemically in the DRN, MnR and locus coeruleus (LC). Neither gentle handling nor wheel running had a large impact on c-fos expression in these areas, although the manipulations were associated with a small increase in c-Fos in TrpOH-like and TrpOH-negative cells, respectively, in the caudal interfascicular DRN region. By contrast, restraint stress significantly increased c-Fos in both TrpOH-like and TrpOH-negative cells in the rostral DRN and LC. c-Fos-positive cells in the DRN did not express tyrosine hydroxylase. These results reveal regionally specific monoaminergic correlates of arousal-induced circadian clock resetting, and suggest a hypothesis that strong activation of some DRN and LC neurons by inescapable stress may oppose clock resetting in response to arousal during the daily sleep period. More generally, these results complement evidence from other rodent species for functional topographic organization of the DRN.
... The rat DR has, however, been indicated to contain a substantial number of nonserotonergic neurons (Descarries et al., 1982). Numerous studies have revealed that nonserotonergic neurons in the rat DR project to various brain regions (Aznar et al., 2004; Beitz et al., 1986; Datiche et al., 1995; Halberstadt and Balaban, 2006, 2007, 2008; Hay-Schmidt et al., 2003; Kim et al., 2004; Kohler and Steinbusch, 1982; Ma et al., 1991; O'Hearn and Molliver, 1984; Petrov et al., 1992, 1994; Van Bockstaele et al., 1993; Villar et al., 1988). These nonserotonergic projection neurons are considered to utilize dopamine (Lindvall and Bjorklund, 1974; Ochi and Shimizu, 1978; Trulson et al., 1985), ␥-aminobutyric acid (GABA; Mugnaini and Oertel , 1985; Stamp and Semba, 1995), glutamate (Kaneko et al., 1989, 1990; Kiss et al., 2002; Ottersen and Storm- Mathisen, 1984; Schwarz and Schwarz, 1992), nitric oxide (Nakamura et al., 1991; Pasqualotto et al., 1991), and neuropeptides such as CRF (Commons et al., 2003) as a neu- rotransmitter. ...
We previously reported that about 80% of vesicular glutamate transporter 3 (VGLUT3)-positive cells displayed immunoreactivity for serotonin, but the others were negative in the rat midbrain raphe nuclei, such as the dorsal (DR) and median raphe nuclei (MnR). In the present study, to investigate the precise distribution of VGLUT3-expressing nonserotonergic neurons in the DR and MnR, we performed double fluorescence in situ hybridization for VGLUT3 and tryptophan hydroxylase 2 (TPH2). According to the distribution of VGLUT3 and TPH2 mRNA signals, we divided the DR into six subregions. In the MnR and the rostral (DRr), ventral (DRV), and caudal (DRc) parts of the DR, VGLUT3 and TPH2 mRNA signals were frequently colocalized (about 80%). In the lateral wings (DRL) and core region of the dorsal part of the DR (DRDC), TPH2-producing neurons were predominantly distributed, and about 94% of TPH2-producing neurons were negative for VGLUT3 mRNA. Notably, in the shell region of the dorsal part of the DR (DRDSh), VGLUT3 mRNA signals were abundantly detected, and about 75% of VGLUT3-expressing neurons were negative for TPH2 mRNA. We then examined the projection of VGLUT3-expressing nonserotonergic neurons in the DRDSh by anterograde and retrograde labeling after chemical depletion of serotonergic neurons. The projection was observed in various brain regions such as the ventral tegmental area, substantia nigra pars compacta, hypothalamic nuclei, and preoptic area. These results suggest that VGLUT3-expressing nonserotonergic neurons in the midbrain raphe nuclei are preferentially distributed in the DRDSh and modulate many brain regions with the neurotransmitter glutamate via ascending axons.
... The tryptophan hydroxylase antibody (T0678, lot # 092K4836, Sigma) is a mouse monoclonal (clone WH-3) antibody, and a recombinant rabbit tryptophan hydroxylase was used as immunogen. This antiserum reacts specifically with tryptophan hydroxylase (55 kDa) in immunoblotting assays (manufacturer's technical information) and stains a pattern of neuronal morphology and distribution identical to previous reports (Datiche et al., 1995). The rabbit polyclonal tyrosine hydroxylase (AB152, lot # 22101229, Chemicon) was generated using sodium dodecyl sulfate-denatured tyrosine hydroxylase from rat pheochromocytoma. ...
Sleep architecture is often disturbed after a stressful event; nevertheless, little is known about the brain circuitry responsible for the sleep perturbations induced by stress. We exposed rats to a psychological stressor (cage exchange) that initially causes an acute stress response, but several hours later generates a pattern of sleep disturbances similar to that observed in stress-induced insomnia in humans: increased sleep latency, decreased non-REM (nREM) and REM sleep, increased fragmentation, and high-frequency EEG activity during nREM sleep. We examined the pattern of Fos expression to identify the brain circuitry activated, and found increased Fos in the cerebral cortex, limbic system, and parts of the arousal and autonomic systems. Surprisingly, there was simultaneous activation of the sleep-promoting areas, most likely driven by ongoing circadian and homeostatic pressure. The activity in the cerebral cortex and arousal system while sleeping generates a novel intermediate state characterized by EEG high-frequency activity, distinctive of waking, during nREM sleep. Inactivation of discrete limbic and arousal regions allowed the recovery of specific sleep components and altered the Fos pattern, suggesting a hierarchical organization of limbic areas that in turn activate the arousal system and subsequently the cerebral cortex, generating the high-frequency activity. This high-frequency activity during nREM was eliminated in the stressed rats after inactivating parts of the arousal system. These results suggest that shutting down the residual activity of the limbic-arousal system might be a better approach to treat stress-induced insomnia, rather than potentiation of the sleep system, which remains fully active.
... The overall concept is supported by a body of evidence including, but not limited to, implications of nucleus accumbens DA involvement in reinforcement of feeding reward (18), and amygdala and hypothalamic 5-HT as integral to conditioned taste aversions (15,28). The dorsal raphe 5-HT system innervates many limbic structures that receive dopaminergic input from the ventral tegmental area (VTA) (the mesolimbic dopaminergic system), including the amygdala, nucleus accumbens, and piriform cortex (3,8,9,24), providing an anatomical rationale for this hypothesis. ...
The serotonin3 (5-HT3) receptor plays an important role in the aminoprivic feeding model. Other neurochemical systems, including cholecystokinin (CCK) and dopamine (DA), are known to affect food intake. We pretreated rats systemically with tropisetron, a 5-HT3 receptor antagonist, alone and combined with antagonists of DA1 and DA2 receptors, and measured intake of an amino acid-imbalanced diet (IMB). As expected, tropisetron significantly increased intake of IMB. SCH-23390, a DA1 antagonist, increased IMB anorexia. When combined with tropisetron, DA2 antagonism with eticlopride reduced short-term intake of both the basal diet (BAS) and IMB. In the IMB model, specificity of 5-HT3-DA2 interactions, and of 5-HT3-CCK(A) interactions from previous studies, prompted investigation of CCK(A)-DA2 interactions; there appeared to be none. SKF-38393, a DA1 agonist, combined with the CCK(A) receptor antagonist, devazepide, increased BAS and tended to increase IMB intake. Thus, CCK(A)-DA1 interactions were not specific for IMB. These data suggest that DA1 receptor activity opposes IMB anorexia, possibly via an interaction with the 5-HT3 receptor.
... These findings are consistent with the present demonstration of limited MR projections to cortex. Along similar lines, injections of retrograde tracers in various regions of cortex have been shown to give rise to relatively small numbers of labeled cells in MR (i.e., considerably fewer than found in DR) ( Deacon et al., 1983; O'Hearn and Molliver, 1984; Room and Groenewegen, 1986; Waterhouse et al., 1986; Newman and Liu, 1987; Datiche et al., 1995). For example, Waterhouse et al. (1986) identified significant numbers of labeled cells in DR, compared with few in MR, after HRP injections in the motor, sensorimotor, and visual cortices of rats. ...
No previous report in any species has examined comprehensively the projections of the median raphe (MR) nucleus with modern tracing techniques. The present report represents an in depth analysis of the projections of MR by use of the anterograde anatomical tracer Phaseolus vulgaris-leucoagglutinin. MR fibers descend along the midline within the brainstem and mainly ascend within the medial forebrain bundle in the forebrain. MR fibers distribute densely to the following brainstem/forebrain sites: caudal raphe nuclei, laterodorsal tegmental nucleus, dorsal raphe nucleus, interpeduncular nucleus, medial mammillary body, supramammillary nucleus, posterior nucleus and perifornical region of the hypothalamus, midline and intralaminar nuclei of thalamus, dopamine-containing cell region of medial zona incerta, lateral habenula, horizontal and vertical limbs of the diagonal band nuclei, medial septum, and hippocampal formation. Virtually all of these structures lie on or close to the midline, indicating that the MR represents a midline/para-midline system of projections. Overall, MR projections to the cortex are light. MR projects moderately to the perirhinal, entorhinal and frontal cortices, but sparingly to remaining regions of cortex. A comparison of MR with dorsal raphe (DR) projections (Vertes RP. 1991. J Comp Neurol 313:643-668) shows that these two major serotonin-containing cell groups of the midbrain distribute to essentially nonoverlapping regions of the forebrain; that is, the MR and DR project to complementary sites in the forebrain. A direct role for the MR in the desynchronization of the electroencephalographic activity of the hippocampus and its possible consequences for memory-associated functions of the hippocampus is discussed.
... For example, the lateral wings receive diverse afferent inputs (Peyron et al. 1998) and provide 5-HT innervation to the thalamus (De Olmos and Heimer 1980;Waterhouse et al. 1993), nucleus accumbens (Van Bockstaele et al. 1993), lateral geniculate nucleus (Pasquier and Villar 1982), and superior colliculus (Waterhouse et al. 1993). The ventromedial DRN receives more restricted afferent input (Peyron et al. 1998) and is a source of 5-HT innnervation to the cortex (Datiche et al. 1995;Waterhouse et al. 1993Waterhouse et al. , 1986, septum , hippocampus , and ventral tegmental area (Simon et al. 1979). This arrangement would allow specific stimuli to differentially alter 5-HT release in selective targets of the DRN through activation of selective CRF afferents. ...
The present study examined the regional localization of corticotropin-releasing factor (CRF)- and 5-hydroxytryptamine (5-HT)-immunoreactive (IR) fibers within the rat dorsal raphe nucleus (DRN) using immunohistochemistry. Additionally, the effects of CRF, administered intracerebroventricularly (0.1-3.0 micrograms) or intraraphe (0.3-30 ng), on discharge rates of putative 5-HT DRN neurons were quantified using in vivo single unit recording in halothane-anesthetized rats. CRF-IR fibers were present at all rostrocaudal levels of the DRN and exhibited a topographical distribution. CRF produced predominantly inhibitory effects on DRN discharge at lower doses and these effects diminished or became excitatory at higher doses. Inhibition of DRN discharge by CRF was attenuated by the nonselective CRF antagonist, DPheCRF12-41 and the CRF-R1-selective antagonist, antalarmin, implicating the CRF-R1 receptor subtype in these electrophysiological effects. The present findings provide anatomical and physiological evidence for an impact of CRF on the DRN-5HT system.
... Anatomical studies have already pointed to differences between anterior and posterior piriform cortices based on the distribution of afferent 52 and associational ®bers, 10,18,32 as well as their connections with higher brain structures 9 and their innervation by central neuromodulatory systems. 11 In addition, electrophysiological studies have further emphasized this dissociation. Indeed, Litaudon and Cattarelli 30 using optical recording of the whole piriform cortex activity showed that following repetitive electrical stimulation of the OB, a higher degree of inhibition was observed in the ventromedial anterior part of the piriform cortex than in the posterior part. ...
In the present work, we investigated lasting changes induced by olfactory learning at different levels of the olfactory pathways. For this, evoked field potentials induced by electrical stimulation of the olfactory bulb were recorded simultaneously in the anterior piriform cortex, the posterior piriform cortex, the lateral entorhinal cortex and the dentate gyrus. The amplitude of the evoked field potential's main component was measured in each site before, immediately after, and 20 days after completion of associative learning. Evoked field potential recordings were carried out under two experimental conditions in the same animals: awake and anesthetized. In the learning task, rats were trained to associate electrical stimulation of one olfactory bulb electrode with the delivery of sucrose (positive reward), and stimulation of a second olfactory bulb electrode with the delivery of quinine (negative reward). In this way, stimulation of the same olfactory bulb electrodes used for inducing field potentials served as a discriminative cue in the learning paradigm. The data showed that positively reinforced learning resulted in a lasting increase in evoked field potential amplitude restricted to posterior piriform cortex and lateral entorhinal cortex. In contrast, negatively reinforced learning was mainly accompanied by a decrease in evoked field potential amplitude in the dentate gyrus. Moreover, the expression of these learning-related changes occurred to be modulated by the animals arousal state. Indeed, the comparison between anesthetized versus awake animals showed that although globally similar, the changes were expressed earlier with respect to learning, under anesthesia than in the awake state.
The olfactory system is an ideal and tractable system for exploring how the brain transforms sensory inputs into behaviour. The basic tasks of any olfactory system include odour detection, discrimination and categorization. The challenge for the olfactory system is to transform the high-dimensional space of olfactory stimuli into the much smaller space of perceived objects and valence that endows odours with meaning. Our current understanding of how neural circuits address this challenge has come primarily from observations of the mechanisms of the brain for processing other sensory modalities, such as vision and hearing, in which optimized deep hierarchical circuits are used to extract sensory features that vary along continuous physical dimensions. The olfactory system, by contrast, contends with an ill-defined, high-dimensional stimulus space and discrete stimuli using a circuit architecture that is shallow and parallelized. Here, we present recent observations in vertebrate and invertebrate systems that relate the statistical structure and state-dependent modulation of olfactory codes to mechanisms of perception and odour-guided behaviour.
Serotonin (5‐HT) plays a trophic role during brain development; chronic changes in cerebral concentration of this neurotransmitter during the critical stage of development can produce severe damage in the formation of the neural circuits. For the present work a hypoproteic (HYP) diet based on corn (CORN) meal which is deficient in tryptophan (TRY) was given to rats before and during pregnancy, which continued to the offspring until they reached 60 days of age. An isocaloric but hypoproteic diet containing normal amount of TRY, and normal chow (Ch) Purina were given with the same scheme to two groups of rats considered as controls. 5‐HT immunohistochemistry was revealed by avidin–biotin complex (ABC) method to quantify serotonergic nerve cells in the nine raphe nuclei. The number of cells immunoreactive to 5‐HT immunoreactive (5‐HTir) were quantified by means of stereological analysis. Results demonstrated a significant variation in 5‐HT expression in the raphe nuclei. Thus, a significant reduction in the number of 5‐HTir cells in the rostral raphe nuclei was seen at all ages studied in the animals fed the corn diet, compared to data obtained from the control groups. This decrease was more evident between the postnatal ages of 30 and 60 days. It is concluded that the variations in the available TRY affect the brain cells producing 5‐HT and the innervation of their target areas.
Muscarinic cholinergic- and metabotropic glutamate receptor-evoked responses of adult and immature olfactory cortical neurones were studied in brain slices using intracellular electrophysiological recordings. An attempt was made to study the ontogeny of the cortical slow post-stimulus afterdepolarization (sADP) induced by muscarinic cholinergic or metabotropic glutamate agonists in immature (14-22 days) and adult (greater than 40 day old) rats. In adult neurones, 10 μM oxotremorine-M (OXO-M, a muscarinic agonist) or 1S,3R- ACPD (10-50 μM, a metabotropic glutamate receptor agonist) displayed postsynaptic excitatory and presynaptic inhibitory effects. In contrast, immature rat olfactory neurones responded to 10 μM OXO-M with spontaneous epileptiform activity which was blocked by pharmacological agents such as atropine (1 μM), pirenzepine (a muscarinic M1 receptor antagonist; 300 nM), tetrodotoxin (1 μM) or the glutamate receptor antagonists DL-APV (100 μM) or CNQX (20 μM). Inhibition of either GABAA or GABAB receptor activity with bicuculline (10 μM) or CGP52432 (1 μM) respectively, actively promoted bursting behaviour, working synergistically with lower doses of OXO-M. Conversely, augmenting GABAA or GABAB receptor function with pentobarbitone (100 μM) or baclofen (10 μM), respectively, blocked the bursting. The induced bursting was accompanied by a dramatic prolongation of evoked postsynaptic potentials (PSPs), exhibiting recurrent superimposed spike discharges, not seen in control. 1S,3R-ACPD (10-50 μM) induced typical adult-type responses in immature cells, i.e. presynaptic inhibition and postsynaptic excitation, with no evidence of epileptiform behaviour. During experiments designed to investigate the possible involvement of release of calcium from intracellular stores in the generation of the cholinergic/metabotropic glutamate agonist-induced sADP and its underlying tail current (IADP) adult guinea- pig olfactory neurones, it was found that the sADP induced by OXO-M (10 μM) or 1S,3R ACPD (10-50 μM), could be reversibly blocked by 0.5-3 mM caffeine, a compound known to promote release of calcium from intracellular stores. However, when this inhibitory action was investigated further using compounds with more specific effects on intracellular calcium release, i.e. ryanodine (10 μM), thapsigargin (3 μM) or dantrolene (10 μM), no consistent inhibition of IADP was observed. In addition, other possible effects of caffeine were discounted as being important by use of more specific pharmacological tools, i.e. IBMX (100 μM; an inhibitor of phosphodiesterase activity) or adenosine (100 μM; to reverse any inhibition of adenosine receptors imposed by caffeine). In caesium-loaded cells, a direct block of voltage-sensitive calcium entry by 3 mM caffeine was found, but this could not account for the full inhibition of IADP in this system; a direct blockade of the proposed IADP K+ channels by caffeine was therefore proposed. In conclusion, the muscarinic response profile of immature rat olfactory neurones was found to differ from adult neurones, since they displayed bursting behaviour in response to muscarinic (but not metabotropic) activation, thought to be generated through a local network mechanism. Furthermore, release of calcium from intracellular stores was unlikely to play an important role in sADP or IADP generation. Caffeine, a known modulator of intracellular calcium release, however, blocked IADP through a mechanism which did not involve intracellular calcium stores, most likely having a direct action on calcium entry and/or the proposed IADP K+ conductance.
Background:
Positron emission tomography (PET) studies in major depressive disorder (MDD) have reported higher serotonin 1A (5-HT1A ) autoreceptor binding in the raphe. In males, the difference is so large that it can potentially be used as the first biological marker for MDD. However, the raphe includes several nuclei, which project to different regions of the brain and spinal cord and may be differentially involved in disease. We aimed to identify 5-HT1A differences in individual raphe nuclei using PET in order to determine whether use of subnuclei would provide greater sensitivity and specificity of diagnosing MDD.
Methods:
We identified individual nuclei using a hybrid set-level technique on an average [11 C]-WAY100635 PET image derived from 52 healthy volunteers (HV). We delineated three nuclei: dorsal raphe nucleus (DRN), median raphe nucleus (MRN), and raphe magnus (RMg). An atlas image of these nuclei was created and nonlinearly warped to each subject (through an associated MRI) in a separate sample of 41 males (25 HV, 16 MDD) who underwent [11 C]-WAY100635 PET.
Results:
5-HT1A binding was elevated in DRN in MDD (P < .01), and was not different in the RMg and MRN between groups. Receiver operating characteristic (ROC) curves showed that combining DRN and MRN produces highest sensitivity (94%) and specificity (84%) to identify MDD.
Conclusions:
In agreement with postmortem studies, we found higher 5-HT1A autoreceptor binding in MDD selectively in the DRN. 5-HT1A autoreceptor binding in the combined DRN and MRN is a better biomarker for MDD than in the raphe as a whole.
No previous report in any species has examined comprehensively the projections of the median raphe (MR) nucleus with modern tracing techniques. The present report represents an in depth analysis of the projections of MR by use of the anterograde anatomical tracer Phaseolus vulgaris-leucoagglutinin. MR fibers descend along the midline within the brainstem and mainly ascend within the medial forebrain bundle in the forebrain. MR fibers distribute densely to the following brainstem/forebrain sites: caudal raphe nuclei, laterodorsal tegmental nucleus, dorsal raphe nucleus, interpeduncular nucleus, medial mammillary body, supramammillary nucleus, posterior nucleus and perifornical region of the hypothalamus, midline and intralaminar nuclei of thalamus, dopamine-containing cell region of medial zona incerta, lateral habenula, horizontal and vertical limbs of the diagonal band nuclei, medial septum, and hippocampal formation. Virtually all of these structures lie on or close to the midline, indicating that the MR represents a midline/para-midline system of projections. Overall, MR projections to the cortex are light. MR projects moderately to the perirhinal, entorhinal and frontal cortices, but sparingly to remaining regions of cortex. A comparison of MR with dorsal raphe (DR) projections (Vertes RP. 1991. J Comp Neurol 313:643–668) shows that these two major serotonin-containing cell groups of the midbrain distribute to essentially nonoverlapping regions of the forebrain; that is, the MR and DR project to complementary sites in the forebrain. A direct role for the MR in the desynchronization of the electroencephalographic activity of the hippocampus and its possible consequences for memory-associated functions of the hippocampus is discussed. J. Comp. Neurol. 407:555–582, 1999.
Because of its anatomical and functional features, the piriform cortex (PC) could play a crucial role
in olfactory learning and memory. We studied its implication in mnesic processes using the
immunodetection of Fos protein in rat. A water-rewarded olfactory discrimination task in a four-arms
maze was used for conditioning. We analysed Fos expression in PC as well as in neocortex and
hippocampus, after completion of the discrimination task, at different time-points throughout learning
and following its reactivation. In PC, we showed that Fos expression was different depending of the
velocity to learning of the conditioned rat. Furthermore, we confirmed the functional heterogeneity of
the PC subdivisions and observed the implication of hippocampal and neocortical areas in the olfactory
memory processes.
The raphe nuclei constitute a collection of cell groups distributed in the midline region of the tegmentum, from the rostral midbrain to the spinal cord transition at the pyramidal tract decussation. In the brain stem, the union of both sides along the midline region is underlined by the crossing of numerous tracts; the most prominent ones are the decussation of the superior cerebellar tract in the mesencephalon, and, in the medulla oblongata, the medial lemniscal and the corticospinal decussations. As a result, cell density in raphe nuclei varies greatly between regions, with sharp cytoarchitectonic boundaries for the dorsal raphe nucleus and ill-defined limits for the midline caudal nuclei. Collectively, the raphe nuclei are part of the reticular formation bordering the midline, and are referred to as the median division of the reticular formation. At variance to the other raphe nuclei, the dorsal raphe nucleus is located dorsal to the medial longitudinal fasciculus, in the ventral periaqueductal gray.
La disfunción serotoninérgica está presente en los trastornos del humor y el suicidio. Los autorreceptores somatodrendríticos 5-HT1A del tronco cerebral regulan la descarga de neuronas serotoninérgicas, pero los estudios efectuados sobre la unión a los autorreceptores en el núcleo dorsal del rafe (NDR) en muestras de tejidos obtenidas en víctimas de suicidio deprimidas describen resultados conflictivos. Tratamos de determinar: 1) la distribución anatómica de la unión al receptor 5-HT1A en el NDR de muestras de tejidos obtenidas de víctimas deprimidas de suicidio y de individuos de control sanos, desde un punto de vista psiquiátrico, fallecidos por causas naturales, y 2) si las diferencias de sexo en la unión a 5-HT1A en el NDR contribuyen a las diferencias observadas en las muestras entre víctimas deprimidas de suicidio y los de control. Se analizaron las autorradiografías cuantitativas del receptor de [3H]8-hidroxi-2-(di-n-propil) aminotetralina ([3H]8-OH-DPAT) en las secciones de tejido post mórtem, obtenidas previamente, que contenían el NDR de víctimas de suicidio libres de fármacos/drogas (n=10) y de controles emparejados (n=10). En el NDR íntegro de los suicidas deprimidos, comparado con los controles, se observó una menor unión total al receptor (fmol/mg de tejido×mm3) (p<0,05). Se observaron diferencias de grupo a lo largo de la extensión rostrocaudal del NDR para la unión transversal a 5-HT1A (fmol/mg de tejido) y la unión al receptor (fmol/mg×mm3, p<0,05). Comparado con controles, en el NDR de suicidas deprimidos la unión transversal a 5-HT1A fue mayor rostralmente y menor, caudalmente. Las diferencias entre suicidas deprimidos y controles estuvieron presentes en hombres y mujeres, aunque en estas se detectó una mayor unión que en hombres. La menor unión al autorreceptor en NDR de suicidas deprimidos podría representar una respuesta homeostática a una menor liberación de serotonina, con un incremento de la descarga de las neuronas serotoninérgicas. La mayor unión al autorreceptor en el NDR rostral podría contribuir a una liberación deficiente de serotonina en la corteza prefrontal ventromedial mediante una menor descarga neuronal.
The raphe nuclei provide serotonergic innervation widely in the brain, thought to mediate a variety of neuromodulatory effects. The mammalian olfactory bulb (OB) is a prominent recipient of serotonergic fibres, particularly in the glomerular layer (GL) where they are thought to gate the incoming signals from the olfactory nerve. The dorsal raphe nucleus (DRN) and the median raphe nucleus (MRN) are known to densely innervate the OB. The majority of such projections are thought to terminate in the GL, but this has not been explicitly tested. We sought to investigate this using rAAV-mediated expression of GFP-synaptophysin targeted specifically in neurons of the DRN or the MRN. With DRN injections, labelled fibres were found mostly in the granule cell layer (GCL), not the GL. Conversely, dense labelling in the GL was observed with MRN injections, suggesting that the source innervation in the GL is the MRN, not the DRN as previously thought. The two raphe nuclei thus give dual innervation within the OB, with distinct innervation patterns. This article is protected by copyright. All rights reserved.
Neurons using serotonin as a neurotransmitter in the central nervous system are restricted to the brainstem, centered on the midline raphe nuclei, extending into the subnuclei of the lateral reticular formation. These neurons divide into two populations. A rostral group resides in the midbrain and rostral pons (caudal linear, dorsal and median raphe nuclei), with a major projection targeted to the forebrain. A caudal group located in the caudal pons and the medulla (raphe magnus, obscurus and pallidus nuclei, lateral medullary reticular formation) has a major projection targeted to the spinal cord. Both groups have projections to the brainstem. Subnuclei of the serotonergic system have distinctive afferent and efferent connections, with some entities more specifically linked to the sensory, motor or limbic systems. Serotonergic neurons segregate, based on their axonal morphology, into neurons with large varicose or small varicose axons, the latter being selectively sensitive to neurotoxins derived from amphetamines. This is the basis for a dual serotonergic projection system, which partially overlaps in certain structures such as the cerebral cortex. Small varicose axons form wide axonal arborization of various densities of synaptic boutons, sustaining a system of volume transmission. Large varicose axons form pericellular arrays of synaptic terminals surrounding the soma and proximal dendrites of selected neurons. Median raphe, raphe obscurus and raphe pallidus nuclei are the sources for serotonergic neurons with large varicose axons. As one of the diffuse aminergic systems of the brain, the serotonergic system segregates clearly into different entities, with subnuclei holding their own efferent and afferent connections, serotonergic neurons having different axonal morphologies and synaptic connections, and, finally, numerous serotonin receptor subtypes having different expression patterns across the brain.
L’area che occupa la porzione centrale del tronco è nota come formazione reticolare. Per gran parte della sua estensione, questa area è occupata da aggregati di cellule sparse, diverse per tipo e dimensioni, e i sistemi di fibre che attraversano questo territorio sono parimenti organizzati in maniera alquanto diffusa. Il termine “formazione reticolare” si riferisce al fatto che i dendriti delle cellule in questa area sono disposti in fasci che insieme formano una struttura a forma di rete [161]. I sistemi di fibre che la attraversano decorrono negli interstizi di questa rete. La formazione reticolare è circondata dai nuclei dei nervi cranici, dai nuclei di proiezione sensoriale e dai lunghi sistemi di fibre ascendenti e discendenti. Per motivi di citoarchitettonica, di chemoarchitettonica e funzionali, la formazione reticolare del bulbo e del ponte è stata divisa in tre zone longitudinali [29] (Figg. 22.1, 22.2):
1.
Una zona mediana e paramediana, costituita dai nuclei del rafe. I neuroni serotoninergici dei gruppi B1–B9 di Dahlström e Fuxe [43, 44] sono disposti nei nuclei del rafe e nell’adiacente formazione reticolare.
Many aspects of animal and human behavior are guided by, or dependent on, the sense of smell‐olfaction. This chapter summarizes
the major facts of the anatomy, neurochemistry, molecular biology and physiology of the olfactory system. We emphasize the
mammalian olfactory system, particularly rodents, because of their widespread use as a laboratory model and the rich database
related to this species.
The morphology and electrophysiological properties of serotonergic and non-serotonergic projection neurons in the dorsal raphe nucleus (DRN) of the rat were examined in frontal brain slices. Biocytin was injected intracellularly into the intracellularly recorded neurons. Then the morphology of the recorded neurons was observed after histochemical visualization of biocytin. The recorded neurons extending their main axons outside the DRN were considered as projection neurons. Subsequently, serotonergic nature of the neurons was examined by serotonin (5-HT) immunohistochemistry. The general form of the dendritic trees is radiant and poorly branching in both 5-HT- and non-5-HT neurons. However, the dendrites of the 5-HT neurons were spiny, whereas those of the non-5-HT neurons were aspiny. The main axons of both 5-HT- and non-5-HT neurons were observed to send richly branching axon collaterals to the DRN, ventrolateral part of the periaqueductal gray and the midbrain tegmentum. In response to weak, long depolarizing current pulses, the 5-HT neurons displayed a slow and regular firing activity. The non-5-HT neurons fired at higher frequencies even when stronger current was injected. Some other differences in electrophysiological properties were also observed between the 5-HT-immunoreactive spiny projection neurons and the 5-HT-immunonegative aspiny projection neurons.
Dysfunction of serotonergic systems is thought to play an important role in a number of neurological and psychiatric disorders. Recent studies suggest that there is anatomical and functional diversity among serotonergic systems innervating forebrain systems involved in the control of physiologic and behavioral responses, including the control of emotional states.
Here, we highlight the methods that have been used to investigate the heterogeneity of serotonergic systems and review the evidence for the unique anatomical, hodological, and functional properties of topographically organized subpopulations of serotonergic neurons in the midbrain and pontine raphe complex.
The emerging understanding of the topographically organized synaptic regulation of brainstem serotonergic systems, the topography of the efferent projections of these systems, and their functional properties, should enable identification of novel therapeutic approaches to treatment of neurological and psychiatric conditions that are associated with dysregulation of serotonergic systems.
Previous studies have shown that amphetamine (AMPH) markedly activates dopaminergic projection areas, together with some important limbic nuclei. However, a global picture of the brain areas activated is lacking and the contribution of the dose of the drug and individual differences to this global brain activation is not known. In the present experiment, we studied in adult male rats the c-fos expression induced by two doses of AMPH (1.5 and 5 mg/kg sc) in a wide range of brain areas, and investigated the possible contribution of novelty-induced activity and anxiety traits. AMPH administration increased Fos+ neurons in an important number of telencephalic, diencephalic and brainstem areas. Interestingly, the ventral tegmental area (VTA) and the dorsal raphe nucleus were activated by the drug, but c-fos expression was restricted to non-dopaminergic and non-serotoninergic neurons, those activated in the VTA being predominantly GABAergic. The use of the factorial analysis, which grouped the areas in function of the correlation between the number of Fos+ neurons observed in each area, revealed three main factors, probably reflecting activation of various relatively independent brain circuits: the first included medial prefrontal cortex regions, most dorsal and ventral striatal subregions and VTA; the second, raphe nuclei; and the third, the different subdivisions of the paraventricular nucleus of the hypothalamus. Other areas such as the central amygdala did not group around any factor. The finding that an important number of activated areas grouped around specific factors is suggestive of activation of partially independent brain circuits. Surprisingly, a minor contribution of novelty-induced activity and anxiety traits on brain activation induced by AMPH was found. It is possible that normal variability in these traits is poorly related to the effects of AMPH or that c-fos expression is not a good tool to reveal such differences.
The regional distribution of the serotonin uptake system was studied in rat brain using a specific polyclonal antibody raised against the putative extracellular loop between transmembrane domains 7 and 8 of the cloned rat serotonin transporter. Light microscope analysis with fluorescence and avidin-biotin-peroxidase techniques revealed a punctate staining as well as numerous labelled thin fibres, which exhibited accumulation of reaction end-product deposit over varicosities. These immunopositive processes were widely and heterogeneously distributed in the rat brain. High densities of immunoreactivity were seen within the caudate-putamen, amygdaloid complex, cortical areas, substantia nigra, ventral pallidum, Islands of Calleja, septal nuclei, interpeduncular nucleus, trigeminal motor nucleus and olfactory nuclei. We also found strong expression of serotonin transporter in the stratum oriens of area CA3 and, to a lesser extent, in the stratum oriens of CA1 and the stratum lacunosum molecular of CA1-CA3 regions of the hippocampus. Within the raphe nuclei, a moderate to high incidence of stained processes was observed, and immunopositive cell bodies were detected in the dorsal raphe nucleus. In addition, some immunoreactive fibres were present in the molecular and granular layers of the cerebellum as well as in the cochlear and olivary nuclei. In none of the regions analysed was evidence for glial staining obtained. The present immunocytochemical data reveal a widespread and heterogeneous distribution of the serotonin transporter in rat brain and suggest that serotoni transporter is preferentially sorted into axons, where it appears concentrated at varicosities and terminal boutons.
By using retrograde and anterograde transport of the B subunit of cholera toxin (CTb), we examined quantitatively the association fiber systems, i.e., the collaterals of pyramidal cell axons, that reciprocally connect both the rostral and the caudal parts of the piriform cortex (PC). Well-defined CTb injections were obtained in layers Ib or II-III of the rostral and the caudal parts of the PC. Using precision counting, we determined the proportion of cellular profiles in layers II and III that gave rise to association fibers and thus demonstrated a predominance of rostrocaudal fibers over the caudorostral ones. Our data also support a precise laminar organization of the PC in which the rostrocaudal fibers originated mainly from layer II and the caudorostral fibers primarily from layer III. Cholera toxin injections into layer Ib produced a peak of labeled profiles 2 mm from the site, indicating that a large proportion of the association fibers from layer II travel for at least 2 mm and then synapse in layer Ib. At either end of the PC, the association projections with respect to olfactory processing, propagation of the activity within the PC, and the possible role of intrinsic fibers in olfactory memory.
In order to determine the origin of the catecholamine innervation of the rat piriform cortex (PC), we combined retrograde transport of the B subunit of the cholera toxin (CTb) with tyrosine hydroxylase (TH) immunohistochemistry. A substantial number of CTb retrogradely labeled cells was found in the parabrachial pigmented, paranigral and interfascicular nuclei of the ventral tegmental area and the dorsal part of the locus coeruleus, whereas nearly no labeling was noted in the substantia nigra. Following TH immunohistochemistry on the same sections, most if not all of the CTb labeled cells were also TH immunoreactive. Occasional double-labeled cells were also observed in the anterior part of the raphe dorsal nucleus. As visualized with dopamine beta-hydroxylase, dopamine or TH immunohistochemistry, the noradrenaline fibers were homogeneously distributed whereas the dopamine fibers showed rostro-caudal and latero-medial differences. The distribution of TH fibers overlapped both patterns. Our report suggests that the heterogeneous distribution of the DA fibers could support a differential centrifugal modulation of the olfactory information processing throughout the PC.
In the present study, the reciprocal connections between the piriform cortex and the prefrontal areas are described on the basis of experiments using the anterograde and the retrograde transport of the cholera toxin B subunit (CTb). Following CTb injections placed in the anterior part of the piriform cortex, retrogradely labeled cells and anterogradely labeled fibers were mainly found in the ventrolateral and lateral orbital areas as well as in the anterior part of the agranular insular cortex. Following injections placed in the posterior part of the piriform cortex, the CTb labeling was primarily observed in the infralimbic area and the posterior part of the agranular insular cortex. Thus, we described a topographical organization of the direct reciprocal connections between the anterior and the posterior parts of the piriform cortex parts and some prefrontal areas. This could support a differential modulation of the olfactory processing along the rostrocaudal dimension of the piriform cortex.
The effect of age on the number of neurons in the nucleus centralis superior (NCS) was determined in 11 behaviorally tested rhesus monkeys of 7-32 years of age, There was a significant age-related decrease in both cell packing density and in the total number of neurons. This decrease in number of neurons appeared to effect two different populations of cell in the NCS, one of which corresponded in size to the serotonergic cells in this nucleus and the other to a smaller-sized cell. Comparisons of the changes in the cell packing density with behavioral testing, showed significant correlations with the overall test performance as well as with individual tests of memory function and of executive system functions. These findings suggest that neuronal loss in the NCS may play a significant role in mediating cognitive changes seen in normal aging.
The piriform cortex (PCx) is a phylogenetically old brain structure which presents characteristics of a content-addressable memory. Taking into account its particular anatomo-functional organization, we hypothesized that this cortex could behave rather as an assembly of different functional units than as a functionally homogeneous structure. This hypothesis was tested by using both anatomical and functional approaches. Immunohistological and tracing experiments demonstrated that both the connections of the PCx with the higher nervous centres, and its monoaminergic and cholinergic modulatory afferents exhibited a heterogeneous distribution. Then, optical monitoring of its neuronal activity with a voltage-sensitive dye pointed out that the PCx is a functionally heterogeneous structure. Electrical stimulations of the olfactory bulb showed that the inhibitory processes which control the cortical responsiveness were not identical in all the PCx area. Two different functional areas at least could be distinguished: in the ventromedial PCx, the afferent activity is privileged since the level of inhibition of disynaptic activation remained large during repetitive stimuli. Contrarily, in the posterior PCx, the disynaptic activity remained unchanged in response to successive stimulations and the responses of neighbouring sites were statistically more synchronized than in its anterior part. Moreover, a late depolarization wave was significantly larger in the posterior PCx. These data are in good agreement with the results provided by computational models of the PCx. In the future, theoretical and experimental investigations of this cortex will be useful for understanding olfactory information processing and as a model of brain functioning at the neocortical level as well.
The dorsal raphe nucleus (DR) harbours the largest single collection of serotonin (5-HT)-containing neurons in the brain but also comprises other types of chemospecific neurons. The aim of the present study was to characterise morphologically and immunohistochemically the DR in the squirrel monkey (Saimiri sciureus). The morphology of the DR 5-HT-immunoreactive (ir) neurons was analysed and their distribution compared to that of neurons displaying immunoreactivity for either tyrosine hydroxylase (TH), gamma-aminobutyric acid (GABA), substance P (SP), calbindin-D28k (CB), calretinin (CR) or parvalbumin (PV). The 5-HT-ir neurons were distributed in a highly heterogeneous manner throughout the rostrocaudal extent of the DR. The morphology and density of the 5-HT neurons were found to vary significantly in the major subdivisions of the primate DR, that is, the median, ventral, dorsal, ventrolateral, lateral and caudal subnuclei. Numerous SP-, GABA- and PV-ir neurons occurred in all six subnuclei of the DR. The distribution of SP-ir neurons was largely in register with that of 5-HT-ir neurons. Neurons expressing the other neuronal markers (TH, CB, CR) were not present in all six DR subnuclei and their distribution was either complementary to, or in register with, that of 5-HT-ir neurons. The median subnucleus was unique because it contained all the different types of chemospecific neurons. This study has revealed that the primate DR is chemically highly heterogeneous, a finding that may explain the multifarious influence that this nucleus exerts upon various forebrain structures.
The evoked potential recorded in the rat piriform cortex in response to electrical stimulation of the olfactory bulb is composed of an early component occasionally followed by a late component (60-70 ms). We previously showed that the late component occurrence was enhanced following an olfactory learning. In the present study carried out in naive rats, we investigated the precise conditions of induction of this late component, and its spatiotemporal distribution along the olfactory pathways. In the anaesthetized rat, a stimulating electrode was implanted in the olfactory bulb. Four recording electrodes were positioned, respectively, in the olfactory bulb, the anterior and posterior parts of the piriform cortex, and the entorhinal cortex. Simultaneous recording of signals evoked in the four sampled structures in response to stimulation of the olfactory bulb revealed that the late component was detected in anterior and posterior piriform cortex as well as in entorhinal cortex, but not in the olfactory bulb. The late component occurred reliably for a narrow range of low intensities of stimulation delivered at frequencies not exceeding 1 Hz. Comparison of late component amplitude and latency across the different recorded sites showed that this component appeared first and with the greatest amplitude in the posterior piriform cortex. In addition to showing a functional dissociation between anterior and posterior parts of the piriform cortex, these data suggest that the posterior piriform cortex could be the locus of generation of this late high amplitude synchronized activity, which would then propagate to the neighbouring regions.
Studies on the neurobiology of cocaine abuse suggest that cocaine directly modifies the activity of dopamine neurons projecting from the dopamine-synthesizing cells of the ventral tegmental area to the nucleus accumbens. The repeated use of cocaine produces persistent adaptations within the mesocorticolimbic system and the resulting changes in monoamine neurotransmission may lead to behavioral sensitization. The present series of experiments sought to determine the effects of the repeated, intermittent challenge that took place two days after discontinuation of the pretreatment regimen; (ii) the ex vivo levels of biogenic monoamines, choline and acetylcholine in the nucleus accumbens, the dorsolateral caudate nucleus, as well as the anterior cingulate, frontal motor, frontal somatosensory and pyriform cortices; and (iii) the degree of neurochemical relationship between the left and right hemispheres. The repeated administration of cocaine produced sensitized behavioral responses to a subsequent challenge. Neurochemical correlates of repeated cocaine administration were observed at the cortical level and included a significant decrease in serotonin levels in the left anterior cingulate and pyriform cortices and an increase in dopamine metabolism in the left pyriform cortex. Furthermore, a shift in the interhemispheric coupling coefficient matrix for dopamine neurotransmission was observed in both the pyriform cortex and nucleus accumbens of cocaine-sensitized animals suggesting that, in these structures, the two hemispheres are operating independently. These results demonstrate that cocaine produces alterations in specific dopaminergic and serotonergic pathways that arise from the mesencephalon and project towards both the anterior cingulate and pyriform cortices.
The ascending serotonergic projections are derived largely from the midbrain median and dorsal raphe nuclei, and contribute to the regulation of many behavioral and physiological systems. Serotonergic innervation of the hamster circadian system has been shown to be substantially different from earlier results obtained with other methods and species. The present study was conducted to determine whether similar differences are observed in other brain regions. Ascending projections from the hamster dorsal or median raphe were identified using an anterograde tracer, Phaseolus vulgans leucoagglutinin, injected by iontophoresis into each nucleus. Brains were processed for tracer immunoreactivity, and drawings were made of the median raphe and dorsal raphe efferent projection patterns. The efferents were also compared to the distribution of normal serotonergic innervation of the hamster midbrain and forebrain. The results show widespread, overlapping projection patterns from both the median and dorsal raphe, with innervation generally greater from the dorsal raphe. In several brain regions, including parts of the pretectum, lateral geniculate and basal forebrain, nuclei are innervated by the dorsal, but not the median, raphe. The hypothalamic suprachiasmatic nucleus is the only site innervated exclusively by the median and not by the dorsal raphe. The pattern of normal serotonin fiber and terminal distribution is generally more robust than would be inferred from the anterograde tracer material. However, there is good qualitative similarity between the two sets of data. The oculomotor nucleus and the medial habenula are unusual to the extent that each has a moderately dense serotonin terminal plexus, although neither receives innervation from the median or dorsal raphe. In contrast, the centrolateral thalamic nucleus and lateral habenula have little serotonergic innervation, but receive substantial other neural input from the raphe nuclei. The normal serotonergic innervation of the hamster brain is similar to that in the rat, although there are exceptions. The anterograde tracing of ascending median or dorsal raphe projections reveals a high, but imperfect, degree of correspondence with the serotonin innervation data, and with data from rats derived from immunohistochemical and autoradiographic tract-tracing techniques.
Over the last 20 years, the choice of neural tracers has increased manyfold, and includes newly introduced anterograde tracers that allow quantitation of single-axon morphologies, and retrograde tracers that can be combined with intracellular fills for the study of dendritic arbors of neurons which have a specific projection pattern. The combination of several different tracers now permits the comparison of multiple connections in the same animal, both quantitatively and qualitatively. Moreover, the finding of new virus strains, which infect neural cells without killing them, provides a tool for studying multisynaptic connections that participate in a circuit. In this paper, the labeling characteristics, mechanism of transport and advantages/disadvantages of use are discussed for the following recently introduced neural tracers: carbocyanine dyes, fluorescent latex microspheres, fluorescent dextrans, biocytin, dextran amines, Phaseolus vulgaris leucoagglutinin, cholera toxin and viruses. We also suggest the choice of specific tracers, depending on the experimental animal, age and type of connection to be studied, and discuss quantitative methodologies.
The primary goals of this study were to: 1) examine the distribution of neurons within the dorsal raphe (DR) nucleus that project to cortical and subcortical sites along the trigeminal somatosensory pathway in rat; 2) determine the extent to which different regions within this ascending sensory system receive collateral projections from the same DR neuron; and 3) identify the putative transmitters contained within these DR projection neurons. Long-Evans hooded rats received pressure injections of various combinations of retrograde fluorescent tracers; into the whisker-related regions of the primary somatosensory cortex (barrel field cortex [BC]), ventral posterior medial thalamus (VPM), and principal nucleus of the trigeminal complex (PrV). The distribution of retrogradely labeled neurons within the DR was examined by fluorescence microscopy. The major finding was that cortically projecting neurons were located within the midline regions of the rostral portion of the DR, whereas cells projecting to subcortical trigeminal somatosensory structures were distributed bilaterally in the lateral wing regions of the DR as well as in the midline portions of the nucleus. Single neurons that send axon collaterals to multiple cortical and subcortical trigeminal somatosensory targets were observed in the dorsomedian and ventromedian regions of the DR. DR neurons that projected to cortical and subcortical sites contained serotonin but not tyrosine hydroxylase, the marker enzyme for catecholamine transmitters. Taken together, these findings provide further evidence of neurochemical specificity and functional anatomical organization within the DR efferent projection system.
The present study was undertaken to further characterize and subdivide the rhinal cortex (insular and perirhinal areas) in the hedgehog tenrec (Echinops telfairi), a placental mammal with a rather low encephalisation index. Injections of wheat germ agglutinin-horseradish peroxidase into the dorsolateral pontine tegmentum revealed a prominent layer 1 projection to several rhinal target areas, while the rhinal cortex only stained weakly for the calcitonin gene-related peptide. Among the regions retrogradely labeled following tracer injections into the rhinal cortex, the parabrachial nucleus was considered the main origin of the tegmento-cortical projection. This conclusion was based on the circumscribed pattern of termination, as well as the differences noted between the pattern of anterograde labeling and the pattern obtained by thyrosine hydroxylase immunohistochemistry. The tracer injections into the dorsolateral tegmentum also revealed numerous retrogradely labeled cells in the layer 5 of the dorsomedial frontal cortex. In contrast, the rhinal cortex only showed few labeled cells and most of these cells were located in the layer 6/7. A comparison with other species indicates that the tenrec's parabrachial nucleus gives rise to the most extensive cortical projections but receives the least prominent input from the lateral cerebral hemisphere. The layer 6/7 projection may be a common mammalian feature but it is overshadowed by the layer 5 projection in higher mammals.
It has been argued that exposure to inescapable shock produces later behavioral changes such as poor shuttle box escape learning because it leads to the conditioning of intense fear, which later transfers to the shuttle box test situation and interferes with escape. Both fear, as assessed by freezing, and escape were measured in Sprague-Dawley rats 24 hrs after exposure to inescapable shock. Lesions of the basolateral region and central nucleus of the amygdala eliminated the fear that transfers to the shuttle box after inescapable shock, as well as the fear conditioned in the shuttle box by the shuttle box shocks. However, the amygdala lesions did not reduce the learning deficit produced by inescapable shock. In contrast, dorsal raphe nucleus lesions did not reduce the fear that transfers to the shuttle box after inescapable shock, but eliminated the enhanced fear conditioning in the shuttle box as well as the escape deficit. The implications of these results for the role of fear and anxiety in mediating inescapable shock effects are discussed. (PsycINFO Database Record (c) 2012 APA, all rights reserved)
A new neuroanatomical method for tracing connections in the central nervous system based on the anterograde axonal transport of the kidney bean lectin,Phaseolus vulgaris-leucoagglutinin (PHA-L) is described. The method, for which a detailed protocol is presented, offers several advantages over present techniques. First, when the lectin is delivered iontophoretically, PHA-L injection sites as small as 50–200 μm in diameter can be produced, and are clearly demarcated since the neurons within the labeled zone are completely filled. Second, many morphological features of such filled neurons are clearly demonstrated including their cell bodies, axons, dendritic arbors and even dendritic spines. Third, there is some evidence to suggest that only the neurons at the injection site that are filled transport demonstrable amounts of the tracer, raising the possibility that the effective injection site can be defined quite precisely. Fourth, even with the most restricted injections, the morphology of the labeled axons and axon terminals is clearly demonstrated; this includes boutons en passant, fine collateral branches, and various terminal specialization, all of which can be visualized as well as in the best rapid Golgi preparations. Fifth, when introduced iontophoretically, PHA-L appears to be transported preferentially in the anterograde direction; only rarely is it transported retrogradely. Sixth, PHA-L does not appear to be taken up and transported effectively by fibers of passage. Seventh, there is no discernible degradation of the transported PHA-L with survival times of up to 17 days. Finally, since the transported marker can be demonstrated with either peroxidase or fluorescent antibody techniques, it may be used in conjunction with other neuroanatomical methods. For example, double anterograde labeling experiments can be done using the autoradiographic method along with immunoperoxidase localization of PHA-L, and the retrogradely transported fluorescent dyes can be visualized in the same tissue sections as PHA-L localized with immunofluorescence techniques.
In this report, we demonstrate that cholera-toxin B subunit (CTb) is a very sensitive retrograde tracer in the central nervous system when recognized by streptavidin-peroxidase immunohistochemistry. We further show that: (1) injection of a small volume of CTb gives rise to small sharply defined injection sites limited to the cell group of interest associated with the labeling of all the known afferent projections, (2) CTb is taken up, and anterogradely as well as retrogradely transported in damaged but not intact fibers of passage, (3) CTb can be applied iontophoretically, allowing us to study the afferents to small cell groups without any evidence of tissue necrosis in the sites and therefore without artefactual labeling due to uptake by damaged fibers of passage, (4) the use of 4% paraformaldehyde fixative ideally suited for the preservation of most neural antigens, the addition of a 48 h colchicine treatment and the development of a double immunohistochemical method allow the biochemical characterization of the cell of origin of particular pathways in the CNS, (5) CTb is also anterogradely transported with an extensive filling of axons and axon terminals and thereby opens up the possibility of identifying simultaneously the afferents as well as the efferents of the group of cells studied and finally (6) the very long conservation of the preparation, the possibility of counterstaining it and of making camera lucida drawings allow easy and precise localization of the retrogradely labeled cells.
The effects of p-chloroamphetamine (PCA), a serotonin (5-HT) releaser, on acquisition and retention were examined in male Sprague-Dawley rats using a one-way active avoidance task. PCA was found to impair avoidance acquisition and retention in a time dependent fashion which followed closely the temporal effects of the drug on 5-HT release in the brain. Thus, the avoidance deficit is related to the rate of change and not to the steady-state levels of 5-HT. The 5-HT releasing effect was most pronounced in the forebrain with less effect in the spinal cord. PCA caused time dependent, regional variations in catecholamine content, which was not related to avoidance performance. The avoidance and retention impairment induced by PCA was blocked by the 5-HT synthesis inhibitor p-chlorophenylalanine (PCPA) but not by depletion of catecholamines with α-methyl-p-tyrosine (H44/68) or by the noradrenergic-selective neurotoxin DSP4. Analysis of the time dependent effects of PCA on monoamine content in saline or PCPA-treated rats indicated that the temporal effects of PCA on avoidance performance is not due to a direct or indirect action on catecholamine neurones. The present experiments support the view that the ascending serotonergic pathways play a significant role in aversive learning in the rat.
The association and commissural fiber systems arising in the olfactory cortical areas caudal to the olfactory peduncle (the piriform cortex, nucleus of the lateral olfactory tract, anterior cortical nucleus of the amygdala, periamygdaloid cortex and entorhinal cortex) have been studied utilizing horseradish peroxidase as both an anterograde and a retrograde axonal tracer.
In the piriform cortex two sublaminae within layer II (IIa and IIb) and layer III have been found to give rise to distinctly different projections. Retrograde cell labeling experiments indicate that the association fiber projection from layer IIb is predominantly caudally directed, while the projection from layer III is predominantly rostrally directed. Cells in layer IIa project heavily to areas both caudal and rostral to the piriform cortex. The commissural fibers from the piriform cortex are largely restricted in their origin to layer IIb of the anterior part of the piriform cortex and in their termination on the contralateral side to the posterior part of the piriform cortex and adjacent olfactory cortical areas. A projection to the olfactory bulb has also been found to arise from cells in layers IIb and III of the ipsilateral piriform cortex, but not in layer IIa.
In addition to those from the piriform cortex, association projections have also been found from other olfactory cortical areas. The nucleus of the lateral olfactory tract has a heavy bilateral projection to the medial part of the anterior piriform cortex and the lateral part of the olfactory tubercle (as well as a lighter projection to the olfactory bulb); both the anterior cortical nucleus of the amygdala and the periamygdaloid cortex project ipsilaterally to several olfactory cortical areas. The entorhinal cortex has been found to project to the medial parts of the olfactory tubercle and the olfactory peduncle. The olfactory tubercle is the only olfactory cortical area from which no association fiber systems (instrinsic or extrinsic) have been found to originate.
A broad topographic organization exists in the distribution of the fibers from several of the olfactory areas. This is most obvious in the anterior part of the olfactory cortex, in which fibers from the more rostral areas (the anterior olfactory nucleus and the anterior piriform cortex) terminate in regions near the lateral olfactory tract, while those from more caudal areas (the posterior piriform cortex and the entorhinal cortex) terminate in areas further removed, both laterally and medially, from the tract.
Projections to olfactory areas from the hypothalamus, thalamus, diagonal band, and biogenic amine cell groups have been briefly described.
Previous reports from this laboratory and elsewhere have provided evidence that the locus coeruleus (LC) and dorsal raphe (DR) nuclei are topographically organized with respect to their efferent targets. Whereas most of these previous studies have focused on relationships between these monoamine-containing brainstem nuclei and cerebral cortex, basal ganglia, and limbic structures, they have not systematically examined the distribution of LC and DR cells that project to multiple structures with common sensory or motor functions. The goal of the present study was to characterize and compare the distributions of LC and DR cells which project to different visual areas of the rat central nervous system. Long-Evans hooded rats received unilateral pressure injections of the retrograde tracer wheat germ agglutinin-horseradish peroxidase in either the dorsal lateral geniculate, ventral lateral geniculate, or lateral posterior nucleus of thalamus; superior colliculus, cortical area 17, cortical area 18a/b cerebellar vermis (lobules VI and VII); or paraflocculus. Transverse sections through the midbrain and pons were examined by light microscopy after performing routine tetramethyl benzidine histochemical procedures. For all cases studied, retrogradely labeled cells were observed throughout the rostrocaudal extent of the LC and DR; however, labeling patterns which were distinctive for different injection sites were noted in each of these brainstem nuclei. The major conclusion drawn from this work is that subsets of LC and DR cells which project to different target structures within the rat visual system are found in overlapping but not necessarily coextensive zones within these nuclei. These studies provide further evidence of a rough topographic ordering within both the LC and DR nuclei, as well as support a new hypothesis that the outputs from each of these nuclei are organized with respect to the sensory related functions of their efferent targets. © 1993 Wiley-Liss, Inc.
The present study was conducted to examine the spatial organization of dorsal (DR) and median (MR) raphe neurons that project to rostrocaudally aligned areas of the rat cerebral cortex. An additional goal was to determine if individual DR cells that send efferents to forelimb sensorimotor or visual regions of the neocortex also send axon collaterals to forelimb (crus II) or visual (paraflocculus) areas of the cerebellum. Long-Evans hooded rats received unilateral pressure injections of horseradish peroxidase (HRP) in either motor n = 4) or sensorimotor (n = 5) or visual (n = 4) cortex to determine the intranuclear location of DR and MR neurons that project to specific neocortical regions. Coronal sections (40-100 μm) through the pons and midbrain were examined by light microscopy after the tetramethyl benzidine reaction and neutral red counterstaining were carried out. The locations of retrogradely labeled cells were recorded relative to a threedimensional biological coordinate system maintained by a computer linked to the light microscope. For double labeling studies, unilateral injections of fast blue and nuclear yellow were made in paired motor (sensorimotor cortex and crus II of the lateral cerebellum) or visual (cortical area 17 and paraflocculus) areas of the CNS. Coronal tissue sections (35 μm) were collected on coverslips and examined on a Leitz fluorescence microscope (wavelength = 365 nm). DR neurons labeled from cerebrocortical injections of HRP were concentrated in the rostral two-thirds of the nucleus. HRP-filled neurons were distributed such that individual groups of neurons projecting to motor, sensorimotor, or visual cortex were aligned in a partially overlapping, rostral to caudal array. In the dorsoventral dimension, retrogradely labeled cells were clustered in three distinct groupings such that neurons projecting to the motor, sensorimotor, and visual areas were concentrated in dorsal, intermediate, and ventral portions of the DR nucleus, respectively. For all cases, the majority of HRP-filled cells were positioned along the midline or displaced to the side of the nucleus that was ipsilateral to the cortical injection site. A small number of retrogradely labeled neurons were observed in the MR following injections in the motor cortex. Computer-assisted reconstruction of th z neuroanatomical data facilitated the visualization of spatial relationships between groups of DR neocortical projection neurons. Twenty to 30% of the cells labeled in the DR after paired injections of fast blue and nuclear yellow in sensorimotor or visual areas of the neocortex and cerebellum were doubled labeled indicating that individual DR neurons can send axon collaterals to functionally analogous regions of the CNS. Overall, the results indicate that, within the DR, a topographic ordering exists with respect to rostrocaudally aligned terminal fields in the neocortex. In addition, a portion of the DR cerebrocortical projection neurons also send axon collaterals to specific regions of the cerebellar cortex. The intranuclear organization observed in this study suggests that neuronal activity in distinct regions of the DR nucleus may independently influence discrete populations of cerebrocortical cells. Furthermore, the possibility must now be considered that a substantial number of single DR neurons provide a common input via axon collerals to portions of the neocortical and cerebellar circuitry that receive afferent information related to the same sensory or motor function.
Neurotensin immunoreactive perikarya, fibers and nerve terminals, visualized by the indirect immunohistofluorescent method in colchicine-pretreated animals, are localized in many discrete regions of the rat brain stem. Cell body groups are found in the inner aspect of the substantia gelatinosa of the caudal trigeminal nuclear complex, the nucleus of the solitary tract, the parabrachial nuclei, the locus coeruleus, the dorsal raphénucleus, the periaqueductal gray matter, and the ventral tegmental area of Tsai. These areas of cell body density are accompanied by concentrations of fibers and terminals, while the occasional positive perikaryon noted in the dorsal cochlear nucleus is accompanied by only sparse fluorescent fiber/terminal patterns. Other brain stem regions, such as the floor of the fourth ventricle and aspects of the caudal ventrolateral reticular formation, possess substantial numbers of fibers and terminals that are not accompanied by cell bodies. Many aspects of this distribution coincide with the brain stem distribution of the enkephalin pentapeptides, though significant differences in localization are also evident. Interactions of neurotensin with other neurotransmitter candidates are also suggested by its presence in areas enriched in norepinephrine, dopamine, serotonin, and substance P. Certain neurotensin localizations suggest an association of the peptide with functional brain systems preferentially involving these regions. In particular periaqueductal gray and substantia gelatinosa neurotensin synapses are plausible sites for the analgesia elicited after intercisternal injection of low doses of neurotensin.
Enkephalin immunoreactive perikarya, fibers and nerve terminals, visualized by the indirect immunohistofluorescent method in colchicine-pretreated animals, are localized in many discrete regions of the rat brain stem. These specific immunohistofluorescent patterns are similar after staining with selective primary antisera directed against either methione-enkephalin or leucine-enkephalin. Cell bodies are found in the substantia gelatinosa and interpolaris zones of the trigeminal nuclear complex, the nucleus of the solitary tract, in the vicinity of the nucleus raphe´magnus, in the dorsal cochlear, medial vestibular, and paraolivary nuclei and, dorsal to this last region, in the parabrachial nuclei and the dorsal tegmental nucleus of Gudden, in the periaqueductal gray matter and interpeduncular nucleus and along the borders of the lateral lemniscus and medial geniculate. In some areas, such as the parabrachial region, nucleus of the solitary tract and substantia gelatinosa of the trigeminal nucleus, these perikarya are associated with densities of fibers and terminals. Other regions, such as the dorsal cochlear nucleus and the vicinity of the nucleus raphe´magnus, contain cell bodies associated with low densities of processes and terminals. In still other nuclei, such as the nucleus of the facial nerve and the locus coeruleus, fiber and terminal densities without associated cell bodies are evident. Many of these enkephalin localizations can be rationalized on the basis of known actions of opiate drugs and the brain stem distribution of opiate receptors.
The serotonin-containing nucleus raphe dorsalis (RD) of the cat contains numerous leucine-enkephalin immunoreactive cells, throughout its rostral-caudal extent. The distribution of the enkephalin neurons closely parallels the cytoarchitectural boundaries of the RD, as described in previous Nissl preparations. Enkephalin perikarya are most numerous along the midline of the RD, but also extend ventrally, into the dorsal portion of the nucleus centralis superior, and laterally, into the ‘wings’ of the rostral RD, at the level of the IV nucleus. The possible contribution of these enkephalin cells to endogenous pain control systems is discussed.
The localization and distribution of serotonin (5-hydroxytryptamine, 5-HT) has been studied with the indirect immunofluorescence technique using a highly specific and well-characterized antibody to 5-HT. In neuron systems 5-HT was found to be primarily present with a distribution similar to that observed in basic mappings carried out with the formaldehyde-induced fluorescence method. In addition to the nine areas originally described, several other areas in the mesencephalon and rhombencephalon appeared to contain widely distributed 5-HT-positive perikarya. In the median eminence 5-HT fluorescent mast cells could be visualized. No 5-HT-positive nerve cell bodies could be observed either in the telencephalon or diencephalon.
Formaldehyde-induced fluorescence histochemistry, by the use of the improved filter system, revealed that dorsal and median raphe nuclei contain in varying numbers blue-green fluorescent neurons among a large number of yellow fluorescent serotonin-containing neurons. Pharmacological treatments indicated the presence of catecholamine in the blue-green fluorescent neurons. Moreover, microspectrofluorometry identified the catecholamine therein as dopamine, based on an excitation maximum shift characteristic of dopamine fluorescence caused by HCl vapor. Significance of these dopaminergic neurons in the raphe nuclei is suggested.
Dorsal raphe unit activity in freely moving cats showed a slow, rhythmic discharge rate during quiet waking (X=2.82 +/- 0.17 spikes/sec), and displayed a strong positive correlation with level of behavioral arousal. Presentation of an auditory stimulus during quiet waking resulted in significant increases in unit activity of 112% and 39% during the first sec and first 10 sec after the stimulus, respectively. This effect rapidly habituated with repeated stimulus presentations. During active waking, unit activity was significantly increased by 22% as compared to quiet waking, but there was no correlation between unit activity and gross body movements. Raphe unit activity showed a significant decrease of 17% during drowsiness (first appearance of EEG synchronization) as compared to quiet waking, and then progressive decreases during the early (--34%), middle (--52%) and late (--68%) phases of slow wave sleep. During all phases of slow wave sleep, the occurrence of sleep spindles was frequently associated with a transitory decrease in unit activity. The discharge rate would typically decrease during the few seconds immediately preceding the spindle, remains at this low level during the occurrence of the spindle, and then increase immediately after the spindle. Raphe unit activity showed decreases of 81% during pre-REM (the 60 sec immediately before REM onset) and 98% during REM, as compared to quiet waking. Unit activity reappeared 3.2 sec before the end of REM, with significant increases in unit activity of 83% and 17% during the first sec and first 10 sec of unit activity, respectively, as compared to quiet waking. The results of these studies are discussed in relation to the hypothesis that serotonin may play a modulatory, rather than mediative, role in behavioral and physiological processes.
The cortical projections of the brain stem were investigated in detail in the cat by means of the horseradish peroxidase (HRP) retrograde axonal transport. Most of the cells providing ascending fibers to the neocortex were located in the pons (locus coeruleus and related structures, central gray substance, dorsal tegmental nucleus, raphe nuclei, reticular nuclei); labeled neurons were also identified in the mesencephalon, mainly in the periaqueductal gray and in the nucleus linearis rostralis. These projections, and particularly the pontine fibers, were diffusely distributed throughout the cerebral cortex.
The results are compared with the data previously obtained by the use of anterograde and retrograde tracing techniques.
The structure and connections of areas within the olfactory peduncle (anterior olfactory nucleus and tenia tecta) have been examined. The anterior olfactory nucleus has been divided into external, lateral, dorsal, medial, and ventro‐posterior parts. In spite of the term nucleus which is applied to these areas, all of them contain pyramidal‐type cells with apical and basal dendrites oriented normal to the surface, and are essentially cortical in organization. Experiments utilizing retrograde and anterograde axonal transport of horseradish peroxidase (HRP) have demonstrated that each of these parts of the anterior olfactory nucleus possesses a unique pattern of afferent and efferent connections with other olfactory areas. All subdivisions have projections to both the ipsilateral and contralateral sides, although the ipsilateral projection of the pars externa (to the olfactory bulb) is extremely light. Interestingly, crossed projections are in each case directed predominantly to areas adjacent to the homotopic areas.
Two primary subdivisions may also be distinguished in the tenia tecta: a dorsal part composed largely of tightly packed neurons which closely resemble the granule cells of the dentate gyrus (bushy apical but no basal dendrites) and a ventral part which contains predominantly pyramidal‐type cells. The connections of these two parts are also very different. The ventral tenia tecta receives substantial projections from the olfactory bulb, pars lateralis of the anterior olfactory nucleus, piriform cortex and lateral entorhinal area. It gives off a heavy return projection to the pars lateralis and lighter projections to the olfactory bulb, piriform cortex and olfactory tubercle. The dorsal tenia tecta receives a heavy projection from the piriform cortex, but none from the olfactory bulb. A few cells in the dorsal tenia tecta are retrogradely labeled from HRP injections into the medial aspect of the olfactory peduncle (involving the ventral tenia tecta and adjacent areas), but none are labeled from the other olfactory areas that have been injected.
An area on the dorsal aspect of the olfactory peduncle that differs significantly from the anterior olfactory nucleus, tenia tecta and piriform cortex in terms of its connections and cytoarchitecture has been termed the dorsal peduncular cortex. The most striking feature of this area is its very heavy reciprocal connection with the entorhinal cortex, although it is also reciprocally connected with the olfactory bulb and piriform cortex and projects to the olfactory tubercle.
Cells in layer I of the medial and ventral aspects of the olfactory peduncle have been retrogradely labeled from HRP injections into the olfactory tubercle and lateral hypothalamic area. These cells overlie the ventral tenia tecta, medial part of the anterior piriform cortex and pars ventro‐posterior and pars lateralis of the anterior olfactory nucleus, but do not appear to be distributed in relation to the cytoarchitectonic boundaries.
Possible functional roles of the areas within the olfactory peduncle have been discussed.
Stereotaxic injections of [14C]leucine were made in nulei raphe centralis superior, raphe dorsalis, raphe magnus and raphe pontis of the cat. The organization of the regional connections was outlined in a stereotaxic atlas using the autoradiographic tracing method: the majority of the ascending pathways from the rostral raphe nuclei are directed mainly through a ventrolateral bundle via the ventral tegmental area of Tsai, with some lateral extensions to the substantia nigra, and then through the fields of Forel and the zona incerta. More rostrally the fibers are joined to the medial forebrain bundle through the hypothalamic region up to the preoptic area or the diagonal band of Broca. Multiple divisions leave this tract towards the epithalamic or the intralaminar thalamic nuclei, the stria terminalis, the septum, the capsula interna and the ansa lenticularis. The bulk of the rostral projections terminates in the frontal lobe, while some labeling is scarcely distributed throughout the rest of the neocortex. The projections of nucleus (n.) raphe centralis superior are specifically associated with the n. interpeduncularis, the mammillary bodies and the hippocampal formation while the n. raphe dorsalis innervates selectively the lateral geniculate bodies, striatus, piriform lobes, olfactory bulb and amygdala. The rest of the ascending fibers form the centrolateral or the dorsal ascending tracts radiating either in the reticular mesencephalic formation or in the periventricular gray matter. On the contrary there are heavy descending projections from n. raphe centralis superior which distribute to the main nuclei of the brain stem, the central gray matter and the cerebellum. The ascending projections form the caudal raphe nuclei are much less dense. They disseminate mainly in the colliculus superior, the pretectum, the nucleus of the posterior commissure, the preoculomotor complex and the intralaminar nuclei of the thalamus. From n. raphe pontis, a dense labeling is selectively localized at the n. paraventricularis hypothalami with some rostral extensions to limbic areas. Diffuse caudal and rostral projections from both nuclei are observed in the mesencephalic, pontobulbar reticular formation and the cerebellum. The main differences come from the specific localization of their descending bulbospinal tracts inside the lateroventral funiculus of the spinal cervical cord.
The present study investigated the distribution of serotonergic and non-serotonergic raphe neurons with direct projections to the visual cortex. The study employed the WGA-apoHRP-Au retrograde transport technique combined with 5-HT immunohistochemical staining. Retrogradely labeled cells were observed in the dorsal raphe nucleus, the median raphe nucleus, and in the B9 and B6 cell groups. One notable finding was the great number of retrogradely labeled, non-5-HT immunoreactive cells. The average percentages of such cells in the various raphe regions were as follows: DR: 52% (n = 401); MR: 35% (n = 311); B9: 24% (n = 129); B6: 95% (n = 200). The present study demonstrated the presence of a significant proportion of non-serotonergic raphe region neurons projecting to the primary visual cortex in the rat. It is suggested that these neurons may complement the aminergic neurons as part of the ascending system which controls the functions of the visual cortex.
Ascending projections from the dorsal raphe nucleus (DR) were examined in the rat by using the anterograde anatomical tracer, Phaseolus vulgaris leucoagglutinin (PHA-L). The majority of labeled fibers from the DR ascended through the forebrain within the medial forebrain bundle. DR fibers were found to terminate heavily in several subcortical as well as cortical sites.
The following subcortical nuclei receive dense projections from the DR: ventral regions of the midbrain central gray including the ‘supraoculomotor central gray’ region, the ventral tegmental area, the substantia nigra-pars compacta, midline and intralaminar nuclei of the thalamus including the posterior paraventricular, the parafascicular, reuniens, rhomboid, intermediodorsal/mediodorsal, and central medial thalamic nuclei, the central, lateral and basolateral nuclei of the amygdala, posteromedial regions of the striatum, the bed nucleus of the stria terminalis, the lateral septal nucleus, the lateral preoptic area, the substantia innominata, the magnocellular preoptic nucleus, the endopiriform nucleus, and the ventral pallidum. The following subcortical nuclei receive moderately dense projections from the DR: the median raphe nucleus, the midbrain reticular formation, the cuneiform/pedunculopontine tegmental area, the retrorubral nucleus, the supramammillary nucleus, the lateral hypothalamus, the paracentral and central lateral intralaminar nuclei of the thalamus, the globus pallidus, the medial preoptic area, the vertical and horizontal limbs of the diagonal band nuclei, the claustrum, the nucleus accumbens, and the olfactory tubercle.
The piriform, insular and frontal cortices receive dense projections from the DR; the occipital, entorhinal, perirhinal, frontal orbital, anterior cingulate, and infralimbic cortices, as well as the hippocampal formation, receive moderately dense projections from the DR.
Some notable differences were observed in projections from the caudal DR and the rostral DR. For example, the hippocampal formation receives moderately dense projections from the caudal DR and essentially none from the rostral DR. On the other hand, virtually all neocortical regions receive significantly denser projections from the rostral than from the caudal DR.
The present results demonstrate that dorsal raphe fibers project significantly throughout widespread regions of the midbrain and forebrain.
Using a double-immunostaining technique with cholera toxin (CT) as a retrograde tracer, the authors examined the cells of origin and the histochemical nature of lower brainstem afferents to the cat posterior hypothalamus. The posterior hypothalamus, in particular the lateral hypothalamic area, receives substantial afferent projections from: substantia nigra, peripeduncular nucleus, ventral tegmental area, periaqueductal grey, mesencephalic reticular formation, peribrachial region including the locus coeruleus complex, rostral raphe nuclei and the rostral part of the nucleus magnus. In addition, a moderate number of retrogradely labeled neurons was found in: Edinger-Westphal nucleus, nucleus reticularis pontis oralis, nucleus reticularis magnocellularis, caudal lateral bulbar reticular formation around the nucleus ambiguus and lateral reticular nucleus and the nucleus of the solitary tract. The posterior hypothalamus receives: 1) dopaminergic inputs from A8, A9 and A10 cell groups; 2) noradrenergic inputs from A6 and A7 pontine, as well as A1 and A2 bulbar cell groups; 3) adrenergic inputs from C1 cell group in the caudal medulla; 4) serotoninergic inputs from the rostral raphe nuclei (B6, B7 and B8 cell groups); 5) cholinergic inputs from the peribrachial region of the dorsal pontine tegmentum as well as from the nucleus reticularis magnocellularis of the medulla; 6) peptidergic inputs such as methionine-enkephalin, substance P, corticotropin-releasing factor and galanin that originate mainly in the mesencephalic periaqueductal grey, the dorsal raphe nucleus and the peribrachial region of the dorsal pontine tegmentum.
In a slice preparation of rat piriform cortex, both intracellular and extracellular techniques were used to examine the pharmacological and electrophysiological actions of serotonin (5-HT). Bath application of 5-HT resulted in either depolarization (57%), hyperpolarization (34%) or no change (9%) in membrane potential of cells in the pyramidal cell layer (layer II) of piriform cortex. Additionally, when KCl-containing electrodes were used, 5-HT induced an increase in depolarizing synaptic potentials in 41% of these cells. It was concluded that these potentials were reverse inhibitory post-synaptic potentials (IPSPs) because they were blocked by bicuculline and tetrodotoxin. The induction of IPSPs by 5-HT was blocked by the 5-HT2-selective antagonist ritanserin. By recording extracellularly in the presence of 5-HT, a group of 5-HT-activated, putative interneurons was found at the border of layers II and III of piriform cortex, 5-HT but not norepinephrine activation was blocked by ritanserin. The actions of 5-HT were mimicked by the 5-HT2 agonist alpha-methyl-5-HT; the 5-HT2 partial agonist, 2,5-dimethoxy-4-methyl-amphetamine had a small agonist action of its own and blunted the effect of 5-HT. Activation of a larger group of putative interneurons by the more universal excitant N-methyl-D-aspartate showed that the 5-HT-activated interneurons represented 23% of the interneurons located on the border between layers II and III. We conclude that 5-HT induces IPSPs in layer II pyramidal cells by activating a subpopulation of interneurons at the border of layers II and III of piriform cortex.
The regional distribution and cellular localization of mRNA coding for the serotonin 1C receptor were investigated in tissue sections of mouse and rat brain by in situ hybridization histochemistry. Several³²P-labelled riboprobes derived from mouse genomic clones were used. The serotonin 1C receptor binding sites were visualized autoradiographically and quantified using [³H]mesulergine as ligand, in the presence of spiperone to block serotonin 1C receptors. Strong hybridization signal was observed in the choroid plexus of all brain ventricles. High levels of hybridization were also seen in the anterior olfactory nucleus, pyriform cortex, amygdala, some thalamic nuclei, especially the lateral habenula, the CA3 area of the hippocampal formation, the cingulate cortex, some components of the basal ganglia and associated areas, particularly the nucleus subthalamicus and the substantia nigra. The midbrain and brainstem showed moderate levels of hybridization. The distribution of the serotonin 1C receptor mRNA corresponded well to that of the serotonin 1C receptors. The highest levels of the serotonin 1C receptor binding were observed in the choroid plexus. In addition, significant levels of the serotonin 1C receptor binding were seen in the anterior olfactory nucleus, pyriform cortex, nucleus accumbens, ventral aspects of the striatum, paratenial and paracentral thalamic nuclei, amygdaloid body and substantia nigra pars reticulata. The cingulate and retrosplenial cortices as well as the caudal aspects of the hippocampus (CA3) were also labelled. Binding in brainstem and medulla was low and homogeneously distributed. No significant binding was seen in the habenular and subthalamic nuclei. Similar findings were obtained in rat brain.
The catecholamines noradrenaline (NA), dopamine (DA), adrenaline (AD), the indoleamine 5-hydroxytryptamine (5-HT; serotonin), as well as some of their major metabolites were assayed by high-performance liquid chromatography (HPLC) with electrochemical detection, in four well-defined areas of the rat cerebral cortex: anterior cingulate (CIN;Cg1 and Cg3), piriform and entorhinal (PiEn), hind-limb primary somatosensory (SSC;HL) and primary visual (VIS; Oc1M and Oc1B). The concentrations of NA and that of its main metabolite 3-methoxy-4-hydroxyphenylglycol were highest in PiEn, had intermediate values in CIN and were lowest for SSC and VIS cortices. The DA levels were also highest in PiEn, intermediate in CIN, while the lowest values were in SSC and VIS cortices. The different DA/NA ratios support the hypothesis that they are indeed independent neurotransmitters. In addition, the levels of 3,4-dihydroxyphenylacetic acid, homovanillic acid and 3-methoxytyramine paralleled the distribution of DA, thus confirming the presence of release sites, even in regions in which the low levels of this catecholamine could be interpreted simply as the precursor of NA. Traces of AD were detected in all the regions examined. The 5-HT contents, as well as that of its precursor 5-hydroxy-1-tryptophan and that of its metabolite 5-hydroxyindole-3-acetic acid were also found to be non-homogenous, with the highest levels measured in the PiEn and CIN regions.
The existence of a dopaminergic projection from the nucleus raphe dorsalis (RD) to the prefrontal cortex of the rat was demonstrated by combining immunohistochemistry with retrograde tracing. Injection of horseradish peroxidase or Fluoro-gold into the medial prefrontal cortex resulted in the appearance of retrogradely labelled neurons in the RD and in other brain regions. Some of these retrogradely labelled RD neurons were shown to be immunoreactive for dopamine. These data support the view that these dopaminergic RD neurons represent an extension of the A10 cell group.
Using immunohistochemical methods with antibodies specific to tyrosine hydroxylase, we examined the distribution of dopaminergic cells in the dorsal and median raphe nucleus of the rat brain. Although dopamine-containing cell bodies were previously thought to be almost exclusively confined to the substantia nigra pars compacta, ventral tegmental area, and tuberoinfundibular system, we found numerous cell bodies which stained for tyrosine hydroxylase in the dorsal and median raphe nuclei.
Glutamate and aspartate are putative excitatory neurotransmitters in the central nervous system. The present study utilized novel monoclonal antibodies against fixative-modified glutamate and aspartate and polyclonal antisera against the amino acid synthesizing enzymes, glutaminase and aspartate aminotransferase, to analyze the distribution of these amino acids in the rodent midbrain periaqueductal gray. Glutamate-, aspartate-, glutaminase- and aspartate aminotransferase-like immunoreactive neurons, fibers and processes are present throughout the rostrocaudal length of the periaqueductal gray. Glutamate- and glutaminase-like immunoreactive neurons displayed a similar homogeneous pattern of distribution, being localized predominantly to the lateral and dorsal subdivisions of the periaqueductal gray. Co-localization experiments suggest that glutamate and glutaminase are in fact co-contained within the same PAG neurons. Aspartate aminotransferase-like immunoreactive neurons were distributed in a pattern similar to glutamate and glutaminase with the exception that fewer cells were stained in the dorsocaudal and the rostral third of the PAG. Aspartate-like immunoreactive neurons were less numerous than glutamate-like immunoreactive cells and were located in the lateral aspect of the PAG. These results demonstrate a specific and distinct distribution of glutamate and aspartate immunoreactive neurons and support recent data suggesting that glutamate and aspartate serve as excitatory neurotransmitters in the PAG.
The ability of the indoleamine serotonin (5-hydroxytryptamine; 5-HT) to alter membrane characteristics of neocortical neurons was analyzed using intracellular recording techniques. The present study demonstrates that 5-HT primarily depolarized 68% of cortical neurons probably by decreasing a resting K+ conductance, an effect blocked by the antagonists ritanserin and cinanserin and apparently mediated by 5-HT2 receptors. A hyperpolarization associated with an increased conductance state and insensitive to 5-HT2 antagonists was observed in 26% of the neurons and could be mimicked by the selective 5-HT1A agonist (+/-)-8-hydroxy-2-(di-N-propyl-amino)tetralin (8-OH-DPAT). Therefore cortical pyramidal neurons contain at least two distinct functional 5-HT receptors whose activation produces opposing effects on membrane potential and conductance.
The distribution of serotonin-2 (5-HT2) receptors in the rat brain was studied by light microscopic quantitative autoradiography. Receptors were labeled with four ligands: [3H]ketanserin, [3H]mesulergine, [3H]LSD and [3H]spiperone, which are reported to show high affinity for 5-HT2 receptors. Co-incubation with increasing concentrations of several well-known 5-HT2-selective drugs, such as pirenperone, cinanserin and ketanserin, resulted in an inhibition of the binding of the four 3H-labeled ligands to the same areas. However, all of them recognized, in addition to 5-HT2 sites, other populations of binding sites. Receptor densities were quantified by microdensitometry with the aid of a computer-assisted image-analysis system. Our results reveal a heterogeneous distribution of 5-HT2 receptor densities in the rat brain. Very high concentrations were localized in the claustrum, olfactory tubercle and layer IV of the neocortex. The anterior olfactory nucleus, piriform cortex and layer I of neocortex were also rich in 5-HT2 receptors. Intermediate concentrations of receptors were found in caudate putamen, nucleus accumbens, layer V of neocortex, ventral dentate gyrus and mammillary bodies. Areas containing only low concentrations of receptors included the thalamus, hippocampus, brainstem, medulla, cerebellum and spinal cord. The specificity of the different ligands used is discussed in terms of the other populations of sites recognized by them. The distribution of 5-HT2 receptors here reported is discussed in correlation with (a) the known distribution of serotoninergic terminals, (b) the specific anatomical systems and (c) the central effects reported to be mediated by 5-HT2-selective drugs.
Recordings were made in opossum prepyriform cortex of summed potential responses to orthodromic volleys in the lateral olfactory tract (LOT). Components of the responses were identified on the basis of time periods and potential peaks, consistent with previous investigations in other mammals. The distribution and time course of the components were determined over the surface of the cortex, and in depth. Effects due to changing stimulus intensity, conditioning by single and repetitive volleys, and level of anesthesia were analyzed. An initial fast transient (t) signals the LOT volley. An A1 wave component was identified with the monosynaptic EPSP generated in the apical dendrites of superficial pyramidal cells by the LOT volley. An S wave component appears to be generated by synchronous action potentials in these cells. A B1 component was identified with EPSPs generated in deep prepyriform neurons by axon collaterals from the superficial pyramidal cells. The axon collaterals of the superficial pyramidal cells appear to traverse a considerable distance before synaptically activating the deep cells. This is consistent with recent anatomical evidence. Alternating summed responses of prepyriform cortex to repetitive LOT volleys were recorded and analyzed. Alternation appears to be due to a rhythmical blockage of the response of the population of deep neurons. Large amplitude summed potentials are generated by antidromic volleys set up by shocks to subcortical efferent fibers or the anterior commissure. The initial portion of these waves appears to be related to antidromic action potentials in the superficial pyramidal cells.
Retrograde transport of a fluorescent dye was employed to study the projections from raphe nuclei to neocortex in the rat. The spatial distributions of labeled raphe cells were analyzed quantitatively to determine whether the nuclei are topographically organized with respect to different cortical targets. The dorsal raphe nucleus (DRN), exclusive of the lateral wing regions, has a predominantly (3:1) ipsilateral projection with decreasing numbers of cells projecting to frontal, parietal, and occipital cortex. Overlapping cell groups within the DRN project differentially to these three cortical areas: DRN cells innervating frontal cortex extend more rostrally and laterally than those to either parietal or occipital cortex. The medium raphe and B9 projections are bilaterally symmetric, with equal cell numbers projecting to frontal, parietal, and occipital cortex. The rostro-caudal distributions of cells that project to disparate cortical areas differ in B9 but not in MR. The percentage of cortically projecting cells that are serotonergic is 80% for the DRN, 60% in the MR and 33% in the B9 cell group. The dorsal raphe nucleus and the B9 cell group are organized heterogeneously, and overlapping sets of neurons project differentially upon particular areas of neocortex. In contrast, the median raphe nucleus projects uniformly upon the neocortex and does not exhibit topographic organization. The three rostral raphe nuclei (DR, MR and B9) are each organized according to different rules with regard to their efferent projections to cortex. The differential organization of the raphe nuclei suggests that groups of cells within these three raphe nuclei are likely to innervate different combinations of cortical targets and thus to have different functional effects.
The serotonergic innervation of the cerebral cortex in the rat has been studied by immunohistochemistry employing an antibody directed against the neurotransmitter, serotonin. The dorsal raphe, median raphe and B9 cell groups contain intensely labelled neuronal perikarya. Bundles of large diameter axons suggestive of fibers of passage are observed in successive sections as they ascend through the midbrain tegmentum, medial forebrain bundle, diagonal band and supracallosal stria en route to the cortex. In addition, a lateral pathway to the cerebral cortex traversing the ansa peduncularis is visualized. All regions of the cerebral cortex appear to be innervated by serotonergic axons which have a distinctive morphology: they are fine (0.1–0.5 μm), varicose, and extremely convoluted. Serotonergic axons of passage are thicker and comparatively straight. Throughout the lateral neocortex, as well as in the anterior cingulate cortex, serotonergic axons form a densely arborizing plexus through all cortical layers. Contrary to earlier reports, based on histofluorescence, describing a sparse innervation of the cortex with most of the fibers found in the molecular layer, the present study reveals that the innervation is relatively uniform across all cortical layers. In most of the cortex the density of serotonin-containing axons exceeds that of noradrenergic fibers. A distinctive and different pattern of serotonin innervation is found in the posterior cingulate cortex (cytoarchitectonic field RSg): the serotonergic axons are restricted largely to lamina I and III. A restricted laminar pattern also characterizes the innervation of the hippocampus; dense axonal plexuses occur in the outer rim of the dentate hilus and in the stratum lacunosum-moleculare. The serotonergic afferents to the cortex appear to have at least two different modes of distribution, a relatively uniform pattern in the anterior cingulate and the lateral neocortex and a restricted, laminar pattern in the posterior cingulate and the hippocampus.The density and extent of the serotonin innervation is such that the raphe neurons may contact every cell in the cortex. The widespread arborization of serotonin axons contrasts with the spatially restricted termination of thalamic afferents. The distribution of serotonin-containing fibers also differs substantially from the terminal patterns of noradrenergic and dopaminergic fibers. The differences in axonal morphology and distribution amongst the monoamine afferents reflect differences in their contributions to cortical circuitry. The present findings indicate that the serotonin-containing neurons may exert a profound and global, but not necessarily uniform, influence upon cortical function.
1. The nucleus raphe dorsalis (RD) and paragiganto-cellularis (PGL) of the cat contain both 5-HT and leucine-enkephalin (ENK) immunoreactive perikarya. 2. Using a sequential immunoperoxidase-immunofluorescence technique for the localization of ENK and 5-HT, respectively, it was demonstrated that some PGL and RD neurons contain both the indoleamine and the peptide. 3. The double labeled neurons in the DR were characteristically small, round cells predominantly located on the midline of the nucleus, dorsal to the medial longitudinal fasciculus. Numerous large 5-HT containing perikarya were also found in the DR, but these cells did not contain ENK immunoreactivity.
We have studied the localization of serotonin- and non-serotonin-containing cell bodies in the midbrain raphe nuclei that project to the entorhinal area and the hippocampal formation in the rat brain, using the technique of combined retrograde fluorescent tracing and immunohistochemistry on the same tissue section. The branching properties of these neurons were studied by retrograde double labelling using two fluorochromes which emit fluorescence with different spectral characteristics. After injections of granular blue or propidium iodide into the medial entorhinal area, retrogradely-labelled cells were found situated bilaterally in the caudal half of the dorsal raphe nucleus, the medial part of the median raphe and throughout the rostrocaudal extension of the nucleus reticularis tegmentipontis. Injections placed successively more laterally in the entorhinal area labelled progressively less cells contralaterally in the dorsal raphe and the reticular tegmental nucleus of the pons. After fluorochrome injections into the dorsal part of the hippocampal formation, retrogradely-labelled cells were found in the caudal part of the dorsal raphe, in the peripheral part of the median raphe and to a minor extent in the medial part of this nucleus, but not in the nucleus reticularis tegmentipontis. The experiments with double retrograde fluorescent tracing showed that the raphe nuclei do not send bilateral projections to the entorhinal area in spite of the fact that many of these cells are located contralateral to the injected hemisphere in single labelling experiments. Injections of the fluorochromes into the entorhinal area and hippocampal formation showed that at least 10% of the raphe cells project to both areas simultaneously. Analysis of sections incubated with antiserum to serotonin showed that a majority of the retrogradely-labelled versus serotonin-immunoreactive cells was found to vary within different parts of the individual raphe nuclei: the ventromedial part of the dorsal, the medial part of the median and the nucleus reticularis tegmentipontis being the highest. The findings indicate that both serotonin- and non-serotonin-containing neurons in the raphe innervate the hippocampal region, that these projections may be crossed but not bilateral, and that the same neuron in the raphe may influence the neural activity in the entorhinal area and the hippocampus simultaneously.
A retrograde labeling procedure utilizing fluorescent substances (Granular Blue, Nuclear Yellow and propidium iodide) was used to establish the presence of branching axons in the ascending raphe system of young rats. After injections in septum, medial thalamus and olfactory cortex, the number of double-labeled cells in various combinations was found to be relatively large in the dorsal raphe nucleus, whereas triple-labeled cells occurred more rarely. Each class of neurons, i.e. single-, double- and triple-labeled, were shown to have a predominant distribution within specific parts of the nucleus.
The serotoninergic nerve cell body population of nucleus raphe dorsalis (RD) was identified by radioautography following cerebroventricular instillation of tritiated serotonin ([3H]5-HT) in adult rats pretreated with a monoamine oxidase inhibitor. Series of histological sections taken throughout the midbrain and upper pons exhibited a similar distribution and number of labeled nerve cell bodies in RD after prolonged administration of either 10-5 or 10-4M [3H]5-HT or 10-4M [3H]5-HT and 10-3M nonradioactive noradrenaline. This allowed systematic mapping and quantification of serotoninergic nerve cell bodies at various levels of the RD. Their extrapolated total number averaged 11,500. Twice as many unreactive (nonserotoninergic) neurons were present within the same region. In electron microscope radioautographs, the labeled cells were usually larger (17.9 micrometer mean diameter) than their unlabeled congeners (13.1 micrometer), but stereological sampling of their perikarial organelle content failed to reveal any difference in cytoplasmic composition. Few [3H]5-HT-labeled axonal varicosities were observed in RD and none were found in close apposition or in synaptic junction with labeled nerve cell bodies, dendrites, or unreactive perikarya. A detailed statistical analysis of silver grain distribution in both labeled and "unlabeled" nerve cell bodies, indicated that in the former, but not in the latter, dense bodies had a relatively high affinity for [3H]5-HT. Mitochondria and the cytoplasmic membrane were the only other organelles to show higher labeling indices in labeled than in unlabeled cells. Other sites of [3H]5-HT localization could be ascribed to artefactitious cross-linkage of the tracer by the fixative, since they had the same relative affinity in the two cell populations. These results provide new insights into the morphology and cytofunctional properties of the 5-HT neurons of rat RD.
It has been established that nerve cell bodies of the nucleus raphe dorsalis (NRD) belong to ascending 5-hydroxytryptamine systems. These neurons could be modulated by GABAergic interneurons or interposed GABA neurons. A high glutamate decarboxylase (GAD) activity in the NRD and a specific high-affinity uptake mechanism for GABA suggest the presence of GABA synthesizing elements in the NRD. Anti-GAD antibodies were used by an immunocytochemical procedure to demonstrate the presence of GABAergic elements. Anti-GAD antibodies were previously tested in the cerebellum and substantia nigra. Large amounts of GAD-positive reaction product were observed in the cytoplasm of some neurons (fusiform, ovoid or multipolar) or appeared as punctate deposits apposed to dendrites, soma and dispersed in the neuropil of the NRD. At the electron microscopic level, GAD-positive reaction product was observed within the cytoplasm of numerous somata in sections from colchicine-treated rats. GAD-positive staining was observed in numerous fibers or axonal terminals and two types of morphologically different fibers could be distinguished. The first displays small clear vesicles and few large granular vesicles (LGV) (80-100 nm), the second displays only clear round vesicles (40-60 nm). After 5,7-dihydroxytryptamine treatment (a neurotoxic for 5-HT terminals), the immunocytochemical labeling is much decreased. Some reactive neurons are still dispersed in the nucleus but the fibers containing LGV are no longer observed. These results strongly suggest that some neuronal elements in the NRD are morphologically, pharmacologically and anatomically similar to 5-HT neurons described at this level. Such cell elements could possess a double GABA and 5-HT potentiality. If this is not the case, a population of GABA neurons could be sensitive to 5,7-DHT and so have the capacity to take up 5-HT. The other reactive elements, insensitive to 5,7-DHT, could represent the GABAergic interneurons postulated at this level. Numerous GAD positive fibers or axon terminals were observed in synaptic contact with dendrites, axons or soma of other neurons. The chemical nature of the neuronal postsynaptic elements remains unknown. These findings strongly support the hypothesis for GABA-mediated inhibition in the NRD.
The distribution of monoaminergic cell bodies in the brainstem of the cat has been examined with Falck‐Hillarp fluorescence histochemical technique. Quantitative determinations indicate that the cat brainstem contains about 60,300 indolaminergic (IA) cells. The majority of these (about 46,700, or 77.5%) are located within raphe nuclei. The largest number is contained within nucleus raphe dorsalis (RD), accounting for around 24,300 IA cells, while raphe pallidus (RP) holds about 8,000, raphe centralis superior (RCS) 7,400, raphe magnus (RM) 2,400, raphe obscurus (RO) 2,300, linearis intermedius (LI) 2,100, and the raphe pontis (RPo) only some 280 IA cells. The IA cells represent, however, only part of the neuronal population of raphe nuclei, which, in addition, hold varying numbers of other medium‐sized and small‐sized neurons. Thus, quantifications in Nissl‐stained material indicate that the IA cells make up about 70% of the medium‐sized cells in RD, 50% in RP, 35% in RCS and RO, 25% in LI, 15% in RM, and only 10% in RPo. The substantial numbers of small‐sized perikarya observed in all raphe nuclei may represent interneurons.
Significant numbers of IA cells were consistently located outside the raphe nuclei at all brainstem levels. In all, these amounted to approximately 13,600, or 22.5% of the total number of IA cells. Thus, IA cells occurred in the myelinated bundles, and sometimes in reticular formation, bordering the raphe nuclei; in the ventral brainstem forming a lateral extension from the ventral raphe (RP, RM, RPo, RCS, and LI) to the position of the rubrospinal bundle; in the periventricular gray and subjacent tegmentum of dorsal pons and caudal mesencephalon; in the locus coeruleus (LC) complex; around the motor trigeminal nucleus; caudal to the red nucleus; and in the interpeduncular and interfascicular nuclei. The wide distribution of IA cells leads to a considerable mixing with catecholaminergic (CA) cell groups.
Our observations on CA cell distribution are essentially in accordance with previous reports. Quantifications indicate that the LC complex contains about 9,150 CA cells, unilaterally. A previously unnoticed group of scattered CA cells was found in relation to the vestibular nuclei and extending dorsally toward the deep cerebellar nuclei.
Diverse physiological actions have been reported for 5-hydroxytryptamine (5-HT, serotonin) in the medial prefrontal cortex (MPFC) and the nucleus accumbens (Acb) suggesting that the 5-HT innervation of these forebrain areas may be derived from different populations of neurons. We examined this possibility by mapping the distribution of 5-HT-immunoreactive (ir) and non-5-HT-ir neurons containing retrograde labeling following injections of different tracers into both these target regions. The analysis was focused in the dorsal raphe nucleus (DRN) of the midbrain, since 5-HT pathways to the MPFC and Acb primarily originate from this area. Volume microinjections of the fluorescent retrograde tracer, Fluoro-Gold (FG), were placed into the MPFC and microinjections of cholera toxin B subunit coupled to 15 nm gold particles (CT-Au) were placed into the Acb of the same animal. Sections through the DRN containing retrogradely labeled neurons were further processed for immunofluorescent localization of 5-HT using a rhodamine marker. Neurons retrogradely labeled from the Acb were greater in number overall than those projecting to the MPFC. In addition, Acb-projecting neurons extended into the lateral wings of the DRN, whereas MPFC-projecting neurons were more restricted to the midline. Both groups of retrogradely labeled neurons, however, were more numerous in the caudal aspect of the dorsal raphe nucleus and were scattered amongst 5-HT immunoreactive perikarya. Of 783 +/- 69 CT-Au labeled cells, 15% also contained the FG label and 11% contained FG and 5-HT immunoreactivity. Of 613 +/- 48 FG labeled cells, 24% also contained the CT-Au label and 21% were also immunoreactive to 5-HT. The results suggest a more prominent input to the Acb from both 5-HT-ir and non-5-HT-ir neurons in the caudal aspect of the DRN and further indicate that while most 5-HT-ir and non-5-HT-ir neurons project differentially to both forebrain regions, a few cells also show collateralization to the MPFC and Acb. Such collateralization of single serotonergic neurons to divergent targets may integrate cognitive and motor activities in response to pharmacological manipulations of ascending serotonergic pathways.
The nucleus submedius in the medial thalamus has been known to receive spinothalamic and trigeminothalamic fibers, and to contain neurons which can be activated by noxious stimuli. These previous findings suggest that the nucleus submedius may be involved in the processing and relay of pain-related information. In the present study, we immunohistochemically observed in the rat and cat that the nucleus submedius was distributed with a considerable amount of serotoninergic fibers. After iontophoretic injection of cholera toxin B subunit into the nucleus submedius, the sequential double-antigen immunofluorescence histochemistry for retrogradely transported cholera toxin B subunit and serotonin revealed that the serotoninergic fibers to the nucleus submedius arose mainly from the dorsal raphe nucleus, and additionally from the ventrolateral and medial parts of the midbrain periaqueductal gray. The direct projections from the dorsal raphe nucleus to the nucleus submedius were confirmed by anterograde axonal tracing after iontophoretic injection of Phaseolus vulgaris-leucoagglutinin into the dorsal raphe nucleus. The disappearance of almost all serotoninergic fibers in the nucleus submedius was also observed after destruction of the dorsal raphe nucleus. The fluorescent retrograde double-labeling with Diamidino Yellow and Fast Blue further revealed that some neurons in the dorsal raphe nucleus projecting directly to the nucleus submedius sent their axon collaterals to the ventrolateral orbital region of the cerebral cortex, nucleus accumbens, amygdala, nucleus raphe magnus, caudal spinal trigeminal nucleus, or spinal cord. The possible roles of the serotoninergic projections from the dorsal raphe nucleus to the nucleus submedius in pain control and/or the olfactolimbic functions are discussed.
Previously, serotonin (5-HT) was shown to increase inhibitory post-synaptic potentials (IPSPs) in layer II pyramidal cells, and excite a subpopulation of interneurons located on the layer II/III border of piriform cortex in rat in vitro brain slices. In the present study, the effects of norepinephrine (NE) and dopamine (DA) on these two populations of neurons were examined in brain slices using intracellular and extracellular recordings. All three monoamines increased GABAergic IPSPs in many pyramidal cells; overall, 5-HT was most effective in eliciting IPSPs (58% of cells), followed by NE (45%), then DA (24%). Commonly, pyramidal cells responded with an increase in IPSPs to more than one of the monoamines. The increase in IPSPs was found to include an increase in the frequency of IPSPs present at baseline, as well as recruitment of additional IPSPs of different amplitudes. In interneurons the effects of the monoamines paralleled that which was found for the pyramidal cells. Thus, all three monoamines increased the firing rate of many interneurons; again 5-HT was most effective (56%), followed by NE (51%), then DA (42%). In about 10% of the interneurons the monoamines inhibited cell firing. Interneurons frequently had responses to more than one of the monoamines. The excitatory amino acid (EAA) antagonist, kynurenic acid (200-400 microM), spared most 5-HT and NE responses on interneurons, suggesting that these effects were directly mediated. We conclude that IPSPs elicited by monoamines in pyramidal cells result from a convergence of inputs from populations of layer II/III interneurons that are activated by one, two or all three of the monoamines.