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Vesicular glutamate transporter 3-expressing nonserotonergic projection neurons constitute a subregion in the rat midbrain raphe nuclei
Abstract
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.
... One particular molecular marker, VGLUT3, has emerged as a particularly straightforward and informative indication of DR subnuclei. In humans and rodents, populations of TPH2 positive neurons in the raphe co-express VGLUT3 to varying degrees (Hioki et al., 2010;Vigneault et al., 2015;Deneris and Gaspar, 2018). ...
... TPH2 characterization Tryptophan hydroxylase 2+ cell distribution and characteristics have been examined extensively in rodents, to our knowledge this study is the first to describe the characteristics of TPH2+ cells within subnuclei of the DR in a non-human primate model. While highly correlated, the DR subnuclei appear differently in rodents and direct equivalences have not been made (Hioki et al., 2010;Calizo et al., 2011;Haugas et al., 2016;Ren et al., 2019). Our detailed characterization will provide a valuable resource to research groups investigating the development of the DR in primates. ...
... This study is the first to detail VGLUT3 expression patterns in the primate DR. We found considerable VGLUT3 signal within (somatic) and outside of TPH2 neurons throughout the DR, consistent with the rodent literature (Gras et al., 2002;Hioki et al., 2010;Ren et al., 2018). ...
Introduction
The neurotransmitter serotonin is a key regulator of neurotransmission, mood, and behavior and is essential in neurodevelopment. Dysfunction in this important neurotransmitter system is connected to behavioral disorders such as depression and anxiety. We have previously shown that the developing serotonin system is sensitive to perinatal exposure to Western-style diet (WSD).
Methods
To advance our hypothesis that perinatal WSD has a long-term impact on the serotonergic system, we designed a fluorescent immunohistochemistry experiment using antibodies against tryptophan hydroxylase 2 (TPH2) and vesicular glutamate transporter 3 (VGLUT3) to probe protein expression in the raphe subnuclei in 13-month-old Japanese macaques (Macaca fuscata; n = 22). VGLUT3 has been shown to be coexpressed in TPH2+ cells in the dorsal raphe (DR) and median raphe nucleus (MnR) of rodent raphe nuclei and may provide information about the projection site of serotonergic fibers into the forebrain. We also sought to improve scientific understanding of the heterogeneity of the serotonin production center for the central nervous system, the midbrain raphe nuclei.
Results
In this immunohistochemical study, we provide the most detailed characterization of the developing primate raphe to date. We utilize multi-level modeling (MLM) to simultaneously probe the contribution of WSD, offspring sex, and raphe anatomical location, to raphe neuronal measurements. Our molecular and morphological characterization revealed that the 13-month-old macaque DR is remarkably similar to that of adult macaques and humans. We demonstrate that vesicular glutamate transporter 3 (VGLUT3), which rodent studies have recently shown can distinguish raphe populations with distinct projection targets and behavioral functions, likewise contributes to the heterogeneity of the primate raphe.
Discussion
This study provides evidence that perinatal WSD has a long-term impact on the density of serotonin-producing neurons, potentially limiting serotonin availability throughout the brain. Due to the critical involvement of serotonin in development and behavior, these findings provide important insight into the mechanisms by which maternal nutrition and metabolic state influence offspring behavioral outcomes. Finally, these findings could inform future research focused on designing therapeutic interventions to optimize neural development and decrease a child’s risk of developing a mental health disorder.
... In the DR, VGLUT3-only and mixed VGLUT3/5-HT populations are mostly segregated. In the ventral part of the DR (DRV or B7v), VGLUT3-positive neurons colocalize extensively with 5-HT (Okaty et al., 2020;Ren et al., 2019) while in the dorsal part of the DR (DRD or B7d) a population of purely VGLUT3-positive neurons has been described (Hioki et al., 2010). DR VGLUT3 neurons send mixed 5-HT/glutamate projections to the amygdala where they can release glutamate in addition to 5-HT, although the contribution of glutamate to amygdala function remains unclear (Sengupta, Bocchio, Bannerman, Sharp, & Capogna, 2017;Sengupta & Holmes, 2019). ...
... Co-localization with 5-HT can be observed in both DR-and MnR-originating VGLUT3 fibers, albeit in variable proportions (Figure 4). In the DR, a large population of VGLUT3-positive neurons co-express serotonergic markers (Hioki et al., 2010;Ren et al., 2019). As expected, we observed overlap between the projection patterns of DR 5-HT neurons (Muzerelle et al., 2014) and VGLUT3 neurons (Figure 1), and frequent co-expression of 5-HT in eYFP-positive fibers from the DR (Figure 4). ...
... Finally, in the entorhinal cortex, MnR VGLUT3 fiber density was high in the lateral entorhinal cortex and sparse in the medial entorhinal cortex (Figure 3B-5). In the hippocampal formation (Figure 3C-F) and all other regions examined, eYFP and VGLUT3 staining showed a high degree of colocalization, as expected for AAV-driven eYFP expression in raphe VGLUT3-positive fibers.Colocalization between VGLUT3 and serotonergic markers varies across different raphe subregions, for example, the most ventral part of the DR exhibits strong overlap between markers while VGLUT3 and 5-HT populations are mostly segregated in the MnR(Hioki et al., 2010; Okaty, Commons, & Dymecki, 2019;Ren et al., 2019;Sos et al., 2017). Therefore, we qualitatively examined the colocalization of eYFP in raphe-originating VGLUT3-positive fibers with 5-HT in the forebrain. ...
The hippocampus (HP) receives neurochemically diverse inputs from the raphe nuclei, including glutamatergic fibers characterized by the expression of the vesicular glutamate transporter VGLUT3. These raphe-HP VGLUT3 (VGLUT3 HP ) projections have been suggested to play a critical role in HP functions, yet a complete anatomical overview of raphe VGLUT3 projections to the forebrain, and in particular the HP, is lacking. Using anterograde viral tracing, we describe largely non-overlapping VGLUT3-positive projections from the dorsal raphe (DR) and median raphe (MnR) to the forebrain, with the HP receiving inputs from the MnR. A limited subset of forebrain regions such as the amygdaloid complex, claustrum and hypothalamus receive projections from both the DR and MnR that remain largely segregated. This highly complementary anatomical pattern suggests contrasting roles for DR and MnR VGLUT3 neurons. To further analyse the topography of VGLUT3 raphe projections to the HP, we used retrograde tracing and found that VGLUT3 HP neurons distribute over several raphe sub-regions (including the MnR, paramedian raphe and B9 nucleus) and lack co-expression of serotonergic markers. Strikingly, two-color retrograde tracing unraveled two parallel streams of VGLUT3-positive projections targeting the dorsal and ventral poles of the HP. These results demonstrate highly organized and segregated VGLUT3-positive projections to the HP, suggesting independent modulation of HP functions such as spatial memory and emotion-related behavior.
... Firstly, its anatomical distribution is unique: while VGLUT1 and 2 show complementary localisation, VG-LUT3 appears intermingled with other transporters, appearing mainly, but not exclusively in subcortical structures. On the mRNA level, it has been shown in neurons of the cortex (layers II, III, and VI), caude putamen, amygdala, hippocampus, hypothalamus, nucleus accumbens, habenula, bed nucleus of stria terminalis (BNST), striatum, ventral tegmental area (VTA), substantia nigra pars compacta, and midbrain raphe nuclei [5,6,11,13,16,[18][19][20][21][22], with controversial results in the cerebellum (in the granular layer, molecular layer, Purkinje cells reported in [5], but not found by others [11,16]). ...
... Moreover, VGLUT3 is not exclusively expressed in the nerve terminals or cell bodies but can also be found in dendrites [5,13]. Interestingly, astrocytes [5,28] and ependymal cell [13,16] were also VGLUT3 positive; however, in situ hybridization did not confirm this on the mRNA level [6,19,29]. ...
... Midbrain raphe nuclei are mostly known for their 5-HT content, which is marked by serotonin transporters (SERT). Interestingly, in these cell groups, SERT+ and VGLUT3+ markers are often co-expressed, but they can also be found separately [5,16,19,20,[45][46][47][48][49][50]. Terminals originating from serotonergic neurons often co-express VGLUT3 in the cortex, especially in layers II/III [20]. ...
Glutamate is the most abundant excitatory amino acid in the central nervous system. Neurons using glutamate as a neurotransmitter can be characterised by vesicular glutamate transporters (VGLUTs). Among the three subtypes, VGLUT3 is unique, co-localising with other “classical” neurotransmitters, such as the inhibitory GABA. Glutamate, manipulated by VGLUT3, can modulate the packaging as well as the release of other neurotransmitters and serve as a retrograde signal through its release from the somata and dendrites. Its contribution to sensory processes (including seeing, hearing, and mechanosensation) is well characterised. However, its involvement in learning and memory can only be assumed based on its prominent hippocampal presence. Although VGLUT3-expressing neurons are detectable in the hippocampus, most of the hippocampal VGLUT3 positivity can be found on nerve terminals, presumably coming from the median raphe. This hippocampal glutamatergic network plays a pivotal role in several important processes (e.g., learning and memory, emotions, epilepsy, cardiovascular regulation). Indirect information from anatomical studies and KO mice strains suggests the contribution of local VGLUT3-positive hippocampal neurons as well as afferentations in these events. However, further studies making use of more specific tools (e.g., Cre-mice, opto- and chemogenetics) are needed to confirm these assumptions.
... To date, diverse lines of studies have focused on the physiological roles of 5-HT neurons (Hornung, 2003;Muller and Jacobs, 2010;Ren et al., 2018). However, the DR not only consists of 5-HT neurons but contains also many tyrosine hydroxylase (TH) positive neurons in rodents (Trulson et al., 1985;Descarries et al., 1986;Hasue and Shammah-Lagnado, 2002;Hioki et al., 2010;Poulin et al., 2014) and primates (Arsenault et al., 1988;Charara and Parent, 1998). Recently, the functional importance of dopaminergic (DA) neurons on affective behaviors in the DR and ventral periaqueductal gray (PAG) regions has been reported in multiple studies in mice. ...
... This is consistent with previous reports that described the presence of DA neurons in this region (Li et al., 2016;Matthews et al., 2016;Cho et al., 2017;Groessl et al., 2018). With anti-TH and anti-VIP antibodies, the TH-positive and VIP-positive cell-bodies were also confirmed, as reported previously ( Fig. 1A; Hioki et al., 2010;Dougalis et al., 2012;Poulin et al., 2014). TH-positive cell bodies were located in the DR and linear nucleus regions. ...
The dorsal raphe (DR) nucleus contains many tyrosine hydroxylase (TH)-positive neurons which are regarded as dopaminergic (DA) neurons. These DA neurons in the DR and periaqueductal gray (PAG) region (DADR-PAG neurons) are a subgroup of the A10 cluster, which is known to be heterogeneous. This DA population projects to the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST) and has been reported to modulate various affective behaviors. To characterize, the histochemical features of DADR-PAG neurons projecting to the CeA and BNST in mice, the current study combined retrograde labeling with Fluoro-Gold (FG) and histological techniques, focusing on TH, dopamine transporter (DAT), vasoactive intestinal peptide (VIP), and vesicular glutamate transporter 2 (VGlut2). To identify putative DA neurons, DAT-Cre::Ai14 mice were used. It was observed that DATDR-PAG neurons consisted of the following two subpopulations: TH+/VIP- and TH-/VIP+ neurons. The DAT+/TH-/VIP+ subpopulation would be non-DA noncanonical DAT neurons. Anterograde labeling of DATDR-PAG neurons with AAV in DAT-Cre mice revealed that the fibers exclusively innervated the lateral part of the CeA and the oval nucleus of the BNST. Retrograde labeling with FG injections into the CeA or BNST revealed that the two subpopulations similarly innervated these regions. Furthermore, using VGlut2-Cre::Ai14 mice, it was turned out that the TH-/VIP+ subpopulations innervating both CeA and BNST were VGlut2-positive neurons. These two subpopulations of DATDR-PAG neurons, TH+/VIP- and TH-/VIP+, might differentially interfere with the extended amygdala, thereby modulating affective behaviors.
... It is well established that the MnR consists of at least three neuronal populations, including 5-HT, Vglut3, and GABAergic neurons (Köhler and Steinbusch, 1982;Gras et al., 2002;Herzog et al., 2004;Jackson et al., 2009;Varga et al., 2009;Hioki et al., 2010;Domonkos et al., 2016;Sos et al., 2017;Senft et al., 2021). The latest studies also revealed the existence of a fourth population of Vglut2 neurons, which, however, constitutes a relatively small population and is more prevalent in the paramedian raphe rather than midline MnR (Hioki et al., 2010;Szo †nyi et al., 2019;Xu et al., 2021; https:// mouse.brain-map.org/experiment/show/73818754). ...
... It is well established that the MnR consists of at least three neuronal populations, including 5-HT, Vglut3, and GABAergic neurons (Köhler and Steinbusch, 1982;Gras et al., 2002;Herzog et al., 2004;Jackson et al., 2009;Varga et al., 2009;Hioki et al., 2010;Domonkos et al., 2016;Sos et al., 2017;Senft et al., 2021). The latest studies also revealed the existence of a fourth population of Vglut2 neurons, which, however, constitutes a relatively small population and is more prevalent in the paramedian raphe rather than midline MnR (Hioki et al., 2010;Szo †nyi et al., 2019;Xu et al., 2021; https:// mouse.brain-map.org/experiment/show/73818754). In our current study, we focused on genetically verified 5-HT, Vglut3, and GABAergic MnR neurons and characterized their activity patterns during the sleep-wake cycle in freely behaving mice. ...
Hippocampal theta oscillations (HTOs) during rapid eye movement (REM) sleep play an important role in mnemonic processes by coordinating hippocampal and cortical activities. However, it is not fully understood how HTOs are modulated by subcortical regions, including the median raphe nucleus (MnR). The MnR is thought to suppress HTO through its serotonergic outputs. Here, our study on male mice revealed a more complex framework indicating roles of nonserotonergic MnR outputs in regulating HTO. We found that nonselective optogenetic activation of MnR neurons at theta frequency increased HTO amplitude. Granger causality analysis indicated that MnR theta oscillations during REM sleep influence HTO. By using three transgenic mouse lines, we found that MnR serotonergic neurons exhibited little or no theta-correlated activity during HTO. Instead, most MnR GABAergic neurons and Vglut3 neurons respectively increased and decreased activities during HTO and exhibited hippocampal theta phase-locked activities. Although MnR GABAergic neurons do not directly project to the hippocampus, they could modulate HTO through local Vglut3 and serotonergic neurons as we found that MnR GABAergic neurons monosynaptically targeted Vglut3 and serotonergic neurons. Additionally, pontine wave recorded from the MnR during REM sleep accompanied nonserotonergic activity increase and HTO acceleration. These results suggest that MnR nonserotonergic neurons modulate hippocampal theta activity during REM sleep, which regulates memory processes.SIGNIFICANCE STATEMENT The MnR is the major source of serotonergic inputs to multiple brain regions including the hippocampus and medial septal area. It has long been thought that those serotonergic outputs suppress HTOs. However, our results revealed that MnR serotoninergic neurons displayed little firing changes during HTO. Instead, MnR Vglut3 neurons were largely silent during HTO associated with REM sleep. Additionally, many MnR GABAergic neurons fired rhythmically phase-locked to HTO. These results indicate an important role of MnR nonserotonergic neurons in modulating HTO.
... By contrast, VGLUT3 was mainly found in discrete populations of 'non-glutamatergic' neurons and only in scattered glutamatergic neurons [2] (Figure 1). VGLUT3 is expressed by subgroups of glutamatergic neurons in the cerebral cortex and raphe nuclei [7,14] and by inner hair cells (IHCs) of the cochlea [15,16] (Figure 1). In addition, central Vesicular glutamate transporters (VGLUTs) ensure excitatory neurotransmission and are topographically arranged in a region-and cell typespecific manner. ...
... VGLUT3 is detected in cholinergic interneurons (ChIs) of the dorsal and ventral striatum [7,17,18], GABAergic cortical and hippocampal interneurons [19][20][21], serotonergic raphe neurons [7,14], and GABAergic neurons in the olfactory bulb [22] (Figure 1). Therefore, VGLUT3-positive neurons act as local interneurons (VGLUT3-interneurons in the cortex, hippocampus, and striatum) and/or as projecting principal neurons (VGLUT3-neurons in the raphe). ...
Vesicular glutamate transporters (VGLUTs) were long thought to be specific markers of glutamatergic excitatory transmission. The discovery, two decades ago, of the atypical VGLUT3 has thoroughly modified this oversimplified view. VGLUT3 is strategically expressed in discrete populations of glutamatergic, cholinergic, serotonergic, and even GABAergic neurons. Recent reports show the subtle, but critical, implications of VGLUT3-dependent glutamate co-transmission and its roles in the regulation of diverse brain functions and dys-functions. Progress in the neuropharmacology of VGLUT3 could lead to decisive breakthroughs in the treatment of Parkinson's disease (PD), addiction, eating disorders, anxiety, presbycusis, or pain. This review summarizes recent findings on VGLUT3 and its vesicular underpinnings as well as on possible ways to target this atypical transporter for future therapeutic strategies.
... There are also dorsal raphe 5HT neurons that innervate the VTA (Van Bockstaele et al., 1994;Pierce et al., 1976). Some of these neurons release only 5HT whereas others release both 5HT and glutamate (Hioki et al., 2010). Receptors localized on VTA DA cell bodies that project to the PFC include the 5HT 1a, 5HT 2c , and 5HT 1b receptors that are localized on GABA terminals in the VTA and activation decreases release of GABA, decreasing inhibitory control and increasing DA release from mesolimbic terminals. ...
... Receptors localized on VTA DA cell bodies that project to the PFC include the 5HT 1a, 5HT 2c , and 5HT 1b receptors that are localized on GABA terminals in the VTA and activation decreases release of GABA, decreasing inhibitory control and increasing DA release from mesolimbic terminals. Glutamate is coreleased by about 60% of DRN 5HT neurons (Hioki et al., 2010) and those 5HT neurons that corelease glutamate primarily project to cortical areas (Ren et al., 2018) and to dopamine cells in the VTA (Qi et al., 2014;Wang et al., 2019). DA and GABA cells in VTA might be particularly interesting since they express 5HT 2c receptors and activation of VTA 5HT 2c receptors inhibits DA neurotransmission (Bubar & Cunningham, 2007). ...
Methylenedioxymethamphetamine (MDMA) is an amphetamine analogue that preferentially stimulates the release of serotonin (5HT) and results in relatively small increases in synaptic dopamine (DA). The ratio of drug‐stimulated increases in synaptic DA, relative to 5HT, predicts the abuse liability; drugs with higher DA:5HT ratios are more likely to be abused. Nonetheless, MDMA is a drug that is misused. Clinical and preclinical studies have suggested that repeated MDMA exposure produces neuroadaptive responses in both 5HT and DA neurotransmission that might explain the development and maintenance of MDMA self‐administration in some laboratory animals and the development of a substance use disorder in some humans. In this paper, we describe the research that has demonstrated an inhibitory effect of 5HT on the acquisition of MDMA self‐administration and the critical role of DA in the maintenance of MDMA self‐administration in laboratory animals. We then describe the circuitry and 5HT receptors that are positioned to modulate DA activity and review the limited research on the effects of MDMA exposure on these receptor mechanisms.
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... Midbrain raphe nuclei, pontine raphe nuclei, pontine reticular formation, and lateral parabrachial nucleus all showed detectable transcripts, while the control cortical samples did not. This is in accordance with the literature on rodents (Fremeau et al., 2002;Gras et al., 2002;Schafer et al., 2002;Herzog et al., 2004;Guo et al., 2005;Stornetta et al., 2005;Hioki et al., 2010). ...
Social behavior is important for our well-being, and its dysfunctions impact several pathological conditions. Although the involvement of glutamate is undeniable, the relevance of vesicular glutamate transporter type 3 (VGluT3), a specific vesicular transporter, in the control of social behavior is not sufficiently explored. Since midbrain median raphe region (MRR) is implicated in social behavior and the nucleus contains high amount of VGluT3+ neurons, we compared the behavior of male VGluT3 knock-out (KO) and VGluT3-Cre mice, the latter after chemogenetic MRR-VGluT3 manipulation. Appropriate control groups were included. Behavioral test battery was used for social behavior (sociability, social discrimination, social interaction, resident intruder test) and possible confounding factors (open field, elevated plus maze, Y-maze tests). Neuronal activation was studied by c-Fos immunohistochemistry. Human relevance was confirmed by VGluT3 gene expression in relevant human brainstem areas. VGluT3 KO mice exhibited increased anxiety, social interest, but also aggressive behavior in anxiogenic environment and impaired social memory. For KO animals, social interaction induced lower cell activation in the anterior cingulate, infralimbic cortex, and medial septum. In turn, excitation of MRR-VGluT3+ neurons was anxiolytic. Inhibition increased social interest 24 h later but decreased mobility and social behavior in aggressive context. Chemogenetic activation increased the number of c-Fos+ neurons only in the MRR. We confirmed the increased anxiety-like behavior and impaired memory of VGluT3 KO strain and revealed increased, but inadequate, social behavior. MRR-VGluT3 neurons regulated mobility and social and anxiety-like behavior in a context-dependent manner. The presence of VGluT3 mRNA on corresponding human brain areas suggests clinical relevance.
... The hypothalamus is directly innervated by the NTS [15,19,21,37] and connects with all other regions of interest in our model [20,22,24,33,44,45]. Another reason to include the hypothalamus is that there is evidence that VNS has an immunomodulatory effect via the hypothalamic-pituitaryadrenal axis [46][47][48]. ...
Vagus Nerve Stimulation (VNS) is an established palliative treatment for drug resistant epilepsy. While effective for many patients, its mechanism of action is incompletely understood. Predicting individuals' response, or optimum stimulation parameters, is challenging. Computational modelling has informed other problems in epilepsy but, to our knowledge, has not been applied to VNS. We started with an established, four-population neural mass model (NMM), capable of reproducing the seizure-like dynamics of a thalamocortical circuit. We extended this to include 18 further neural populations, representing nine other brain regions relevant to VNS, with connectivity based on existing literature. We modelled stimulated afferent vagal fibres as projecting to the nucleus tractus solitarius (NTS), which receives input from the vagus nerve in vivo. Bifurcation analysis of a deterministic version of the model showed higher background NTS input made the model monostable at a fixed point (FP), representing normal activity, while lower inputs produce bistability between the FP and a limit cycle (LC), representing the seizure state. Adding noise produced transitions between seizure and normal states. This stochastic model spent decreasing time in the seizure state with increasing background NTS input, until seizures were abolished, consistent with the deterministic model. Simulated VNS stimulation, modelled as a 30 Hz square wave, was summed with the background input to the NTS and was found to reduce total seizure duration in a dose-dependent manner, similar to expectations in vivo. We have successfully produced an in silico model of VNS in epilepsy, capturing behaviour seen in vivo. This may aid understanding therapeutic mechanisms of VNS in epilepsy and provides a starting point to (i) determine which patients might respond best to VNS, and (ii) optimise individuals' treatments.
... Next, we investigated whether swim stress increased c-Fos immunoreactivity specifically in 5-HT-glutamate co-releasing neurons, using the same sections examined for c-Fos alone. Previous studies have revealed that VGLUT3-expressing neurons in the midbrain raphe nuclei comprise two sub-populations, one colocalizing 5-HT and the other lacking 5-HT 16,27 . Here, the 5-HTspecific marker tryptophan hydroxylase 2 (TPH2) was used to distinguish these two populations (Fig. 3A). ...
The majority of midbrain 5-hydroxytryptamine (5-HT) neurons express the vesicular glutamate transporter 3 (VGLUT3) and co-release 5-HT and glutamate, but the function of this co-release is unclear. Given the strong links between 5-HT and uncontrollable stress, we used a combination of c-Fos immunocytochemistry and conditional gene knock out in mice to test the hypothesis that glutamate co-releasing 5-HT neurons would be activated by stress and involved in stress coping.
Acute, uncontrollable swim stress increased c-Fos immunoreactivity in neurons co-expressing VGLUT3 and the 5-HT marker tryptophan hydroxylase 2 (TPH2) in the dorsal raphe nucleus (DRN). This effect was localised in the ventral DRN subregion and prevented by the antidepressant fluoxetine. In contrast, a more controllable stressor, acute social defeat, had no effect on c-Fos immunoreactivity in VGLUT3-TPH2 co-expressing neurons in the DRN.
To test whether activation of glutamate co-releasing 5-HT neurons was causally linked to stress coping, mice with a specific deletion of VGLUT3 in 5-HT neurons were exposed to acute swim stress. Compared to wildtype controls, the mutant mice showed increased climbing behaviour, a measure of active coping. Wildtype mice also showed increased climbing when administered fluoxetine, revealing an interesting parallel between the behavioural effects of genetic loss of VGLUT3 in 5-HT neurons and 5-HT reuptake inhibition.
We conclude that 5-HT-glutamate co-releasing neurons are recruited by exposure to uncontrollable stress. Furthermore, natural variation in the balance of 5-HT and glutamate released at the 5-HT synapse may impact on stress susceptibility.
... Moreover, DRN is best known as the origin of extensive serotonergic projections to the forebrain [22][23][24][25][26], whose involvement in reward processing has been extensively demonstrated. Apart from 5-HT neurons, the DRN contains a large proportion (estimated 50-75%) of non-serotonergic neurons comprised primarily of GABA-and glutamate-producing neurons [23,27]. ...
The pathology of depression is related to the imbalance of various neurotransmitters. The dorsal raphe nucleus (DRN), the main brain region producing 5-HT, is crucially involved in the pathophysiology of depression. It contains several neuron types, in which GABAergic neurons are activated by stimuli associated with negative experiences and 5-HT neurons are activated by reward signals. However, little is known about its underlying molecular mechanisms. Here, we found that p11, a multifunctional protein associated with depression, was down-regulated by chronic social defeat stress in 5-HTDRN neurons. Knockdown of p11 in DRN induced depression-like behaviors, while its overexpression in 5-HTDRN neurons alleviated depression-like behavior caused by chronic social defeat stress. Further, p11 regulates membrane trafficking of glutamate receptors in 5-HTDRN neurons, suggesting a possible molecular mechanism underlying the participation of p11 in the pathological process of depression. This may facilitate the understanding of the molecular and cellular basis of depression.
... The sections were further incubated with Alexa488-conjugated streptavidin (2.5 mg/ml). For detection of axon terminals of neurons transduced with iChloC-mCherry, sections were incubated overnight with an anti-monomeric RFP guinea pig antibody (0.1 mg/ml) (Hioki et al., 2010). After a rinse, the sections were incubated for 1 h with a biotinylated donkey antibody to guinea pig IgG (10 mg/ml; 706-065-148, Jackson ImmunoResearch Laboratories). ...
Thermoregulatory behavior in homeothermic animals is an innate behavior to defend body core temperature from environmental thermal challenges in coordination with autonomous thermoregulatory responses. In contrast to the progress in understanding the central mechanisms of autonomous thermoregulation, those of behavioral thermoregulation remain poorly understood. We have previously shown that the lateral parabrachial nucleus (LPB) mediates cutaneous thermosensory afferent signaling for thermoregulation. To understand the thermosensory neural network for behavioral thermoregulation, in the present study, we investigated the roles of ascending thermosensory pathways from the LPB in avoidance behavior from innocuous heat and cold in male rats. Neuronal tracing revealed two segregated groups of LPB neurons projecting to the median preoptic nucleus (MnPO), a thermoregulatory center (LPB→MnPO neurons), and those projecting to the central amygdaloid nucleus (CeA), a limbic emotion center (LPB→CeA neurons). While LPB→MnPO neurons include separate subgroups activated by heat or cold exposure of rats, LPB→CeA neurons were only activated by cold exposure. By selectively inhibiting LPB→MnPO or LPB→CeA neurons using tetanus toxin light chain or chemogenetic or optogenetic techniques, we found that LPB→MnPO transmission mediates heat avoidance, whereas LPB→CeA transmission contributes to cold avoidance. In vivo electrophysiological experiments showed that skin cooling-evoked thermogenesis in brown adipose tissue requires not only LPB→MnPO neurons but also LPB→CeA neurons, providing a novel insight into the central mechanism of autonomous thermoregulation. Our findings reveal an important framework of central thermosensory afferent pathways to coordinate behavioral and autonomous thermoregulation and to generate the emotions of thermal comfort and discomfort that drive thermoregulatory behavior.
Significance Statement
Coordination of behavioral and autonomous thermoregulation is important for maintaining thermal homeostasis in homeothermic animals. However, the central mechanism of thermoregulatory behaviors remains poorly understood. We have previously shown that the lateral parabrachial nucleus (LPB) mediates ascending thermosensory signaling that drives thermoregulatory behavior. In this study, we found that one pathway from the LPB to the median preoptic nucleus mediates heat avoidance, whereas the other pathway from the LPB to the central amygdaloid nucleus is required for cold avoidance. Surprisingly, both pathways are required for skin cooling-evoked thermogenesis in brown adipose tissue, an autonomous thermoregulatory response. This study provides a central thermosensory network that coordinates behavioral and autonomous thermoregulation and generates thermal comfort and discomfort that drive thermoregulatory behavior.
... The tissue was cut into 30μm-thick frontal sections on a freezing microtome. The primary antibodies used are anti-EP3R rabbit antibody (1 μg/ml) (14,15), anti-Fos goat antibody (1:1000; sc-52G, Santa Cruz Biotechnology), anti-CTb goat serum (1:5000; #703, List Biological Laboratories), anti-GFP mouse antibody (1:200; A11120, Thermo Fisher Scientific), anti-GFP rabbit antibody (0.5 μg/ml) (60), anti-VGAT guinea pig serum (1:1000; 131004, Synaptic Systems), anti-VGLUT2 rabbit antibody (0.5 μg/ml) (61), anti-GAD67 mouse antibody (1:300; MAB5406, Sigma-Aldrich), anti-synaptophysin mouse antibody (1:1000; S5768, Sigma-Aldrich), and anti-monomeric red fluorescent protein (mRFP) guinea pig antibody (1 μg/ml) (62). The anti-mRFP guinea pig antibody and the anti-GFP mouse antibody showed cross-reactivity to mCherry and EYFP, respectively. ...
The bidirectional controller of the thermoregulatory center in the preoptic area (POA) is unknown. Using rats, here, we identify prostaglandin EP3 receptor–expressing POA neurons (POA EP3R neurons) as a pivotal bidirectional controller in the central thermoregulatory mechanism. POA EP3R neurons are activated in response to elevated ambient temperature but inhibited by prostaglandin E 2 , a pyrogenic mediator. Chemogenetic stimulation of POA EP3R neurons at room temperature reduces body temperature by enhancing heat dissipation, whereas inhibition of them elicits hyperthermia involving brown fat thermogenesis, mimicking fever. POA EP3R neurons innervate sympathoexcitatory neurons in the dorsomedial hypothalamus (DMH) via tonic (ceaseless) inhibitory signaling. Although many POA EP3R neuronal cell bodies express a glutamatergic messenger RNA marker, their axons in the DMH predominantly release γ-aminobutyric acid (GABA), and their GABAergic terminals are increased by chronic heat exposure. These findings demonstrate that tonic GABAergic inhibitory signaling from POA EP3R neurons is a fundamental determinant of body temperature for thermal homeostasis and fever.
... Combination of single FISH and immunofluorescence labeling was performed as previously described (Hioki et al., 2010;Ma et al., 2011;Sohn et al., 2014) with some modification. Briefly, sense and antisense single-stranded riboprobes for vesicular glutamate transporter 1 (VGluT1; nucleotides 855-1788 in GenBank accession No: XM_133432.2) (Nakamura et al., 2007) and glutamic acid decarboxylase 67 kDa isoform (GAD67; nucleotides 43-661 in GenBank accession No: Y12257.1) ...
The claustrum coordinates the activities of individual cortical areas through abundant reciprocal connections with the cerebral cortex. Although these excitatory connections have been extensively investigated in three subregions of the claustrum—core region and dorsal and ventral shell regions—the contribution of GABAergic neurons to the circuitry in each subregion remains unclear. Here, we examined the distribution of GABAergic neurons and their dendritic and axonal arborizations in each subregion. Combining in situ hybridization with immunofluorescence histochemistry showed that approximately 10% of neuronal nuclei-positive cells expressed glutamic acid decarboxylase 67 mRNA across the claustral subregions. Approximately 20%, 30%, and 10% of GABAergic neurons were immunoreactive for parvalbumin (PV), somatostatin (SOM), and vasoactive intestinal polypeptide, respectively, in each subregion, and these neurochemical markers showed little overlap with each other. We then reconstructed PV and SOM neurons labeled with adeno-associated virus vectors. The dendrites and axons of PV and SOM neurons were preferentially localized to their respective subregions where their cell bodies were located. Furthermore, the axons were preferentially extended in a rostrocaudal direction, whereas the dendrites were relatively isotropic. The present findings suggest that claustral PV and SOM neurons might execute information processing separately within the core and shell regions.
... The dorsal raphe nucleus (DRN) is a nucleus located in the dorsal region of the midbrain and comprised of at least four principal cell types that release different neurotransmitters, such as serotonin, glutamate, gamma-aminobutyric acid (GABA), and dopamine [1,2]. DRN has long been suggested to play a key role in sleep-wake [3]. ...
The dorsal raphe nucleus (DRN) is involved in regulating the sleep–wake behavior. DRN includes several neuron types, such as 5-HTergic and GABAergic neurons. GABAergic neurons, the second most common neurons in the DRN, participate in a variety of neurobiological processes. However, their role in sleep–wake regulation and the underlying neural circuitry remains unclear. Herein, we used fiber photometry and synchronous electromyography (EEG)/electroencephalogram (EMG) recording to demonstrate that DRN GABAergic neurons exhibit high activities during wakefulness and low activities during NREM sleep. Brief optogenetic activation of DRN GABAergic neurons reduced the latency of NREM-to-wake transition and increased the probability of wakefulness, while long-term optogenetic activation of these neurons significantly increased the amount of wakefulness. Chemogenetic activation of DRN GABAergic neurons increased wakefulness for almost 2 h and maintained long-lasting arousal. In addition, inhibition of DRN GABAergic neurons with chemogenetics caused a reduction in the amount of wakefulness. Finally, similar to the effects of activating the soma of DRN GABAergic neurons, optogenetic stimulation of their terminals in the ventral tegmental area (VTA) induced instant arousal and promoted wakefulness. Taken together, our results illustrate that DRN GABAergic neurons are vital to the induction and maintenance of wakefulness, which promote wakefulness through the GABAergic DRN-VTA pathway.
... The Gfap-positive processes were extended from the subgranular zone of DG that was determined by FT-GO IF for Rbfox3 ( Fig. 5f,i), indicating Gfap-positive processes emanating from neural stem cells 26 . Multiplex mRNA FISH histochemistry facilitates identifying molecular signatures of individual cell populations in fixed tissues [27][28][29] . Neocortical interneurons contain a tremendous diversity in term of their gene expressions [30][31][32] . ...
Tyramide signal amplification (TSA) is a highly sensitive method for histochemical analysis. Previously, we reported a TSA system, biotinyl tyramine-glucose oxidase (BT-GO), for bright-filed imaging. Here, we develop fluorochromized tyramide-glucose oxidase (FT-GO) as a multiplex fluorescent TSA system. FT-GO involves peroxidase-catalyzed deposition of fluorochromized tyramide (FT) with hydrogen peroxide produced by enzymatic reaction between glucose and glucose oxidase. We showed that FT-GO enhanced immunofluorescence signals while maintaining low background signals. Compared with indirect immunofluorescence detections, FT-GO demonstrated a more widespread distribution of monoaminergic projection systems in mouse and marmoset brains. For multiplex labeling with FT-GO, we quenched antibody-conjugated peroxidase using sodium azide. We applied FT-GO to multiplex fluorescent in situ hybridization, and succeeded in labeling neocortical interneuron subtypes by coupling with immunofluorescence. FT-GO immunofluorescence further increased the detectability of an adeno-associated virus tracer. Given its simplicity and a staining with a high signal-to-noise ratio, FT-GO would provide a versatile platform for histochemical analysis.
... We can only speculate about the factors behind these conflicting results. It is now well established that neurons with a 5-HT-only phenotype form a small minority within MnR, whereas neurons with a glutamatergic (VGLUT3), mixed 5-HT/VGLUT3, GABA-only or unknown phenotype are more common (Hioki et al. 2010;Ren et al. 2019;Sos et al. 2017). Presumably, 5-HT cell-specific viral tracing studies missed mPFC inputs to the abundant non-5-HT neuronal populations within MnR, whereas our findings include them. ...
Anatomical and functional evidence suggests that the PFC is fairly unique among all cortical regions, as it not only receives input from, but also robustly projects back to mesopontine monoaminergic and cholinergic cell groups. Thus, the PFC is in position to exert a powerful top-down control over several state-setting modulatory transmitter systems that are critically involved in the domains of arousal, motivation, reward/aversion, working memory, mood regulation, and stress processing. Regarding this scenario, the origin of cortical afferents to the ventral tegmental area (VTA), laterodorsal tegmental nucleus (LDTg), and median raphe nucleus (MnR) was here compared in rats, using the retrograde tracer cholera toxin subunit b (CTb). CTb injections into VTA, LDTg, or MnR produced retrograde labeling in the cortical mantle, which was mostly confined to frontal polar, medial, orbital, and lateral PFC subdivisions, along with anterior- and mid-cingulate areas. Remarkably, in all of the three groups, retrograde labeling was densest in layer V pyramidal neurons of the infralimbic, prelimbic, medial/ventral orbital and frontal polar cortex. Moreover, a lambda-shaped region around the apex of the rostral pole of the nucleus accumbens stood out as heavily labeled, mainly after injections into the lateral VTA and LDTg. In general, retrograde PFC labeling was strongest following injections into MnR and weakest following injections into VTA. Altogether, our findings reveal a fairly similar set of prefrontal afferents to VTA, LDTg, and MnR, further supporting an eminent functional role of the PFC as a controller of major state-setting mesopontine modulatory transmitter systems.
... Additionally, different sub-regions of RN have been associated with depression and anxiety (Commons, 2016). Rodent studies reveal that 5-HT neurons often co-express other neurotransmitters, for example, glutamate and GABA (Hioki et al., 2010). ...
Humans are inherently social beings. Being suggestible to each other's expectations enables pro‐social skills that are crucial for social learning and adaptation. Despite their high relevance for psychiatry, the neurobiological mechanisms underlying social adaptation are still not well understood. This review, therefore, provides a conceptual framework covering various distinct mechanisms underlying social adaptation and explores the neuropharmacology — in particular the role of the serotonin (5‐HT) system — in modulating these mechanisms. This article reviews empirical results on social influence processing and reconciles them with recent findings from psychedelic research on social processing to elucidate neurobiological and neuropharmacological underpinnings of social adaptation. Various computational, neurobiological, and neurochemical processes are involved in distinct mechanisms underlying social adaptation such as the multisensory process of social information integration that is crucial for the forming of self‐representation and representations of social norms. This is again associated with self‐ and other‐perception during social interactions as well as value‐based decision‐making that guides our behavior in daily interactions. We highlight the critical role of 5‐HT in these processes and suggest that 5‐HT can facilitate social learning and may represent an important target for treating psychiatric disorders characterized by impairments in social functioning. This framework also has important implications for psychedelic‐assisted therapy as well as for the development of novel treatment approaches and future research directions.
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... To directly investigate the effect of local GABAergic cells on the spike output of non-GABAergic putative projection neurons (Jacobs and Azmitia, 1992;Hioki et al., 2009;Jackson et al., 2009;Varga et al., 2009;Szőnyi et al., 2014Szőnyi et al., , 2019, we combined cell type-selective optogenetic manipulation of the former with whole-cell patch clamp recordings from MRR neurons. In several cases, we abruptly lost the cells at the middle of the stimulation because of movement, therefore some of the experiments (n = 6/6 at ChR2 and n = 3/11 at ArchT) were carried out under urethane anesthesia. ...
Ascending serotonergic/glutamatergic projection from the median raphe region (MRR) to the hippocampal formation regulates both encoding and consolidation of memory and the oscillations associated with them. The firing of various types of MRR neurons exhibits rhythmic modulation coupled to hippocampal oscillatory activity. A possible intermediary between rhythm-generating forebrain regions and entrained ascending modulation may be the GABAergic circuit in the MRR, known to be targeted by a diverse array of top-down inputs. However, the activity of inhibitory MRR neurons in an awake animal is still largely unexplored. In this study, we utilized whole cell patch-clamp, single cell, and multichannel extracellular recordings of GABAergic and non-GABAergic MRR neurons in awake, head-fixed mice. First, we have demonstrated that glutamatergic and serotonergic neurons receive both transient, phasic, and sustained tonic inhibition. Then, we observed substantial heterogeneity of GABAergic firing patterns but a marked modulation of activity by brain states and fine timescale coupling of spiking to theta and ripple oscillations. We also uncovered a correlation between the preferred theta phase and the direction of activity change during ripples, suggesting the segregation of inhibitory neurons into functional groups. Finally, we could detect complementary alteration of non-GABAergic neurons’ ripple-coupled activity. Our findings support the assumption that the local inhibitory circuit in the MRR may synchronize ascending serotonergic/glutamatergic modulation with hippocampal activity on a subsecond timescale.
... DR is a neurochemically heterogeneous structure containing distinct clusters of 5-HT neurons and several other differentially distributed major neurotransmitters and neuropeptides (Calizo et al., 2011). Some DR subregions display large proportions of GABAergic, dopaminergic, glutamatergic and neurons with a mixed glutamatergic/serotonergic phenotype (Hioki et al., 2010;Calizo et al., 2011;Soiza-Reilly and Commons, 2011). It has been reported that ICV injection of AD 20 nmol evoked serotonergic and non-serotonergic neuronal activation in the DR (Flores et al., 2018). ...
The dorsal raphe (DR) nucleus is involved in a myriad of physiological functions, such as the control of sleep-wake cycle, motivation, pain, energy balance, and food intake. We have previously demonstrated that in ad libitum fed rats the intra-DR administration of phenylephrine, an α-1 receptor agonist, does not affect food intake, whereas clonidine, an α-2 receptor agonist, potently stimulates food intake. These results indicated that in fed rats an increased adrenergic tonus blocked food intake, since the activation of α-2 auto-receptors, which decreases pre-synaptic release of adrenaline/noradrenaline, affected food intake. Thus, in this study we assessed whether the response to adrenergic stimuli would differ after overnight fasting, a situation of low adrenergic activity in the DR. Intra-DR administration of adrenaline and noradrenaline blocked food intake evoked by overnight fasting. Similarly, phenylephrine administration decreased hunger-induced food intake. These changes in food intake were accompanied by changes in other behaviors, such as increased immobility time and feeding duration. On the other hand, intra-DR administration of clonidine did not affect food-intake or associated behaviors. These results further support the hypothesis that in fed animals, increased adrenergic tonus in DR neurons inhibiting feeding, while in fasted rats the adrenergic tonus decreases and favors food intake. These data indicate a possible mechanism through which adrenergic input to the DRN contributes to neurobiology of feeding.
... The palmitoylated fluorescent protein, palGFP or pal-mRFP1, is targeted to the plasma membrane and can clearly visualize the edges of neuronal cells. Taken together, Sindbis virus vector expressing palGFP or pal-mRFP1 works as a highly sensitive anterograde tracer [2][3][4][5][6][7][8]. ...
Elucidating neuronal circuits is fundamental issue for understanding how the brain works and implements higher-order functions. Various viral vectors have been developed and become valuable tools for the analysis of neuronal circuits in the central nervous system. Sindbis virus vector is very useful for anterograde labeling of neurons, since the vector expresses reporter protein rapidly and strongly. Furthermore, the vector makes it possible to visualize whole structures of a single neuron by injecting adequately diluted virus solution. After immunoperoxidase staining with a tyramine-based signal amplification technique, two-dimensional reconstruction of a single neuron is performed with a virtual slide system and graphic software. In this chapter, we describe a set of single-neuron tracing method in exact detail. On the other hand, Sindbis virus vector shows very high cytotoxicity by shutting off host cellular transcription and translation, and is not suitable for the experiments requiring long-term expression of transgene. In the previous study, we developed novel lentivirus vector and succeeded in neuron-specific and high-level sustained gene expression. This novel vector is expected to be applied as a sensitive anterograde tracer in addition to Sindbis virus vector.
... Cognitive and behavioural changes following VGLUT3 knockout/downregulation may not be regulated by serotonergic neurons. Neuronal projections from the dorsal raphe that contain VGLUT3, but not 5-HT have been previously documented (Commons, 2009;Hioki et al., 2010). These non-serotonergic neurons have also been implicated in reward (McDevitt et al., 2014) and have even been shown to mimic addictive phenotypes produced by VGLUT3 knockout. ...
The overconsumption of sugar-sweetened food and beverages underpins the current rise in obesity rates. Sugar overconsumption induces maladaptive neuroplasticity to decrease dietary control. Although serotonin and glutamate co-localisation has been implicated in reward processing, it is still unknown how chronic sucrose consumption changes this transmission in regions associated with executive control over feeding—such as the prefrontal cortex (PFC) and dentate gyrus (DG) of the hippocampus. To address this, a total of 16 C57Bl6 mice received either 5% w/v sucrose or water as a control for 12 weeks using the Drinking-In-The-Dark paradigm (n = 8 mice per group). We then examined the effects of chronic sucrose consumption on the immunological distribution of serotonin (5-HT), vesicular glutamate transporter 3 (VGLUT3) and 5-HT+/VGLUT3+ co-localised axonal varicosities. Sucrose consumption over 12 weeks decreased the number of 5-HT–/VGLUT3+ and 5-HT+/VGLUT3+ varicosities within the PFC and DG. The number of 5-HT+/VGLUT3– varicosities remained unchanged within the PFC but decreased in the DG following sucrose consumption. Given that serotonin mediates DG neurogenesis through microglial migration, the number of microglia within the DG was also assessed in both experimental groups. Sucrose consumption decreased the number of DG microglia. Although the DG and PFC are associated with executive control over rewarding activities and emotional memory formation, we did not detect a subsequent change in DG neurogenesis or anxiety-like behaviour or depressive-like behaviour. Overall, these findings suggest that the chronic consumption of sugar alters serotonergic neuroplasticity within neural circuits responsible for feeding control. Although these alterations alone were not sufficient to induce changes in neurogenesis or behaviour, it is proposed that the sucrose consumption may predispose individuals to these cognitive deficits which ultimately promote further sugar intake.
... Different groups have recently demonstrated that stimulation of 5-HT cells can be reinforcing and that this effect is mediated through activation of VTA-DA cells via glutamatergic co-transmission (Cunha et al., 2020;Liu et al., 2014;Luo et al., 2015;Wang et al., 2019). Indeed, it was demonstrated that DRN neurons express VGlut3 and that both 5-HT only and 5-HT-glutamatergic neurons from DRN project to VTA neurons (Cunha et al., 2020;Hioki et al., 2010;Liu et al., 2014;Wang et al., 2019). Paralleling these results, a co-transmission of 5-HT and glutamate in the basal amygdala and orbitofrontal cortex following 5-HT fibers stimulation was demonstrated (Ren et al., 2018;Sengupta et al., 2017). ...
Serotonin is a critical neuromodulator involved in development and behavior. Its role in reward is however still debated. Here, we first review classical studies involving electrical stimulation protocols and pharmacological approaches. Contradictory results on the serotonergic’ involvement in reward emerge from these studies. These differences might be ascribable to either the diversity of cellular types within the raphe nuclei or/and the specific projection pathways of serotonergic neurons. We continue to review more recent work, using optogenetic approaches to activate serotonergic cells in the Raphe to VTA pathway. From these studies, it appears that activation of this pathway can lead to reinforcement learning mediated through the excitation of dopaminergic neurons by serotonergic neurons co-transmitting glutamate. Finally, given the importance of serotonin during development on adult emotion, the effect of abnormal early-life levels of serotonin on the dopaminergic system will also be discussed. Understanding the interaction between the serotonergic and dopaminergic systems during development and adulthood is critical to gain insight into the specific facets of neuropsychiatric disorders.
... In addition to serotonin neurons (positive for tryptophan hydroxylase-2, TPH2+), DR contains non-serotonin neurons. Many of these express vesicular glutamate transporter 3 (VGLUT3+, presumably glutamatergic), and are located within approximately 200 μm of the midline (Hioki et al., 2010;McDevitt et al., 2014;Qi et al., 2014;Wang et al., 2019;Cunha et al., 2020). Co-transmission is ubiquitous: a large fraction of DR VGLUT3+ neurons is also serotonergic (Shutoh et al., 2008). ...
The midbrain dorsal raphe (DR) and ventral tegmental area (VTA) contain two of the brain's main ascending neuromodulatory transmitters: serotonin and dopamine. We studied the pathway from DR to VTA using single-cell RNA sequencing, anatomical tracing, and electrophysiology and behavior in mice. Single-cell sequencing confirmed a differential distribution of dopamine cell types between medial and lateral aspects of the VTA. This molecular diversity included differential expression of a subset of glutamatergic and serotonergic receptors. Anatomical data showed that distinct serotonergic and glutamatergic populations of DR neurons project to distinct medial-lateral locations in VTA. Physiological data showed that serotonergic neurons are positioned to excite putative dopaminergic neurons in lateral VTA on short timescales (within trial), and inhibit them on long timescales (on the next trial). Our results reveal precise anatomical specificity of DR projections to VTA, and suggest a functional role for serotonergic modulation of dopaminergic function across multiple timescales.
... Briefly, conditioned media-derived pellets containing DsRed2-mitochondria fixed in 4% paraformaldehyde (Nacalai Tesque, Inc., 30525-89-4) in 0.1 M PB (pH7.2) overnight at 4°C were washed with PBS, embedded in 12% gelatin (Rousselot) in 0.1 M PB, immersed in 2.3 M sucrose in 0.1 M PB overnight and plunged into liquid nitrogen. Sections approximately 60 nm thick were cut with a cryo-ultramicrotome (UC7/FC7, Leica Microsystems, Vienna, Austria) and reacted overnight at 4°C with mouse anti-TOMM20 antibody (1:50) (Santa Cruz Biotechnology, sc-17764) and 1 µg/ml rabbit anti-mRFP antibody (kindly gifted by Dr. Hiroyuki Hioki, Juntendo University, Japan) [40], followed by donkey anti-mouse IgG conjugated with 12 nm colloidal gold particles (1:40; Jackson ImmunoResearch Laboratories, Inc., 115-205-146) and donkey anti-rabbit IgG conjugated with 6 nm colloidal gold particles (1:40;Jackson ImmunoResearch Laboratories,Inc.,. The grids with sections were embedded in a thin layer of 2% methylcellulose (Sigma-Aldrich, M-6385) with 0.4% uranyl acetate (pH 4.0), air-dried, and observed with a Hitachi HT7700 electron microscope (Hitachi, Tokyo, Japan). ...
Mitochondrial quality control, which is crucial for maintaining cellular homeostasis, has been considered to be achieved exclusively through mitophagy. Here we report an alternative mitochondrial quality control pathway mediated by extracellular mitochondria release. By performing time-lapse confocal imaging on a stable cell line with fluorescent-labeled mitochondria, we observed release of mitochondria from cells into the extracellular space. Correlative light-electron microscopy revealed that majority of the extracellular mitochondria are in free form and, on rare occasions, some are enclosed in membrane-surrounded vesicles. Rotenone- and carbonyl cyanide m-chlorophenylhydrazone-induced mitochondrial quality impairment promotes the extracellular release of depolarized mitochondria. Overexpression of PRKN (parkin RBR E3 ubiquitin protein ligase), which has a pivotal role in mitophagy regulation, suppresses the extracellular mitochondria release under basal and stress condition, whereas its knockdown exacerbates it. Correspondingly, overexpression of PRKN-independent mitophagy regulators, BNIP3 (BCL2 interacting protein 3) and BNIP3L/NIX (BCL2 interacting protein 3 like), suppress extracellular mitochondria release. Autophagy-deficient cell lines show elevated extracellular mitochondria release. These results imply that perturbation of mitophagy pathway prompts mitochondria expulsion. Presence of mitochondrial protein can also be detected in mouse sera. Sera of PRKN-deficient mice contain higher level of mitochondrial protein compared to that of wild-type mice. More importantly, fibroblasts and cerebrospinal fluid samples from Parkinson disease patients carrying loss-of-function PRKN mutations show increased extracellular mitochondria compared to control subjects, providing evidence in a clinical context. Taken together, our findings suggest that extracellular mitochondria release is a comparable yet distinct quality control pathway from conventional mitophagy.
Abbreviations: ACTB: actin beta; ANXA5: annexin A5; ATP5F1A/ATP5A: ATP synthase F1 subunit alpha; ATG: autophagy related; BNIP3: BCL2 interacting protein 3; BNIP3L/NIX: BCL2 interacting protein 3 like; CCCP: carbonyl cyanide m-chlorophenylhydrazone; CM: conditioned media; CSF: cerebrospinal fluid; DMSO: dimethyl sulfoxide; EM: electron microscopy; HSPD1/Hsp60: heat shock protein family D (Hsp60) member 1; KD: knockdown; KO: knockout; MAP1LC3A/LC3: microtubule associated protein 1 light chain 3 alpha; MT-CO1: mitochondrially encoded cytochrome c oxidase I; NDUFB8: NADH:ubiquinone oxidoreductase subunit B8; OE: overexpression; OPA1: OPA1 mitochondrial dynamin like GTPase; OXPHOS: oxidative phosphorylation; PBS: phosphate-buffered saline; PB: phosphate buffer; PD: Parkinson disease; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SDHB: succinate dehydrogenase complex iron sulfur subunit B; TOMM20: translocase of outer mitochondrial membrane 20; TOMM40: translocase of outer mitochondrial membrane 40; UQCRC2: ubiquinol-cytochrome c reductase core protein 2; WT: wild-type
... Nissl stained cells, of 33,008 [± 2,345] obtained in juvenile rats by King et al. (2002). This is problematic, given that the DRN consists primarily of neuropil, having a high density of glial, dopaminergic, GABAergic, and glutamatergic cells, on top of 5-HT cells (Adell et al., 2002;Allers and Sharp, 2003;Belin et al., 1979;Commons, 2009;Fu et al., 2010;Hioki et al., 2010;Jacobs and Azmitia, 1992;Jahanshahi et al., 2013;Jolas and Aghajanian, 1997;Molliver, 1987;Soiza-Reilly and Commons, 2014). One other stereological study was identified, reporting an estimate of roughly 18,500 5-HT cells in the DRN of Wistar rats, with relatively high CEs ranging from 0.07 to 0.09 for various DRN sub-regions (Maia et al., 2016). ...
Serotonin (5-HT) is a common neurotransmitter in mammals, playing a central role in the regulation of various processes such as sleep, perception, cognitive and autonomic functions in the nervous system. Previous studies have demonstrated that 5-HT type 3 (5-HT3) receptors are expressed in either or both the substantia nigra (SN) and the dorsal raphe nucleus (DRN) in humans, marmosets, rats and Syrian hamsters. Here, we quantify the distribution of 5-HT3 receptors across these regions in the adult rat. Fluorescent immunohistochemistry was performed on sections of rat brain covering the entire rostro-caudal extent of the SN and DRN with antibodies specific to the 5-HT3A receptor subunit, as well as others targeting the monoaminergic markers tyrosine hydroxylase (TH) and the 5-HT transporter (SERT). The number of 5-HT3A receptor-positive, TH-positive (n = 28,428 ± 888, Gundersen’s m = 1 coefficient of error [CE] = 0.05) and SERT-positive (n = 12,852 ± 462, CE = 0.06) cells were estimated in both the SN and the DRN using stereology. We found that 5-HT3A receptor-positive cells are present in the SNr (n = 1250 ± 64, CE = 0.24), but they did not co-localise with TH-positive cells, nor were they present in the SNc. In contrast, no 5-HT3A receptor-positive cells were found in the DRN. These results support the presence of 5-HT3 receptors in the SN, but not in the DRN, and do not support their expression on monoaminergic cells within these two brain areas.
... The DRN has reciprocal connections with numerous hypothalamic nuclei, known to control feeding (Petrovický et al., 1981). The DRN contains different types of neurons, such as neurons expressing serotonin (5-HT), dopamine, glutamate, and GABA (Hioki et al., 2010). Furthermore, the DRN contains topographically organized subpopulations of 5-HT, neurons that provide the majority of serotonergic inputs to limbic forebrain structures involved in regulating complex responses to stress. ...
Major depressive disorder is associated with weight loss and decreased appetite; however, the signaling that connects these conditions is unclear. Here, we show that MC4R signaling in the dorsal raphe nucleus (DRN) affects feeding, anxiety, and depression. DRN infusion of α-MSH decreases DRN neuronal activation and feeding. DRN MC4R is expressed in GABAergic PRCP-producing neurons. DRN selective knockdown of PRCP (PrcpDRNKD), an enzyme inactivating α-MSH, decreases feeding and DRN neuronal activation. Interestingly, PrcpDRNKD mice present lower DRN serotonin levels and depressive-like behavior. Similarly, PRCP-ablated MC4R mice (PrcpMC4RKO) show metabolic and behavioral phenotypes comparable to those of PrcpDRNKD mice. Selective PRCP re-expression in DRN MC4R neurons of PrcpMC4RKO mice partially reverses feeding, while fully restoring mood behaviors. Chemogenetic inhibition of DRN MC4R neurons induces anxiety, depression, and reduced feeding, whereas chemogenetic activation reverses these effects. Our results indicate that MC4R signaling in DRN plays a role in feeding, anxiety, and depression.
A major subpopulation of midbrain 5-hydroxytryptamine (5-HT) neurons expresses the vesicular glutamate transporter 3 (VGLUT3) and co-releases 5-HT and glutamate, but the function of this co-release is unclear. Given the strong links between 5-HT and uncontrollable stress, we used a combination of c-Fos immunohistochemistry and conditional gene knockout mice to test the hypothesis that glutamate co-releasing 5-HT neurons are activated by stress and involved in stress coping. Acute, uncontrollable swim stress increased c-Fos immunoreactivity in neurons co-expressing VGLUT3 and the 5-HT marker tryptophan hydroxylase 2 (TPH2) in the dorsal raphe nucleus (DRN). This effect was localized in the ventral DRN subregion and prevented by the antidepressant fluoxetine. In contrast, a more controllable stressor, acute social defeat, had no effect on c-Fos immunoreactivity in VGLUT3-TPH2 co-expressing neurons in the DRN. To test whether activation of glutamate co-releasing 5-HT neurons was causally linked to stress coping, mice with a specific deletion of VGLUT3 in 5-HT neurons were exposed to acute swim stress. Compared to wildtype controls, the mutant mice showed increased climbing behavior, a measure of active coping. Wildtype mice also showed increased climbing when administered fluoxetine, revealing an interesting parallel between the behavioral effects of genetic loss of VGLUT3 in 5-HT neurons and 5-HT reuptake inhibition. We conclude that 5-HT-glutamate co-releasing neurons are recruited by exposure to uncontrollable stress. Furthermore, natural variation in the balance of 5-HT and glutamate co-released at the 5-HT synapse may impact stress susceptibility.
Aggressive behavior is instinctively driven behavior that helps animals to survive and reproduce and is closely related to multiple behavioral and physiological processes. The dorsal raphe nucleus (DRN) is an evolutionarily conserved midbrain structure that regulates aggressive behavior by integrating diverse brain inputs. The DRN consists predominantly of serotonergic (5‐HT:5‐hydroxytryptamine) neurons and decreased 5‐HT activity was classically thought to increase aggression. However, recent studies challenge this 5‐HT deficiency model, revealing a more complex role for the DRN 5‐HT system in aggression. Furthermore, emerging evidence has shown that non‐5‐HT populations in the DRN and specific neural circuits contribute to the escalation of aggressive behavior. This review argues that the DRN serves as a multifaceted modulator of aggression, acting not only via 5‐HT but also via other neurotransmitters and neural pathways, as well as different subsets of 5‐HT neurons. In addition, we discuss the contribution of DRN neurons in the behavioral and physiological aspects implicated in aggressive behavior, such as arousal, reward, and impulsivity, to further our understanding of DRN‐mediated aggression modulation.
The dorsal raphe nucleus (DRN) is an important nucleus in pain regulation. However, the underlying neural pathway and the function of specific cell types remain unclear. Here, we report a previously unrecognized ascending facilitation pathway, the DRN to mesoaccumbal dopamine (DA) circuit, for regulating pain. Chronic pain increased the activity of DRN glutamatergic, but not serotonergic, neurons projecting to the ventral tegmental area (VTA) (DRN Glu -VTA) in male mice. Optogenetic activation of DRN Glu -VTA circuit induced a pain-like response in naïve male mice and its inhibition produced analgesic effect in male mice with neuropathic pain. Furthermore, we discovered that DRN ascending pathway regulated pain through strengthened excitatory transmission onto the VTA DA neurons projecting to the ventral part of nucleus accumbens medial shell (vNAcMed), thereby activated the mesoaccumbal DA neurons. Correspondingly, optogenetic manipulation of this three-node pathway bilaterally regulated pain behaviors. These findings identified a DRN ascending excitatory pathway that is crucial for pain sensory processing, which can potentially be exploited toward targeting pain disorders.
Significance Statement The dorsal raphe nucleus (DRN) in the midbrain contributes to pain processing, yet the detailed cellular and circuitry mechanisms remain largely unknown. Here, we report that chronic pain increases the activity of a specific subpopulation of DRN glutamatergic neurons, which project to the ventral tegmental area (VTA). The elevated excitability of DRN glutamatergic neurons causes the increased excitatory inputs to VTA dopamine neurons that selectively innervate the ventral part of the nucleus accumbens medial shell (vNAcMed). Optogenetic activation of the DRN-VTA-vNAcMed pathway induced neuronal plasticity in the VTA and resulted in pain hypersensitivity. These findings shed light on how ascending DRN excitatory circuit is involved in the sensory modulation of pain.
Chronic pain causes both physical suffering and comorbid mental symptoms such as anhedonia. However, the neural circuits and molecular mechanisms underlying these maladaptive behaviors remain elusive. Here using a mouse model, we report a pathway from vesicular glutamate transporter 3 neurons in the dorsal raphe nucleus to dopamine neurons in the ventral tegmental area (VGluT3 DRN → DA VTA ) wherein population-level activity in response to innocuous mechanical stimuli and sucrose consumption is inhibited by chronic neuropathic pain. Mechanistically, neuropathic pain dampens VGluT3 DRN → DA VTA glutamatergic transmission and DA VTA neural excitability. VGluT3 DRN → DA VTA activation alleviates neuropathic pain and comorbid anhedonia-like behavior (CAB) by releasing glutamate, which subsequently promotes DA release in the nucleus accumbens medial shell (NAcMed) and produces analgesic and anti-anhedonia effects via D2 and D1 receptors, respectively. In addition, VGluT3 DRN → DA VTA inhibition produces pain-like reflexive hypersensitivity and anhedonia-like behavior in intact mice. These findings reveal a crucial role for VGluT3 DRN → DA VTA → D2/D1 NAcMed pathway in establishing and modulating chronic pain and CAB.
Serotonin is a neurotransmitter that is synthesized and released from the brainstem raphe nuclei to affect many brain functions. It is well known that the activity of raphe serotonergic neurons is changed in response to the changes in feeding status to regulate appetite via the serotonin receptors. Likewise, changes in volume status are known to alter the activity of raphe serotonergic neurons and drugs targeting serotonin receptors were shown to affect sodium appetite. Therefore, the central serotonin system appears to regulate ingestion of both food and salt, although neural mechanisms that induce appetite in response to hunger and sodium appetite in response to volume depletion are largely distinct from each other. In this review, we discuss our current knowledge regarding the regulation of ingestion - appetite and sodium appetite - by the central serotonin system.
Background:
Mu opioid receptors (MORs) are key for reward processing, mostly studied in dopaminergic pathways. MORs are also expressed in the dorsal raphe nucleus (DRN), central for the modulation of reward and mood, but MOR function in the DRN remains underexplored. Here, we investigated whether MOR-expressing neurons of the DRN (DRN-MOR neurons) participate to reward and emotional responses.
Methods:
We characterized DRN-MOR neurons anatomically using immunohistochemistry, and functionally using fiber photometry in responses to morphine and rewarding/aversive stimuli. We tested the effect of opioid uncaging on the DRN on place conditioning. We examined the effect of DRN-MOR neuron opto-stimulation on positive reinforcement and mood-related behaviors. We mapped their projections, and selected DRN-MOR neurons projecting to the lateral hypothalamus (DRN/LH-MOR) for a similar optogenetic experimentation.
Results:
DRN-MOR neurons form a heterogeneous neuronal population, essentially composed of GABAergic and glutamatergic neurons. Calcium activity of DRN-MOR neurons was inhibited by rewarding stimuli and morphine. Local photo-uncaging of oxymorphone in DRN produced conditioned place preference. DRN-MOR neuron opto-stimulation triggered real-time place preference and was self-administered, promoted social preference; reduced anxiety and passive coping. Finally, specific opto-stimulation of DRN/LH-MOR neurons recapitulated reinforcing effects of total DRN-MOR neuron stimulation.
Conclusions:
Our data show that DRN-MOR neurons respond to rewarding stimuli, and that their opto-activation has reinforcing effects and promotes positive emotional responses, an activity partially mediated by their projections to the LH. Our study also suggests a complex regulation of DRN activity by MOR opioids, involving mixed inhibition/activation mechanisms that fine-tune DRN function.
Parkinson’s disease (PD) is a common neurodegenerative disease implicated in multiple interacting neurotransmitter pathways. Glutamate is the central excitatory neurotransmitter in the brain and plays critical influence in the control of neuronal activity. Impaired Glutamate homeostasis has been shown to be closely associated with PD. Glutamate is synthesized in the cytoplasm and stored in synaptic vesicles by vesicular glutamate transporters (VGLUTs). Following its exocytotic release, Glutamate activates Glutamate receptors (GluRs) and mediates excitatory neurotransmission. While Glutamate is quickly removed by excitatory amino acid transporters (EAATs) to maintain its relatively low extracellular concentration and prevent excitotoxicity. The involvement of GluRs and EAATs in the pathophysiology of PD has been widely studied, but little is known about the role of VGLUTs in the PD. In this review, we highlight the role of VGLUTs in neurotransmitter and synaptic communication, as well as the massive alterations in Glutamate transmission and VGLUTs levels in PD. Among them, adaptive changes in the expression level and function of VGLUTs may exert a crucial role in excitatory damage in PD, and VGLUTs are considered as novel potential therapeutic targets for PD.
The hippocampus (HP) receives neurochemically diverse inputs from the raphe nuclei, including glutamatergic axons characterized by the expression of the vesicular glutamate transporter type 3 (VGLUT3). These raphe-HP VGLUT3 projections have been suggested to play a critical role in HP functions, yet a complete anatomical overview of raphe VGLUT3 projections to the forebrain, and in particular to the HP, is lacking. Using anterograde viral tracing, we describe largely nonoverlapping VGLUT3-positive projections from the dorsal raphe (DR) and median raphe (MnR) to the forebrain, with the HP receiving inputs from the MnR. A limited subset of forebrain regions such as the amygdaloid complex, claustrum, and hypothalamus receives projections from both the DR and MnR that remain largely segregated. This highly complementary anatomical pattern suggests contrasting roles for DR and MnR VGLUT3 neurons. To further analyze the topography of VGLUT3 raphe projections to the HP, we used retrograde tracing and found that HP-projecting VGLUT3-positive neurons (VGLUT3HP ) distribute over several raphe subregions (including the MnR, paramedian raphe, and B9 cell group) and lack co-expression of serotonergic markers. Strikingly, double retrograde tracing experiments unraveled two parallel streams of VGLUT3-positive projections targeting the dorsal and ventral poles of the HP. These results demonstrate highly organized and segregated VGLUT3-positive projections to the HP, suggesting independent modulation of HP functions such as spatial memory and emotion-related behavior.
Anatomical and functional evidence suggests that the PFC is fairly unique among all cortical regions, as it not only receives input from, but also robustly projects back to mesopontine monoaminergic and cholinergic cell groups. Thus the PFC is in a position to exert a powerful top-down control over several state setting modulatory transmitter systems that are critically involved in the domains of arousal, motivation, reward/aversion, working memory, mood regulation, and stress processing. Regarding this scenario, the origin of cortical afferents to the ventral tegmental area (VTA), laterodorsal tegmental nucleus (LDTg), and median raphe nucleus (MnR) was here compared, by using the retrograde tracer cholera toxin subunit b (CTb). CTb injections into VTA, LDTg, or MnR produced retrograde labeling in the cortical mantle, which was mostly confined to medial, orbital, and lateral PFC subdivisions, along with rostral and mid-cingulate areas. Remarkably, in all of the three groups, retrograde labeling was densest in layer V pyramidal neurons of the infralimbic, prelimbic, medial orbital and frontal polar cortex. Moreover, a conspicuous lambda-shaped region around the apex of the rostral pole of the nucleus accumbens stood consistently out as heavily labeled. At almost all rostrocaudal levels through the PFC analyzed, retrograde labeling was strongest following injections into MnR and weakest following injections into VTA. Altogether, our findings reveal a broadly similar set of prefrontal afferents to VTA, LDTg, and MnR, further supporting an eminent functional role of the PFC as a controller of all major state setting mesopontine modulatory transmitter systems.
Given its limited accessibility, the CA2 area has been less investigated compared to other subregions of the hippocampus. While the development of transgenic mice expressing Cre recombinase in the CA2 has revealed unique features of this area, the use of mouse lines has several limitations, such as lack of specificity. Therefore, a specific gene delivery system is required. Here, we confirmed that the AAV-PHP.eB capsid preferably infected CA2 pyramidal cells following retro-orbital injection and demonstrated that the specificity was substantially higher after injection into the lateral ventricle. In addition, a tropism for the CA2 area was observed in organotypic slice cultures. Combined injection into the lateral ventricle and stereotaxic injection into the CA2 area specifically introduced the transgene into CA2 pyramidal cells, enabling us to perform targeted patch-clamp recordings and optogenetic manipulation. These results suggest that AAV-PHP.eB is a versatile tool for specific gene transduction in CA2 pyramidal cells.
It has been known that a number of tyrosine hydroxylase (TH)-positive neurons, which are regarded as dopaminergic (DA) neurons, exist in the dorsal raphe (DR). These DA neurons in the DR and periaqueductal gray (PAG) region (DADR-PAG neurons) are thought to belong to the A10 cluster, which is known to be heterogeneous. This DA population projects to the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST) and has been reported to modulate various affective behaviors. The DA transporter (DAT) neurons, which are well overlapping with DA neurons, in the DR-PAG region are also expected to be heterogeneous. However, even though the heterogeneity of DA/DATDR-PAG neurons has been suggested, the characteristics of each DA/DATDR-PAG neuron subpopulation are not well investigated. In this paper, we summarize the previous reports investigating the heterogeneity of DA/DATDR-PAG neurons and the functional importance of DA/DATDR-PAG neurons on various affective behaviors and introduce our recent findings that DATDR-PAG neurons consist of two subpopulations: TH+/vasoactive intestinal peptide (VIP)− putative DA neurons and TH-/VIP+ putative glutamatergic neurons.
In the avian ascending auditory pathway, the nucleus mesencephalicus lateralis pars dorsalis (MLd; the auditory midbrain center) receives inputs from virtually all lower brainstem auditory nuclei and sends outputs bilaterally to the nucleus ovoidalis (Ov; the auditory thalamic nucleus). Axons from part of the MLd terminate in a particular domain of Ov, thereby suggesting a formation of segregated pathways point-to-point from lower brainstem nuclei via MLd to the thalamus. However, it has not yet been demonstrated whether any spatial clustering of thalamic neurons that receive inputs from specific domains of MLd exists. Ov neurons receive input from bilateral MLds; however, the degree of laterality has not been reported yet. In this study, we injected a recombinant avian adeno-associated virus, a transsynaptic anterograde vector into the MLd of the chick, and analyzed the distribution of labeled postsynaptic neurons on both sides of the Ov. We found that fluorescent protein-labeled neurons on both sides of the Ov were clustered in domains corresponding to subregions of the MLd. The laterality of projections was calculated as the ratio of neurons labeled by comparing ipsilateral to contralateral projections from the MLd, and it was 1.86 on average, thereby indicating a slight ipsilateral projection dominance. Bilateral inputs from different subdomains of the MLd converged on several single Ov neurons, thereby implying a possibility of a de novo binaural processing of the auditory information in the Ov.
The circuit mechanism of the thermoregulatory center in the preoptic area (POA) is unknown. Using rats, here we show prostaglandin EP3 receptor-expressing POA neurons (POA EP3R neurons) as a pivotal bidirectional controller in the central thermoregulatory mechanism. POA EP3R neurons are activated in response to elevated ambient temperature, but inhibited by prostaglandin E 2 , a pyrogenic mediator. Chemogenetic stimulation of POA EP3R neurons at room temperature reduces body temperature by enhancing heat dissipation, whereas inhibition of them elicits hyperthermia involving brown fat thermogenesis, mimicking fever. POA EP3R neurons innervate sympathoexcitatory neurons in the dorsomedial hypothalamus (DMH) via tonic inhibitory signaling. Although many POA EP3R neuronal cell bodies express a glutamatergic mRNA marker, paradoxically, their axons in the DMH predominantly contain terminals with GABAergic presynaptic proteins, which are increased by chronic heat exposure. These findings indicate that tonic GABAergic inhibitory signaling from POA EP3R neurons is a fundamental determinant of body temperature for thermal homeostasis and fever.
The striatum is one of the key nuclei for adequate control of voluntary behaviors and reinforcement learning. Two striatal projection neuron types, expressing either dopamine receptor D1 (D1R) or dopamine receptor D2 (D2R) constitute two independent output routes: the direct or indirect pathways, respectively. These pathways co-work in balance to achieve coordinated behavior. Two projection neuron types are equivalently intermingled in most striatal space. However, recent studies revealed two atypical zones in the caudal striatum: the zone in which D1R-neurons are the minor population (D1R-poor zone) and that in which D2R-neurons are the minority (D2R-poor zone). It remains obscure as to whether these imbalanced zones have similar properties on axonal projections and electrophysiology compared to other striatal regions. Based on morphological experiments in mice using immunofluorescence, in situ hybridization, and neural tracing, here, we revealed that the poor zones densely projected to the globus pallidus and substantia nigra pars lateralis, with a few collaterals in substantia nigra pars reticulata and compacta. Similar to that in other striatal regions, D1R-neurons were the direct pathway neurons. We also showed that the membrane properties of projection neurons in the poor zones were largely similar to those in the conventional striatum using in vitro electrophysiological recording. In addition, the poor zones existed irrespective of the age or sex of mice. We also identified the poor zones in the common marmoset as well as other rodents. These results suggest that the poor zones in the caudal striatum follow the conventional projection patterns irrespective of the imbalanced distribution of projection neurons. The poor zones could be an innate structure and common in mammals. The unique striatal zones possessing highly restricted projections could relate to functions different from those of motor-related striatum.
Energy balance is orchestrated by an extended network of highly interconnected nuclei across the central nervous system. While much is known about the hypothalamic circuits regulating energy homeostasis, the ‘extra-hypothalamic’ circuits involved are relatively poorly understood. In this review, we focus on the brainstem’s dorsal raphe nucleus (DRN), integrating decades of research linking this structure to the physiologic and behavioral responses that maintain proper energy stores. DRN neurons sense and respond to interoceptive and exteroceptive cues related to energy imbalance and in turn induce appropriate alterations in energy intake and expenditure. The DRN is also molecularly differentiable, with different populations playing distinct and often opposing roles in controlling energy balance. These populations are integrated into the extended circuit known to regulate energy balance. Overall, this review summarizes the key evidence demonstrating an important role for the DRN in regulating energy balance.
Post-Traumatic Stress Disorder (PTSD), characterized by re-experiencing, avoidance, negative affect, and impaired memory processing, may develop after traumatic events. PTSD is complicated by impaired plasticity and medial prefrontal cortex (mPFC) activity, hyperactivity of the amygdala, and impaired fear extinction. Cannabidiol (CBD) is a promising candidate for treatment due to its multimodal action that enhances plasticity and calms hyperexcitability. CBD’s mechanism in the mPFC of PTSD patients has been explored extensively, but literature on the mechanism in the dorsal raphe nucleus (DRN) is lacking. Following the PRISMA guidelines, we examined current literature regarding CBD in PTSD and overlapping symptomologies to propose a mechanism by which CBD treats PTSD via corticoraphe circuit. Acute CBD inhibits excess 5-HT release from DRN to amygdala and releases anandamide (AEA) onto amygdala inputs. By first reducing amygdala and DRN hyperactivity, CBD begins to ameliorate activity disparity between mPFC and amygdala. Chronic CBD recruits the mPFC, creating harmonious corticoraphe signaling. DRN releases enough 5-HT to ameliorate mPFC hypoactivity, while the mPFC continuously excites DRN 5-HT neurons via glutamate. Meanwhile, AEA regulates corticoraphe activity to stabilize signaling. AEA prevents DRN GABAergic interneurons from inhibiting 5-HT release so the DRN can assist the mPFC in overcoming its hypoactivity. DRN-mediated restoration of mPFC activity underlies CBD’s mechanism on fear extinction and learning of stress coping.
Significance
Depression is a serious disease afflicting an increasing number of people, including adolescents. Animal studies indicate that the neuropeptide galanin and its three receptors, GalR1 to 3, are involved in depression-like behavior, evidenced by increased GalR1 transcript levels in the ventral periaqueductal gray of “depressed” rats. Here, we show coexistence of Scratch2 and GalR1, identifying a possible mechanism underlying control of GalR1 expression in this region: the transcription factor Scratch2 lowers, via binding to an E-box in the GalR1 promoter, expression of the GalR1 receptor. This may represent a further role for Scratch2, earlier associated with embryonic development, cell migration, and neurogenesis. Because GalR1 seems involved in major depression, we discuss whether or not Scratch2 also may play a role in the human disease.
Background
Retrograde and anterograde transsynaptic viral vectors are useful tools for studying the input and output organization of neuronal circuitry, respectively. While retrograde transsynaptic viral vectors are widely used, viral vectors that show anterograde transsynaptic transduction are not common.
New Method
We chose recombinant avian adeno-associated virus (A3V) carrying the mCherry gene and injected it into the eyeball, cochlear duct, and midbrain auditory center of chickens. We observed different survival times to examine the virus transcellular transport and the resulting mCherry expression. To confirm the transcellular transduction mode, we co-injected A3V and cholera toxin B subunit.
Results
Injecting A3V into the eyeball and cochlea labeled neurons in the visual and auditory pathways, respectively. Second-, and third-order labeling occurred approximately two and seven days, respectively, after injection into the midbrain. The distribution of labeled neurons strongly suggests that A3V transport is preferentially anterograde and transduces postsynaptic neurons.
Comparison with Existing Method(s)
A3V displays no extrasynaptic leakage and moderate speed of synapse passage, which is better than other viruses previously reported. Compared with AAV1&9, which have been shown to pass one synapse anterogradely, A3V passes several synapses in the anterograde direction.
Conclusions
A3V would be a good tool to study the topographic organization of projection axons and their target neurons.
Dopamine (DA), serotonin (5-hydroxytryptamine, 5-HT), and endocannabinoids (ECs) are key neuromodulators involved in many aspects of motivated behavior, including reward processing, reinforcement learning, and behavioral flexibility. Among the longstanding views about possible relationships between these neuromodulators is the idea of DA and 5-HT acting as opponents. This view has been challenged by emerging evidence that 5-HT supports reward seeking via activation of DA neurons in the ventral tegmental area. Adding an extra layer of complexity to these interactions, the endocannabinoid system is uniquely placed to influence dopaminergic and serotonergic neurotransmission. In this review we discuss how these three neuromodulatory systems interact at the cellular and circuit levels. Technological advances that facilitate precise identification and control of genetically targeted neuronal populations will help to achieve a better understanding of the complex relationship between these essential systems, and the potential relevance for motivated behavior.
We developed an adeno-associated virus (AAV) vector-based technique to label mouse neostriatal neurons comprising direct and indirect pathways with different fluorescent proteins and analyze their axonal projections. The AAV vector expresses GFP or RFP in the presence or absence of Cre recombinase and should be useful for labeling two cell populations exclusively dependent on its expression. Here, we describe the AAV vector design, stereotaxic injection of the AAV vector, and a highly sensitive immunoperoxidase method for axon visualization.
For complete details on the use and execution of this protocol, please refer to Okamoto et al. (2020).
The present study aimed to evaluate the effects of pharmacological manipulation of α-adrenergic agonists in the dorsal raphe nucleus (DR) on food intake in satiated rats. Adult male Wistar rats with chronically implanted cannula in the DR were injected with adrenaline (AD) or noradrenaline (NA) (both at doses of 6, 20 and 60 nmol), or α-1 adrenergic agonist phenylephrine (PHE) or α-2 adrenergic agonist clonidine (CLO) (both at doses of 6 and 20 nmol). The injections were followed by the evaluation of ingestive behaviors. Food and water intake were evaluated for 60 min. Administration of AD and NA at 60 nmol and CLO at 20 nmol increased food intake and decreased latency to start consumption in satiated rats. The ingestive behavior was not significantly affected by PHE treatment in the DR. CLO treatment increased Fos expression in the arcuate nucleus (ARC) and paraventricular nucleus of the hypothalamus (PVN) in rats that were allowed to eat during the experimental recording (AF group). However, when food was not offered during the experiment (WAF group), PVN neurons were not activated, whereas, neuronal activity remained high in the ARC when compared to control group. Noteworthy, ARC POMC neurons expressed Fos in the AF group. However, double-labeled POMC/Fos cells were absent in the ARC of the WAF group, although an increase in Fos expression was observed in non-POMC cells after CLO injections in the WAF group. In conclusion, the data from the present study highlight that the pharmacological activation of DR α-adrenoceptors affects food intake in satiated rats. The feeding response evoked by CLO injections into DR was similar to that induced by NA or AD injections, suggesting that the hyperphagia after NA or AD treatment depends on α-2 adrenoceptors activation. Finally, we have demonstrated that CLO injections into DR impact neuronal activity in the ARC, possibly evoking a homeostatic response toward food intake.
The serotonergic precursor tryptophan and the dopaminergic precursor tyrosine have been shown to be important modulators of mood, behaviour and cognition. Specifically, research on the function of tryptophan has characterised this molecule as particularly relevant in the context of pathological disorders such as depression. Moreover, a large body of evidence has now been accumulated to suggest that tryptophan may also be involved in executive function and reward processing. Despite some clear differentiation with tryptophan, the data reviewed in this paper illustrates that tyrosine shares similar functions with tryptophan in the regulation of executive function and reward, and that these processes in turn, rather than acting in isolation, causally influence each other.
We developed novel lentiviral vectors by using "Tet-Off system" and succeeded in achieving high-level and neuron-specific gene transduction in vivo. One week after viral injection into the rat neostriatum, the GFP expression was almost completely neuron-specific and about 40 times higher than the expression of a conventional lentiviral vector. High transcriptional activity and neuronal specificity were sustained for up to 8 weeks. Furthermore, neuronal processes of the infected neurons were efficiently visualized by adding a plasma membrane-targeting signal to GFP. These results suggest that the present method is valuable for strong gene transduction and clear visualization of neurons in vivo.
The dorsal raphe nucleus (DRN) is a heterogeneous brainstem nucleus located in the midbrain and pons. Via widespread projections, which target a multitude of brain areas, its neurons utilize many transmitters to control various physiological functions, including learning, memory and affect. Accordingly, the DRN has been strongly associated with brain dysfunction, especially mood disorders such as depression, but also Alzheimer's disease. The DRN's most abundant transmitter, serotonin, has received the most attention in studies on both normal brain function and disease, and lately its involvement in the regulation of neuroplasticity has been under particular scrutiny. This chapter begins with a systematic overview of what we currently know about the anatomy of the DRN and its neurons, including their ascending projections. It continues with a review of the transmitters of the DRN, followed by a discussion on the connection between the DRN and neuroplasticity. Special emphasis is put on serotonin and its central role in neuroplasticity, which is proving to be of high priority in unraveling the full picture of the cellular mechanisms and their interconnections in the etiology of major depression and Alzheimer's disease.
Autosomal-dominant sensorineural hearing loss is genetically heterogeneous, with a phenotype closely resembling presbycusis, the most common sensory defect associated with aging in humans. We have identified SLC17A8, which encodes the vesicular glutamate transporter-3 (VGLUT3), as the gene responsible for DFNA25, an autosomal-dominant form of progressive, high-frequency nonsyndromic deafness. In two unrelated families, a heterozygous missense mutation, c.632C-->T (p.A211V), was found to segregate with DFNA25 deafness and was not present in 267 controls. Linkage-disequilibrium analysis suggested that the families have a distant common ancestor. The A211 residue is conserved in VGLUT3 across species and in all human VGLUT subtypes (VGLUT1-3), suggesting an important functional role. In the cochlea, VGLUT3 accumulates glutamate in the synaptic vesicles of the sensory inner hair cells (IHCs) before releasing it onto receptors of auditory-nerve terminals. Null mice with a targeted deletion of Slc17a8 exon 2 lacked auditory-nerve responses to acoustic stimuli, although auditory brainstem responses could be elicited by electrical stimuli, and robust otoacoustic emissions were recorded. Ca(2+)-triggered synaptic-vesicle turnover was normal in IHCs of Slc17a8 null mice when probed by membrane capacitance measurements at 2 weeks of age. Later, the number of afferent synapses, spiral ganglion neurons, and lateral efferent endings below sensory IHCs declined. Ribbon synapses remaining by 3 months of age had a normal ultrastructural appearance. We conclude that deafness in Slc17a8-deficient mice is due to a specific defect of vesicular glutamate uptake and release and that VGLUT3 is essential for auditory coding at the IHC synapse.
Injections of a mixture of tritiated amino acids were made into the posterior hypothalamus in a series of rats and cats. In every case in which the injection involved a significant proportion of the cells in the supramammillary region, labeled fibers could be followed to the dentate gyrus, the anterior hippocampal rudiment and the induseum griseum of both sides. In the dentate gyrus the hypothalamic afferents terminate in a narrow band in the outer half of the stratum gramulosum and the inner 20 micron or so, of the stratum moleculare, immediately deep to the zone of termination of the associational and commissural afferents. As judged by silver grain counts across the width of the zone of labeled terminals, the projection to the ipsilateral side is several times as heavy as that to the contralateral side, and although it involves the entire septo-temporal (=rostro-caudal) extent of the gyrus on both sides, the projection to the suprapyramidal (inner) blade of the dentate gyrus is approximately twice as heavy as that to the infrapyramidal (outer) blade.
Fluorescence-activated cell sorting (FACS) was used to identify and isolate permeabilized dopaminergic nerve terminals from rat striatum based on immunofluorescent labeling of tyrosine hydroxylase (TH). Striatal synaptosomes were permeabilized by fixation with modified Zamboni fluid. A highly fluorescent subpopulation of particles was detected by FACS following sequential incubation with a monoclonal antibody to TH (LNC 1) and a fluorescein-conjugated secondary antibody. After correcting for nonsynaptosomal particles present in the synaptosomal fraction, analysis of these data suggested that approximately 12-15% of striatal synaptosomes were dopaminergic, consistent with previous estimates. Specific labelling by LNC 1 was decreased if synaptosomes were prepared from rats that had received intraventricular injections of 6-hydroxydopamine. The decrease in labeling was highly correlated with the extent of the lesion as determined by measurement of striatal dopamine levels, suggesting that LNC 1-labeled synaptosomes were derived from nigrostriatal dopamine terminals. In order to verify that LNC 1-labeled synaptosomes were enriched for TH, equal numbers of labeled and control synaptosomes were isolated by FACS and analyzed by SDS-PAGE. LNC 1-labeled synaptosomes were shown by Western blot techniques to be enriched 6-fold for TH compared with control synaptosomes, suggesting that the labeled population consisted almost entirely of dopaminergic synaptosomes.
The serotoninergic (5-HT) input from the dorsal raphe nucleus (DRN) to midbrain dopamine (DA) neurons is one of the most prominent. In this study, using standard extracellular single cell recording techniques we investigated the effects of electrical stimulation of the DRN on the spontaneous activity of substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA) DA neurons in anesthetized rats. Poststimulus time histograms (PSTH) revealed two different types of response in both SNpc and VTA. Some cells exhibited an inhibition-excitation response while in other DA neurons the initial response was an excitation followed by an inhibition. In SNpc, 56% of the DA cells recorded were initially inhibited and 31% of the DA cells were initially excited. In contrast, 63% of VTA DA cells were initially excited and 34% were initially inhibited. Depletion of endogenous 5-HT by the neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT), and the 5-HT synthesis inhibitor para-chlorophenylalanine (PCPA), almost completely eliminated the inhibition-excitation response in both SNpc and VTA DA cells, without changing the percentage of DA cells initially excited. Consequently, the proportion of DA neurons that were not affected by DR stimulation increased after 5-HT depletion (from 13% to 60% in SNpc and from 6% to 31% in VTA). In several DA cells, DRN stimulation caused important changes in firing rate and firing pattern. These data strongly suggest that the 5-HT input from the DRN is mainly inhibitory. It also suggests that 5-HT afferences modulate SNpc and VTA DA neurons in an opposite manner. Our results also suggest that non-5-HT inputs from DR can also modulate mesencephalic DA neurons. A differential modulation of VTA and SNpc DA neurons by 5-HT afferences from the DRN could have important implications for the development of drugs to treat schizophrenia or other neurologic and psychiatric diseases in which DA neurons are involved. Synapse 35:281–291, 2000. © 2000 Wiley-Liss, Inc.
To understand the heterogeneity of γ-aminobutyric acid type B receptor (GABABR)- mediated events, we investigated expression of GABABR1a and 1b mRNA variants in GABA and non-GABAergic neurons of the rat central nervous system (CNS), by using nonradioactive in situ hybridization histochemistry and, in combination with GABA immunocytochemistry, double labeling. In situ hybridization with a pan probe, which recognizes a common sequence of both GABABR1a and GABABR1b mRNA variants, demonstrated widespread expression of GABABR1 mRNA at various levels in the CNS. Both GABABR1a and GABABR1b were expressed in the neocortex, hippocampus, dorsal thalamus, habenula, and septum, but only GABABR1a was detected in cerebellar granule cells, in caudate putamen, and most hindbrain structures. A majority of GABA neurons in cerebral cortex showed hybridization signals for both GABABR1a and GABABR1b, whereas those in most subcortical structures expressed either or neither of the two. GABA neurons in thalamic reticular nucleus and caudate putamen hybridized primarily for GABABR1a. Purkinje cells in the cerebellar cortex expressed predominantly GABABR1b. GABA neurons in dorsal lateral geniculate nucleus did not display significant levels of either GABABR1a or GABABR1b mRNAs. These data suggested widespread availability of GABABR-mediated inhibition in the CNS. The differential but overlapping expression of GABABR1 mRNA variants in different neurons and brain structures may contribute to the heterogeneity of GABABR-mediated inhibition. Some GABA neurons possessed, but others might lack the molecular machinery for GABABR-mediated disinhibition, autoinhibition, or both. J. Comp. Neurol. 416:475–495, 2000.
The role of serotonergic systems in regulation of behavioral arousal and sleep-wake cycles is complex and may depend on both the receptor subtype and brain region involved. Increasing evidence points toward the existence of multiple topographically organized subpopulations of serotonergic neurons that receive unique afferent connections, give rise to unique patterns of projections to forebrain systems, and have unique functional properties. A better understanding of the properties of these subpopulations of serotonergic neurons may aid in the understanding of the role of serotonergic systems in regulation of behavioral arousal, sleep-wake cycles and other physiological and behavioral responses attributed to serotonin. In this chapter, we outline evidence for multiple serotonergic systems within the midbrain and pontine raphe complex that can be defined based on cytoarchitectonic and hodological properties. In addition, we describe how these topographically organized groups of serotonergic neurons correspond to the six major ascending serotonergic tracts innervating the forebrain.
This chapter reviews the distribution of glutaminase in the brain and describes the glutamate synthesis and metabolism in glial cells. It discusses the role of glutamate and aspartate aminotransferase (AAT) in gamma-aminobutyric acid (GABAergic) neurons. Glutamate is synthesized directly from L-glutamine, 1-pyrroline-5-carboxylate (P5C) or ketoglutarate (2-oxoglutarate) in the central nervous system (CNS). The formation of glutamate from glutamine is an energy-saving process catalyzed by phosphate-activated glutaminase (PAG), which plays a major role in the production of transmitter glutamate. P5C is derived from ornithine through glutamic semialdehyde by the catalysis of ornithine-aminotransferase (OAT) or from proline by proline oxidase (PO), and then converted to glutamate by P5C dehydrogenase (P5CDH). α-Ketoglutarate is transformed to glutamate through reductive amination catalyzed by the reverse reaction of glutamate dehydrogenase (GDH). Ketoglutarate is also converted to glutamate through transamination reaction catalyzed by several aminotransferases such as aspartate aminotransferase (AAT) and alanine aminotransferase.
The reviewed literature leaves us with an impression of the cellular interchange of Glu and its precursors and metabolites (Fig.. 2) that is somewhat different from the classical idea of the 'glutamine" cycle with its near-stoichiometric exchange of Glu and glutamine between neurons and astrocytes. (1) Glu is synthesized from glutamine, as previously thought, but also from neuronal precursors supplied by neuronal pyruvate carboxylation. (2) Transmitter Glu is mostly taken up into astrocytes for conversion to glutamine, but an unknown fraction is metabolized by the astrocytic TCA cycle, either fully to CO2 and water, or only partially, to malate which is converted to pyruvate and hence lactate. (3) Uptake of Glu into astrocytes stimulates astrocytic uptake of serum glucose and export of lactate to the extracellular fluid. (4) Lactate, whether formed from transmitter Glu or serum glucose, may increase regional cerebral blood flow and act as a main neuronal energy substrate. (5) Glutamine is shunted to neurons where it to a large extent is metabolized to CO2 and water and, possibly to a lesser extent, is converted to transmitter Glu. (6) The uptake processes related to the handling of transmitter Glu and glutamine together with the formation of glutamine cause the expenditure of % of the total energy of the serum glucose taken up by the brain.
A battery of monoclonal antibodies (mAbs) against brain cell nuclei has been generated by repeated immunizations. One of these, mAb A60, recognizes a vertebrate nervous system- and neuron-specific nuclear protein that we have named NeuN (Neuronal Nuclei). The expression of NeuN is observed in most neuronal cell types throughout the nervous system of adult mice. However, some major cell types appear devoid of immunoreactivity including cerebellar Purkinje cells, olfactory bulb mitral cells, and retinal photoreceptor cells. NeuN can also be detected in neurons in primary cerebellar cultures and in retinoic acid-stimulated P19 embryonal carcinoma cells. Immunohistochemically detectable NeuN protein first appears at developmental timepoints which correspond with the withdrawal of the neuron from the cell cycle and/or with the initiation of terminal differentiation of the neuron. NeuN is a soluble nuclear protein, appears as 3 bands (46-48 x 10(3) M(r)) on immunoblots, and binds to DNA in vitro. The mAb crossreacts immunohistochemically with nervous tissue from rats, chicks, humans, and salamanders. This mAb and the protein recognized by it serve as an excellent marker for neurons in the central and peripheral nervous systems in both the embryo and adult, and the protein may be important in the determination of neuronal phenotype.
Gamma-aminobutyric acid (GABA)ergic neurons in the central nervous system regulate the activity of other neurons and play a crucial role in information processing. To assist an advance in the research of GABAergic neurons, here we produced two lines of glutamic acid decarboxylase-green fluorescence protein (GAD67-GFP) knock-in mouse. The distribution pattern of GFP-positive somata was the same as that of the GAD67 in situ hybridization signal in the central nervous system. We encountered neither any apparent ectopic GFP expression in GAD67-negative cells nor any apparent lack of GFP expression in GAD67-positive neurons in the two GAD67-GFP knock-in mouse lines. The timing of GFP expression also paralleled that of GAD67 expression. Hence, we constructed a map of GFP distribution in the knock-in mouse brain. Moreover, we used the knock-in mice to investigate the colocalization of GFP with NeuN, calretinin (CR), parvalbumin (PV), and somatostatin (SS) in the frontal motor cortex. The proportion of GFP-positive cells among NeuN-positive cells (neocortical neurons) was approximately 19.5%. All the CR-, PV-, and SS-positive cells appeared positive for GFP. The CR-, PV, and SS-positive cells emitted GFP fluorescence at various intensities characteristics to them. The proportions of CR-, PV-, and SS-positive cells among GFP-positive cells were 13.9%, 40.1%, and 23.4%, respectively. Thus, the three subtypes of GABAergic neurons accounted for 77.4% of the GFP-positive cells. They accounted for 6.5% in layer I. In accord with unidentified GFP-positive cells, many medium-sized spherical somata emitting intense GFP fluorescence were observed in layer I.
Brain-specific Na+-dependent inorganic phosphate cotransporter (BNPI) was recently reported to serve as a vesicular glutamate transporter (VGluT), and was renamed VGluT1 (Bellocchio et al. [ 2000] Science 289:957–960; Takamori et al. [2000] Nature 407:189–194). Ahead of these reports, cDNA encoding another brain-specific inorganic phosphate transporter, which showed 82% amino acid identity to VGluT1, was cloned and designated differentiation-associated Na+-dependent inorganic phosphate cotransporter (DNPI; Aihara et al. [2000] J Neurochem 74:2622–2625). In the present study, we produced a specific antibody against a C-terminal portion of DNPI, and studied the immunohistochemical localization of DNPI in the rat cerebral cortex in comparison with that of VGluT1. DNPI immunoreactivity was enriched in neuropil of layers I and IV and to a lesser extent in the upper portion of layer VI of the cerebral neocortex, whereas VGluT1 immunoreactivity was distributed more evenly in neuropil of the neocortex. Electron microscopic observation revealed that both DNPI and VGluT1 immunoreactivities were mainly located on synaptic vesicles in nerve terminals which made asymmetrical contacts in the neocortex. Furthermore, neither DNPI nor VGluT1 immunoreactivity in the neocortex was colocalized with gamma aminobutyric acid (GABA)ergic axon terminal markers, immunoreactivity for glutamic acid decarboxylase or vesicular GABA transporter. Neuronal depletion in the ventrobasal thalamic nuclei produced by the kainic acid injection resulted in a clear reduction of DNPI immunoreactivity in layers I, IV, and VI of the somatosensory cortex. These results indicate that DNPI is located on the membrane of synaptic vesicles in thalamocortical axon terminals, and that it may be a candidate for VGluT of thalamocortical glutamatergic neurons. J. Comp. Neurol. 435:379–387, 2001. © 2001 Wiley-Liss, Inc.
The indoleamme serotonin (5-hydroxytryptamine, 5-HT) was described in the central nervous system (CNS) more than 30 years
ago (Amin et al., 1954; Bogdanski et al., 1956; De Robertis, 1964; Twarog and Page, 1953; Zieher and De Robertis, 1963) based
on biochemical determinations of this amine in different brain regions. These studies were rapidly followed by the use of
histofluorescent microscopy (Anden et al., 1965, 1966, 1967; Dahlstrom and Fuxe, 1964; Fuxe, 1965; Moore et al., 1978; Ungerstedt,
1971) and measurements of the activity of tryptophan hydroxylase and 5-HT uptake (Kuhar et al., 1972). This work was later
complemented by anatomical descriptions of the now chemically identified neurotransmitter systems using radioautographic tracing
techniques that take advantage of the uptake properties of the 5-HT system (Azmitia, 1978; Azmitia and Segal, 1978; Beaudet
and Descarrres, 1981, Parent et al, 1981) as well as by the use of immunocytochemrcal methods with antibodies against 5-HT
itself (Steinbusch, 1981, Steinbusch et al , 1978; Steinbusch and Nreuwenhuys, 1983).
We have analyzed expression of a gene encoding a brain-specific Na+-dependent inorganic phosphate cotransporter (DNPI), which was recently cloned from human brain, in rat forebrain using in situ hybridization. The expression of DNPI mRNA showed a widespread but highly heterogeneous pattern of distribution in the forebrain, where hybridization signals were observed in neurons but not in any other types of cells. Neurons expressing the mRNA were far more numerous in the diencephalon than in the telencephalon. In the thalamus, a number of neurons with high levels of signals were localized to all nuclei of the dorsal thalamus, habenular nuclei and subthalamic nucleus, but not the reticular nucleus and zona incerta. Moderate signal levels were seen in many neurons throughout the hypothalamus, particularly the ventromedial, paraventricular, supraoptic and arcuate nuclei, lateral hypothalamic area and mammillary complex. In contrast, expression of DNPI mRNA in the telencephalon was generally at a low level and occurred locally in some restricted regions within the neocortex, retrosplenial cortex, piriform cortex, olfactory regions, hippocampal formation and medial amygdaloid nucleus. The present results suggest that DNPI functions in heterogeneous neuron populations as a neuron-specific Na+-dependent inorganic phosphate cotransport system predominantly expressed in the diencephalon of the rat.
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.
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.
L-glutamic acid is a key chemical transmitter of excitatory signals in the nervous system. The termination of glutamatergic transmission occurs via uptake of glutamate by a family of high-affinity glutamate transporters that utilize the Na+/K+ electrochemical gradient as a driving force. The stoichiometry of a single translocation cycle is still debatable, although all proposed models stipulate an inward movement of a net positive charge. This electrogenic mechanism is capable of translocating the neurotransmitter against its several thousand-fold concentration gradient, therefore, keeping the resting glutamate concentration below the treshold levels. The five cloned transporters (GLAST/EAAT1, GLT1/EAAT2, EAAC1/EAAT3, EAAT4, and EAAT5) exhibit distinct distribution patterns and kinetic properties in different brain regions, cell types, and reconstitution systems. Moreover, distinct pharmacological profiles were revealed among the species homologues. GLAST and GLT1, the predominant glutamate transporters in the brain, are coexpressed in astroglial processes, whereas neuronal carriers are mainly located in the dendrosomatic compartment. Some of these carrier proteins may possess signal transducing properties, distinct from their transporter activity. Some experimental conditions and several naturally occurring and synthetic compounds are capable of regulating the expression of glutamate transporters. However, selective pharmacological tools interfering with the individual glutamate carriers have yet to be developed.
The third vesicular glutamate transporter, VGLUT3, is distributed in cell bodies of neocortical neurons and axon terminals mainly in the superficial part of layer II/III of the cerebral cortex. We examined the chemical characteristics of VGLUT3-expressing neurons by immunohistochemistry in the rat neocortex. Since the vast majority of VGLUT3-immunoreactive neurons showed immunoreactivities for GABA, preprotachykinin B (PPTB) and cholecystokinin, VGLUT3-immunoreactive neocortical neurons were considered to constitute a subgroup of GABAergic interneurons. VGLUT3-immunoreactive axon terminals were immunopositive for either vesicular GABA transporter (VGAT) or serotonin. These results together with anterograde tracer injection and chemical lesion experiments in the dorsal and median raphe nuclei revealed that the neocortex contains at least two kinds of VGLUT3-laden axon terminals: one is serotonergic and derived from the raphe nuclei, and the other is GABAergic and intrinsic in the neocortex. Furthermore, many VGLUT3/VGAT-immunoreactive terminals formed axon baskets and made axosomatic symmetric synapses on neocortical neurons, most of which were immunoreactive for PPTB. VGLUT3-immunopositive axon baskets surrounded about a half of PPTB-positive and almost all VGLUT3-positive neurons. Thus, VGLUT3-expressing GABAergic interneurons form a chemically specific circuit within the PPTB-producing interneuron group and it is likely that glutamate is used within the chemically specific circuit.
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.
The periaqueductal gray (PAG)-nucleus retroambiguus (NRA) pathway has been known to be involved in the control of vocalization and sexual behavior. To know how the amygdaloid complex influences the PAG-NRA pathway, here we first examined the synaptic organization between the central amygdaloid nucleus (CeA) fibers and the PAG neurons that project to the NRA by using anterograde and retrograde tract-tracing techniques in the rat. After ipsilateral injections of biotinylated dextran amine (BDA) into the CeA and cholera toxin B subunit (CTb) into the NRA, the prominent overlapping distribution of BDA-labeled axon terminals and CTb-labeled neurons was found ipsilaterally in the lateral/ventrolateral PAG, where some of the BDA-labeled terminals made symmetrical synaptic contacts with somata and dendrites of the CTb-labeled neurons. After CTb injection into the lateral/ventrolateral PAG, CTb-labeled neurons were distributed mainly in the medial division of the CeA. After BDA injection into the lateral/ventrolateral PAG, BDA-labeled fibers were distributed mainly in and around the NRA within the medulla oblongata. Using a combined retrograde tracing and in situ hybridization technique, we further demonstrated that more than half of the CeA neurons labeled with Fluoro-Gold (FG) injected into the lateral/ventrolateral PAG were positive for glutamic acid decarboxylase 67 mRNA and that the vast majority of PAG neurons labeled with FG injected into the NRA expressed vesicular glutamate transporter 2 mRNA. The present results suggest that the glutamatergic PAG-NRA pathway is under the inhibitory influence of the GABAergic CeA neurons.
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.
Vesicular glutamate transporter 1 (VGLUT1) and VGLUT2 show complementary distribution in neocortex; VGLUT1 is expressed mainly in axon terminals of neocortical neurons, whereas VGLUT2 is located chiefly in thalamocortical axon terminals. However, we recently reported a frequent colocalization of VGLUT1 and VGLUT2 at a subset of axon terminals in postnatal developing neocortex. We here quantified the frequency of colocalization between VGLUT1 and VGLUT2 immunoreactivities at single axon terminals by using the correlation coefficient (CC) as an indicator in order to determine the time course and spatial extent of the colocalization during postnatal development of mouse neocortex. The colocalization was more frequent in the primary somatosensory (S1) area than in both the primary visual (V1) and the motor areas; of area S1 cortical layers, colocalization was most evident in layer IV barrels at postnatal day (P) 7 and in adulthood. CC in layer IV showed a peak at P7 in area S1, and at P10 in area V1 though the latter peak was much smaller than the former. These results suggest that thalamocortical axon terminals contained not only VGLUT2 but also VGLUT1, especially at P7–10. Double fluorescence in situ hybridization confirmed coexpression of VGLUT1 and VGLUT2 mRNAs at P7 in the somatosensory thalamic nuclei and later in the thalamic dorsal lateral geniculate nucleus. As VGLUT1 is often used in axon terminals that show synaptic plasticity in adult brain, the present findings suggest that VGLUT1 is used in thalamocortical axons transiently during the postnatal period when plasticity is required.
The connexions between the dorsal raphe nucleus and the nucleus locus coeruleus were studied in urethane anaesthetized rats. 1. Cells in the locus coeruleus gave an excitatory response to a noxious stimulus, e.g. leg pinch. 2. This excitatory response was blocked by either a parenteral or an ionophoretic injection of morphine and recovered after an injection of naloxone. 3. Electrical stimulation in the region of the dorsal raphe blocked excitatory locus coeruleus responses to noxious stimuli. 4. While naloxone did not antagonize the effects of the dorsal raphe stimulation towards locus coeruleus activity, these effects were absent in rats pretreated with a serotonin synthesis inhibitor, PCPA or with 5,7-DHT which destroys serotonin-containing terminals, and were reduced by the serotonin antagonist methysergide. 5. A serotonin-containing inhibitory pathway between the dorsal raphe and the locus coeruleus is proposed to account for these results.
It is important to know if the transmission of sound signals through the inferior colliculus is mediated by the transmitters glutamate or aspartate because of pharmacological consequences for auditory perception. In order to identify candidate's neurons, the retrograde transport for [3H]-D-aspartate, injected into the left inferior colliculus, was studied in cats. Labelled cells were found in the dorsal and intermediate lateral lemniscal nuclei, mainly on the contralateral side. The cochlear nuclei, superior olivary nuclei and the auditory cortex were not labelled in brains containing other labelled neurons at greater distances from the injection site. Labelled cells were found in the reticular formation and adjacent nucleus coeruleus, the parabrachial nuclei, raphe nuclei (magnus, dorsalis and centralis superior), nucleus prepositus hypoglossi, lateral hypothalamus and hippocampal CA1.
A battery of monoclonal antibodies (mAbs) against brain cell nuclei has been generated by repeated immunizations. One of these, mAb A60, recognizes a vertebrate nervous system- and neuron-specific nuclear protein that we have named NeuN (Neuronal Nuclei). The expression of NeuN is observed in most neuronal cell types throughout the nervous system of adult mice. However, some major cell types appear devoid of immunoreactivity including cerebellar Purkinje cells, olfactory bulb mitral cells, and retinal photoreceptor cells. NeuN can also be detected in neurons in primary cerebellar cultures and in retinoic acid-stimulated P19 embryonal carcinoma cells. Immunohistochemically detectable NeuN protein first appears at developmental timepoints which correspond with the withdrawal of the neuron from the cell cycle and/or with the initiation of terminal differentiation of the neuron. NeuN is a soluble nuclear protein, appears as 3 bands (46-48 x 10(3) M(r)) on immunoblots, and binds to DNA in vitro. The mAb crossreacts immunohistochemically with nervous tissue from rats, chicks, humans, and salamanders. This mAb and the protein recognized by it serve as an excellent marker for neurons in the central and peripheral nervous systems in both the embryo and adult, and the protein may be important in the determination of neuronal phenotype.
Retrograde tracer injections of fluorescein- and rhodamine-labelled latex microspheres centered in the parvicellular zone of the hypothalamic paraventricular nucleus and pontine lateral parabrachial nucleus revealed that 36% of the labelled neurons in the dorsal raphe nucleus send collaterals to both structures. These cells were organized in a well-distinguishable cluster within the dorsal raphe nucleus. By combining retrograde tracing with immunocytochemistry, it was found that less than 8% of the double-labelled cells stained positively for serotonin. Of the remaining raphe nuclei that were examined, only the median raphe nucleus contributed a minor nonserotoninergic projection to the paraventricular or lateral parabrachial nuclei. Few of the retrogradely labelled cells in the median raphe nucleus contained both tracers. These results suggest that nonserotoninergic and serotoninergic neurons in the dorsal raphe nucleus, via collateral branching, may simultaneously influence the activity of two central nervous system nuclei involved in autonomic control.
The presence in the brain of the urea cycle intermediate citrulline in the absence of a complete urea cycle has never been adequately explained. In an attempt to clarify this problem, we developed antibodies to citrulline and determined the distribution of citrulline-immunoreactivity in fixed sections of rat brain using immunoperoxidase and indirect immunofluorescence techniques. Citrulline-positive neurons were found to have a restricted distribution within the brain. A few cells were present in the cortex and corpus callosum. A large population of strongly stained cells was diffusely scattered throughout the striatum, nucleus accumbens and olfactory tubercle. Less strongly stained cells were detected in the supraoptic and paraventricular nuclei of the hypothalamus, the dorsal raphe, and the laterodorsal and pedunculopontine tegmental nuclei of the pons. The citrulline-immunoreactive cells were similar to those previously shown to contain NADPH-diaphorase activity, and double staining experiments indicated that citrulline-immunoreactivity was present in a subpopulation of NADPH-diaphorase-positive neurons. We have recently identified NADPH-diaphorase as a nitric oxide synthase. Thus the presence of citrulline in these cells suggests that it is formed within the brain as a coproduct during nitric oxide formation from arginine.
The neuronal distribution of argininosuccinate synthetase (ASS) was mapped in the rat brain. Argininosuccinate synthetase is one of the enzymes of the arginine metabolic pathway and catabolizes the synthesis of argininosuccinate from aspartate and citrulline. Since arginine is the precursor of nitric oxide, argininosuccinate synthetase may act as part of the nitric oxide producing pathway. Argininosuccinate is also suggested to have a messenger function in the nervous system. Therefore, the localization of ASS is of great interest.
Polyclonal antisera against purified rat liver argininosuccinate synthetase revealed a characteristic distribution pattern of argininosuccinate synthetase-like immunoreactivity: (1) many neurons with strong argininosuccinate synthetase-like immunoreactivity were observed in the septal area, basal forebrain, anterior medial and premammillary nuclei of the hypothalamus, anterior and midline thalamic nuclei, dorsal endopiriform nucleus of the amygdala, basal nucleus of Meynert, subthalamic nucleus, laterodorsal tegmental nucleus, raphe nuclei, nucleus ambiguus, and the area postrema, (2) neuropile staining was dense in the septal areas, hypothalamus, area postrema, nucleus of the solitary tract, and the laminae I and II of the caudal subnucleus of the spinal trigeminal nucleus and the spinal dorsal horn, (3) relay nuclei of the specific sensory systems such as the dorsal lateral geniculate nucleus and the ventral nuclei of the thalamus were devoid of argininosuccinate synthetase-like immunoreactivity, (4) no staining was seen in the large white matter structures such as the internal capsule, corpus callosum, and the anterior commissure, and (5) most of the neurons stained were small or medium in size and appeared to be interneurons. The results suggest that argininosuccinate synthetase affects the widely distributed, neuromodulatory system in the brain.
Previous studies have shown a possible connection between the nucleus raphe dorsalis (NRD) and the amygdala in mediating opioid analgesia. In the present study, horseradish peroxidase (HRP) retrograde tracing was used in combination with serotonin (5-HT) immunocytochemical staining in an attempt to search for serotonergic projections from the NRD to the amygdala. In rats which received an injection of HRP into the amygdala, HRP retrogradely labelled 5-HT-immunoreactive cells were observed in the NRD. About 10% of the 5-HT-immunoreactive neurons in the NRD give rise to axons to the amygdala. These cells are predominantly situated in the ipsilateral wing and ventromedial part of the NRD. These data indicate the existence of serotonergic projections from the NRD to the amygdala, providing a morphological substrate for the putative antinociceptive pathway from the NRD to the amygdala.
The presence of phosphate-activated glutaminase in monoaminergic neurons was shown in the rat brain by a double-staining method using a mouse monoclonal anti-glutaminase antibody combined with rabbit antisera against tyrosine hydroxylase, dopamine-beta-hydroxylase, phenylethanolamine-N-methyltransferase or serotonin. Glutaminase-like immunoreactivity was detected within perikarya of many monoaminergic neurons in the substantia nigra pars compacta, locus ceruleus, raphe nuclei, etc. Possible functional significance of glutaminase in monoamine neurons was discussed.
Serial 50 microns Nissl-stained sections through the midbrain and pontine central gray of four adult humans (mean age 56 years, mean postmortem delay 3 hours) were analysed and the subnuclei of the dorsal raphe nucleus (DR) delineated on the basis of neuronal morphology and density. Five subnuclei were apparent: the interfascicular, ventral, ventrolateral, dorsal, and caudal. The area of each subnucleus was measured in sections selected at regular intervals throughout the length of the DR. The number of neurons was counted and their density within each subnucleus calculated. The dorsal subnucleus was the largest and contained the majority of neurons but had the lowest neuronal density. The ventrolateral subnucleus had the highest density of neurons. A total of 235,000 +/- 15,000 neurons (average of 1,200 +/- 200 neurons per section) were found within a volume of 71.3 +/- 4.5 mm3 of DR with a mean neuronal density of 3,300 +/- 200 neurons/mm3. Morphometric and morphological analysis of DR neurons revealed four distinct neuron types: round, ovoid, fusiform, and triangular. These types of neurons characterized particular subnuclei. The location and boundaries of the subnuclei of the human dorsal raphe are presented in the form of an atlas. The subdivisions described are similar to that described in other mammals. On the basis of this information the location of particular projection neurons within the human dorsal raphe can be predicted and the effects of disease on this nucleus may be forecast.
In order to study the morphological substrate of possible thalamic influence on the cells of origin and area of termination of the projection from the entorhinal cortex to the hippocampal formation, we examined the pathways, terminal distribution, and ultrastructure of the innervation of the hippocampal formation and parahippocampal region by the nucleus reuniens of the thalamus (NRT). We employed anterograde tracing with Phaseolus vulgaris ‐leucoagglutinin (PHA‐L). Injections of PHA‐L in the NRT produce fiber and terminal labeling in the stratum lacunosum‐moleculare of field CA1 of the hippocampus, the molecular layer of the subiculum, layers I and III/IV of the dorsal subdivision of the lateral entorhinal area (DLEA), and layers I and III‐VI of the ventral lateral (VLEA) and medial (MEA) divisions of the entorhinal cortex. Terminal labeling is most dense in the stratum lacunosum‐moleculare of field CA1, the molecular layer of the ventral part of the subiculum, MEA, and layer I of the perirhinal cortex. In layer I of the caudal part of DLEA and in MEA, terminal labeling is present in clusters. Injections in the rostral half of the NRT produce the same distribution in the hippocampal region as those in the caudal half of the NRT, although the projections from the rostral half of the NRT are much stronger. A topographical organization is present in the projections from the head of the NRT, so that the dorsal part projects predominantly to dorsal parts of field CA1 and the subiculum and to lateral parts of the entorhinal cortex, whereas the ventral part projects in greatest volume to ventral parts of field CA1 and the subiculum and to medial parts of the entorhinal cortex.
The distribution of the reuniens fibers coursing in the cingulate bundle was determined by comparing cases with and without transections of this bundle. The fibers carried by the cingulate bundle exclusively innervate field CA1 of the hippocampus, the dorsal part of the subiculum, and the presubiculum and parasubiculum. They participate in the innervation of the ventral part of the subiculum and MEA.
Electron microscopy was used to visualize the axon terminals of PHA‐L‐labeled reuniens fibers. These terminals possess spherical synaptic vesicles and form asymmetric synaptic contacts with dendritic spines or with thin shafts of spinous dendrites. Following lesions of the cingulate bundle, large numbers of degenerating axon terminals are present in MEA. Scattered degenerating axon terminals are visible in the areas that in the light microscopic tracing/lesion experiments are depleted of PHA‐L‐labeled fibers, i.e., field CA1 of the hippocampus, the dorsal part of the subiculum, and MEA. Also, the molecular layer of the ventral part of the subiculum contains degenerating terminals.
The results of the present study suggest that the NRT may simultaneously influence the parent cell bodies of the perforant pathway in the entorhinal cortex and the target neurons of this pathway in field CA1 and the subiculum of the hippocampal formation. The perforantpathway constitutes the major route along which cortical input is mediated to the hippocampal formation.
In an attempt to identify glutamatergic neurons, the cerebral cortex and thalamus of the rat were examined immunohistochemically by using a monoclonal antibody against phosphate-activated glutaminase (PAG), a major synthetic enzyme of transmitter glutamate in the central nervous system. In both the neocortex and mesocortex, pyramidal cells in layers V and VI showed intense PAG-like immunoreactivity (PAG-LI), whereas neuronal cell bodies in layers I–IV showed weak PAG-LI. At the deep border of layer VI, neurons with horizontally elongated cell bodies showed PAG-LI. In the pyriform and entorhinal cortices, neurons with intense to moderate PAG-LI were seen in layer II as well as in the deeper layers. In the hippocampal formation, pyramidal cells in CA1, CA2, and CA3 and polymorphic cells in CA4 showed PAG-LI; PAG-LI was most intense in pyramidal cells of CA3. Fine granules with weak PAG-LI were also seen on and/or within the cell bodies of granule cells in the dentate gyrus. In the thalamus, neurons with PAG-LI were distributed in all nuclei, although regional differences were observed in the distribution pattern of neurons with PAG-LI and in the intensity of PAG-LI in individual neurons. The largest neurons in each thalamic nucleus showed intense PAG-LI; these were considered to be projection neurons. In addition to perikaryal labeling, many fine, PAG-like immunoreactive granules were distributed in the neuropil of both the cerebral cortex and thalamic nuclei. Some of these fine granules with PAG-LI in the neuropil were assumed to represent fiber terminals with PAG-LI, because the distribution pattern of the deposits in the primary somatosensory and primary visual cortices resembled that of thalamocortical fiber terminals.
Distribution of putative glutamatergic neurons in the lower brainstem and cerebellum of the rat was examined immunocytochemically by using a monoclonal antibody against phosphate-activated glutaminase, which has been proposed to be a major synthetic enzyme of transmitter glutamate and so may serve as a marker for glutamatergic neurons in the central nervous system. Intensely-immunolabeled neuronal cell bodies were densely distributed in the main precerebellar nuclei sending mossy fibers to the cerebellum; in the pontine nuclei, pontine tegmental reticular nucleus of Bechterew, external cuneate nucleus, and lateral reticular nucleus of the medulla oblongata. Phosphate-activated glutaminase-immunoreactive granular deposits were densely seen in the brachium pontis and restiform body, suggesting the immunolabeling of mossy fibers of passage. In the cerebellum, neuropil within the granule cell layer of the cerebellar cortex displayed intense phosphate-activated glutaminase-immunoreactivity, and that within the deep cerebellar nuclei showed moderate immunoreactivity. These results indicate that many mossy fiber terminals originate from phosphate-activated glutaminase-containing neurons and utilize phosphate-activated glutaminase for the synthesis of transmitter glutamate. Intensely-immunostained neuronal cell bodies were further observed in other regions which have been reported to contain neurons sending mossy fibers to the cerebellum; in the dorsal part of the principal sensory trigeminal nucleus, dorsomedial part of the oral subnucleus of the spinal trigeminal nucleus, interpolar subnucleus of the spinal trigeminal nucleus, paratrigeminal nucleus, supragenual nucleus, regions dorsal to the abducens nucleus and genu of the facial nerve, superior and medial vestibular nuclei, cell groups f, x and y, hypoglossal prepositus nucleus, intercalated nucleus, nucleus of Roller, reticular regions intercalated between the motor trigeminal and principal sensory trigeminal nuclei, linear nucleus, and gigantocellular and paramedian reticular formation. Neuronal cell bodies with intense phosphate-activated glutaminase-immunoreactivity were also found in other brainstem regions, such as the paracochlear glial substance, posterior ventral cochlear nucleus, and cell group e. Although it is still controversial whether all glutamatergic neurons use phosphate-activated glutaminase in a transmitter-related process and whether phosphate-activated glutaminase is involved in other metabolism-related processes, the neurons showing intense phosphate-activated glutaminase-immunoreactivity in the present study were suggested to be putative glutamatergic neurons.
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.
Injections of HRP into the superior colliculus labelled cells in the lateral cell groups of the dorsal raphe nucleus. The cytoarchitectural features and location of these cells showed remarkable similarities with those known to project to the lateral geniculate body, and, therefore, the possible existence of branching neurons in the dorsal raphe nucleus projecting to these two visual structures was tested. Injections into the lateral geniculate body and the superior colliculus of several fluorescent tracers—namely, Fast Blue, Fluoro‐Gold, propidium iodide, rhodamine‐B‐isothiocyanate, and Diamidino Yellow, used in different combinations, showed single‐ and double‐labelled neurons in the lateral wings of the dorsal raphe nucleus. In order to verify the chemical nature of these cells, the tissue was processed for immunofluorescence with serotonin antibodies. The results obtained showed several triple‐labelled cells exhibiting two fluorescent tracers as well as 5‐hydroxytryptamine‐like immunoreactivity. Some immunonegative tracer‐positive cells were also observed, suggesting their nonserotoninergic nature. Finally, electrolytic lesions of the lateral wings of the dorsal raphe nucleus caused a gradual disappearance of serotonin‐immunoreactive fibers in these visual areas following different survival times. This correlated well with a decrease in the serotonin content studied by high‐pressure liquid chromatography.
These results support a role of the serotoninergic dorsal raphe projection to the lateral geniculate body and to the superior colliculus in the processing of visual information, and they suggest that serotonin may have a coordinating influence on primary visual centers.
Previous studies have shown that both the midbrain periaqueductal gray (PAG) and the superior colliculus receive a significant serotoninergic (5-HT) innervation. In the present study the origins of these 5-HT projections to the rodent PAG and superior colliculus were analyzed by using a combined immunohistochemical-retrograde transport technique. Thirteen brainstem regions were found to contain double-labelled 5-HT-like immunoreactive neurons following HRP injections into the PAG while only four brainstem nuclei contained double-labelled neurons following superior collicular injections. After HRP deposits into the ventral PAG, the largest percentage of double-labelled neurons was identified in nucleus raphe magnus, pars alpha of the nucleus gigantocellularis, and the paragigantocellular nucleus. The dorsal PAG, on the other hand, received the largest percentage of its 5-HT projections from nuclei raphe dorsalis, raphe obscurus, raphe pontis, and raphe medianis. The 5-HT input to the superior colliculus was found to arise exclusively from nuclei raphe dorsalis, raphe medianis, and raphe pontis and from the contralateral periaqueductal gray. Raphe nuclei were found to contribute serotoninergic projections to both the PAG and the superior colliculus while reticular nuclei contributed 5-HT projections only to the PAG. Injections of the fluorescent retrograde tracers true blue and nuclear yellow were then made into the PAG and superior colliculus to ascertain if neurons located in raphe nuclei that projected to both structures provided axon collaterals to both areas. Generally, less than 10% of raphe neurons projecting to the superior collicuius were identified as providing axon collaterals to the PAG.
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.