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Morphological features and electrophysiological properties of serotonergic and non-serotonergic projection neurons in the dorsal raphe nucleus: An intracellular recording and labeling study in rat brain slices
Abstract
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.
... We identified 5-HT neurons using the following criteria based on their electrophysiological properties: [17][18][19] wide half width of action potentials [>0.8 ms, duration at half peak amplitude measured from the baseline membrane potential (% À60 mV)], slow time constants (s > 20 ms) of voltage responses to injection of a hyperpolarizing current of 100 pA, no hyperpolarization-activated potentials and no rebound depolarizations after the offset of hyperpolarizing currents. Most neurons that satisfy these criteria are 5-HT neurons, 18,19 with the exception of a very minor population of non-5-HT neurons with similar electrophysiological properties. ...
... We identified 5-HT neurons using the following criteria based on their electrophysiological properties: [17][18][19] wide half width of action potentials [>0.8 ms, duration at half peak amplitude measured from the baseline membrane potential (% À60 mV)], slow time constants (s > 20 ms) of voltage responses to injection of a hyperpolarizing current of 100 pA, no hyperpolarization-activated potentials and no rebound depolarizations after the offset of hyperpolarizing currents. Most neurons that satisfy these criteria are 5-HT neurons, 18,19 with the exception of a very minor population of non-5-HT neurons with similar electrophysiological properties. 17 If the electrode potential polarization at the end of the recording was over 65 mV from the initial value, the recording was omitted from the analysis. ...
... The resting membrane potential, input resistance and time constants of hyperpolarizing potentials were identical among the naïve, non-LH and LH groups (Table 1 and Supplementary Table 1) and to those reported in previous experiments. 17,19 These results suggest that the subthreshold electrophysiological properties were identical among the three rat groups. ...
Animals suffering from uncontrollable stress sometimes show low effort to escape stress [learned helplessness (LH)]. Changes in serotonin (5-HT) signaling are thought to underlie this behavior. Although the release of 5-HT is triggered by the action potential firing of dorsal raphe nuclei 5-HT neurons, the electrophysiological changes induced by uncontrollable stress are largely unclear. Herein, we examined electrophysiological differences among 5-HT neurons in naïve rats, LH rats and rats resistant to inescapable stress (non-LH). Five-week-old male Sprague–Dawley rats were exposed to inescapable foot shocks. After an avoidance test session, rats were classified as LH or non-LH. Activity-dependent 5-HT release induced by the administration of high potassium solution was slower in free-moving LH rats. Subthreshold electrophysiological properties of 5-HT neurons were identical among the three rat groups, but the depolarization-induced spike firing was significantly attenuated in LH rats. To clarify the underlying mechanisms, potassium (K+) channels regulating the spike firing were initially examined using naïve rats. K+ channels sensitive to 500 μM tetraethylammonium caused rapid repolarization of the action potential and the small conductance calcium-activated K+ channels (SK channels) produced afterhyperpolarization. Additionally, dendrotoxin-I (DTX-I), a blocker of Kv1.1 (encoded by Kcna1), Kv1.2 (encoded by Kcna2) and Kv1.6 (encoded by Kcna6) voltage-dependent K+ channels, weakly enhanced the spike firing frequency during depolarizing current injections without changes in individual spike waveforms in naïve rats. We found that DTX-I significantly enhanced the spike firing of 5-HT neurons in LH rats. Consequently, the difference in spike firing among the three rat groups was abolished in the presence of DTX-I. These results suggest that the upregulation of DTX-I-sensitive Kv1 channels underlies the firing attenuation of 5-HT neurons in LH rats. We also found that the antidepressant ketamine facilitated the spike firing of 5-HT neurons and abolished the firing difference between LH and non-LH by suppressing DTX-I-sensitive Kv1 channels. The DTX-I-sensitive Kv1 channel may be a potential target for developing drugs to control activity of 5-HT neurons.
... Later work described a broad action potential, large slow afterhyperpolarization (AHP) potential, slow interspike depolarization, and high input resistance (Vandermaelen and Aghajanian, 1983). Subsequently, electrophysiological recordings coupled with post-hoc immunohistochemical identification of 5-HT neurons revealed that 5-HT neurons themselves exhibit a range of characteristics, overlapping with those of non-5-HT neurons (Calizo et al., 2011;Kirby et al., 2003;Li et al., 2001;Marinelli et al., 2004). 5-HT neurons tended to have a broader action potential and greater input resistance than non-5-HT neurons, but these characteristics do not definitively identify 5-HT neurons. ...
... Scn3b, when expressed with the alpha subunit Nav1.3, promotes slower channel inactivation, which would increase the duration of the action potential (Cusdin et al., 2010). This finding raises the possibility that expression of these subunits may contribute to the broad action potential associated primarily, but not exclusively, with 5-HT neurons (Calizo et al., 2011;Kirby et al., 2003;Li et al., 2001;Marinelli et al., 2004). ...
... C4. non-5-HT neurons in the DRN (Kirby et al., 2003;Li et al., 2001;Marinelli et al., 2004), and this is consistent with our finding that the majority of potassium channels that could contribute to these characteristics did not have robustly patterned expression. ...
The dorsal raphe nucleus (DR) is the major source of serotonin (5-hydroxytryptamine, 5-HT) in the forebrain and dysfunction of this midbrain structure is implicated in affective disorders. The DR is composed of several types of 5-HT and non-5-HT neurons and their excitable-membrane properties are heterogeneous and overlapping. In order to understand how these properties may be generated, we examined the mRNA expression patterns of voltage- and ligand-gated ion channels in the DR using the Allen Mouse Brain Atlas. Since DR cytoarchitecture is organized with respect to the midline, we sought to identify genes that were expressed in a pattern with respect to the midline, either enriched or depleted, rather than those that were homogenously expressed throughout the DR. Less than 10% of the screened genes for voltage-gated ion channels showed patterned expression within the DR. Identified genes included voltage-gated sodium channel beta subunits, potassium channels, P/Q-, N-type calcium channels, as well as the alpha2/delta-1 calcium channel. Several voltage-gated chloride channels were also identified, although these may function within intracellular compartments. Of the ligand-gated ion channels examined, 20% showed patterned expression. These consisted primarily of glutamate and GABA-A receptor subunits. The identified genes likely contribute to unique excitable properties of different groups of neurons in the DR and may include novel pharmacologic targets for affective disorders.
... In humans, 5-HT neurons are round, ovoid, fusiform, and triangular with a size between 15 and 20 μm (Baker et al., 1990), while in rats, these cells are ovoid and fusiform (Steinbusch et al., 1981;Harandi et al., 1987). In rats, GABAergic neurons are smaller than most of 5-HT cells (Harandi et al., 1987;Johnson, 1994;Li et al., 2001). However, GAD67-positive cells from 10 to 20 μm have been reported in mice (Bang and Commons, 2012). ...
... GABAergic neurons are widespread across the DRN. On the opposite, 5-HT neurons are commonly seen forming clusters (Harandi et al., 1987) with a widely dispersed arbor that extends from the ventral part of the nucleus to the periaqueductal gray (Li et al., 2001;Allers and Sharp, 2003;Calizo et al., 2011). ...
The dorsal raphe nucleus (DRN), located in the brainstem, is involved in several functions such as sleep, temperature regulation, stress responses, and anxiety behaviors. This nucleus contains the largest population of serotonin expressing neurons in the brain. Serotonergic DRN neurons receive tonic γ-aminobutyric acid (GABA)inhibitory inputs from several brain areas, as well as from interneurons within the same nucleus. Serotonergic and GABAergic neurons in the DRN can be distinguished by their size, location, pharmacological responses, and electrophysiological properties. GABAergic neurons regulate the excitability of DRN serotonergic neurons and the serotonin release in different brain areas. Also, it has been shown that GABAergic neurons can synchronize the activity of serotonergic neurons across functions such as sleep or alertness. Moreover, dysregulation of GABA signaling in the DRN has been linked to psychiatric disorders such as anxiety and depression. This review focuses on GABAergic transmission in the DRN. The interaction between GABAergic and serotonergic neurons is discussed considering some physiological implications. Also, the main electrophysiological and morphological characteristics of serotonergic and GABAergic neurons are described.
... The properties of neurons in the raphe nuclei are diverse and heterogeneous, including metabolism, anatomy, neurochemistry and physiology (Andrade and Haj-Dahmane, 2013;Beck et al., 2004;Fernandez et al., 2017;Gaspar and Lillesaar, 2012;Muzerelle et al., 2016;Okaty et al., 2015). Electrophysiologically, 5-HT and non-5-HT neurons are heterogeneous in terms of resting membrane potentials, input resistances, spike amplitudes and spike thresholds (Allers and Sharp, 2003;Kocsis et al., 2006;Li et al., 2001;Marinelli et al., 2004). There are also regional excitability differences among subnuclei within the DRN. ...
... For example, the lateral wings contain neurons with higher membrane excitability (Crawford et al., 2010; but see Shikanai et al., 2012). 5-HT neurons have been found to exhibit classical regular-spiking and bursting behavior (Cohen et al., 2015;Hajós et al., , 2007Kirby et al., 2003;Li et al., 2001) and are also associated with a characteristic slow after-hyperpolarization (AHP) (Kirby et al., 2003). The variety of 5-HT neuronal spiking behavior has been suggested to be due to the interplay among multiple ion channel currents (Aghajanian and Sanders-Bush, 2002). ...
Despite its importance in regulating emotion and mental well-being, the complex structure and function of the serotonergic system present formidable challenges toward understanding its mechanisms. In this paper, we review studies investigating the interactions between serotonergic and related brain systems and their behavior at multiple scales, with a focus on biologically based computational modeling. We first discuss serotonergic intracellular signaling and neuronal excitability, followed by neuronal circuit and systems levels. At each level of organization, we will discuss the experimental work accompanied by related computational modeling work. We then suggest that a multiscale modeling approach that integrates the various levels of neurobiological organization could potentially transform the way we understand the complex functions associated with serotonin.
... This notion was further extended by antidromic invasion experiments [17]. More recently, single-cell recording and labeling were conducted in the rat DRN, but without providing entire axon reconstructions [18,19]. Similar approach was also used to gather morphological data on 5-HT neurons of the rat medulla [20]. ...
... Such elongated unit may cover the whole rostrocaudal extent of the DRN indicating that a single neuron is able to receive and integrate most of the DRN afferent projections. This columnar arrangement of DRN neurons is in accordance with previously published descriptions [19,36,37], including the seminal paper of Cajal [38]. ...
This study aimed at providing the first detailed morphological description, at the single-cell level, of the rat dorsal raphe nucleus neurons, including the distribution of the VGLUT3 protein within their axons. Electrophysiological guidance procedures were used to label dorsal raphe nucleus neurons with biotinylated dextran amine. The somatodendritic and axonal arborization domains of labeled neurons were reconstructed entirely from serial sagittal sections using a computerized image analysis system. Under anaesthesia, dorsal raphe nucleus neurons display highly regular (1.72±0.50 Hz) spontaneous firing patterns. They have a medium size cell body (9.8±1.7 µm) with 2-4 primary dendrites mainly oriented anteroposteriorly. The ascending axons of dorsal raphe nucleus are all highly collateralized and widely distributed (total axonal length up to 18.7 cm), so that they can contact, in various combinations, forebrain structures as diverse as the striatum, the prefrontal cortex and the amygdala. Their morphological features and VGLUT3 content vary significantly according to their target sites. For example, high-resolution confocal analysis of the distribution of VGLUT3 within individually labeled-axons reveals that serotonin axon varicosities displaying VGLUT3 are larger (0.74±0.03 µm) than those devoid of this protein (0.55±0.03 µm). Furthermore, the percentage of axon varicosities that contain VGLUT3 is higher in the striatum (93%) than in the motor cortex (75%), suggesting that a complex trafficking mechanism of the VGLUT3 protein is at play within highly collateralized axons of the dorsal raphe nucleus neurons. Our results provide the first direct evidence that the dorsal raphe nucleus ascending projections are composed of widely distributed neuronal systems, whose capacity to co-release serotonin and glutamate varies from one forebrain locus to the other.
... Various electrophysiological, pharmacological, immunohistochemical and morphological studies of the raphe nuclei neurons have been conducted (Beck, Pan, Akanwa, & Kirby, 2004;Calizo et al., 2011;Giovanni et al., 2008;Hajós, Sharp, & Newberry, 1996;Kirby, Pernar, Valentino, & Beck, 2003;Kocsis, Varga, Dahan, & Sik, 2006;Li, Li, Kaneko, & Mizuno, 2001;Marinelli et al., 2004;Vandermaelen & Aghajanian, 1983). According to the classical and convenient identification, 5-HT neurons are presumed to have relatively slow and regular firing, broad action potentials, affinity to inhibitory feedback from 5-HT 1A autoreceptors, or slow after-hyperpolarization (AHP) after a spike (Aghajanian & Vandermaelen, 1982;Hajós et al., 1996;Sprouse & Aghajanian, 1987;Vandermaelen & Aghajanian, 1983). ...
... In our model, we could conveniently capture such neuronal properties (e.g. Kirby et al., 2003;Li et al., 2001) by simply setting the reset membrane potential c at ∼−60 mV or below (no bursting) while having some form of a spike frequency adaptation as shown in Fig. 3(a). The change in initial instantaneous firing rate was more gradual than its steady state over time (Fig. 3(b)) and applied current amplitude (Fig. 3(c)). ...
Serotonin (5-HT) plays an important role in regulating mood, cognition and behaviour. The midbrain dorsal raphe nucleus (DRN) is one of the primary sources of 5-HT. Recent studies show that DRN neuronal activities can encode rewarding (e.g., appetitive) and unrewarding (e.g., aversive) behaviours. Experiments have also shown that DRN neurons can exhibit heterogeneous spiking behaviours. In this work, we build and study a basic spiking neuronal network model of the DRN constrained by neuronal properties observed in experiments. We use an efficient adaptive quadratic integrate-and-fire neuronal model to capture slow afterhyperpolarization current, occasional bursting behaviours in 5-HT neurons, and fast spiking activities in the non-5-HT inhibitory neurons. Provided that our noisy and heterogeneous spiking neuronal network model adopts a feedforward inhibitory network architecture, it is able to replicate the main features of DRN neuronal activities recorded in monkeys performing a reward-based memory-guided saccade task. The model exhibits theta band oscillation, especially among the non-5-HT inhibitory neurons during the rewarding outcome of a simulated trial, thus forming a model prediction. By varying the inhibitory synaptic strengths and the afferent inputs, we find that the network model can oscillate over a range of relatively low frequencies, allow co-existence of multiple stable frequencies, and spike synchrony can spread from within a local neural subgroup to global. Our model suggests plausible network architecture, provides interesting model predictions that can be experimentally tested, and offers a sufficiently realistic multi-scale model for 5-HT neuromodulation simulations.
... A recent study directly compared the properties of 5-HT-containing and non-5-HT-containing neurons in the DRN using in vitro intracellular electrophysiological recording techniques followed by neurochemical identification of the recorded cell. Li et al., (2001) found that, morphologically, 5-HT-containing cells were distinguished by their spiny dendrites as compared with non-5-HT-containing cells whose dendrites were primarily aspiny. Electrophysiologically, 5-HT-containing cells had a larger membrane time constant and larger action potential (amplitude and duration) than non-5-HT-containing cells. ...
... Cell surface area and cell morphology are known to affect the tau characteristic, thus these data may indicate that serotonergic cells have a distinct morphology from non-serotonergic cells. Consistent with this, a recent report noted that serotonin-containing DRN cells have dendritic spines whereas dendrites of non-serotonincontaining DRN neurons are aspiny (Li et al., 2001), a distinction that could contribute to the difference in tau. ...
The membrane properties and receptor-mediated responses of rat dorsal raphe nucleus neurons were measured using intracellular recording techniques in a slice preparation. After each experiment, the recorded neuron was filled with neurobiotin and immunohistochemically identified as 5-hydroxytryptamine (5-HT)-immunopositive or 5-HT-immunonegative. The cellular characteristics of all recorded neurons conformed to previously determined classic properties of serotonergic dorsal raphe nucleus neurons: slow, rhythmic activity in spontaneously active cells, broad action potential and large afterhyperpolarization potential. Two electrophysiological characteristics were identified that distinguished 5-HT from non-5-HT-containing cells in this study. In 5-HT-immunopositive cells, the initial phase of the afterhyperpolarization potential was gradual (tau=7.3+/-1.9) and in 5-HT-immunonegative cells it was abrupt (tau=1.8+/-0.6). In addition, 5-HT-immunopositive cells had a shorter membrane time constant (tau=21.4+/-4.4) than 5-HT-immunonegative cells (tau=33.5+/-4.2). Interestingly, almost all recorded neurons were hyperpolarized in response to stimulation of the inhibitory 5-HT(1A) receptor. These results suggested that 5-HT(1A) receptors are present on non-5-HT as well as 5-HT neurons. This was confirmed by immunohistochemistry showing that although the majority of 5-HT-immunopositive cells in the dorsal raphe nucleus were double-labeled for 5-HT(1A) receptor-IR, a small but significant population of 5-HT-immunonegative cells expressed the 5-HT(1A) receptor. These results underscore the heterogeneous nature of the dorsal raphe nucleus and highlight two membrane properties that may better distinguish 5-HT from non-5-HT cells than those typically reported in the literature. In addition, these results present electrophysiological and anatomical evidence for the presence of 5-HT(1A) receptors on non-5-HT neurons in the dorsal raphe nucleus.
... The forebrain 5-HT system is implicated in many aspects of cerebral function, including emotion and fear processing, cognition, movement and regulation of the sleep-wake cycle (Dugovic, 2001;Fornal, 1991, 1999;Meneses, 1999). The electrophysiological properties of 5-HT neurones in the DRN has been investigated using in vivo extracellular or intracellular recording combined with histofluorescence (Aghajanian and Haigler, 1974;Aghajanian and Vandermaelen, 1982), as well as more recently using in vitro intracellular recording combined with immunocytochemistry (Li et al., 2001). It is generally agreed that in vivo 5-HT neurones spontaneously fire broad spikes in a slow, and regular firing pattern (Aghajanian et al., 1978;Hajó s et al., 1998;Jacobs and Fornal, 1991;Sawyer et al., 1985). ...
... Thus, the examples of 5-HT/TrH-ir neurones analysed were spiny whilst the non-5-HT/TrH-ir neurones were aspiny. Non-5-HT containing, aspiny DRN neurones have been reported previously (Li et al., 2001). Finally, a population of slow, irregular firing DRN neurones were reportedly resistant to 5-HT lesions (Aghajanian et al., 1978). ...
GABA neurones in the dorsal raphe nucleus (DRN) influence ascending 5-hydroxytryptamine (5-HT) neurones but are not physiologically or anatomically characterised. Here, in vivo juxtacellular labelling methods in urethane-anaesthetised rats were used to establish the neurochemical and morphological identity of a fast-firing population of DRN neurones, which recent data suggest may be GABAergic. Slow-firing, putative 5-HT DRN neurones were also identified for the first time using this approach. Fast-firing, DRN neurones were successfully labelled with neurobiotin (n=10) and the majority (n=8/10) were immunoreactive for the GABA synthetic enzyme glutamic acid decarboxylase. These neurones were located in the DRN (mainly lateral regions), and consistently fired spikes with short width (1.1+/-0.1 ms) and high frequency (12.1+/-2.0 Hz). In most cases spike trains were regular but displayed low frequency oscillations (1-2 Hz). These neurones were morphologically heterogeneous but commonly had branching axons with varicosities and dendrites that extended across DRN subregions and the midline. Slow-firing DRN neurones were also successfully labelled with neurobiotin (n=24). These neurones comprised a population of neurones immunopositive for 5-HT and/or tryptophan hydroxylase (n=12) that fired broad spikes (2.2+/-0.2 ms) with high regularity and low frequency (1.7+/-0.2 Hz). However, a slow-firing, less regular population of neurones immunonegative for 5-HT/tryptophan hydroxylase (n=12) was also apparent. In summary, this study chemically identifies fast- and slow-firing neurones in the DRN and establishes for the first time that fast-firing DRN neurones are GABAergic. The electrophysiological and morphological properties of these neurones suggest a novel function involving co-ordination between GABA and 5-HT neurones dispersed across DRN subregions.
... Neurons from multiple brain areas expressing monoamines [dopamine (DA), noradrenaline (NE)], histamine (HA), gamma-aminobutyric acid (GABA), glutamate (GLU), acetylcholine (ACh) or neuropeptides (orexin, melaninconcentrating hormone among other), project to the DRN, and directly or indirectly through local circuits control the activity of 5HT neurons [1,19,25]. ...
The neural systems involved in the generation and maintenance of wakefulness (W) and REM sleep (REMS) include dopaminergic (DAergic) neurons in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc), as well as serotonergic (5HT) neurons in the dorsal raphe nucleus (DRN). There is also a small group of DAergic neurons in the ventral periaqueductal gray or rostral DRN (also called A10dc region). Retrograde tracer techniques have established that the DRN receives DAergic inputs from the VTA and SNc. Furthermore, autoradiographic and in situ hybridization techniques have shown that dopamine D1 and D2 receptor subtypes are distributed within the DRN. Microinjection of D1 or D2 receptor subtype agonists in the DRN increases the excitability of 5-HT neurons, and intra-DRN administration of D1 and D2 selective receptor agonists SKF38393, bromocriptine and quinpirole to rats, induced a significant reduction of REMS and the number of REM periods. In addition, bromocriptine and quinpirole increased W. Furthermore, microinjection into the DRN of the D1 and D2 receptor antagonists SCH23390 and sulpiride significantly augmented REMS and the number of REM periods. Pretreatment with the above-mentioned antagonists prevented the effects of SKF38393 and bromocriptine on sleep variables. Thus, DAergic signal in the DRN contributes to the regulation of W and REMS.
... Stimulation and data acquisition were controlled by the PULSE/PULSEFIT software package (HEKA) on a Macintosh computer, and data analysis was performed with IGOR software (WaveMetrics, Lake Oswego, OR, USA). Properties of 5-HT specific neurons were analyzed according to existing electrophysiological criteria (de Kock et al. 2006;Li et al. 2001; Vandermaelen and Aghajanian 1983), such as amplitude, halfheight width (HHW) and action potential frequency. Our main criteria for 5-HT specific neuron identity were action potential HHW of > 1.5 ms and maximal sustained firing rate of < 12 Hz (Mlinar et al. 2015). ...
Human induced pluripotent stem cells (hiPSCs) have revolutionized the generation of experimental disease models, but the development of protocols for the differentiation of functionally active neuronal subtypes with defined specification is still in its infancy. While dysfunction of the brain serotonin (5-HT) system has been implicated in the etiology of various neuropsychiatric disorders, investigation of functional human 5-HT specific neurons in vitro has been restricted by technical limitations. We describe an efficient generation of functionally active neurons from hiPSCs displaying 5-HT specification by modification of a previously reported protocol. Furthermore, 5-HT specific neurons were characterized using high-end fluorescence imaging including super-resolution microscopy in combination with electrophysiological techniques. Differentiated hiPSCs synthesize 5-HT, express specific markers, such as tryptophan hydroxylase 2 and 5-HT transporter, and exhibit an electrophysiological signature characteristic of serotonergic neurons, with spontaneous rhythmic activities, broad action potentials and large afterhyperpolarization potentials. 5-HT specific neurons form synapses reflected by the expression of pre- and postsynaptic proteins, such as Bassoon and Homer. The distribution pattern of Bassoon, a marker of the active zone along the soma and extensions of neurons, indicates functionality via volume transmission. Among the high percentage of 5-HT specific neurons (~ 42%), a subpopulation of CDH13 + cells presumably designates dorsal raphe neurons. hiPSC-derived 5-HT specific neuronal cell cultures reflect the heterogeneous nature of dorsal and median raphe nuclei and may facilitate examining the association of serotonergic neuron subpopulations with neuropsychiatric disorders.
... Further, it was reported that NPS could modulate the Ih current, a hyperpolarization-activated mixed cationic current carried by HCN channels, in cells of the rat amygdala (Zhang et al., 2016). As there is a prominent Ih current in DR and LDT cells evidenced during protocols stepping the holding voltage negative than À80 mV (Biel et al., 2009;Li et al., 2001), we examined the effect of NPS on this current. However, in voltage clamp experiments, we failed to detect any effect of NPS application on the amplitude of Ih in these neurons, which suggests that the HCN channel does not play a role in NPS-mediated membrane currents in these two nuclei (personal observations). ...
Neuropeptide S (NPS) is a peptide recently recognized to be present in the CNS, and believed to play a role in vigilance and mood control, as behavioral studies have shown it promotes arousal and has an anxiolytic effect. Although NPS precursor is found in very few neurons, NPS positive fibers are present throughout the brain stem. Given the behavioral actions of this peptide and the wide innervation pattern, we examined the cellular effects of NPS within two brain stem nuclei known to play a critical role in anxiety and arousal: the dorsal raphe (DR) and laterodorsal tegmentum (LDT). In mouse brain slices, NPS increased cytoplasmic levels of calcium in DR and LDT cells. Calcium rises were independent of action potential generation, reduced by low extracellular levels of calcium, attenuated by IP3 - and ryanodine (RyR)-dependent intracellular calcium store depletion, and eliminated by the receptor (NPSR) selective antagonist, SHA 68. NPS also exerted an effect on the membrane of DR and LDT cells inducing inward and outward currents, which were driven by an increase in conductance, and eliminated by SHA 68. Membrane actions of NPS were found to be dependent on store-mediated calcium as depletion of IP3 and RyR stores eliminated NPS-induced currents. Finally, NPS also had actions on synaptic events, suggesting facilitation of glutamatergic and GABAergic presynaptic transmission. When taken together, actions of NPS influenced the excitability of DR and LDT neurons, which could play a role in the anxiolytic and arousal-promoting effects of this peptide.
... In midbrain serotonergic raphe neurons, Kocsis et al. (2006) obtained in vivo rates with mean 5.4 Hz. For experiments with depolarizing current injection, Li and Bayliss (1998) obtained initial firing rates of around 8 Hz for caudal raphe with an injected current of 60 pA; Li et al. (2001) reported firing of rat DRN SE cells at frequencies as high as 35 Hz with current injection, and Ohliger-Frerking et al. (2003) found rates as high as 8 Hz and 11 Hz with 100 pA current injection in lean and obese Zucker rats, respectively. See also subsection 4.1 on firing rates. ...
Serotonergic, noradrenergic and dopaminergic brainstem (including midbrain) neurons, often exhibit spontaneous and fairly regular spiking with frequencies of order a few Hz, though dopaminergic and noradrenergic neurons only exhibit such pacemaker-type activity in vitro or in vivo under special conditions. A large number of ion channel types contribute to such spiking so that detailed modeling of spike generation leads to the requirement of solving very large systems of differential equations. It is useful to have simplified mathematical models of spiking in such neurons so that, for example, features of inputs and output spike trains can be incorporated including stochastic effects for possible use in network models. In this article we investigate a simple two-component conductance-based model of the Hodgkin-Huxley type. Solutions are computed numerically and with suitably chosen parameters mimic features of pacemaker-type spiking in the above types of neurons. The effects of varying parameters is investigated in detail, it being found that there is extreme sensitivity to eight of them. Transitions from non-spiking to spiking are examined for two of these, the half-activation potential for an activation variable and the added (depolarizing) current and contrasted with the behavior of the classical Hodgkin-Huxley system. The plateaux levels between spikes can be adjusted, by changing a set of voltage parameters, to agree with experimental observations. Experiment has shown that in, in vivo, dopaminergic and noradrenergic neurons' pacemaker activity can be induced by the removal of excitatory inputs or the introduction of inhibitory ones. These properties are confirmed by mimicking opposite such changes in the model, which resulted in a change from pacemaker activity to bursting-type phenomena.
... The exact source(s) of endogenously released galanin activating the up-regulated GALR1 receptors in vPAG remains to be established. There is a galanin terminal network of varying density in the entire rat vPAG (89), and some of these fibers could originate from galanin + 5-HT neurons because the 5-HT neurons send richly branching axon collaterals within the DR (104,127,152); there are also VGLUT3 + 5-HT collaterals and at least some may contain galanin (140). Nevertheless, the galanin input to the GALR1 + neurons is probably mainly from nonserotonergic neurons. ...
Significance
The pathophysiology of depression remains unclear, but accumulated evidence implicates disturbances in monoaminergic transmission in the brain. Several studies suggest that members of the diverse family of neuropeptides may also be involved. In the rat, the neuropeptide galanin is coexpressed with noradrenaline and serotonin, and modulates the signaling of these neurotransmitters. Here, we explored a possible role of galanin and its receptors in a rat model of depression based on chronic mild stress using quantitative real-time PCR combined with viral-mediated delivery of galanin receptor 1 (Galr1) siRNA. Our results indicate involvement of the GALR1 receptor subtype in the ventral periaqueductal gray in depression-like behavior, possibly representing a novel target for antidepressant therapy.
... Electrophysiological and pharmacological criteria including low frequency firing rate, wide spikes, and a hyperpolarizing response to 5-HT 1A receptor agonists, initially used to distinguish between 5-HT and unidentified cells (Aghajanian and Lakoski, 1984;Aghajanian and Vandermaelen, 1982;Vandermaelen and Aghajanian, 1983) have become inadequate for proper categorization of DR neurons (Calizo et al., 2011;Kocsis et al., 2006). A number of ultrastructural studies reported that identification of 5-HT neurons based on these criteria alone produced a number of false-negative and false-positive results (Allers and Sharp, 2003;Kirby et al., 2003;Li et al., 2001). ...
... In midbrain serotonergic raphe neurons, Kocsis et al. (2006) obtained in vivo rates with mean 5.4 Hz. For experiments with depolarizing current injection, Li and Bayliss (1998) obtained initial firing rates of around 8 Hz for caudal raphe with an injected current of 60 pA; Li et al. (2001) reported firing of rat DRN SE cells at frequencies as high as 35 Hz with current injection, and Ohliger-Frerking et al. (2003) found rates as high as 8 Hz and 11 Hz with 100 pA current injection in lean and obese Zucker rats, respectively. ...
Many central neurons, and in particular certain brainstem aminergic neurons
exhibit spontaneous and fairly regular spiking with frequencies of order a few
Hz. A large number of ion channel types contribute to such spiking so that
accurate modeling of spike generation leads to the requirement of solving very
large systems of differential equations, ordinary in the first instance. Since
analysis of spiking behavior when many synaptic inputs are active adds further
to the number of components, it is useful to have simplified mathematical
models of spiking in such neurons so that, for example, stochastic features of
inputs and output spike trains can be incorporated. In this article we
investigate two simple two-component models which mimic features of spiking in
serotonergic neurons of the dorsal raphe nucleus and noradrenergic neurons of
the locus coeruleus. The first model is of the Fitzhugh-Nagumo type and the
second is a reduced Hodgkin-Huxley model. For each model solutions are computed
with two representative sets of parameters. Frequency versus input currents are
found and reveal Hodgkin type 2 behavior. For the first model a bifurcation and
phase plane analysis supports these findings. The spike trajectories in the
second model are very similar to those in DRN SE pacemaker activity but there
are more parameters than in the Fitzhugh-Nagumo type model. The article
concludes with a brief review of previous modeling of these types of neurons
and its relevance to studies of serotonergic involvement in spatial working
memory and obsessive-compulsive disorder.
... Neuronal types (5-HTergic or GABAergic neurons) were identified based on their distinct morphological [19], electrophysiological [19,36,37,38,39,40] and pharmacological properties [41,42]. According to previous studies by others [19,43], we initially considered the neuron with a large fusiform or multipolar cell body as a putative 5-HTergic neuron, and the neuron with a small cell body and aspiny dendrites as a putative GABAergic neuron ( Fig 1B). The neuronal types were further confirmed by a set of electrophysiological and pharmacological criteria as detailed in Table 1 Shoulder was a delayed repolarization on the single action potential. ...
Sodium salicylate (NaSal), a tinnitus inducing agent, can activate serotonergic (5-HTergic) neurons in the dorsal raphe nucleus (DRN) and can increase serotonin (5-HT) level in the inferior colliculus and the auditory cortex in rodents. To explore the underlying neural mechanisms, we first examined effects of NaSal on neuronal intrinsic properties and the inhibitory synaptic transmissions in DRN slices of rats by using whole-cell patch-clamp technique. We found that NaSal hyperpolarized the resting membrane potential, decreased the input resistance, and suppressed spontaneous and current-evoked firing in GABAergic neurons, but not in 5-HTergic neurons. In addition, NaSal reduced GABAergic spontaneous and miniature inhibitory postsynaptic currents in 5-HTergic neurons. We next examined whether the observed depression of GABAergic activity would cause an increase in the excitability of 5-HTergic neurons using optogenetic technique in DRN slices of the transgenic mouse with channelrhodopsin-2 expressed in GABAergic neurons. When the GABAergic inhibition was enhanced by optical stimulation to GABAergic neurons in mouse DRN, NaSal significantly depolarized the resting membrane potential, increased the input resistance and increased current-evoked firing of 5-HTergic neurons. However, NaSal would fail to increase the excitability of 5-HTergic neurons when the GABAergic synaptic transmission was blocked by picrotoxin, a GABA receptor antagonist. Our results indicate that NaSal suppresses the GABAergic activities to raise the excitability of local 5-HTergic neural circuits in the DRN, which may contribute to the elevated 5-HT level by NaSal in the brain. © 2015 Jin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
... Classic electrophysiological criteria first used to identify putative 5-HT neurons according to their slowfiring, regular patterns and wide spikes (Vandermaelen & Aghajanian, 1983) have become insufficient for gross classification of raphe cells (Allers & Sharp, 2003;Kocsis et al., 2006;Calizo et al., 2011). Several intracellular (Li et al., 2001;Kirby et al., 2003) and juxtacellular labelling (Allers & Sharp, 2003) studies reported that neuronal identification based on electrophysiological criteria alone produced a significant number of false-negative and false-positive results. Allers & Sharp (2003) found that half of the slow-firing DR neurons were non-serotonergic and 20% of fast-spiking putative non-5-HT neurons were actually serotonergic (Allers & Sharp, 2003). ...
High-frequency deep brain stimulation (HFS-DBS) of the subcallosal cingulate (SCC) region has been investigated as a treatment for refractory forms of depression with a ~50% remission rate in open label studies. However, the therapeutic mechanisms of DBS are still largely unknown. Using anaesthetized Sprague Dawley rats, we recorded neuronal spiking activity in 102 neurons of the dorsal raphe (DR) before, during and after the induction of a 5-min HFS train in the infralimbic region (IL) of the medial prefrontal cortex (mPFC), the rodent homologue of the human SCC. The majority of DR cells (82%) significantly decreased firing rate during HFS (P < 0.01, 55.7 ± 4.5% of baseline, 35 rats). To assess whether mPFC-HFS mediates inhibition of DR cellular firing by stimulating local GABAergic interneurons, the GABAA antagonist bicuculline (Bic, 100 μm) was injected directly into the DR during HFS. Neurons inhibited by HFS recovered their firing rate during Bic+HFS (P < 0.01, n = 15, seven rats) to levels not different from baseline. Cells that were not affected by HFS did not change firing rate during Bic+HFS (P = 0.968, n = 7, three rats). These results indicate that blocking GABAA reverses HFS-mediated inhibition of DR neurons. As the cells that were not inhibited by HFS were also unaffected by HFS+Bic, they are probably not innervated by local GABA. Taken together, our results suggest that mPFC-HFS may exert a preferential effect on DR neurons with GABAA receptors.
© 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.
... Immunohistochemical studies have shown that these two raphe nuclei not only contain cell bodies and dendrites of 5-HT neurons but also contain a network of 5-HT fibres (Kapadia et al. 1985;Leger et al. 2001;Li et al. 2001). The raphe nuclei also contain high levels of 5-HT, which is released from 5-HT neurons within these nuclei, and/or other raphe nuclei, in a concentration relatively greater than in the other forebrain regions (Adell et al. 2002). ...
... For the former case, with g N a,max =0, in simulations a hyperpolarizing current µ = 0.1 nA was applied for 100 ms using the same parameter set as in run F10 of Table 27. The result is shown in the left panel of Figure 29 where it is seen that during the application of the hyperpolarizing current, V decreases from resting level of -60 mV to about -79 mV with a relatively small time constant of about 7 ms, which is similar to the average value reported in Li et al. (2001). In another run a time constant of about 30 ms was obtained, which is more in the range of reported values for the majority of DRN SE neurons. ...
Serotonergic neurons of the dorsal raphe nucleus, with their extensive innervation of limbic and higher brain regions and interactions with the endocrine system have important modulatory or regulatory effects on many cognitive, emotional and physiological processes. They have been strongly implicated in responses to stress and in the occurrence of major depressive disorder and other pyschiatric disorders. In order to quantify some of these effects, detailed mathematical models of the activity of such cells are required which describe their complex neurochemistry and neurophysiology. We consider here a single-compartment model of these neurons which is capable of describing many of the known features of spike generation, particularly the slow rhythmic pacemaking activity often observed in these cells in a variety of species. Included in the model are 11 kinds of ion channels: a fast sodium current INa, a delayed rectifier potassium current IKDR, a transient potassium current IA, a slow non-inactivating potassium current IM, a low-threshold calcium current IT, two high threshold calcium currents IL and IN, small and large conductance potassium currents ISK and IBK, a hyperpolarization-activated cation current IH and a leak current ILeak. In Sections 3-8, each current type is considered in detail and parameters estimated from voltage clamp data where possible. Three kinds of model are considered for the BK current and two for the leak current. Intracellular calcium ion concentration Cai is an additional component and calcium dynamics along with buffering and pumping is discussed in Section 9. The remainder of the article contains descriptions of computed solutions which reveal both spontaneous and driven spiking with several parameter sets. Attention is focused on the properties usually associated with these neurons, particularly long duration of action potential, steep upslope on the leading edge of spikes, pacemaker-like spiking, long-lasting afterhyperpolarization and the ramp-like return to threshold after a spike. In some cases the membrane potential trajectories display doublets or have humps or notches as have been reported in some experimental studies. The computed time courses of IA and IT during the interspike interval support the generally held view of a competition between them in influencing the frequency of spiking. Spontaneous activity was facilitated by the presence of IH which has been found in these neurons by some investigators. For reasonable sets of parameters spike frequencies between about 0.6 Hz and 1.2 Hz are obtained, but frequencies as high as 6 Hz could be obtained with special parameter choices. Topics investigated and compared with experiment include shoulders, notches, anodal break phenomena, the effects of noradrenergic input, frequency versus current curves, depolarization block, effects of cell size and the effects of IM. The inhibitory effects of activating 5-HT1A autoreceptors are also investigated. There is a considerable discussion of in vitro versus in vivo firing behavior, with focus on the roles of noradrenergic input, corticotropin-releasing factor and orexinergic inputs. Location of cells within the nucleus is probably a major factor, along with the state of the animal.
... Serotonergic REM-off neurons in DRN play suppressive role in REM sleep genesis by inhibiting cholinergic REM-on neurons in laterodorsal and pedunculopontine tegmental nuclei (PPT/ LDT) (McCarley, 2007). On the other hand, DRN GABAergic interneurons make synaptic contacts with cholinergic cells of the LDT/PPT nuclei (Ford et al., 1995;Li et al., 2001). The activation of DRN GABAergic interneurons will lead to the inhibition of the cholinergic neuron and the suppression of REM sleep (McCarley, 2007;Monti, 2010). ...
... Visualizations of complete serotonergic (SE) neurons are not feasible because of their extensive axonal arborizations, but a useful schematic is shown inFigure 1 taken from Maeda et al. (1989). There have been numerous morphological studies of the somadendritic parts of these cells (for example, Pfister and Danner, 1980; Diaz-Cintra et al., 1981; Descarries et al., 1982; Park et al., 1982; Imai et al., 1986; Park, 1987; Li et al., 2001; Allers and Sharp, 2003; Hajós et al., 2007; Figure 1: Illustration of the extensive innervation of brain ans spinal cord regions by serotonergic neurons of raphe nuclei. From Maeda et al. (1989). ...
Serotonergic neurons of the dorsal raphe nuclei, with their extensive innervation of nearly the whole brain have important modulatory effects on many cognitive and physiological processes. They play important roles in clinical depression and other psychiatric disorders. In order to quantify the effects of serotonergic transmission on target cells it is desirable to construct computational models and to this end these it is necessary to have details of the biophysical and spike properties of the serotonergic neurons. Here several basic properties are reviewed with data from several studies since the 1960s to the present. The quantities included are input resistance, resting membrane potential, membrane time constant, firing rate, spike duration, spike and afterhyperpolarization (AHP) amplitude, spike threshold, cell capacitance, soma and somadendritic areas. The action potentials of these cells are normally triggered by a combination of sodium and calcium currents which may result in autonomous pacemaker activity. We here analyse the mechanisms of high-threshold calcium spikes which have been demonstrated in these cells the presence of TTX (tetrodotoxin). The parameters for calcium dynamics required to give calcium spikes are quite different from those for regular spiking which suggests the involvement of restricted parts of the soma-dendritic surface as has been found, for example, in hippocampal neurons.
... These studies are consistent with anatomical studies showing that there are many interconnections between the different raphe nuclei 180 and axon collaterals of serotonin neurons travel for some distance within the dorsal raphe nuclei. 186 There are also a number of distinct serotonin subsystems with unique genetic programming and functions. 187 Recent studies have shown that, in raphe nuclei, there are at least three different serotonin cell-types grouped by anatomical, physiological, and molecular characteristics, and their distribution transcends the traditional anatomical classification of raphe subfields. ...
The complexities of the involvement of the serotonin transmitter system in numerous biological processes and psychiatric disorders is, to a substantial degree, attributable to the large number of serotonin receptor families and subtypes that have been identified and characterized for over four decades. Of these, the 5-HT(1A) receptor subtype, which was the first to be cloned and characterized, has received considerable attention based on its purported role in the etiology and treatment of mood and anxiety disorders. 5-HT(1A) receptors function both at presynaptic (autoreceptor) and postsynaptic (heteroreceptor) sites. Recent research has implicated distinct roles for these two populations of receptors in mediating emotion-related behavior. New concepts as to how 5-HT(1A) receptors function to control serotonergic tone throughout life were highlights of the proceedings of the 2012 Serotonin Club Meeting in Montpellier, France. Here, we review recent findings and current perspectives on functional aspects of 5-HT(1A) auto- and heteroreceptors with particular regard to their involvement in altered anxiety and mood states.
... To date, many investigations have described the properties of DRN 5-HT cells through in vivo or in vitro electrophysiological recordings with neurochemical identification [5][6][7][8]. However, these studies did not directly characterize the properties of DRN GABAergic cells but described GABAergic cells as putative non-5-HT cells that do not exhibit tryptophan hydroxylase (TPH) immunoreactivity. ...
The dorsal raphe nucleus (DRN) is the origin of the central serotonin [5-hydroxytryptamine (5-HT)] system and plays an important role in the regulation of many physiological functions such as sleep/arousal, food intake and mood. In order to understand the regulatory mechanisms of 5-HT system, characterization of the types of neurons is necessary. We performed electrophysiological recordings in acute slices of glutamate decarboxylase 67-green fluorescent protein knock-in mice. We utilized this mouse to identify visually GABAergic cells. Especially, we examined postsynaptic responses mediated by 5-HT receptors between GABAergic and serotonergic cells in the DRN. Various current responses were elicited by 5-HT and 5-HT(1A) or 5-HT(2A/2C) receptor agonists in GABAergic cells. These results suggested that multiple 5-HT receptor subtypes overlap on GABAergic cells, and their combination might control 5-HT cells. Understanding the postsynaptic 5-HT feedback mechanisms may help to elucidate the 5-HT neurotransmitter system and develop novel therapeutic approaches.
... Modified from AmargosBosch et al., 2004 A second possibility entails an activation of non-serotonergic inhibitory afferents to the prefrontal cortex from the raphe nuclei. GABAergic projection neurons have been found in the dorsal raphe nucleus, and many inhibitions have an initial component independent of 5- HT1A receptors (Li et al., 2001; Puig et al., 2005). Moreover, the involvement of direct GABAergic projections to the prefrontal cortex is suggested by the short-latency (≤ 8 ms) inhibitions recorded in prefrontal pyramidal neurons that cannot be accounted for by the slow conduction velocity of serotonergic axons (Puig et al., 2005). ...
... This suggests that nearby neurons are likely to coordinate their activation states. Consistent with this finding, individual neurons in the DR are known to have local axon collaterals ramifying within the DR (Li et al., 2001). If functionally similar neurons cluster together, proximally localized axon collaterals are likely to participate in autoregulatory feedback networks, potentially playing a homeostatic function. ...
Brain serotonin neurons are heterogeneous and can be distinguished by several anatomical and physiological characteristics. Toward resolving this heterogeneity into classes of functional relevance, subtypes of mature serotonin neurons were previously identified based on gene expression differences initiated during development in different rhombomeric (r) segments of the hindbrain. This redefinition of mature serotonin neuron subtypes based on the criteria of genetic lineage, along with the enabling genetic fate mapping tools, now allows various functional properties, such as axonal projections, to be allocated onto these identified subtypes. Furthermore, our approach uniquely enables interconnections between the different serotonin neuron subtypes to be determined; this is especially relevant because serotonin neuron activity is regulated by several feedback mechanisms. We used intersectional and subtractive genetic fate mapping tools to generate three independent lines of mice in which serotonin neurons arising in different rhombomeric segments, either r1, r2 or both r3 and r5, were uniquely distinguished from all other serotonin neurons by their expression of enhanced green fluorescent protein. Each of these subgroups of serotonergic neurons had a unique combination of forebrain projection targets. Typically more than one subgroup innervated an individual target area. Unique patterns of interconnections between the different groups of serotonin neurons were also observed and these pathways could subserve feedback regulatory circuits. Overall, the current findings suggest that activation of subsets of serotonin neurons could result in topographic serotonin release in the forebrain coupled with feedback inhibition of serotonin neurons with alternative projection targets.
... These nuclei are considered to be the main source of prosencephalic serotonin (5hydroxytryptamine) (5-HT) [1][2][3][4]. The dorsal raphe nucleus and the median raphe nucleus (MnR) also contain distinct subpopulations of non-serotonergic neurons that occur in equal or greater numbers compared to the serotonergic neurons [5][6][7][8][9]. In the MnR, GABAergic neurons are located in both midline and lateral regions across the rostro-caudal extent of the MnR. ...
This study investigated the participation of median raphe nucleus (MnR) α1-adrenergic receptors in the control of feeding behaviour. The α1-adrenergic agonist phenylephrine (PHE) and α2-adrenergic agonist clonidine (CLON) (at equimolar doses of 0, 6 and 20 nmol) were injected into the MnR of: a) rats submitted to overnight fasting (18 h); or b) rats maintained with 15 g of lab chow/day for 7 days. Immediately after the drug injections, the animals were placed in the feeding chamber and feeding and non-ingestive behaviours such as grooming, rearing, resting, sniffing and locomotion were recorded for 30 min. The results showed that both doses of PHE injected into the MnR of overnight fasted animals decreased food intake accompanied by an increase in the latency to start feeding. A reduction in feeding duration was observed only after treatment of the MnR with the 20 nmol dose of PHE. Both locomotion duration and sniffing frequency increased after injection with the highest dose PHE into the MnR. Feeding frequency and the other non-ingestive behaviours remained unchanged after PHE treatment in the MnR. Both doses of PHE injected into the MnR of food-restricted rats decreased food intake. This hypophagic response was accompanied by a decrease in feeding duration only after treatment of the MnR with the highest dose of PHE. The latency to start feeding and feeding frequency were not affected by injection of either dose of PHE into the MnR. While both doses of PHE increased sniffing duration, the highest dose of PHE increased resting duration and resting frequency. Treatment with CLON into the MnR did not affect feeding behaviour in either of the food deprivation conditions. The present results indicate the inhibitory functional role of α1-adrenergic receptors within the MnR on feeding behaviour.
... For technical reasons, dendritic spines are difficult to study in the DR; however, 5-HT neurons are known to have dendritic spines, sparsely on their primary and the secondary dendrites but with progressively high density on higher order dendrites (Li et al., 2001). Dendritic spines are of particular interest for their association with synaptic plasticity. ...
The dorsal raphe nucleus (DR) contains the majority of serotonin (5-hydroxytryptamine, 5-HT) neurons in the brain that regulate neural activity in forebrain regions through their widespread projections. DR function is linked to stress and emotional processing, and is implicated in the pathophysiology of affective disorders. Glutamatergic drive of the DR arises from many different brain areas with the capacity to inform the nucleus of sensory, autonomic, endocrine and metabolic state as well as higher order neural function. Imbalance of glutamatergic neurotransmission could contribute to maladaptive 5-HT neurotransmission and represents a potential target for pharmacotherapy. Within the DR, glutamate-containing axon terminals can be identified by their content of one of three types of vesicular glutamate transporter, VGLUT1, 2 or 3. Each of these transporters is heavily expressed in particular brain areas such that their content within axons correlates with the afferent's source. Cortical sources of innervation to the DR including the medial prefrontal cortex heavily express VGLUT1 whereas subcortical sources primarily express VGLUT2. Within the DR, many local neurons responsive to substance P contain VGLUT3, and these provide a third source of excitatory drive to 5-HT cells. Moreover VGLUT3 is present, with or without 5-HT, in output pathways from the DR. 5-HT and non-5-HT neurons receive and integrate glutamatergic neurotransmission through multiple subtypes of glutamate receptors that have different patterns of expression within the DR. Interestingly, excitatory drive provided by glutamatergic neurotransmission is closely opposed by feedback inhibition mediated by 5-HT1A receptors or local GABAergic circuits. Understanding the intricacies of these local networks and their checks and balances, may help identify how potential imbalances could cause psychopathology and illuminate strategies for therapeutic manipulation.
... Non-5-HT containing neurons are present and occur in equal or greater number to 5-HT neurons in DR and MR (Descarries et al., 1982;Kiss et al., 2002;Kohler and Steinbusch, 1982;Li et al., 2001;Van Bockstaele et al., 1993). For any given neurotransmitter, however, the number of neurons is lower, i.e., one third to one tenth less, than the number of 5-HT neurons. ...
The median (MR) and dorsal raphe (DR) nuclei contain the majority of the 5-hydroxytryptamine (5-HT, serotonin) neurons that project to limbic forebrain regions, are important in regulating homeostatic functions and are implicated in the etiology and treatment of mood disorders and schizophrenia. The primary synaptic inputs within and to the raphe are glutamatergic and GABAergic. The DR is divided into three subfields, i.e., ventromedial (vmDR), lateral wings (lwDR) and dorsomedial (dmDR). Our previous work shows that cell characteristics of 5-HT neurons and the magnitude of the 5-HT(1A) and 5-HT(1B) receptor-mediated responses in the vmDR and MR are not the same. We extend these observations to examine the electrophysiological properties across all four raphe subfields in both 5-HT and non-5-HT neurons. The neurochemical topography of glutamatergic and GABAergic cell bodies and nerve terminals were identified using immunohistochemistry and the morphology of the 5-HT neurons was measured. Although 5-HT neurons possessed similar physiological properties, important differences existed between subfields. Non-5-HT neurons were indistinguishable from 5-HT neurons. GABA neurons were distributed throughout the raphe, usually in areas devoid of 5-HT neurons. Although GABAergic synaptic innervation was dense throughout the raphe (immunohistochemical analysis of the GABA transporters GAT1 and GAT3), their distributions differed. Glutamate neurons, as defined by vGlut3 anti-bodies, were intermixed and co-localized with 5-HT neurons within all raphe subfields. Finally, the dendritic arbor of the 5-HT neurons was distinct between subfields. Previous studies regard 5-HT neurons as a homogenous population. Our data support a model of the raphe as an area composed of functionally distinct subpopulations of 5-HT and non-5-HT neurons, in part delineated by subfield. Understanding the interaction of the cell properties of the neurons in concert with their morphology, local distribution of GABA and glutamate neurons and their synaptic input, reveals a more complicated and heterogeneous raphe. These results provide an important foundation for understanding how specific subfields modulate behavior and for defining which aspects of the circuitry are altered during the etiology of psychological disorders.
... This raises the possibility that 5-HT1A receptors could function in a region-non-autonomous fashion to mediate communication between different groups of neurons. Consistent with the possibility, it is known that there are many interconnections between the different raphe nuclei (Frazer & Hensler, 1999) as well as local axon collaterals of 5-HT neurons that travel for some distance within the dorsal raphe nucleus (Li et al., 2001). Understanding the organization of these recurrent axon collaterals may be a future avenue of research. ...
Nicotine activates serotonin [5-hydroxytryptamine (5-HT)] neurons innervating the forebrain, and this is thought to reduce anxiety. Nicotine withdrawal has also been associated with an activation of 5-HT neurotransmission, although withdrawal increases anxiety. In each case, 5-HT1A receptors have been implicated in the response. To determine whether there are different subgroups of 5-HT cells activated during nicotine administration and withdrawal, we mapped the appearance of Fos, a marker of neuronal activation, in 5-HT cells of the dorsal raphe nucleus (DR) and median raphe nucleus (MR). To understand the role of 5-HT1A receptor feedback inhibitory pathways in 5-HT cell activity during these conditions, we administered a selective 5-HT1A receptor antagonist and measured novel disinhibited Fos expression within 5-HT cells. Using these approaches, we found evidence that acute nicotine exposure activates 5-HT neurons rostrally and in the lateral wings of the DR, whereas there is 5-HT1A receptor-dependent inhibition of cells located ventrally at both the rostral level and mid-level. Previous chronic nicotine exposure did not modify the pattern of activation produced by acute nicotine exposure, but increased 5-HT1A receptor-dependent inhibition of 5-HT cells in the caudal DR. This pattern was nearly reversed during nicotine withdrawal, when there was evidence for caudal activation and mid-level and rostral 5-HT1A receptor-dependent inhibition. These results suggest that the distinct behavioral states produced by nicotine exposure and withdrawal correlate with reciprocal rostral-caudal patterns of activation and 5-HT1A receptor-mediated inhibition of DR 5-HT neurons. The complementary patterns of activation and inhibition suggest that 5-HT1A receptors may help to shape distinct topographic patterns of activation within the DR.
... 39,43 In addition, the subpopulations of 5-HT neurons coexpress different neurotransmitters and neuromodulators including DA, GLU, GABA, substance P, and corticotropin releasing factor. 41,42,142 Thus, the possibility exists that efferent projections from each subpopulation of 5-HT neurons form synapses with only one subtype of serotonin receptor. This would allow different 5-HT subsystems to influence specifically and separately the neurons involved in the regulation of W and REMS. ...
... Moreover, numerous brain areas have neurons that project to the DRN and express monoamines (NE, HA), amino acids (GABA, GLU), ACh or neuropeptides (OX, MCH, CRF and SP among others) that directly or indirectly, through local circuits, regulate the activity of 5-HT cells. [13][14][15][16][17][18][19][20][21][22] Based on cellular morphology, 23 expression of other neurotransmitters, 16 afferent and efferent connections, 24,25 and functional properties, 21 5-HT neurons of the DRN have been grouped into several subnuclei or cell clusters. These differences among subpopulations of 5-HT neurons may have important implications for neural mechanisms underlying 5-HT modulation of sleep and wakefulness. ...
Serotonergic (5-HT) cells in the rat dorsal raphe nucleus (DRN) appear in topographically organized groups. Based on cellular morphology, expression of other neurotransmitters, afferent and efferent connections and functional properties, 5-HT neurons of the DRN have been grouped into six cell clusters. The subdivisions comprise the rostral, ventral, dorsal, lateral, caudal and interfascicular parts of the DRN. In addition to 5-HT cells, neurons containing γ-aminobutyric acid (GABA), glutamate, dopamine, nitric oxide and the neuropeptides corticotropin-releasing factor, substance P, galanin, cholecystokinin, neurotensin, somatostatin, vasoactive intestinal peptide, neuropeptide Y, thyrotropin-releasing hormone, growth hormone, leu-enkephalin, met-enkephalin and gastrin have been characterized in the DRN. Moreover, numerous brain areas have neurons that project to the DRN and express monoamines (norepinephrine, histamine), amino acids (GABA, glutamate), acetylcholine or neuropeptides (orexin, melanin-concentrating hormone, corticotropin-releasing factor and substance P) that directly or indirectly, through local circuits, regulate the activity of 5-HT cells. The 5-HT cells predominate along the midline of the rostral, dorsal and ventral subdivisions of the DRN and outnumber the non-5-HT cells occurring in the raphe nucleus. The GABAergic and glutamatergic neurons are clustered mainly in the lateral and dorsal subdivisions of the DRN, respectively. The 5-HT(1A) receptor is located on the soma and the dendrites of 5-HT neurons and at postsynaptic sites (outside the DRN). It is expressed, in addition, by non-5-HT cells of the DRN. The 5-HT(1B) receptor is located at presynaptic and postsynaptic sites (outside the boundaries of the DRN). It has been described also in the ventromedial DRN where it is expressed by non-5-HT cells. The 5-HT(2A) and 5-HT(2C) receptors are located within postsynaptic structures. At the level of the DRN the 5-HT(2A) and 5-HT(2C) receptor-containing cells are predominantly GABAergic interneurons and projection neurons. Within the boundaries of the DRN the 5-HT(3) receptor is expressed by, among others, glutamatergic interneurons. 5-HT(7) receptors in the DRN are not localized to serotonergic neurons but, at least in part, to GABAergic cells and terminals. The complex structure of the DRN may have important implications for neural mechanisms underlying 5-HT modulation of wakefulness and REM sleep.
Parkinson’s disease (PD) is characterized by motor impairments caused by degeneration of dopamine neurons in the substantia nigra pars compacta. In addition to these symptoms, PD patients often suffer from non-motor co-morbidities including sleep and psychiatric disturbances, which are thought to depend on concomitant alterations of serotonergic and noradrenergic transmission. A primary locus of serotonergic neurons is the dorsal raphe nucleus (DRN), providing brain-wide serotonergic input. Here, we identified electrophysiological and morphological parameters to classify serotonergic and dopaminergic neurons in the murine DRN under control conditions and in a PD model, following striatal injection of the catecholamine toxin, 6-hydroxydopamine (6-OHDA). Electrical and morphological properties of both neuronal populations were altered by 6-OHDA. In serotonergic neurons, most changes were reversed when 6-OHDA was injected in combination with desipramine, a noradrenaline reuptake inhibitor, protecting the noradrenergic terminals. Our results show that the depletion of both noradrenaline and dopamine in the 6-OHDA mouse model causes changes in the DRN neural circuitry.
Parkinson's disease (PD) is characterized by motor impairments caused by degeneration of dopamine neurons in the substantia nigra pars compacta. In addition to these symptoms, PD patients often suffer from non-motor co-morbidities including sleep and psychiatric disturbances, which are thought to depend on concomitant alterations of serotonergic and noradrenergic transmission. A primary locus of serotonergic neurons is the dorsal raphe nucleus (DRN), providing brain-wide serotonergic input. Here, we identified electrophysiological and morphological parameters to classify serotonergic and dopaminergic neurons in the murine DRN under control conditions and following striatal injection of the catecholamine toxin, 6-hydroxydopamine (6-OHDA). Electrical and morphological properties of both neuronal populations were altered by 6-OHDA. In serotonergic neurons, most changes were reversed when 6-OHDA was injected in combination with desipramine, a noradrenaline reuptake inhibitor, protecting the noradrenergic terminals. Our results show that the depletion of both noradrenaline and dopamine in the 6-OHDA mouse model causes changes in the DRN neural circuitry.
Activity of dorsal raphe neurons is controlled by noradrenaline afferents. In this brain region, noradrenaline activates Gaqcoupled a1-adrenergic receptors (a1-AR), causing action potential (AP) firing and serotonin release. In vitro, electrical stimulation elicits vesicular noradrenaline release and subsequent activation of a1-AR to produce an EPSC (a1-AR-EPSC). The duration of the a1-AR-EPSC (;27 s) is much longer than that of most other synaptic currents, but the factors that govern the spatiotemporal dynamics of a1-AR are poorly understood. Using an acute brain slice preparation from adult male and female mice and electrophysiological recordings from dorsal raphe neurons, we found that the time course of the a1-AREPSC was slow, but highly consistent within individual serotonin neurons. The amount of noradrenaline released influenced the amplitude of the a1-AR-EPSC without altering the time constant of decay suggesting that once released, extracellular noradrenaline was cleared efficiently. Reuptake of noradrenaline via noradrenaline transporters was a primary means of terminating the a1-AR-EPSC, with little evidence for extrasynaptic diffusion of noradrenaline unless transporter-dependent reuptake was impaired. Taken together, the results demonstrate that despite slow intrinsic signaling kinetics, noradrenaline-dependent synaptic transmission in the dorsal raphe is temporally and spatially controlled and noradrenaline transporters are critical regulators of serotonin neuron excitability. Given the functionally distinct types of neurons intermingled in the dorsal raphe nucleus and the unique roles of these neural circuits in physiological responses, transporters may preserve independence of each synapse to encode a long-lasting but discrete signal.
The development of antidepressant drugs, in the last 6 decades, has been associated with theories based on a deficiency of serotonin (5-HT) and/or noradrenaline (NA) systems. Although the pathophysiology of major depression (MD) is not fully understood, numerous investigations have suggested that treatments with various classes of antidepressant drugs may lead to an enhanced 5-HT and/or adapted NA neurotransmissions. In this review, particular morpho-physiological aspects of these systems are first considered. Second, principal features of central 5-HT/NA interactions are examined. In this regard, the effects of the acute and sustained antidepressant administrations on these systems are discussed. Finally, future directions including novel therapeutic strategies are proposed.
Serotonergic neurons of the median raphe nucleus (MnR) and hypothalamic melanin-concentrating hormone (MCH)-containing neurons, have been involved in the control of REM sleep and mood.
In the present study, we examined in rats and cats the anatomical relationship between MCH-containing fibers and MnR neurons, as well as the presence of MCHergic receptors in these neurons. In addition, by means of in vivo unit recording in urethane anesthetized rats, we determined the effects of MCH in MnR neuronal firing.
Our results showed that MCH-containing fibers were present in the central and paracentral regions of the MnR. MCHergic fibers were in close apposition to serotonergic and non-serotonergic neurons. By means of an indirect approach, we also analyzed the presence of MCHergic receptors within the MnR. Accordingly, we microinjected MCH conjugated with the fluorophore rhodamine (R-MCH) into the lateral ventricle. R-MCH was internalized into serotonergic and non-serotonergic MnR neurons; some of these neurons were GABAergic. Furthermore, we determined that intracerebroventricular administration of MCH induced a significant decrease in the firing rate of 53 % of MnR neurons, while the juxtacellular administration of MCH reduced the frequency of discharge in 67 % of these neurons. Finally, the juxtacellular administration of the MCH-receptor antagonist ATC-0175 produced an increase in the firing rate in 78 % of MnR neurons. Hence, MCH produces a strong regulation of MnR neuronal activity. We hypothesize that MCHergic modulation of the MnR neuronal activity may be involved in the promotion of REM sleep and in the pathophysiology of depressive disorders.
Sudden unexpected death in epilepsy (SUDEP) is a devastating epilepsy complication. Seizure-induced respiratory arrest (S-IRA) occurs in many witnessed SUDEP patients and animal models as an initiating event leading to death. Thus, understanding the mechanisms underlying S-IRA will advance the development of preventive strategies against SUDEP. Serotonin (5-HT) is an important modulator for many vital functions, including respiration and arousal, and a deficiency of 5-HT signaling is strongly implicated in S-IRA in animal models, including the DBA/1 mouse. However, the brain structures that contribute to S-IRA remain elusive. We hypothesized that the dorsal raphe (DR), which sends 5-HT projections to the forebrain, is implicated in S-IRA. The present study used optogenetics in the DBA/1 mouse model of SUDEP to selectively activate 5-HT neurons in the DR. Photostimulation of DR 5-HT neurons significantly and reversibly reduced the incidence of S-IRA evoked by acoustic stimulation. Activation of 5-HT neurons in the DR suppressed tonic seizures in most DBA/1 mice without altering the seizure latency and duration of wild running and clonic seizures evoked by acoustic stimulation. This suppressant effect of photostimulation on S-IRA is independent of seizure models, as optogenetic stimulation of DR also reduced S-IRA induced by pentylenetetrazole, a proconvulsant widely used to model human generalized seizures. The S-IRA-suppressing effect of photostimulation was increased by 5-hydroxytryptophan, a chemical precursor for 5-HT synthesis, and was reversed by ondansetron, a specific 5-HT3 receptor antagonist, indicating that reduction of S-IRA by photostimulation of the DR is specifically mediated by enhanced 5-HT neurotransmission. Our findings suggest that deficits in 5-HT neurotransmission in the DR are implicated in S-IRA in DBA/1 mice, and that targeted intervention in the DR is potentially useful for prevention of SUDEP.
Based on electrophysiological, neurochemical, and neuropharmacological approaches, it is currently accepted that serotonin (5-HT) functions to promote waking (W) and to inhibit (permissive role) REM sleep (REMS). Serotonergic neurons of the dorsal raphe nucleus (DRN) fire at a steady rate during W, decrease their firing during slow-wave sleep (SWS), and virtually cease activity during REMS. Serotonin released during W activates 5-HT1A somatodendritic receptors and 5-HT2A/2C receptors expressed by GABAergic interneurons, and induces a decrease of the firing rate of 5-HT cells characteristic of SWS. In addition to local inhibitory circuits, GABAergic neurons of the ventrolateral preoptic nucleus play a role in the deactivation of the 5-HT and all other arousal systems, which results in the occurrence of REMS. Studies on the effects on REMS of direct administration of selective 5-HT1A (8-OH-DPAT, flesinoxan), and 5-HT2A/2C (DOI) receptor agonists into the DRN tend to indicate that quite different mechanisms are involved in their effects. Direct infusion of 8-OH-DPAT or flesinoxan into the DRN significantly enhances REMS, and this effect is prevented by local infusion of the selective 5-HT1A receptor antagonist WAY 100635. In agreement with the reciprocal interaction hypothesis of REMS generation, inhibition of DRN serotonergic neurons following somatodendritic 5-HT 1A receptor stimulation suppressed 5-HT inhibition of mesopontine cholinergic neurons and increased REMS. Infusion of DOI into the DRN induces a significant reduction of REMS in the rat. Pretreatment with selective 5-HT 2A and 5-HT2C receptor antagonists prevents the DOIinduced suppression of REMS. Serotonin-containing neurons of the DRN do not express 5-HT2A or 5-HT2C receptors. The 5-HT2A and 5-HT2C receptor-containing neurons are predominantly GABAergic interneurons and projection neurons. Since DOI inhibits the firing of serotonergic neurons in the DRN and reduces the extracellular concentration of 5-HT, it can be proposed that the DOI activation of long-projection GABAergic neurons that express 5-HT2A/2C receptors would be responsible for the inhibition of cholinergic cells in the laterodorsal tegmental and pedunculopontine tegmental nuclei (LDT/PPT) and the suppression of REMS. Microinjection of 8-OH-DPAT or flesinoxan into the LDT/PPT induces an inhibitory response on target neurons and the suppression of REMS. Moreover, infusion of DOI into the LDT/PPT selectively decreases REMS. In this respect, activation of 5-HT2A/2C receptors expressed by GABAergic interneurons in the LDT/PPT would produce the local release of GABA and the reduction of the behavioral state.
The firing activity of serotonergic neurons in raphe nuclei is regulated by negative feedback exerted by extracellular serotonin (5-HT)o acting through somatodendritic 5-HT1A autoreceptors. The steady-state [5-HT]o, sensed by 5-HT1A autoreceptors, is determined by the balance between the rates of 5-HT release and reuptake. Although it is well established that reuptake of 5-HTo is mediated by 5-HT transporters (SERT), the release mechanism has remained unclear. It is also unclear how selective 5-HT reuptake inhibitor (SSRI) antidepressants increase the [5-HT]o in raphe nuclei and suppress serotonergic neuron activity, thereby potentially diminishing their own therapeutic effect. Using an electrophysiological approach in a slice preparation, we show that, in the dorsal raphe nucleus (DRN), continuous nonexocytotic 5-HT release is responsible for suppression of phenylephrine-facilitated serotonergic neuron firing under basal conditions as well as for autoinhibition induced by SSRI application. By using 5-HT1A autoreceptor-activated G protein-gated inwardly rectifying potassium channels of patched serotonergic neurons as 5-HTo sensors, we show substantial nonexocytotic 5-HT release under conditions of abolished firing activity, Ca(2+) influx, vesicular monoamine transporter 2-mediated vesicular accumulation of 5-HT, and SERT-mediated 5-HT transport. Our results reveal a cytosolic origin of 5-HTo in the DRN and suggest that 5-HTo may be supplied by simple diffusion across the plasma membrane, primarily from the dense network of neurites of serotonergic neurons surrounding the cell bodies. These findings indicate that the serotonergic system does not function as a sum of independently acting neurons but as a highly interdependent neuronal network, characterized by a shared neurotransmitter pool and the regulation of firing activity by an interneuronal, yet activity-independent, nonexocytotic mechanism.
© 2015 Mlinar et al.
Numerous studies link decreased serotonin metabolites with increased impulsive and aggressive traits. However, although pharmacological depletion of serotonin is associated with increased aggression, interventions aimed at directly decreasing serotonin neuron activity have supported the opposite association. Furthermore, it is not clear if altered serotonin activity during development may contribute to some of the observed associations. Here, we used two pharmacogenetic approaches in transgenic mice to selectively and reversibly reduce the firing of serotonin neurons in behaving animals. Conditional overexpression of the serotonin 1A receptor (Htr1a) in serotonin neurons showed that a chronic reduction in serotonin neuron firing was associated with heightened aggression. Overexpression of Htr1a in adulthood, but not during development, was sufficient to increase aggression. Rapid suppression of serotonin neuron firing by agonist treatment of mice expressing Htr1a exclusively in serotonin neurons also led to increased aggression. These data confirm a role of serotonin activity in setting thresholds for aggressive behavior and support a direct association between low levels of serotonin homeostasis and increased aggression.
The dorsal raphe nucleus contains one of the largest groups of serotonergic neurons in the mammalian brain and is the main site of origin of the serotonergic projection to the cerebral cortex. Early electrophysiological studies suggested that serotonergic neurons in this cell group formed a homogeneous cell class. More recent studies however have reported heterogeneity among the core anatomical and electrophysiological properties of these neurons, thus raising the possibility that serotonergic neurons of this cell group may form two or more distinct cell classes. In this Viewpoint, we review these findings and suggest ways to look at cellular heterogeneity among serotonergic neurons.
The raphe nucleus in the brain is the main source of serotonin (5-HT), an important brain chemical in regulating mood, cognition and behavior. This paper presents a spiking neuronal network model of the dorsal region of the raphe nucleus (DRN). We solve the perplexing problem of heterogeneous spiking neuronal behavior observed in the DRN by using an adaptive quadratic integrate-and-fire neuronal model and varying only its membrane potential reset after a spike, suggesting a potential role of certain recovery ionic currents. Specifically, the model can mimic the effects of slow afterhyperpolarization current and control the production of spikes per burst as found in experiments. Our model predicts specific input-output functions of the neurons which can be experimentally tested. Phase-plane analysis confirms their spiking dynamics. By coupling the 5-HT neurons with non-5-HT inhibitory neurons, we show that the DRN neuronal spiking activities recorded in behaving monkeys can generally be reproduced by adopting a feedforward inhibitory network architecture. Our model further predicts a low frequency network oscillation (about 8 Hz) among non-5-HT neurons around the rewarding epoch of a simulated experimental trial, which can be verified through direct recordings in behaving animals. Our computational model of the DRN accounts for the heterogeneous spiking patterns found in experiments, suggests plausible network architecture, and provides model predictions which can be directly tested in experiments. The model conveniently forms the basis for building extended network models to study complex interactions of the 5-HT system with other brain regions.
Higher-order executive tasks such as learning, working memory, and behavioral flexibility depend on the prefrontal cortex (PFC), the brain region most elaborated in primates. The prominent innervation by serotonin neurons and the dense expression of serotonergic receptors in the PFC suggest that serotonin is a major modulator of its function. The most abundant serotonin receptors in the PFC, 5-HT1A, 5-HT2A and 5-HT3A receptors, are selectively expressed in distinct populations of pyramidal neurons and inhibitory interneurons, and play a critical role in modulating cortical activity and neural oscillations (brain waves). Serotonergic signaling is altered in many psychiatric disorders such as schizophrenia and depression, where parallel changes in receptor expression and brain waves have been observed. Furthermore, many psychiatric drug treatments target serotonergic receptors in the PFC. Thus, understanding the role of serotonergic neurotransmission in PFC function is of major clinical importance. Here, we review recent findings concerning the powerful influences of serotonin on single neurons, neural networks, and cortical circuits in the PFC of the rat, where the effects of serotonin have been most thoroughly studied.
We have recorded, for the first time, in non-anesthetized, head-restrained mice, a total of 407 single units throughout the dorsal raphe nucleus (DR), which contains serotonin (5-hydroxytryptamine, 5-HT) neurons, during the complete wake-sleep cycle. The mouse DR was found to contain a large proportion (52.0%) of waking (W)-active neurons, together with many sleep-active (24.8%) and W/paradoxical sleep (PS)-active (18.4%) neurons and a few state-unrelated neurons (4.7%). The W-active, W/PS-active, and sleep-active neurons displayed a biphasic narrow or triphasic broad action potential. Of the 212 W-active neurons, 194 were judged serotonergic (5-HT W-active neurons) because of their triphasic long-duration action potential and low rate of spontaneous discharge, while the remaining 18 were judged non-serotonergic (non-5-HT W-active neurons) because of their biphasic narrow action potential and higher rate of spontaneous discharge. The 5-HT W-active neurons were subdivided into four groups, types I, II, III, and IV, on the basis of differences in firing pattern during wake-sleep states, their waking selectivity of discharge being in the order type I>type II>type III>type IV. During the transition from sleep to waking, the vast majority of waking-specific or waking-selective type I and II neurons discharged after onset of waking, as seen with non-5-HT W-specific neurons. Triphasic DR W/PS-active neurons were characterized by a low rate of spontaneous discharge and a similar distribution to that of tyrosine hydroxylase-immunoreactive, dopaminergic neurons. Triphasic DR slow-wave sleep (SWS)-active and SWS/PS neurons were also characterized by slow firing. At the transition from sleep to waking, sleep-selective neurons with no discharge activity during waking ceased firing before onset of waking, while, at the transition from waking to sleep, they fired after onset of sleep. The present study shows a marked heterogeneity and functional topographic organization of both serotonergic and non-serotonergic mouse DR neurons and suggests that they play different roles in behavioral state control and the sleep/waking switch.
The serotonin (5HT) system of the brain is involved in many CNS functions including sensory perception, stress responses and psychological disorders such as anxiety and depression. Of the nine 5HT nuclei located in the mammalian brain, the dorsal raphe nucleus (DRN) has the most extensive forebrain connectivity and is implicated in the manifestation of stress-related psychological disturbances. Initial investigations of DRN efferent connections failed to acknowledge the rostrocaudal and mediolateral organization of the nucleus or its neurochemical heterogeneity. More recent studies have focused on the non-5HT contingent of DRN cells and have revealed an intrinsic intranuclear organization of the DRN which has specific implications for sensory signal processing and stress responses. Of particular interest are spatially segregated subsets of nitric oxide producing neurons that are activated by stressors and that have unique efferent projection fields. In this regard, both the midline and lateral wing subregions of the DRN have emerged as prominent loci for future investigation of nitric oxide function and modulation of sensory- and stressor-related signals in the DRN and coinciding terminal fields.
Based on electrophysiological, neurochemical, genetic and neuropharmacological approaches it is currently accepted that serotonin (5-HT) functions to promote waking (W) and to inhibit rapid-eye movement sleep (REMS). The serotonin-containing neurons of the dorsal raphe nucleus (DRN) provide part of the serotonergic innervation of the telencephalon, diencephalon, mesencephalon and rhombencephalon of laboratory animals and man. The DRN has been subdivided into several clusters on the basis of differences in cellular morphology, expression of other neurotransmitters and afferent and efferent connections. These differences among subpopulations of 5-HT neurons may have important implications for neural mechanisms underlying 5-HT modulation of sleep and waking. The DRN contains 5-HT and non-5-HT neurons. The latter express a variety of substances including dopamine, γ-aminobutyric acid (GABA) and glutamate. In addition, nitric oxide and a number of neuropeptides have been characterized in the DRN. Available evidence tends to indicate that non-5-HT cells contribute to the regulation of the activity of 5-HT neurons during the sleep-wake cycle through local circuits and/or their mediation of the effects of afferent inputs. Mutant mice that do not express 5-HT(1A) or 5-HT(1B) receptor exhibit greater amounts of REMS than their wild-type couterparts. 5-HT(2A) and 5-HT(2C) receptor knockout mice show a significant increase of W and a reduction of slow wave sleep that is related, at least in part, to the increased release of norepinephrine and dopamine. A normal circadian sleep pattern is observed in 5-HT(7) receptor knockout mice; however, the mutants spend less time in REMS. Local microinjection of 5-HT(1B), 5-HT(2A/2C), 5-HT(3) and 5-HT(7) receptor agonists into the DRN selectively suppresses REMS in the rat. In contrast, microinjection of 5-HT(1A) receptor agonists promotes REMS. Similarly, local administration of the melanin-concentrating hormone or the GABA(A) receptor agonist muscimol produces an increase of REMS in the rat. Presently, there are no data on the effect of local infusion into the DRN of noradrenergic, dopaminergic, histaminergic, orexinergic and cholinergic agonists on sleep variables in laboratory animals.
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.
There is considerable interest in the regulation of the extracellular compartment of the transmitter serotonin (5-hydroxytryptamine, 5-HT) in the midbrain raphe nuclei because it can control the activity of ascending serotonergic systems and the release of 5-HT in terminal areas of the forebrain. Several intrinsic and extrinsic factors of 5-HT neurons that regulate 5-HT release in the dorsal (DR) and median (MnR) raphe nucleus are reviewed in this article. Despite its high concentration in the extracellular space of the raphe nuclei, the origin of this pool of the transmitter remains to be determined. Regardless of its origin, is has been shown that the release of 5-HT in the rostral raphe nuclei is partly dependent on impulse flow and Ca(2+) ions. The release in the DR and MnR is critically dependent on the activation of 5-HT autoreceptors in these nuclei. Yet, it appears that 5-HT autoreceptors do not tonically inhibit 5-HT release in the raphe nuclei but rather play a role as sensors that respond to an excess of the endogenous transmitter. Both DR and MnR are equally responsive to the reduction of 5-HT release elicited by the local perfusion of 5-HT(1A) receptor agonists. In contrast, the effects of selective 5-HT(1B) receptor agonists are more pronounced in the MnR than in the DR. However, the cellular localization of 5-HT(1B) receptors in the raphe nuclei remains to be established. Furthermore, endogenous noradrenaline and GABA tonically regulate the extracellular concentration of 5-HT although the degree of tonicity appears to depend upon the sleep/wake cycle and the behavioral state of the animal. Glutamate exerts a phasic facilitatory control over the release of 5-HT in the raphe nuclei through ionotropic glutamate receptors. Overall, it appears that the extracellular concentration of 5-HT in the DR and the MnR is tightly controlled by intrinsic serotonergic mechanisms as well as afferent connections.
The median raphe nucleus is involved in controlling and maintaining hippocampal activity through its projection to inhibitory neurons in medial septum and hippocampus. It has been shown that anterogradely axonal-traced fibers originating in the median raphe nucleus project onto calbindin-containing neurons in hippocampus and parvalbumin-containing neurons in medial septum. Parallel immunohistochemistry studies showing serotonin fibers contacting calbindin- and parvalbumin-positive neurons have led to the assumption that raphe fibers projecting on these types of neurons are mainly serotonergic. However, in both dorsal and median raphe nucleus there is a large amount of non-serotonergic neurons which also are projecting neurons, indicating that a part of the raphe fibers projecting to hippocampus and septum may be non-serotonergic. Our aim was to determine whether there is a non-serotonergic projection from the raphe nucleus onto calbindin- and parvalbumin-containing neurons in hippocampus and septum. Biotin dextran amine was used as the anterograde neuronal tracer and injected into either dorsal or median raphe nucleus. By use of triple immunofluorescence-labeling we analyzed the serotonergic content of the biotin dextran amine-labeled fibers contacting parvalbumin- and calbindin-positive neurons. Surprisingly, we found a significant non-serotonergic projection from both dorsal and median raphe nuclei onto calbindin- and parvalbumin-containing interneurons in septum and hippocampus, with a preference in hippocampus for projecting onto calbindin-positive neurons. These results indicate that the raphe nuclei may exert their control on hippocampal and septal activity not only through a serotonergic projection, but also through a significant non-serotonergic pathway.
Three types of neurons, distinguished on the basis of their spontaneous firing rates and patterns, extracellularly recorded waveforms and responses to neostriatal stimulation, were observed in the dorsal raphe nucleus in urethane-anesthetized rats. Type 1 neurons (presumed to be serotonergic) fired spontaneously from 0.1 to 3 spikes/s in a regular pattern, with initial positive-going bi- or triphasic action potentials. Type 1 cells exhibited long-latency antidromic responses to neostriatal stimulation (mean +/- S.E.M. 24.9 +/- 0.3 ms) that sometimes occurred at discrete multiple latencies, and supernormal periods persisting up to 100 ms following spontaneous spikes. Type 2 cells fired spontaneously in an irregular, somewhat bursty pattern from 0 to 2 spikes/s with initial negative-going biphasic spikes, and were antidromically activated from neostriatal stimulation at shorter latencies than Type 1 cells (21.8 +/- 0.9 ms). Type 3 cells were characterized by initial positive-going biphasic waveforms and displayed a higher discharge rate (5-30 spikes/s) than Type 1 or Type 2 cells. Type 3 cells could not be antidromically activated from neostriatal stimulation. The relatively long conduction time to neostriatum of the Type 1 presumed serotonergic neuron is discussed with respect to previous interpretations of the synaptic action of serotonin in the neostriatum. In conjunction with these antidromic activation studies, the neurophysiological consequences of serotonergic terminal autoreceptor activation were examined by measuring changes in the excitability of serotonergic terminal fields in the neostriatum following administration of the serotonin autoreceptor agonist, 5-methoxy-N,N-dimethyltryptamine (5-MeODMT). The excitability of serotonergic terminal fields was decreased by intravenous injection of 40 micrograms/kg 5-MeODMT, and by infusion of 10-50 microM 5-MeODMT directly into the neostriatum. These results are interpreted from the perspective of mechanisms underlying autoreceptor-mediated regulation of serotonin release.
The purpose of this study was to ascertain the identity of presumed noradrenergic or serotonergic neurons recorded by single cell techniques in the mammalian brain. A double labeling method was developed in which intracellular injections of a red fluorescing dye (ethidium bromide) could be co-localized with the formaldehyde-induced green fluorescence of norepinephrine or yellow fluorescence of serotonin. By this method, neurons of the rat locus coeruleus that display a characteristic activation-inhibition response to noxious stimuli were confirmed to be noradrenergic; the slow, rhythmically firing neurons of the dorsal raphe nucleus were confirmed to be serotonergic.
Pyramidal neurons in layer VI of the primary motor and somatosensory cortices were examined by a combined method of intracellular recording, biocytin injection, and immunocytochemistry using in vitro slice preparations of rat brain immunofluorescence staining revealed that biocytin-injected pyramidal cells in layer VI were separated into glutaminase (PAG)-immunopositive and PAG-immunonegative cells. Although the two groups of pyramidal cells showed no statistically significant differences in passive membrane properties and spike characteristics, a clear difference was found in spike afterpotentials. Ten of 12 PAG-positive pyramidal cells showed no or a small fast afterhyperpolarization (fAHP), whereas 10 of 11 PAG-negative pyramidal cells displayed a large fAHP. Depolarizing afterpotentials were observed only in PAG-positive pyramidal cells than in PAG-negative cells. In contrast, the arborization of basal dendrites was more developed in PAG-positive pyramidal cells than in PAG-negative cells. The main axons of all the pyramidal cells entered the subcortical axons of all the pyramidal cells entered the subcortical white matter. The local axon collaterals of PAG-positive pyramidal cells were widely spread in the horizontal direction, whereas those of PAG-negative cells were distributed vertically along the dendritic tree. Since PAG is considered to be a marker of glutamatergic neurons in the cerebral cortex, the present results indicate that layer VI pyramidal cells are separated into glutamatergic and nonglutamatergic neurons that have different electrical properties and input-output organizations. Thus, cortical outputs from layer VI are suggested to use at least two distinct systems.
Previous electrophysiological studies have shown that spontaneously active mesencephalic 5-hydroxytryptaminergic neurons of anaesthetized or freely moving animals fire solitary spikes in a slow, regular pattern. In the present study, using extracellular single unit recordings from dorsal and median raphe neurons of the anaesthetized rat, an additional electrophysiological property of a sub-population of presumed 5-hydroxytryptaminergic neurons was observed. These neurons, during their otherwise regular firing pattern, repeatedly fired two (or occasionally three or even four) spikes where only one was expected. Spikes in this burst-like repetitive firing mode (spikes in doublets or triplets) occurred in a short time interval (range: 2.4-11.5 ms), and with a diminishing spike amplitude. Cross-correlation analysis of spikes in doublets revealed a very high interdependency between them. The proportion of spikes in doublets to solitary spikes showed great variation between different neurons, ranging from 5 to 95% of the total spikes displayed. However, for each neuron the proportion of spikes in doublets to solitary spikes, and the time interval between the spikes in doublets, remained constant during control recordings. All these features are characteristic of single neurons firing in a repetitive firing pattern rather than simultaneous recordings of two separate 5-hydroxytryptaminergic neurons. Repetitive firing neurons were recorded with a similar frequency in both chloral hydrate and Saffan anaesthetized rats, and were detected using both glass and metal electrodes. Furthermore, neurons with a repetitive firing pattern were inhibited by intravenous administration of a selective 5-hydroxytryptamine1A receptor agonist and a 5-hydroxytryptamine reuptake inhibitor, thus displaying responses typical of 5-hydroxytryptaminergic neurons. Repetitive firing neurons occurred in both the dorsal and median raphe nuclei, although they were much more frequent in the dorsal raphe nucleus (91 of 332 neurons). The occurrence of repetitive firing neurons in the midbrain raphe nuclei is a newly described phenomenon which may indicate unique properties of a sub-population of 5-hydroxytryptaminergic neurons. In functional terms, it could modify both axonal and dendritic 5-hydroxytryptamine release, and provide an additional option for neuronal information signalling.
This study examined the electrophysiological and morphological characteristics of layers V-VI pyramidal prefrontal cortex (PFC) neurons. In vitro intracellular recordings coupled with biocytin injections that preserved some of the PFC efferents to the nucleus accumbens (NAc) were made in brain slices. Four principal pyramidal cell types were identified and classified as regular spiking (RS) (19%), intrinsic bursting (IB) (64%), repetitive oscillatory bursting (ROB) (13%), and intermediate (IM) (4%) types. All PFC cells exhibited either subthreshold oscillation in membrane voltage or pacemaker-like rhythmic firing. IB neurons were demonstrated electrophysiologically and cytochemically to be PFC-->NAc neurons. In all IB and some RS neurons, a tetrodotoxin-sensitive, slowly inactivating Na+ current and a transient Ni(2+)-sensitive, low-threshold Ca2+ current mediated subthreshold inward rectification. During sustained membrane depolarization, the Na+ current was opposed by a 4-aminopyridine-sensitive, outwardly rectifying, slowly inactivating K+ current. Together, these three currents controlled the firing threshold of the PFC neurons. All IB and ROB cells also had postspike Ca(2+)-mediated depolarizing afterpotentials, postburst Ca(2+)-dependent after hyperpolarizations, and low- and high-threshold Ca2+ spikes. In addition, ROB cells had a hyperpolarizing "sag" mediated by the cationic conductance, Ih. IB and ROB neurons had extensive dendritic trees and radially ascending or tangentially projecting axon collaterals. RS and IM cells had comparatively simpler morphological profiles. These electrophysiological and morphological properties of the four principal pyramidal PFC cell types have provided valuable details for understanding further how PFC processes input and transmit outputs to regions such as the NAc.
Pyramidal cells of layer V in rat prefrontal cortex display a prominent fast afterdepolarization (fADP) following an action potential. This ADP is blocked by replacing extracellular calcium with magnesium, by the application of the calcium-channel blocker cadmium, and by buffering intracellular calcium at near physiological levels. Thus this fast ADP appears mediated by a calcium-activated current. A prominent ADP is also observed following a calcium spike recorded in the presence of tetrodotoxin. The current underlying this ADP was recorded using a hybrid current-voltage protocol. A strong ADP could be observed in the presence of potassium channel blockers as well as at ECl. Furthermore, the current underlying the ADP increased with hyperpolarization in the subthreshold range and displayed an extrapolated reversal potential near +30 mV. Reducing the ratio of extracellular to intracellular sodium inhibited the current underlying the ADP and caused a hyperpolarizing shift in its reversal potential. We conclude that these cells express a calcium-activated cation nonselective current whose activation contributes to the generation of the fADP. This current could play an important role in determining the firing properties of pyramidal cells in cortex.
Morphology and electrical membrane properties of neurons in the superficial part of the magnocellular layer of the rat medullary dorsal horn (MDH: caudal subnucleus of the spinal trigeminal nucleus) were examined by using horizontal slice preparations. Intracellular recording and biocytin-injection combined with histochemical and immunohistochemical staining were done. Twenty-four neurons were examined successfully and classified into projection neurons (PNs) and intrinsic neurons (INs). The PNs were further divided into type I PNs (I-PNs) and type II PNs (II-PNs). The I-PNs sent axons to the medullary reticular formation; the II-PNs sent axons to the interpolar subnucleus of the spinal trigeminal nucleus but had no axons extending to the medullary reticular formation. The INs that sent no axons to the brain regions outside the MDH were also divided into small INs with spiny dendrites (INSSs) and large INs with aspiny dendrites (INLAs). The dendritic fields of the PNs extended to laminae I and II of the MDH and occasionally further to the spinal tract of the trigeminal nerve, whereas those of the INs were confined within the magnocellular layer of the MDH. The axonal branches of each IN formed a dense axonal mesh around the cell body of the parent neuron. Although the main bodies of the axonal fields of the INs were located in the magnocellular layer, some axonal branches extended to laminae I and II of the MDH. Immunoreactivity for NK1 receptor (substance P receptor) was found in approximately half of the PNs but not in the INs. Although no strong correlation was found between morphology and electrical membrane properties, there were some differences in electrical properties among the morphologically classified neuron groups, e.g., hyperpolarizing sag was observed in some PNs but not in the Ins; inward rectification was observed in some of the INSSs and INLAs but not in the PNs; the slow ramp depolarization and the slow afterdepolarization were observed in all INSSs examined but not in the PNs or INLAs. J. Comp. Neurol. 428:641–655, 2000. © 2000 Wiley-Liss, Inc.
Neurons of the mesencephalic trigeminal nucleus (MTN) are considered to be homologous to mechanosensitive neurons in the sensory ganglia. The sites of origin of serotonin (5HT)-immunoreactive axons on neuronal cell bodies in the MTN were studied in the rat by combining immunofluorescence histochemical techniques with retrograde tracing of Fluoro-Gold (FG) and anterograde tracing of biotin-conjugated dextran amine (BDA). The tracing studies, which were combined with multiple-labeling immunohistochemistry and confocal microscopy, indicated that 5HT-immunoreactive axon terminals on the cell bodies of MTN neurons originated from the medullary raphe nuclei, such as the nucleus raphes magmus (RMg), alpha part of the nucleus reticularis gigantocellularis (GiA) and nucleus raphes obscurus (ROb), as well as from the mesopontine raphe nuclei, such as the nucleus raphes dorsalis (DR), nucleus raphes pontis (PnR) and nucleus raphes medianus (MnR); mainly from the RMg, GiA and DR, and additionally from the ROb, PnR and MnR. The cell bodies in close apposition to 5HT-immunoreactive axon terminals were found through the whole rostrocaudal extent of the MTN. Electron microscopically a number of axon terminals that were labeled with BDA injected into the raphe nuclei were confirmed to be in asymmetric synaptic contact with the cell bodies of MTN neurons. It was also indicated that substance P existed in some of the 5HT-containing axosomatic terminals arising from the ROb, RMg and GiA. The present results indicated that proprioceptive sensory signals from the muscle spindles or periodontal ligament might be modulated at the level of the primary afferent cell bodies in the MTN by 5HT-containing axons from the mesopontine and medullary raphe nuclei including the GiA.
Ascending projections from the dorsal raphe nucleus (DR) were examined in the rat by using the anterograde anatomical tracer, Phaseolus vulgaris leucoagglutinin (PHA-L). The majority of labeled fibers from the DR ascended through the forebrain within the medial forebrain bundle. DR fibers were found to terminate heavily in several subcortical as well as cortical sites.
The following subcortical nuclei receive dense projections from the DR: ventral regions of the midbrain central gray including the ‘supraoculomotor central gray’ region, the ventral tegmental area, the substantia nigra-pars compacta, midline and intralaminar nuclei of the thalamus including the posterior paraventricular, the parafascicular, reuniens, rhomboid, intermediodorsal/mediodorsal, and central medial thalamic nuclei, the central, lateral and basolateral nuclei of the amygdala, posteromedial regions of the striatum, the bed nucleus of the stria terminalis, the lateral septal nucleus, the lateral preoptic area, the substantia innominata, the magnocellular preoptic nucleus, the endopiriform nucleus, and the ventral pallidum. The following subcortical nuclei receive moderately dense projections from the DR: the median raphe nucleus, the midbrain reticular formation, the cuneiform/pedunculopontine tegmental area, the retrorubral nucleus, the supramammillary nucleus, the lateral hypothalamus, the paracentral and central lateral intralaminar nuclei of the thalamus, the globus pallidus, the medial preoptic area, the vertical and horizontal limbs of the diagonal band nuclei, the claustrum, the nucleus accumbens, and the olfactory tubercle.
The piriform, insular and frontal cortices receive dense projections from the DR; the occipital, entorhinal, perirhinal, frontal orbital, anterior cingulate, and infralimbic cortices, as well as the hippocampal formation, receive moderately dense projections from the DR.
Some notable differences were observed in projections from the caudal DR and the rostral DR. For example, the hippocampal formation receives moderately dense projections from the caudal DR and essentially none from the rostral DR. On the other hand, virtually all neocortical regions receive significantly denser projections from the rostral than from the caudal DR.
The present results demonstrate that dorsal raphe fibers project significantly throughout widespread regions of the midbrain and forebrain.
Conventional intracellular and single-electrode voltage-clamp recordings were obtained from rat brain slices containing dorsolateral septal nucleus (DLSN) neurons in vitro. We observed a slow afterdepolarizing potential (slow-ADP) that lasted up to several seconds (half-decay time was in the range of 0.7.-1.4 s) in almost 15% of DLSN neurons; these same neurons could exhibit burst firing activity. The amplitude of this slow-ADP was not affected by hyperpolarization of the membrane potential. The slow-ADP was associated with an increased membrane conductance. Hybrid voltage clamping of the slow-ADP revealed a transient slow inward current (slow-ADC). The current-voltage relationship of the slow-ADC was linear between -40 and -100 mV and generated an extrapolated reversal potential of -30 mV. We investigated the ionic mechanism of the slow-ADP in the rat DLSN. Slow-ADPs were not blocked by 1 μM tetrodotoxin (TTX) but were markedly depressed by 200 μM Cd2+, Ca2+-free, low-NA+ solutions, and the intracellular injection of ethylene glycol-bis(B-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). Neither diltiazam (10 μM), an L-type Ca2+ channel blocker nor omega-conatoxin (0.2-2.5 μM), an N-type Ca2+ channel blocker affected the slow-ADP. Similarly, the slow-ADP was not affected in a low-Cl- solution. On the other hand, the slow-ADP was enhanced in a K+-free solution. In addition, the slow-ADP was not affected by 1 mM kynurenic acid, a broad-spectrum excitatory amino acid antagonist. We conclude that the slow-ADP in the rat DLSN is mediated by a novel Ca2+-dependent, Na+-dependent, and nonsynaptic inward current that may be similar to the Ca2+-activated nonspecific cation channel currents (i.e., CAN-currents) described in various tissues. This current appears to underlie some forms of spontaneous bursting activity recorded from rat DLSN neurons. It may also be responsible for some types of bursting activity recorded in other CNS neurons.
The class of rapidly firing neurons in the dorsal raphe of the rat was examined using extracellular recording and intracellular injection of horseradish peroxidase. Rapidly firing neurons (termed F-cells in this report) continue to fire at high spontaneous rates during intracellular recording. This and their brief (ca. 1 ms) and symmetrical action potentials distinguish them from the slowly firing, presumably serotonergic neurons in dorsal raphe. Intracellular labeling with horseradish peroxidase reveals that F-cells have small (10-15 microns) spherical, multipolar or piriform somata. Somatic spines are sparse or entirely absent. The general form of the dendritic tree is radiant and poorly branching. However, the dendrites of F-cells take two forms, with both forms being present on the same neuron so that besides having a complement of poorly branching dendrites, each F-cell has at least one dendrite with a slightly tufted branching pattern: a short primary dendrite gives rise to 3 or 4 secondary branches. The axons of F-cells project from the nucleus. They align themselves along the trajectories of known dorsal raphe efferent pathways, coursing laterally and ventrorostrally beyond the bounds of the nucleus. Morphometric measurements of retrogradely labeled dorsal raphe projection neurons provide additional evidence that small projection neurons exist.
1. The electrophysiological and pharmacological properties of slow afterpotentials in large layer V neurons from cat sensorimotor cortex were studied in an in vitro slice preparation using intracellular recording and single-microelectrode voltage clamp. These properties were used to assess the role of afterpotential mechanisms in prolonged excitability changes. 2. The mean duration of a slow afterhyperpolarization (sAHP) was 13.5 s following 100 spikes evoked at 100 Hz. Its time course was best described by two exponential components, which decayed with time constants of several hundred milliseconds (the early sAHP) and several seconds (the late sAHP). The amplitude of both the early and late components were sensitive to membrane potential and raised extracellular K+ concentration [( K+]o). 3. The early sAHP was reduced when divalent cations were substituted for Ca2+, whereas the late sAHP was unaffected. We conclude that a Ca2+-mediated K+ conductance is responsible for much of the early sAHP. In the presence of tetrodotoxin (TTX), 1-s voltage-clamp steps were used to evoke slow AHPs or outward ionic currents. These AHPs and currents were abolished in Ca2+-free perfusate, but they had a maximum duration of only a few seconds. Thus the slowest outward currents we could observe during voltage clamp in TTX were responsible only for the early sAHP. 4. The possible role of an electrogenic Na+-K+ pump in the late sAHP was examined by applying ouabain to the slice. Ouabain did not reduce selectively the late sAHP, and its effect was best explained by a decrease in intracellular K+ concentration and an increase in [K+]o. 5. Muscarinic and beta-adrenergic agonists reduced or abolished the entire (early and late) sAHP. Neither type of agonist affected the Ca2+-dependent, apamin-sensitive medium-duration afterhyperpolarization (35). We conclude that both the Ca2+-mediated K+ conductance underlying the early sAHP and the Ca2+-independent mechanisms underlying the late sAHP are sensitive to at least two classes of transmitter agonists. 6. We focused on the muscarinic effects. When concentrations greater than 5 microM were employed, the entire (early and late) sAHP was replaced by a slow afterdepolarization (sADP). Muscarine reduced the sAHP directly by reducing the underlying outward ionic currents and indirectly by causing the sADP. The sADP was Ca2+-mediated, since it was abolished by Ca2+-free perfusate but not by TTX. 7. The ionic currents underlying the sAHP and the sADP influenced excitability for seconds following evoked repetitive firing.(ABSTRACT TRUNCATED AT 400 WORDS)
The nucleus raphe dorsalis of the rat was investigated by means of the Golgi rapid impregnation technique: a) Type 1 neurons: polygonal neurons with somatic spines. The axons which course towards the ventral tegmental area, there by giving off a few collaterals. b( Type 2 neurons: fusiform neurons the axons of which course in a latero-dorsal direction, emitting a few collaterals, too. c) Type 3 neurons: small pyriform neurons. The axons of these cells do not show any favoured route within the nucleus. The type 1 and type 2 neurons are considered to be efferent neurons having different modes of projection and termination. The type 1 neurons are supposed to represent the 5-HT ergic raphe dorsalis neurons. The type 3 neurons, are in probability, raphe interneurons. The findings presented here are indicative of the nucleus raphe dorsalis of the rat to have a more intricate cytoarchitecture as has been thought previously.
Intracellular recordings in vivo from serotonergic dorsal raphe neurons of the rat brain reveal that these cells undergo a pronounced postspike hyperpolarization followed by a gradual interspike depolarization leading to the succeeding spike. Such repetitive cycles of interspike hyperpolarization and depolarization, which can be termed "pacemaker potentials', can account for the automaticity of these cells. When serotonergic neuronal firing is inhibited by LSD, such pacemaker potentials no longer occur and the cells remain in an hyperpolarized state.
Three similar neuron types (polygonal, fusiform and pyriform neurons) were found to exist in populations of the nucleus raphe dorsalis and of nucleus raphe medianus, resp. The neuron types of both subnuclei were compared morphometrically using sections prepared by means of the Einarson--technique. The comparison between morphometrical values (size and breadth of the somata and nuclei, resp.) of the neuron types didn't show significant differences.
Stimulation of ventral medial tegmentum elicits an antidromic action potential and an inhibitory postsynaptic potential in dorsal raphe projection neurons. The inhibitory postsynaptic potential is not wholly due to activation of recurrent inhibitory circuits as it is not monosynaptic and its onset precedes the antidromic action potential. Light microscopic examination of horseradish peroxidase-filled projection neurons reveals a neuron type with radiating, poorly branched dendrites and terminal dendritic thickets. The axon of the exemplary neuron presented is seen to leave the nucleus but gives off a single collateral while still within the parent cell's dendritic domain.
The serotoninergic nerve cell body population of nucleus raphe dorsalis (RD) was identified by radioautography following cerebroventricular instillation of tritiated serotonin ([3H]5-HT) in adult rats pretreated with a monoamine oxidase inhibitor. Series of histological sections taken throughout the midbrain and upper pons exhibited a similar distribution and number of labeled nerve cell bodies in RD after prolonged administration of either 10-5 or 10-4M [3H]5-HT or 10-4M [3H]5-HT and 10-3M nonradioactive noradrenaline. This allowed systematic mapping and quantification of serotoninergic nerve cell bodies at various levels of the RD. Their extrapolated total number averaged 11,500. Twice as many unreactive (nonserotoninergic) neurons were present within the same region. In electron microscope radioautographs, the labeled cells were usually larger (17.9 micrometer mean diameter) than their unlabeled congeners (13.1 micrometer), but stereological sampling of their perikarial organelle content failed to reveal any difference in cytoplasmic composition. Few [3H]5-HT-labeled axonal varicosities were observed in RD and none were found in close apposition or in synaptic junction with labeled nerve cell bodies, dendrites, or unreactive perikarya. A detailed statistical analysis of silver grain distribution in both labeled and "unlabeled" nerve cell bodies, indicated that in the former, but not in the latter, dense bodies had a relatively high affinity for [3H]5-HT. Mitochondria and the cytoplasmic membrane were the only other organelles to show higher labeling indices in labeled than in unlabeled cells. Other sites of [3H]5-HT localization could be ascribed to artefactitious cross-linkage of the tracer by the fixative, since they had the same relative affinity in the two cell populations. These results provide new insights into the morphology and cytofunctional properties of the 5-HT neurons of rat RD.
Using Rapid Golgi and Nissl techniques, three major cell types: fusiform, multipolar and ovoid-shaped were identified in the nucleus raphe dorsalis of male rats at 30, 90, and 220 days of age. We have described the orientation and dendritic architecture of raphe cells as to type and the relationships of these cells to blood vessels and surrounding structures. For each cell type, the origin of the axon is characteristic. One hundred neurons per age group were measured at their maximal linear extent and the number of spines on the somal surface was counted. Dendritic number, linear extent, diameter and the number of spines along a 50 micron segment near the mid-point of dendritic length in an equal number of primary and secondary dendrites were quantified in each age group. The most striking age-related changes in the multipolar and ovoid-shaped cells were dendritic number, diameter and spine number as well as the number of perisomatic spines. The fusiform cells showed the least age-related changes. In general, the nucleus raphe dorsalis is organized as a reticular nucleus with neurons having few, straight and poorly ramified dendrites.
Retrograde axonal transport of the cholera toxin B subunit (CTb) was combined with 5-HT immunohistochemistry to determine the origin of the serotonergic innervation of the piriform cortex (PC) in the rat. After iontophoretic CTb injections in the PC, a substantial number of retrogradely labeled cells were found in the middle and medio-ventral part of the dorsal raphe nucleus (RD). A few retrogradely labeled cells were also observed in the median raphe nucleus (MnR) and the B9 serotonergic cell groups. Following CTb and 5-HT immunohistochemistry on the same sections, double-labeled cells were observed in the RD, MnR and B9 groups. In the RD, 30% of CTb stained cells were immunoreactive to 5-HT. After colchicine or nialamide (a monoamine oxidase inhibitor) pretreatment the percentage of these double-labeled cells reached 70%. These results indicate that both 5-HT and non-5-HT neurons in the RD innervate the PC and that the percentage of double-labeled cells is influenced by drug pretreatment. To determine the terminal fields of the RD efferent fibers in the PC, injections of the anterograde tracer PHA-L were also performed. Analysis of the fiber distribution in the PC further revealed some medio-lateral and antero-posterior differences.
Serotonergic neurons are thought to play a role in depression and obsessive compulsive disorder. However, their functional transmitter repertoire is incompletely known. To investigate this repertoire, intracellular recordings were obtained from 132 cytochemically identified rat mesopontine serotonergic neurons that had re-established synapses in microcultures. Approximately 60% of the neurons evoked excitatory glutamatergic potentials in themselves or in target neurons. Glutamatergic transmission was frequently observed in microcultures containing a solitary serotonergic neuron. Evidence for co-release of serotonin and glutamate from single raphe neurons was also obtained. However, evidence for gamma-aminobutyric acid release by serotonergic neurons was observed in only two cases. These findings indicate that many cultured serotonergic neurons form glutamatergic synapses and may explain several observations in slices and in vivo.
Early studies that used older tracing techniques reported exceedingly few projections from the dorsal raphe nucleus (DR) to the brainstem. The present report examined DR projections to the brainstem by use of the anterograde anatomical tracer Phaseolus vulgaris leucoagglutinin (PHA-L). DR fibers were found to terminate relatively substantially in several structures of the midbrain, pons, and medulla. The following pontine and midbrain nuclei receive moderate to dense projections from the DR: pontomesencephalic central gray, mesencephalic reticular formation, pedunculopontine tegmental nucleus, medial and lateral parabrachial nuclei, nucleus pontis oralis, nucleus pontis caudalis, locus coeruleus, laterodorsal tegmental nucleus, and raphe nuclei, including the central linear nucleus, median raphe nucleus, and raphe pontis. The following nuclei of the medulla receive moderately dense projections from the DR: nucleus gigantocellularis, nucleus raphe magnus, nucleus raphe obscurus, facial nucleus, nucleus gigantocellularis-pars alpha, and the rostral ventrolateral medullary area. DR fibers project lightly to nucleus cuneiformis, nucleus prepositus hypoglossi, nucleus paragigantocellularis, nucleus reticularis ventralis, and hypoglossal nucleus.
Some differences were observed in projections from rostral and caudal parts of the DR. The major difference was that fibers from the rostral DR distribute more widely and heavily than do those from the caudal DR to structures of the medulla, including raphe magnus and obscurus, nucleus gigantocellularis-pars alpha, nucleus paragigantocellularis, facial nucleus, and the rostral ventrolateral medullary area. A role for the dorsal raphe nucleus in several brainstem controlled functions is discussed, including REM sleep and its events, nociception, and sensory motor control. © Wiley-Liss, Inc.
Here we report the existence of burst-firing neurones in the rat dorsal raphe as detected in vivo using intracellular electrophysiological techniques. These neurones discharged single action potentials and doublets or triplets of action potentials in a slow and regular pattern. The apparent input resistance, action potential width and firing threshold of these burst-firing raphe neurones were indistinguishable from classical 5-HT neurones. Spike doublets were evoked by depolarising DC currents, but only in burst-firing neurones. These findings provide further evidence to support the hypothesis that 5-HT neurones (or a sub-set of them) are capable of burst-firing activity.
Recently, we described neurones in the rat dorsal raphe nucleus (DRN) with electrophysiological characteristics typical of 5-hydroxytryptamine (5-HT) neurones except that the neurones fired brief bursts. Here we report the effect of 5-HT lesions on the incidence of these burst-firing neurones. In vivo extracellular recordings revealed that in rats pretreated with the selective 5-HT neurotoxin, 5,7-dihydroxytryptamine, the occurrence of typical 5-HT neurones was significantly decreased (80%) compared to controls. In these 5-HT lesioned animals, the number of the burst-firing DRN neurones was also significantly reduced (91%). Neurones previously characterised as not containing 5-HT, were not altered by the lesion. These data are further evidence to support our hypothesis that burst-firing neurones in the DRN contain 5-HT.
We recently reported raphe neurones which frequently fired spikes in short bursts. However, the action potentials were broad and the neurones fired in a slow and regular pattern, suggesting they were an unusual type of 5-hydroxytryptamine (5-HT) neurone. In the present study, we investigated whether these putative burst-firing 5-HT neurones project to the forebrain and whether all spikes fired in bursts propagate along the axon. In anaesthetised rats, electrical stimulation of the medial forebrain bundle evoked antidromic spikes in both burst-firing neurones and in single-spiking, classical 5-HT neurones recorded in the dorsal raphe nucleus. Although the antidromic spike latency of the single-spiking and burst-firing neurones showed a clear overlap, burst-firing neurones had a significantly shorter latency than single-spiking neurones. For both burst-firing neurones and classical 5-HT neurones, antidromic spikes made collisions with spontaneously occurring spikes. Furthermore, in all burst-firing neurones tested, first, second and third order spikes in a burst could be made to collide with antidromic spike. Interestingly, in a small number of burst-firing neurones, antidromic stimulation evoked spike doublets, similar to those recorded spontaneously. From these data we conclude that burst-firing neurones in the dorsal raphe nucleus project to the forebrain, and each spike generated by the burst propagates along the axon and could thereby release transmitter (5-HT).
Morphologic features and electrical membrane properties of neurons in the substantia gelatinosa (SG) of the caudal spinal trigeminal nucleus (the medullary dorsal horn; MDH) were examined in the rat. Intracellular recording and biocytin-injection combined with histochemical staining were performed in horizontal slices. Twenty-four SG (lamina II) neurons were recorded stably and stained successfully. Both projection neurons (PNs; n = 9) that sent axons to regions outside the MDH and intrinsic neurons (INs; n = 15) that sent axons only to the MDH were observed. The INs were divided into those with dense axonal arborization (INDAs; n = 7) and those with sparse axonal arborization (INSAs; n = 8). In the PNs, the dendrites with spines spread to all MDH layers (laminae I-III). The main axons sent collaterals within the SG and rostrally, caudally, or medially to laminae I and III of the MDH, interpolar spinal trigeminal nucleus, spinal tract of the trigeminal nerve, or upper cervical cord segments. In the INDAs, the dendrites arising from the rostral and caudal pole of the cell bodies mainly extended rostrally and caudally parallel to the rostrocaudal axis of the SG: the dendritic trees were elongated and oval in shape and were confined within the SG. The axonal field of each INDA, a dense mesh of axonal processes, was elongated and oval in shape and almost was confined within the SG. In the INSAs, a small, round cell body was located in the center of each dendritic field, which usually was limited within the SG. Axonal processes ran radially to spread to all layers of the MDH, constituting round or oval axonal fields. The three groups of SG neurons showed more or less different intracellular responses to current injections. In particular, adaptation of spike frequency, hyperpolarizing sag, and rebound excitation were observed in the PNs and INSAs but not in the INDAs. Slow ramp depolarization and slow afterdepolarization were recorded only in INDAs.
Brain serotonergic neurons display a distinctive slow and regular discharge pattern in behaving animals. This activity gradually declines across the arousal-waking sleep cycle, becoming virtually silent during rapid eye movement sleep. The activity of these neurons, in both the pontine and medullary groups, is generally unresponsive to a variety of physiological challenges or stressors. However, these neurons are activated in association with increased muscle tone/tonic motor activity, especially if the motor activity is in the repetitive or central pattern generator mode. We interpret these data within the following theoretical framework. The primary function of the brain serotonergic system is to facilitate motor output. Concurrently, the system coordinates autonomic and neuroendocrine function with the present motor demand, and inhibits information processing in various sensory pathways. Reciprocally, when the serotonin system is briefly inactivated (e.g., during orientation to salient stimuli), this disfacilitates motor function and disinhibits sensory information processing. It is within this context that serotonin exerts its well-known effects on pain, feeding, memory, mood, etc.
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