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Neurochemical identification of stereotypic burst-firing neurons in the rat dorsal raphe nucleus using juxtacellular labelling methods: Burst firing 5HT neurons
Abstract
Recent electrophysiological studies have discovered evidence of heterogeneity of 5-hydroxytryptamine (5-HT) neurons in the mesencephalic raphe nuclei. Of particular interest is a subpopulation of putative 5-HT neurons that display many of the electrophysiological properties of presumed 5-HT-containing neurons (regular and slow firing of single spikes with a broad waveform) but fire spikes in short, stereotyped bursts. In the present study we investigated the chemical identity of these neurons in rats utilizing in vivo juxtacellular labelling methods. Of ten dorsal raphe nucleus (DRN) neurons firing short stereotyped bursts within an otherwise regular firing pattern, all exhibited immunoreactivity for either 5-HT (n = 6) or the 5-HT synthesizing enzyme, tryptophan hydroxylase (TRH; n = 2) or both (n = 2). Supporting pharmacological experiments demonstrated that the burst firing DRN neurons demonstrated equal sensitivity to 5-HT(1A) agonism and alpha(1)-adrenoceptor antagonism to single spiking DRN neurons that we have previously identified as 5-HT-containing. Collectively these data provide direct evidence that DRN neurons that exhibit stereotyped burst firing activity are 5-HT containing. The presence of multiple types of electrophysiologically distinct midbrain 5-HT neurons is discussed.
... To date, most studies characterizing neuronal groups using electrophysiological extracellular recordings focus on: the frequency of neuronal discharge, the rhythmicity (measured by the auto-correlation of events), the regularity (measured by the coefficient of variation), and the phase coherence (or phase locking) with hippocampal, cortical or other biological rhythms [32][33][34][35][36][37][38][39][40]. For example, serotonergic neurons from the DRN have been described as clock-like, exhibiting spontaneous activity, a slow 1 − 5 Hz firing-rate [41], regular spiking activity (coefficients of variation close to zero), wide action potentials (>1.4 ms), and to have rhythmic patterns of discharge [41,42], suggesting a "neuronal signature" of this neuro-chemical group. ...
... 79% percent of these neurons exhibit uni-modal interval histogram (IH), 26% have a rhythmic pattern of discharge in the ACH, and 20% have a predominant interval in the auto-correlation histogram (ACH). Moreover, a burst firing pattern is observed in 4 neurons (5%), i.e., showing doublets or triplets with < 20 ms intervals and a prominent decrease in the amplitude of higher order spikes [32]. On the other hand, the recorded neurons in the MRN (n = 92) display the following electrophysiological characteristics. ...
... Hajos et al. in 1995 [33] described that during their regular discharge these neurons show spike bursts (from 2 to 4 spikes) [32,33]. In these bursts, the spikes have a short interval (range: 2.4-11.5 ms), and the secondary spikes show a decrease in amplitude. ...
The dorsal (DRN) and median (MRN) raphe are important nuclei involved in similar functions, including mood and sleep, but playing distinct roles. These nuclei have a different composition of neuronal types and set of neuronal connections, which among other factors, determine their neuronal dynamics. Most works characterize the neuronal dynamics using classic measures, such as using the average spiking frequency (FR), the coefficient of variation (CV), and action potential duration (APD). In the current study, to refine the characterization of neuronal firing profiles, we examined the neurons within the raphe nuclei. Through the utilization of nonlinear measures, our objective was to discern the redundancy and complementarity of these measures, particularly in comparison with classic methods. To do this, we analyzed the neuronal basal firing profile in both nuclei of urethane-anesthetized rats using the Shannon entropy (Bins Entropy) of the inter-spike intervals, permutation entropy of ordinal patterns (OP Entropy), and Permutation Lempel-Ziv Complexity (PLZC). Firstly, we found that classic (i.e., FR, CV, and APD) and nonlinear measures fail to distinguish between the dynamics of DRN and MRN neurons, except for the OP Entropy. We also found strong relationships between measures, including the CV with FR, CV with Bins entropy, and FR with PLZC, which imply redundant information. However, APD and OP Entropy have either a weak or no relationship with the rest of the measures tested, suggesting that they provide complementary information to the characterization of the neuronal firing profiles. Secondly, we studied how these measures are affected by the oscillatory properties of the firing patterns, including rhythmicity, bursting patterns, and clock-like behavior. We found that all measures are sensitive to rhythmicity, except for the OP Entropy. Overall, our work highlights OP Entropy as a powerful and useful quantity for the characterization of neuronal discharge patterns.
... To date, most studies characterising neuronal groups using electrophysiological extracellular recordings focus on: the frequency of neuronal discharge, the rhythmicity (measured by the auto-correlation of events), the regularity (measured by the coefficient of variation), and the phase coherence (or phase locking) with hippocampal, cortical or other biological rhythms [31][32][33][34][35][36][37][38][39]. For example, serotonergic neurons from the DRN have been described as clock-like, exhibiting spontaneous activity, a slow 1 − 5 Hz firing-rate [40], regular spiking activity (coefficients of variation close to zero), wide action potentials (> 1.4 ms), and to have rhythmic patterns of discharge [40,41], suggesting a "neuronal signature" of this neuro-chemical group. ...
... The action potential (AP) of each neuron were averaged and analyzed in shape and duration. The APs were mostly triphasic, and the duration of the first two phases was considered as the AP duration (APD) [31]. The frequency of spontaneous activity (FR) and standard deviation was also calculated. ...
... 79% percent of these neurons exhibit uni-modal IH, 26% have a rhythmic pattern of discharge in the ACH, and 20% have a predominant interval in the ACH. Moreover, a burst firing pattern is observed in 4 neurons (5%), i.e., showing doublets or triplets with < 20 ms intervals and a prominent decrease in the amplitude of higher order spikes [31]. On the other hand, the recorded neurons in the MRN (n = 92) display the following electrophysiological characteristics. ...
The dorsal (DRN) and median (MRN) raphe are the main serotonergic nuclei, being implicated in sleep and mood regulation. The DRN is mainly serotonergic, where neurons have regular spiking activity, slow firing rate (FR), and long action potential duration (APD). The MRN is divided in a median serotonergic region and a paramedian region, containing principally GABAergic neurons, resulting in more diverse neurochemical and electrophysiological features. In the present study, we aimed to enrich the characterization of the raphe nuclei neurons by using non-linear metrics. This was done by analyzing the neuronal basal firing profile in both nuclei of urethane-anesthetized rats using Ordinal Patterns (OP) Entropy, Bins Entropy, and Permutation Lempel-Ziv Complexity (PLZC). In a first step, we found that typical linear metrics – such as FR, coefficient of variation (CV), and APD – fail to distinguish between MRN and DRN neurons, while OP entropy is significantly different between these nuclei. We also found that the FR has a strong linear relationship with CV, Bins Entropy, and PLZC. Similarly, CV has a strong correlation with FR and Bins Entropy, whereas PLZC shows a strong linear fit with Bins Entropy. However, OP Entropy has either a weak or no linear relationship with the rest of the metrics tested, suggesting that OP Entropy is a good metric to differentiate neuronal firing profiles. In a second step, we studied how these metrics are affected by the oscillatory properties of the firing patterns. We found that all metrics are sensitive to rhythmicity – with the exception of OP Entropy. Again, this highlights OP Entropy as a powerful and useful quantity for the characterization of neuronal discharge patterns.
... Glutamate injections, inducing phasic bursts of LC activity and NA release in the hippocampus, were administered in both anesthetized and awake animals, inducing clear PS potentiation and variable effects on the EPSP (94). Similar as described above, prior administration of propranolol, a non-selective β-receptor antagonist, blocked all potentiating effects, confirming the β-receptor dependent character of NA potentiation (94). Similar effects were observed in awake animals, with a clear potentiation of the population spike, with absence of consistent effects on the EPSP (95). ...
... They hypothesized that due to the temporal resolution an initial rapid increase in NA was not detected as it was followed by a decrease due to local breakdown at the synapse or the inability of LC to restore NA while firing at frequencies above baseline levels. 94 An alternative for microdialysis is voltammetry which has a higher temporal resolution but lacks good distinction between the different catecholamines (92). ...
... We have to point out that our study has some caveats, such as low expression levels and the use of single tungsten electrodes, making it hard to record and impossible to identify the transduction character of a recorded neuron. However, in animals injected with the CAV2 achieving high and specific hM3Dq expression it would be possible to achieve a dose-response curve for clozapine and juxtacellular labelling with neurobiotin, which could give confirmation about the transduction characteristics of the recorded neurons as described by multiple other groups performing unit recording in LC(93)(94)(95)(96). ...
... However, differences in excitability and membrane properties were also noted between cells in different raphe subnuclei (Trulson and Frederickson 1987;Beck et al. 2004a;Crawford et al. 2010). In vivo recordings further showed the existence of fast, slow and bursting firing 5-HT neurons (Kocsis et al. 2006;Hajós et al. 2007) and a diversity of responses of DR neurons to salient stimuli (Ranade and Mainen 2009;Schweimer and Ungless 2010). Neurochemically, all 5-HT raphe neurons share a common identity (Deneris and Wyler 2012), but they also differ in their co-neurotransmission properties (Gaspar and Lillesaar 2012). ...
... Accumulating evidence suggests that 5-HT heterogeneity is key to understand how this small group of cells participates in many different biological functions (Calizo et al. 2011;Hale and Lowry 2011;Gaspar and Lillesaar 2012). Previous efforts to characterize 5-HT neurons have relied on independent morphological (Molliver 1987), molecular (Larm et al. 2003;Aznar et al. 2005;Lacoste et al. 2006;Amilhon et al. 2010;Fu et al. 2010;Spaethling et al. 2014) or electrophysiological features (Kirby et al. 2003;Hajós et al. 2007;Calizo et al. 2011). Here, we carried out a characterization and classification of 5-HT neurons based on a multi-scale analysis. ...
... This is in accordance with recent studies showing that this pathway is under inhibitory control by reciprocal amygdala monosynaptic inputs to GABAergic neurons in the DR (Pollak Dorocic et al. 2014;Weissbourd et al. 2014). Though most 5-HT neurons are regular, slow-spiking, some neurons in the DR can also discharge high frequency bursts of action potentials (Kocsis et al. 2006;Hajós et al. 2007). Tonic pacemaker activity in serotonergic neurons serves primarily to maintain 5-HT levels throughout the brain, while phasic bursts are stimulusspecific responses delivering fine temporal and spatial synaptic information (McQuade and Sharp 1995;Gartside et al. 2000;Hajós et al. 2007;Ranade and Mainen 2009;Schweimer and Ungless 2010;Miyazaki et al. 2011). ...
Serotonergic neurons of the raphe nuclei exhibit anatomical, neurochemical and elecrophysiological heterogeneity that likely underpins their specific role in multiple behaviors. However, the precise organization of serotonin (5-HT) neurons to orchestrate 5-HT release patterns throughout the brain is not well understood. We compared the electrophysiological and neurochemical properties of dorsal and median raphe 5-HT neurons projecting to the medial prefrontal cortex (mPFC), amygdala (BLA) and dorsal hippocampus (dHP), combining retrograde tract tracing with brain slice electrophysiology and single-cell RT-PCR in Pet1-EGFP mice. Our results show that 5-HT neurons projecting to the dHP and the mPFC and the BLA form largely non-overlapping populations and that BLA-projecting neurons have characteristic excitability and membrane properties. In addition, using an unbiased clustering method that correlates anatomical, molecular and electrophysiological phenotypes, we find that 5-HT neurons with projections to the mPFC and the dHP segregate from those projecting to the BLA. Single-cell gene profiling showed a restricted expression of the peptide galanin in the population of 5-HT neurons projecting to the mPFC. Finally, cluster analysis allowed identifying an atypical subtype of 5-HT neuron with low excitability, long firing delays and preferential expression of the vesicular glutamate transporter type 3. Overall, these findings allow to define correlated anatomical and physiological identities of serotonin raphe neurons that help understanding how discrete raphe cells subpopulations account for the heterogeneous activities of the midbrain serotonergic system.
... The 5-HT neurons in the DRN display diverse in vivo spiking behaviors [63][64][65][66] . One subgroup of 5-HT neurons could be identified by electrophysiological characteristics, including firing rate, firing rhythmicity, and spike distribution, which displayed slowfiring clock-like pattern 52,67,68 . In our studies, the putative 5-HT neurons showed the low frequency (1.65 Hz) with a highly regular pattern that was revealed by the narrow interspike interval (Fig. 3j, m). ...
... The MC4R neurons were identified by the short latencies of evoked spikes accurately following high-frequency photostimulation, as well as the identical waveforms of evoked and spontaneous spikes. The putative 5-HT neurons were identified by the electrophysiological characteristics, including firing rate, firing rhythmicity, and spike distribution, which displayed slow-firing clock-like pattern 52,67,68 . To ablate AgRP → dlDRN circuit the DT (0.4 ng) was infused to the dlDRN using a needle (Hamilton Small Hub RN 33 G, Reno, NV) connected with a 10 μl syringe (Hamilton 700 Microliter, Reno, NV), at a rate of 0.01 μl/min. ...
Contrasting to the established role of the hypothalamic agouti-related protein (AgRP) neurons in feeding regulation, the neural circuit and signaling mechanisms by which they control energy expenditure remains unclear. Here, we report that energy expenditure is regulated by a subgroup of AgRP neurons that send non-collateral projections to neurons within the dorsal lateral part of dorsal raphe nucleus (dlDRN) expressing the melanocortin 4 receptor (MC4R), which in turn innervate nearby serotonergic (5-HT) neurons. Genetic manipulations reveal a bi-directional control of energy expenditure by this circuit without affecting food intake. Fiber photometry and electrophysiological results indicate that the thermo-sensing MC4RdlDRN neurons integrate pre-synaptic AgRP signaling, thereby modulating the post-synaptic serotonergic pathway. Specifically, the MC4RdlDRN signaling elicits profound, bi-directional, regulation of body weight mainly through sympathetic outflow that reprograms mitochondrial bioenergetics within brown and beige fat while feeding remains intact. Together, we suggest that this AgRP neural circuit plays a unique role in persistent control of energy expenditure and body weight, hinting next-generation therapeutic approaches for obesity and metabolic disorders. Neuronal signaling has an important role in the regulation of energy expenditure and body weight, however, the underlying mechanisms are incompletely understood. Here, the authors report a AgRP-MC4R-serotonin expressing neuronal circuit that regulate energy expenditure without affecting feeding.
... Tonic firing is characterized by a highly regular firing rate at frequencies comprised between 0.1 Hz and 3 Hz in brain slices from rats as well as in behaving cats and rats (Vandermaelen and Aghajanian, 1983;Veasey et al., 1997;Ranade and Mainen, 2009). A fast-firing population of DR neurons has been described, exhibiting tonic firing frequencies up to 17 Hz in rodents (Allers and Sharp, 2003;Kocsis et al., 2006;Hajós et al., 2007). Phasic firing consists in a burst of 2-4 AP with an frequency above 100 Hz (Allers and Sharp, 2003;Kirby et al., 2003;Kocsis et al., 2006;Hajós et al., 2007;Schweimer and Ungless, 2010). ...
... A fast-firing population of DR neurons has been described, exhibiting tonic firing frequencies up to 17 Hz in rodents (Allers and Sharp, 2003;Kocsis et al., 2006;Hajós et al., 2007). Phasic firing consists in a burst of 2-4 AP with an frequency above 100 Hz (Allers and Sharp, 2003;Kirby et al., 2003;Kocsis et al., 2006;Hajós et al., 2007;Schweimer and Ungless, 2010). ...
Elaboration of appropriate responses to behavioral situations rests on the ability of selecting appropriate motor outcomes in accordance to specific environmental inputs. To this end, the primary motor cortex (M1) is a key structure for the control of voluntary movements and motor skills learning. Subcortical loops regulate the activity of the motor cortex and thus contribute to the selection of appropriate motor plans. Monoamines are key mediators of arousal, attention and motivation. Their firing pattern enables a direct encoding of different states thus promoting or repressing the selection of actions adapted to the behavioral context. Monoaminergic modulation of motor systems has been extensively studied in subcortical circuits. Despite evidence of converging projections of multiple neurotransmitters systems in the motor cortex pointing to a direct modulation of local circuits, their contribution to the execution and learning of motor skills is still poorly understood. Monoaminergic dysregulation leads to impaired plasticity and motor function in several neurological and psychiatric conditions, thus it is critical to better understand how monoamines modulate neural activity in the motor cortex. This review aims to provide an update of our current understanding on the monoaminergic modulation of the motor cortex with an emphasis on motor skill learning and execution under physiological conditions.
... The DRN neurons were characteristic as 5-HT-containing if they had off-line analysis-neurons with slow firing rates (0.1-4 spikes/second), long spike durations (2-4 milliseconds), single or bursting firing patterns, and action potentials with biphasic or triphasic extracellular waveforms. [23][24][25] The GABA interneurons in the DRN were characterized by their shorter action potential durations (<1 millisecond), higher firing rates (5-40 spikes/second), and irregular firing patterns. The locations of all recorded neurons were histologically confirmed to be in the DRN. ...
... The 5-HT neurons exhibited a typical long-duration action potential of 2 to 4 milliseconds that displayed a prominent positive deflection followed by a negative or negative/positive transient ( Figure 1B). 23 The GABA interneurons in the DRN were characterized by their shorter action potential durations (<1 millisecond; Figure 1C). [26][27][28][29] ...
The 5-hydroxytryptamine (5-HT; serotonin) neurotransmission is severely affected by the degeneration of nigrostriatal dopaminergic neurons. Here, we report the effects of the systemic administration of the 5-HT7 receptor agonist AS-19. In sham rats, the mean response of the 5-HT neurons in the dorsal raphe nucleus (DRN) to systemic AS-19 was excitatory and the mean response of the γ-aminobutyric acid (GABA) interneurons was inhibitory. In Parkinson disease (PD) rats, the same dose did not affect the 5-HT neurons and only high doses (640 μg/kg intravenous) were able to the increase GABA interneuron activity. These results indicate that DRN 5-HT neurons and GABA interneurons are regulated by the activation of 5-HT7 receptors and that the degeneration of the nigrostriatal pathway leads to decreased responses of these neurons to AS-19, which in turn suggests that the 5-HT7 receptors on 5-HT neurons and GABA interneurons in PD rats are dysfunctional and downregulated.
... By using a juxtacellular labeling method (Pinault, 1996), which greatly improved neuron type identification, it was confirmed that most DRN serotonergic neurons exhibit slow and regular spiking (Allers and Sharp, 2003) and that a subset discharges in burst-like repetitive mode (Hajós et al., 2007). Further studies using juxtacellular labeling revealed both a subset of fast-firing (>8 Hz) serotonergic neurons (Kocsis et al., 2006) and functional differences between single spike and burst firing serotonergic neurons (Schweimer and Ungless, 2010;Schweimer et al., 2011). ...
... The fact that LFO-type neurons have been rarely observed does not preclude the possibility that in vivo a higher fraction, or even all serotonergic neurons in the DRN can discharge in LFO mode. In that respect an analogy can be drawn with spike doublets firing mode, which has not been observed in vitro, but has been observed in recordings from serotonergic neurons in anesthetized rats and mice (Hajós et al., 1995(Hajós et al., , 2007Montalbano et al., 2015). The functional implications of LFO spiking mode are currently unclear. ...
Tonic spiking of serotonergic neurons establishes serotonin levels in the brain. Since the first observations, slow regular spiking has been considered as a defining feature of serotonergic neurons. Recent studies, however, have revealed the heterogeneity of serotonergic neurons at multiple levels, comprising their electrophysiological properties, suggesting the existence of functionally distinct cellular subpopulations. In order to examine in an unbiased manner whether serotonergic neurons of the dorsal raphe nucleus (DRN) are heterogeneous, we used a non-invasive loose-seal cell-attached method to record α1 adrenergic receptor-stimulated spiking of a large sample of neurons in brain slices obtained from transgenic mice lines that express fluorescent marker proteins under the control of serotonergic system-specific Tph2 and Pet-1 promoters. We found wide homogeneous distribution of firing rates, well fitted by a single Gaussian function (r2 = 0.93) and independent of anatomical location (P = 0.45), suggesting that in terms of intrinsic firing properties, serotonergic neurons in the DRN represent a single cellular population. Characterization of the population in terms of spiking regularity was hindered by its dependence on the firing rate. For instance, the coefficient of variation of the interspike intervals (ISI), a common measure of spiking irregularity, is of limited usefulness since it correlates negatively with the firing rate (r = −0.33, P < 0.0001). Nevertheless, the majority of neurons exhibited regular, pacemaker-like activity, with coefficient of variance of the ISI lower than 0.5 in ~97% of cases. Unexpectedly, a small percentage of neurons (~1%) exhibited a particular spiking pattern, characterized by low frequency (~0.02–0.1 Hz) oscillations in the firing rate. Transitions between regular and oscillatory firing were observed, suggesting that the oscillatory firing is an alternative firing pattern of serotonergic neurons.
... 68 Most of DRN 5-HT neurons are quiescent or fire at slow and regular frequency, 36,37 and the probability to encounter burst firing neurons in the DRN is very low. 71 The OXT-induced switch from low regular to high burst firing mode combined with the increased excitability could mediate the reported in vivo increase of 5-HT release and contribute, at least in part, to anxiolytic-like effects induced by activation of OXTRs. 13,72 Furthermore, because activation of DRN 5-HT neurons increases sociability, 30,66 it is possible that the OXT-induced excitation of these neurons could encode the prosocial effects of OXT, though additional studies are required to directly test this notion. ...
Oxytocin (OXT) modulates wide spectrum of social and emotional behaviors via modulation of numerous neurotransmitter systems, including serotonin (5-HT). However, how OXT controls the function of dorsal raphe nucleus (DRN) 5-HT neurons remains unknown. Here, we reveal that OXT excites and alters the firing pattern of 5-HT neurons via activation of postsynaptic OXT receptors (OXTRs). In addition, OXT induces cell-type-specific depression and potentiation of DRN glutamate synapses by two retrograde lipid messengers, 2-arachidonoylglycerol (2-AG) and arachidonic acid (AA), respectively. Neuronal mapping demonstrates that OXT preferentially potentiates glutamate synapses of 5-HT neurons projecting to medial prefrontal cortex (mPFC) and depresses glutamatergic inputs to 5-HT neurons projecting to lateral habenula (LHb) and central amygdala (CeA). Thus, by engaging distinct retrograde lipid messengers, OXT exerts a target-specific gating of glutamate synapses on the DRN. As such, our data uncovers the neuronal mechanisms by which OXT modulates the function of DRN 5-HT neurons.
... The onset of a burst was signified by the occurrence of two spikes with an interspike interval (ISI) < 0.16 s for hippocampal glutamate, ISI < 0.01 s for DRN 5-HT, and ISI < 0.08 s for VTA dopamine neurons. The termination of a burst was defined as an ISI > 0.16 s for hippocampal glutamate [28], ISI > 0.01 s for DRN 5-HT neurons [29], and ISI > 0.16 s for VTA dopamine neurons [30,31]. ...
Background:
Short-term treatment with non-peptide agonists of delta-opioid receptors, such as agonist SNC80, induced behavioral effects in rodents, which could be modulated via changes in central neurotransmission. The present experiments aimed at testing the hypothesis that chronic treatment with SNC80 induces anxiolytic effects associated with changes in hippocampal glutamate and brainstem monoamine pathways.
Methods:
Adult male Wistar rats were used in experiments. Rats were treated with SNC80 (3 mg/kg/day) for fourteen days. Neuronal excitability was assessed using extracellular in vivo single-unit electrophysiology. The behavioral parameters were examined using the elevated plus maze and open field tests.
Results:
Chronic SNC80 treatment increased the excitability of hippocampal glutamate and ventral tegmental area dopamine neurons and had no effect on the firing activity of dorsal raphe nucleus serotonin cells. Chronic SNC80 treatment induced anxiolytic effects, which were, however, confounded by increased locomotor activity clearly confirmed in an open field test. The ability to cope with stressful situations and habituation processes in a novel environment was not influenced by chronic treatment with SNC80.
Conclusion:
Our study suggests that the psychoactive effects of SNC80 might be explained by its ability to stimulate hippocampal glutamate and mesolimbic dopamine transmission.
... The onset of a burst was signified by the occurrence of two spikes with ISI < 0.08 s for noradrenaline and dopamine neurons, and ISI < 0.01 s for 5-HT neurons. The termination of a burst was defined as an ISI > 0.16 s for noradrenaline and dopamine neurons [23,24] and ISI > 0.010 s for 5-HT neurons [25]. Statistical assessments were performed using SigmaPlot 12.5 software D. Grinchii et al. ...
Trace amine-associated receptor 1 (TAAR1) has been recently identified as a target for the future antidepressant, antipsychotic, and anti-addiction drugs. Full (e.g. RO5256390) and partial (e.g. RO5263397) TAAR1 agonists showed antidepressant-, antipsychotic- and anti-addiction-like behavioral effects in rodents and primates. Acute RO5256390 suppressed, and RO5263397 stimulated serotonin (5-HT) neurons of the dorsal raphe nucleus (DRN) and dopamine neurons of the ventral tegmental area (VTA) in brain slices, suggesting that the behavioral effects of TAAR1 ligands involve 5-HT and dopamine. For more comprehensive testing of this hypothesis, we examined acute and chronic effects of RO5256390 and RO5263397 on monoamine neurons in in vivo conditions. Excitability of 5-HT neurons of the DRN, noradrenaline neurons of the locus coeruleus (LC), and dopamine neurons of the VTA was assessed using single-unit electrophysiology in anesthetized rats. For acute experiments, RO5256390 and RO5263397 were administered intravenously; neuronal excitability after RO5256390 and RO5263397 administration was compared to the basal activity of the same neuron. For chronic experiments, RO5256390 was administered orally for fourteen days prior to electrophysiological assessments. The neuronal excitability in RO5256390-treated rats was compared to vehicle-treated controls. We found that acute RO5256390 inhibited 5-HT and dopamine neurons. This effect of RO5256390 was reversed by the subsequent and prevented by the earlier administration of RO5263397. Acute RO5256390 and RO5263397 did not alter the excitability of LC noradrenaline neurons in a statistically significant way. Chronic RO5256390 increased excitability of 5-HT neurons of the DRN and dopamine neurons of the VTA. In conclusion, the putative antidepressant and antipsychotic effects of TAAR1 ligands might be mediated, at least in part, via the modulation of excitability of central 5-HT and dopamine neurons.
... Tonic (basal) neurotransmitter levels arise from clocklike neural firing over minutes to hours to days. Phasic (transient) changes in neurotransmitter levels are rapid (tens of milliseconds to seconds) and are hypothesized to result from synchronized bursts of neural firing in response to evoked or naturally occurring stimuli [29][30][31][32][33]. The ability to monitor transitory neurochemical events, in conjunction with changes in tonic signaling, will enable a more comprehensive understanding of how chemical neurotransmission encodes behaviorally relevant information [34,35]. ...
Many voltammetry methods have been developed to monitor brain extracellular dopamine levels. Fewer approaches have been successful in detecting serotonin in vivo. No voltammetric techniques are currently available to monitor both neurotransmitters simultaneously across timescales, even though they play integrated roles in modulating behavior. We provide proof-of-concept for rapid pulse voltammetry coupled with partial least squares regression (RPV-PLSR), an approach adapted from multi-electrode systems (i.e., electronic tongues) used to identify multiple components in complex environments. We exploited small differences in analyte redox profiles to select pulse steps for RPV waveforms. Using an intentionally designed pulse strategy combined with custom instrumentation and analysis software, we monitored basal and stimulated levels of dopamine and serotonin. In addition to faradaic currents, capacitive currents were important factors in analyte identification arguing against background subtraction. Compared to fast-scan cyclic voltammetry-principal components regression (FSCV-PCR), RPV-PLSR better differentiated and quantified basal and stimulated dopamine and serotonin associated with striatal recording electrode position, optical stimulation frequency, and serotonin reuptake inhibition. The RPV-PLSR approach can be generalized to other electrochemically active neurotransmitters and provides a feedback pipeline for future optimization of multi-analyte, fit-for-purpose waveforms and machine learning approaches to data analysis.
Graphical abstract
... De cette manière, les neurones 5-HT du RM et du RD présentent une signature électrophysiologique différente (Beck et al., 2004) alors qu'au sein même du RD, les neurones 5-HT peuvent également présenter des différences de propriétés intrinsèques selon leur localisation topographique (Calizo et al., 2011;Crawford et al., 2010). Enfin, les neurones 5-HT du RD sont également doués d'une activité de type phasique sous forme de bouffées (Allers and Sharp, 2003;Hajós et al., 2007). Ce type d'activité permet notamment une augmentation transitoire de la libération de sérotonine dans les aires post-synaptiques (Gartside et al., 2000). ...
Les études épidémiologiques mettent en évidence que les patients atteint de diabète de type 2 (DT2) ont deux fois plus de risque de souffrir de dépression majeure (DM), un trouble mental caractérisé par une tristesse intense et/ou une anhédonie. Plus précisément, les données de la littérature indiquent que la résistance à l’insuline, qui est la caractéristique majeure du DT2, est positivement corrélée à la sévérité des symptômes dépressifs. Etant donné le rôle essentiel que joue la neurotransmission sérotoninergique (5-HT) dans la physiopathologie de la DM, nous avons émis l’hypothèse que la résistance à l’insuline sélective de ce système neuronal est responsable des troubles de l’humeur associés au DT2. En ce sens, des altérations du système 5-HT ont été observées dans un modèle de troubles émotionnels associés à un DT2. En effet, le régime obésogène utilisé dans ce modèle induit une altération significative des propriétés électrophysiologiques des neurones 5-HT du Raphé dorsal (DR) ainsi qu’une diminution des taux de sérotonine. Cette étude vise également à déterminer l’action de l’insuline cérébrale sur le comportement émotionnel et le système sérotoninergique. En utilisant une approche par électrophysiologie ex-vivo, nous avons pu observer que l’insuline modulait positivement l’activité des neurones 5-HT du RD. Nous avons également pu mettre en évidence un effet de type anxiolytique de l’action de l’insuline sur le cerveau par voie intranasale. Cet effet est accompagné de diminution des taux de sérotonine (5-HT) tissulaires dans certaines structures cérébrales impliquées dans la régulation de l’anxiété. Enfin, nous avons utilisé un modèle transgénique grâce auquel nous avons invalidé sélectivement le récepteur à l’insuline dans les neurones 5-HT. L’étude comportementale a permis de mettre en évidence une diminution du comportement de type anxieux chez ces animaux, associé à une diminution de l’activité des neurones sérotoninergiques du RD. Ensemble ces données suggèrent que l’insuline peut moduler le comportement émotionnel notamment via le système sérotoninergique. Ces éléments pourront contribuer à la possible découverte de nouveaux traitements et à la prise en charge des troubles de l’humeur chez les patients atteint de DT2.
... Most of the studies classified putative DRN 5-HT neurons based on their electrophysiological properties (Aghajanian and Vandermaelen, 1982). However, these criteria might be insufficient to clearly identify 5-HT neurons as demonstrated by juxtacellular experiments (Allers and Sharp, 2003;Hajos et al., 2007;Kocsis et al., 2006). We will thus also highlight recent evidence obtained with recordings of genetically identified 5-HT neurons during reward-related tasks. ...
Serotonin is a critical neuromodulator involved in development and behavior. Its role in reward is however still debated. Here, we first review classical studies involving electrical stimulation protocols and pharmacological approaches. Contradictory results on the serotonergic’ involvement in reward emerge from these studies. These differences might be ascribable to either the diversity of cellular types within the raphe nuclei or/and the specific projection pathways of serotonergic neurons. We continue to review more recent work, using optogenetic approaches to activate serotonergic cells in the Raphe to VTA pathway. From these studies, it appears that activation of this pathway can lead to reinforcement learning mediated through the excitation of dopaminergic neurons by serotonergic neurons co-transmitting glutamate. Finally, given the importance of serotonin during development on adult emotion, the effect of abnormal early-life levels of serotonin on the dopaminergic system will also be discussed. Understanding the interaction between the serotonergic and dopaminergic systems during development and adulthood is critical to gain insight into the specific facets of neuropsychiatric disorders.
... The onset of a burst was signified by the occurrence of two spikes with ISI < 0.08 s for NE and DA neurons, and ISI < 0.01 s for 5-HT neurons. The termination of a burst was defined as an ISI > 0.16 s for NE and DA neurons ( Dawe et al., 2001 ;Grace and Bunney, 1984 ) and ISI > 0.01 s for 5-HT neurons ( Hajós et al., 2007 ). Coefficients of variation were calculated as ( σ / μ) × 100%, where μ is the mean, and σ is the standard deviation. ...
Higher risk of depression and schizophrenia in descendants of mothers experienced acute infection during the pregnancy has been reported. Since monoamines are fundamental in mentioned psychopathologies, it is possible that maternal immune activation leads to impaired functioning of serotonin (5-HT), noradrenaline, and dopamine neurons in offspring. To test this hypothesis, we examined the effect of maternal immune activation by lipopolysaccharide (LPS) in rats on the excitability of monoamine-secreting neurons in the offspring. LPS was administered during days 15–19 of the gestation in the rising doses of 20–80 µg/kg; control dams received vehicle. During days 53–63 postpartum, rats were anesthetized and electrodes were inserted into the dorsal raphe nucleus, locus coeruleus, and ventral tegmental area for in vivo excitability assessment of 5-HT, noradrenaline, and dopamine neurons. Maternal immune activation suppressed the firing rate of 5-HT neurons in both sexes and stimulated the firing rate of dopamine neurons in males. Decrease in the firing rate of 5-HT neurons was accompanied with an increase, and increase in the firing rate of dopamine neurons with a decrease, in the density of spontaneously active cells. Maternal immune activation also decreased the variability of interspike intervals in 5-HT and dopamine neurons. It is possible that the alteration of excitability of 5-HT and dopamine neurons by maternal immune activation is involved in the psychopathologies induced by infectious disease during the pregnancy. Stimulation of dopamine excitability in males might be a compensatory mechanism secondary to the maternal immune challenge-induced suppression of 5-HT neurons.
... 2012), and in turn display excitatory effects on most elements of the reticular activating system (Brown et al., 2001;Haas et al., 2008). Similar to NE and HA neurons, 5HT neurons are also wake-active, and exhibit slow, tonic firing patterns across the wakeful states, with subpopulations demonstrating burst firing capabilities (Hajós et al., 2007). Unlike NE and HA neurons, most dorsal raphe-5HT neurons do not fire spontaneously and require afferent input from NE neurons to maintain their tonic output (Vandermaelen and Aghajanian, 1983). ...
It was long thought that astrocytes, given their lack of electrical signaling, were not involved in communication with neurons. However, we now know that one astrocyte on average maintains and regulates the extracellular neurotransmitter and potassium levels of more than 140,000 synapses, both excitatory and inhibitory, within their individual domains, and form a syncytium that can propagate calcium waves to affect distant cells via release of “gliotransmitters” such as glutamate, ATP, or adenosine. Neuromodulators can affect signal-to-noise and frequency transmission within cortical circuits by effects on inhibition, allowing for the filtering of relevant vs. irrelevant stimuli. Moreover, synchronized “resting” and desynchronized “activated” brain states are gated by short bursts of high-frequency neuromodulatory activity, highlighting the need for neuromodulation that is robust, rapid, and far-reaching. As many neuromodulators are released in a volume manner where degradation/uptake and the confines of the complex CNS limit diffusion distance, we ask the question—are astrocytes responsible for rapidly extending neuromodulator actions to every synapse? Neuromodulators are known to influence transitions between brain states, leading to control over plasticity, responses to salient stimuli, wakefulness, and sleep. These rapid and wide-spread state transitions demand that neuromodulators can simultaneously influence large and diverse regions in a manner that should be impossible given the limitations of simple diffusion. Intriguingly, astrocytes are ideally situated to amplify/extend neuromodulator effects over large populations of synapses given that each astrocyte can: (1) ensheath a large number of synapses; (2) release gliotransmitters (glutamate/ATP/adenosine) known to affect inhibition; (3) regulate extracellular potassium that can affect excitability and excitation/inhibition balance; and (4) express receptors for all neuromodulators. In this review article, we explore the hypothesis that astrocytes extend and amplify neuromodulatory influences on neuronal networks via alterations in calcium dynamics, the release of gliotransmitters, and potassium homeostasis. Given that neuromodulatory networks are at the core of our sleep-wake cycle and behavioral states, and determine how we interact with our environment, this review article highlights the importance of basic astrocyte function in homeostasis, general cognition, and psychiatric disorders.
... 5-HT neurons in the DRN typically exhibit slow regular or irregular firing characteristics [6], [40], [41], and so the identified slow-firing DRN neurons could potentially be 5-HT neurons. Future work will confirm this. ...
... 5-HT neurons were identified according to the following electrophysiological properties: 1) a firing rate of 0.5-3 Hz, 2) a biphasic or triphasic action potential with steady, regular firing and, 3) a spike duration of 1.5-3.0 ms [32,33]. Four to five electrode descents were carried out in each rat and neurons were recorded for 2 min after stabilization. ...
Previous research has implicated the serotonin-2B (5-HT2B) receptor as a possible contributor to the antidepressant-like response. Aripiprazole has been successfully used in combination with selective serotonin reuptake inhibitors (SSRIs) in treatment-resistant depression and it, among all receptors, exhibits the highest affinity for the 5-HT2B receptor. However, the potential contribution of such an antagonistic action on 5-HT2B receptors in the context of adjunct therapy is not known. In vivo electrophysiological recordings of ventral tegmental area (VTA) dopamine (DA) neurons, dorsal raphe nucleus (DRN) 5-HT neurons and pyramidal neurons in the medial prefrontal cortex (mPFC), and the hippocampus were conducted in anaesthetized Sprague-Dawley rats after the administration of 5-HT2B receptor ligands alone or in combination with the SSRI escitalopram. An escitalopram-induced decrease in DA, but not 5-HT firing activity, was rescued by 2-day co-administration of the selective 5-HT2B receptor antagonist LY266097. In the mPFC, 14-day escitalopram administration alone had no effect on pyramidal neuron firing and burst activity, whereas, aripiprazole administered alone or in combination with escitalopram for 14 days increased pyramidal neuron firing and burst activity. Likewise, the administration of LY266097 alone or its addition on the last 3 days of a 14-day escitalopram regimen increased pyramidal neuron firing and burst activity. These results indicated that 5-HT2B receptors play, at least in part, a role in this enhancement. In the hippocampus, 5-HT2B receptor activation by BW723c86 decreased escitalopram-induced inhibition of 5-HT reuptake, which was reversed by a 5-HT2B receptor antagonist. Altogether, these results put into evidence the possibility that 5-HT2B receptor blockade contributes to the therapeutic effect of aripiprazole addition to SSRIs in depression.
... (2) a lower titer to avoid aspecific expression due to possible promoter leakage resulting from high MOI and/or (3) higher injected volumes to cover more LC cells (Bruinstroop et al., 2012;Vazey and Aston-Jones, 2014). To allow confirmation on the transduction characteristics of the recorded LC neurons in anesthetized rats juxtacellular labeling with neurobiotin can be performed as proven by other groups performing LC unit recording (Allers and Sharp, 2003;Hajós et al., 2007;Bangasser et al., 2011;Dempsey et al., 2015). ...
Aim
Selective chemogenetic modulation of locus coeruleus (LC) neurons would allow dedicated investigation of the role of the LC-NA pathway in brain excitability and disorders such as epilepsy. This study investigated the feasibility of an experimental set-up where chemogenetic modification of the brainstem locus coeruleus NA neurons is aimed at and followed by LC unit activity recording in response to clozapine.
Methods
The LC of male Sprague-Dawley rats was injected with 10 nl of adeno-associated viral vector AAV2/7-PRSx8-hM3Dq-mCherry (n = 19, DREADD group) or AAV2/7-PRSx8-eGFP (n = 13, Controls). Three weeks later, LC unit recordings were performed in anesthetized rats. We investigated whether clozapine, a drug known to bind to modified neurons expressing hM3Dq receptors, was able to increase the LC firing rate. Baseline unit activity was recorded followed by subsequent administration of 0.01 and 0.1 mg/kg of clozapine in all rats. hM3Dq-mcherry expression levels were investigated using immunofluorescence staining of brainstem slices at the end of the experiment.
Results
Unit recordings could be performed in 12 rats and in a total of 12 neurons (DREADDs: n = 7, controls: n = 5). Clozapine 0.01 mg/kg did not affect the mean firing rate of recorded LC-neurons; 0.1 mg/kg induced an increased firing rate, irrespective whether neurons were recorded from DREADD or control rats (p = 0.006). Co-labeling of LC neurons and mCherry-tag showed that 20.6 ± 2.3% LC neurons expressed the hM3Dq receptor. Aspecific expression of hM3Dq-mCherry was also observed in non-LC neurons (26.0 ± 4.1%).
Conclusion
LC unit recording is feasible in an experimental set-up following manipulations for DREADD induction. A relatively low transduction efficiency of the used AAV was found. In view of this finding, the effect of injected clozapine on LC-NA could not be investigated as a reliable outcome parameter for activation of chemogenetically modified LC neurons. The use of AAV2/7, a vector previously applied successfully to target dopaminergic neurons in the substantia nigra, leads to insufficient chemogenetic modification of the LC compared to transduction with AAV2/9.
... Indeed a chemically defined 5-HT neuron can send several types of axonal projections with different morphologies to different brain regions (Gagnon and Parent, 2014). Hence, research has been rather devoted to studying the molecular or physiological diversity of 5-HT neurons, identifying various 5-HT neuronal subtypes that differentially express the 5-HT1A autoreceptor (Sotelo et al., 1990;Kirby et al., 2003;Bonnavion et al., 2010;Kiyasova et al., 2013;Fernandez et al., 2016), substance P/neurokinin receptor 1 (NK1; Lacoste et al., 2006), galanin and its receptor (Xu and Hökfelt, 1997;Larm et al., 2003), neuronal nitric oxide synthase (nNOS; Xu and Hökfelt, 1997), gamma-aminobutyric acid (GABA)-synthesizing enzyme glutamic acid decarboxylase (GAD; Fu et al., 2010), alpha7 nicotinic receptor (Aznar et al., 2005), MET receptor tyrosine kinase (Kast et al., 2017) or display different pharmacological and electrophysiological properties (Kirby et al., 2003;Hajós et al., 2007;Calizo et al., 2011). This heterogeneity appears to be target-specific (Fernandez et al., 2016;Prouty et al., 2017) and could therefore be used to establish a specific anatomy/function cartography of raphe serotonin sub-systems (Ren et al., 2018). ...
... Indeed a chemically defined 5-HT neuron can send several types of axonal projections with different morphologies to different brain regions ( Gagnon and Parent, 2014). Hence, research has been rather devoted to studying the molecular or physiological diversity of 5-HT neurons, identifying various 5-HT neuronal subtypes that differentially express the 5-HT1A autoreceptor ( Sotelo et al., 1990;Kirby et al., 2003;Bonnavion et al., 2010;Kiyasova et al., 2013;Fernandez et al., 2016), substance P/neurokinin receptor 1 (NK1; Lacoste et al., 2006), galanin and its receptor ( Xu and Hökfelt, 1997;Larm et al., 2003), neuronal nitric oxide synthase (nNOS; Xu and Hökfelt, 1997), gamma-aminobutyric acid (GABA)-synthesizing enzyme glutamic acid decarboxylase (GAD; Fu et al., 2010), alpha7 nicotinic receptor ( Aznar et al., 2005), MET receptor tyrosine kinase (Kast et al., 2017) or display different pharmacological and electrophysiological properties ( Kirby et al., 2003;Hajós et al., 2007;Calizo et al., 2011). This heterogeneity appears to be target-specific ( Fernandez et al., 2016;Prouty et al., 2017) and could therefore be used to establish a specific anatomy/function cartography of raphe serotonin sub-systems ( Ren et al., 2018). ...
A subpopulation of raphe 5-HT neurons expresses the vesicular glutamate transporter VGLUT3 with the co-release of glutamate and serotonin proposed to play a pivotal role in encoding reward- and anxiety-related behaviors. Serotonin axons are identifiable by immunolabelling of either serotonin (5-HT) or the plasma membrane 5-HT transporter (SERT), with SERT labeling demonstrated to be only partially overlapping with 5-HT staining. Studies investigating the colocalization or segregation of VGLUT3 within SERT or 5-HT immunolabeled boutons have led to inconsistent results. Therefore, we combined immunohistochemistry, high resolution confocal imaging and 3D-reconstruction techniques to map and quantify the distribution of VGLUT3 immunoreactive boutons within 5-HT vs SERT- positive axons in various regions of the mouse forebrain, including the prefrontal cortex, nucleus accumbens core and shell, bed nucleus of the stria terminalis, dorsal striatum, lateral septum, basolateral and central amygdala and hippocampus. Our results demonstrate that about 90% of 5-HT boutons are colocalized with SERT in almost all the brain regions studied, which therefore reveals that VGLUT3 and SERT do not segregate. However, in the posterior part of the NAC shell, we confirmed the presence of a subtype of 5-HT immunoreactive axons that lack the SERT. Interestingly, about 90% of the 5-HT/VGLUT3 boutons were labelled for the SERT in this region, suggesting that VGLUT3 is preferentially located in SERT immunoreactive 5-HT boutons. This work demonstrate that VGLUT3 and SERT cannot be used as specific markers to classify the different subtypes of 5-HT axons.
... Putative 5-HT neurons were identified based initially on their firing characteristics (e.g., long-duration action potentials, regular firing pattern interrupted with burst activity). Next, neurons were identified as serotonergic based on well characterized responses to systemic administration (i.v.) of 5HT1AR agonist (8-OH-DPAT) and reversal with antagonist (WAY-100635) which restored 5-HT neuron firing to that of baseline (Fig. 6b-d) [15,33]. Figure 6a shows typical traces of 5-HT and non-5HT DR neurons. ...
Abstract Levodopa-induced dyskinesias (LID) are a prevalent side effect of chronic treatment with levodopa (L-DOPA) for the motor symptoms of Parkinson’s disease (PD). It has long been hypothesized that serotonergic neurons of the dorsal raphe nucleus (DRN) are capable of L-DOPA uptake and dysregulated release of dopamine (DA), and that this “false neurotransmission” phenomenon is a main contributor to LID development. Indeed, many preclinical studies have demonstrated LID management with serotonin receptor agonist treatment, but unfortunately, promising preclinical data has not been translated in large-scale clinical trials. Importantly, while there is an abundance of convincing clinical and preclinical evidence supporting a role of maladaptive serotonergic neurotransmission in LID expression, there is no direct evidence that dysregulated DA release from serotonergic neurons impacts LID formation. In this study, we ectopically expressed the DA autoreceptor D2Rs (or GFP) in the DRN of 6-hydroxydopamine (6-OHDA) lesioned rats. No negative impact on the therapeutic efficacy of L-DOPA was seen with rAAV-D2Rs therapy. However, D2Rs treated animals, when subjected to a LID-inducing dose regimen of L-DOPA, remained completely resistant to LID, even at high doses. Moreover, the same subjects remained resistant to LID formation when treated with direct DA receptor agonists, suggesting D2Rs activity in the DRN blocked dyskinesogenic L-DOPA priming of striatal neurons. In vivo microdialysis confirmed that DA efflux in the striatum was reduced with rAAV-D2Rs treatment, providing explicit evidence that abnormal DA release from DRN neurons can affect LID. This is the first direct evidence of dopaminergic neurotransmission in DRN neurons and its modulation with rAAV-D2Rs gene therapy confirms the serotonin hypothesis in LID, demonstrating that regulation of serotonergic neurons achieved with a gene therapy approach offers a novel and potent antidyskinetic therapy.
... Tonic firing is characterized by low frequency (0.1-3 Hz), and is classically defined as having clock-like, pace-maker regularity. Phasic firing characterized with burst of higher firing rates (up to 17 Hz) has indeed been reported in serotonin neurons (Allers and Sharp, 2003;Kocsis et al., 2006;Hajós et al., 2007). The precise control of neuronal activity that differentiates these two modes of release is not yet well understood. ...
Several lines of evidence implicate serotonin (5-hydroxytryptamine, 5-HT)in regulating personality traits and mood control. Serotonergic neurons are classically thought to be tonic regular-firing, “clock-like” neurons. Neurotransmission by serotonin is tightly regulated by the serotonin transporter (SERT) and by autoreceptors (serotonin receptors expressed by serotonin neurons) through negative feedback inhibition at the cell bodies and dendrites (5-HT1A receptors) of the dorsal raphe nuclei or at the axon terminals (5-HT1B receptors). In dorsal raphe neurons, the release of serotonin from vesicles in the soma, dendrites, and/or axonal varicosities is independent of classical synapses and can be induced by neuron depolarization, by the stimulation of L-type calcium channels, by activation of glutamatergic receptors, and/or by activation of 5-HT2 receptors. The resulting serotonin release displays a slow kinetic and a large diffusion. This process called volume transmission may ultimately affect the rate of discharge of serotonergic neurons, and their tonic activity. The therapeutic effects induced by serotonin-selective reuptake inhibitor (SSRI) antidepressants are initially triggered by blocking SERT but rely on consequences of chronic exposure, i.e., a selective desensitization of somatodendritic 5-HT1A autoreceptors. Agonist stimulation of 5-HT2B receptors mimicked behavioral and neurogenic SSRI actions, and increased extracellular serotonin in dorsal raphe. By contrast, a lack of effects of SSRIs was observed in the absence of 5-HT2B receptors (knockout-KO), even restricted to serotonergic neurons (Htr2b5-HTKO mice). The absence of 5-HT2B receptors in serotonergic neurons is associated with a higher 5-HT1A-autoreceptor reactivity and thus a lower firing activity of these neurons. In agreement, mice with overexpression of 5-HT1A autoreceptor show decreased neuronal activity and increased depression-like behavior that is resistant to SSRI treatment. We propose thus that the serotonergic tone results from the opposite control exerted by somatodendritic (Gi-coupled) 5-HT1A and (Gq-coupled) 5-HT2B receptors on dorsal raphe neurons. Therefore, 5-HT2B receptors may contribute to SSRI therapeutic effects by their positive regulation of adult raphe serotonergic neurons. Deciphering the molecular mechanism controlling extrasynaptic release of serotonin, and how autoreceptors interact in regulating the tonic activity of serotonergic neurons, is critical to fully understand the therapeutic effect of SSRIs.
... However, we found that an early adolescence exposure to MPH leads to increased burst activities of DRN serotonin neurons (Figure 3(b)). In a similar way to dopamine neurons (Overton and Clark, 1997), bursting activity of DRN serotonin neurons is known to result in a greater efficiency of serotonin release in serotonin-projecting-region (Gartside et al., 2000;Hajos and Sharp, 1996;Hajos et al., 2007;Hurtig et al., 2007). Some serotonin neurons in the DRN may have adapted their firing mode Figure 6. ...
Background:
Psychostimulants like methylphenidate or D-amphetamine are often prescribed for attention deficit and hyperactivity disorders in children. Whether such drugs can be administered into a developing brain without consequences in adulthood is still an open question.
Methods:
Here, using in vivo extracellular electrophysiology in anesthetised preparations, combined with behavioural assays, we have examined the long-term consequences in adulthood of a chronic methylphenidate oral administration (5 mg/kg/day, 15 days) in early adolescent (post-natal day 28) and late adolescent (post-natal day 42) rats, by evaluating body weight change, sucrose preference (indicator of anhedonia), locomotor sensitivity to D-amphetamine and electrical activities of ventral tegmental area dopamine and dorsal raphe nucleus serotonin neurons.
Results:
Chronic methylphenidate treatment during early or late adolescence did not induce weight deficiencies and anhedonia-like behaviours at adulthood. However, it increased bursting activities of dorsal raphe nucleus serotonin neurons. Furthermore, chronic methylphenidate treatment during early but not during late adolescence enhanced D-amphetamine-induced rearing activity, as well as ventral tegmental area dopamine cell excitability (firing, burst and population activity), associated with a partial desensitisation of dopamine D2 auto-receptors.
Conclusions:
We have demonstrated here that early, but not late, adolescent exposure to oral methylphenidate may induce long-lasting effects on monoamine neurotransmission. The possible clinical implication of these data will be discussed.
... Rates as high as 20 Hz in cat DRN were reported under glutamate application by Levine and Jacobs (1992). Bursting is also sometimes reported in these cells (Hájós et al., 1996;Hájós et al., 2007). As remarked above, DA cells in vivo generally exhibit burst firing and in vitro pacemaking was found to have an average value of 5.4 Hz in one experiment (Grenhoff et al., 1988). ...
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.
... 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.
... Extracellular in vivo recordings in the DR paired with juxtacellular labeling has revealed small subsets of 5-HT neurons with unique properties, including fast firing rates, bursting activity, and synchronization with theta rhythm (Allers and Sharp, 2003; Kocsis et al., 2006; Hajos et al., 2007). Previously we found differences between median raphe and vmDR 5-HT membrane properties in the rat (). ...
Anxiety disorders are prevalent in human and veterinary medicine yet the underlying mechanism is poorly understood. Because serotonin (5-HT) neurons of the dorsal raphe (DR) are thought to play a prominent role, my goal was to understand the changes in DR 5-HT neurons that underlie anxiety and other stress-related disorders. Two DR subdivisions were studied in a series of experiments: the ventromedial DR (vmDR), a well characterized subfield with a high density of 5-HT neurons, and the lateral wing DR (lwDR), a largely uncharacterized subfield with a more sparse distribution of 5-HT neurons. Many stress paradigms activate 5-HT neurons of the lwDR more so than 5-HT neurons of the vmDR, suggesting a unique role for lwDR 5-HT cells in stress circuits. However, it is not known if lwDR 5-HT neurons possess physiological characteristics that contribute to their increased propensity to be activated by a stressor. I found that lwDR 5-HT neurons demonstrated increased intrinsic excitability, increased glutamatergic input, and similar GABAergic input when compared to vmDR 5-HT neurons. Using the chronic social defeat model of anxiety, the distinctions between lwDR and vmDR neurons were explored further. Social defeat induced anxious behavior and stress-associated pathological changes in the peripheral organs of intruder mice. For the first time, investigation into the neural mechanisms of social defeat has focused on 5-HT neuron physiology, revealing subregion-specific effects within the DR. Increased excitability was seen in the vmDR neurons of the most anxious mice. This was accompanied by a decrease in GABAergic input to vmDR 5-HT neurons potentially mediated by both presynaptic and postsynaptic changes. The lwDR 5-HT neurons demonstrated distinct stress-induced changes limited to the slower kinetics of postsynaptic GABAAR. The differential effect of social stress on inhibitory input to vmDR or lwDR neurons suggest that the 5-HT output in brain regions targeted by each subfield is differentially affected in anxiety disorders. Collectively these findings help fill the gap in our understanding of local DR circuitry, the heterogeneity of 5-HT neurons, and the distinct regulation of vmDR and lwDR neurons in the circuits that mediate stress and contribute to the pathophysiology of anxiety.
... Information regarding the neurochemical identity of the recorded neurons can be obtained using a variety of strategies, including post-mortem immunohistochemistry combined with juxtacellular recording/labeling, but usually with a trade-off between reliability and difficulty. Indeed, various parameters of the recorded physiological signals is thought to provide information about the cell type being recorded, for example the shape and duration of action potentials, but the reliability of this simple method to define the neurochemical identity of the targeted neuron remains debatable (Allers and Sharp, 2003;Hajos et al., 2007;Sapin et al., 2010). Pharmacological tools can also provide information on the identity of the neurons being recorded, for example the purportedly selective expression of inhibitory autoreceptors on serotonergic (Courtney and Ford, 2016) and noradrenergic (Schlicker and Gothert, 1998) neurons or SK channels on dopaminergic neurons (Koulchitsky et al., 2012) makes them identifiable using a systemic drug administration while recording single unit activity. ...
Monoamines are key neuromodulators involved in a variety of physiological and pathological brain functions. Classical studies using physiological and pharmacological tools have revealed several essential aspects of monoaminergic involvement in regulating the sleep-wake cycle and influencing sensory responses but many features have remained elusive due to technical limitations. The application of optogenetic tools led to the ability of monitoring and controlling neuronal populations with unprecedented temporal precision and neurochemical specificity. Here, we focus on recent advances in revealing the roles of some monoamines in brain state control and sensory information processing. We summarize the central position of monoamines in integrating sensory processing across sleep-wake states with an emphasis on research conducted using optogenetic techniques. Finally, we discuss the limitations and perspectives of new integrated experimental approaches in understanding the modulatory mechanisms of monoaminergic systems in the mammalian brain.
... Recent work has also described the heterogeneity in physiological properties of 5-HT neurons in different DRN subfields that have known projections to dissimilar forebrain regions that regulate different types of behavior . Therefore, it will be important to probe a In agreement with the findings here, previous studies had suggested preferential innervation of DRN GABA neurons by vmPFC terminals Hajós et al., 2007;. Given these reports and our previous work showing that DRN GABA neurons locally synapse on and inhibit 5-HT neurons , we are presented with a putative circuit whereby DRN GABA neurons are positioned critically to gate top-down drive of the DRN and 5-HT output that would subsequently influence affective regulation. ...
Regulation of social behaviors is necessary to achieve social inclusion, establish relationships and sustain those relationships through adversity. Impairments in socio-emotional function and competence are prominent and debilitating features of major depression, yet are not traditionally recognized as cardinal symptoms of the disease. However, these deficits often persist in patients whose other mood symptoms have remitted and can predict risk of relapse, indicating an important role as a vulnerability factor. Understanding the neurobiology of socioaffective dysfunction in depression is thus important for determining the pathology of the disorder and developing effective treatments. Human imaging studies of depressive patients have consistently reported abnormal activity in the ventromedial prefrontal cortex (vmPFC), an area important for emotional processing and social cognition. Tracing studies in animals and tractography in humans have shown that the dorsal raphe nucleus (DRN) is a major projection target of the vmPFC. The DRN contains the most serotonin (5-HT) producing neurons in the brain and its output has been shown to regulate behaviors along an affiliative-agonistic axis, however it is neuronally heterogeneous. This thesis investigated the cytoarchitecture of the vmPFC-DRN microcircuit and its relevance to socioaffective behaviors using genetic mapping, whole cell electrophysiology and optogenetics. I showed that GABAergic neurons, which are the primary non-serotonergic neuronal population in the DRN, mediated top-down projections from the vmPFC onto mood-regulating 5-HT neurons and demonstrated the relevance of this pathway in mediating socioaffective decisions using the chronic social defeat stress (CSDS) paradigm. In addition, I used deep brain stimulation of the vmPFC as an antidepressant model to show that therapeutic response may rely on restoring the excitatory/inhibitory balance of inputs to 5-HT neurons. Together, these results will provide a better understanding of socioaffective circuitry and could lead to the development of more effective and efficient strategies to treat mood disorders.
... Hz were recorded (Vandermaelen and Aghajanian, 1983). The start of a burst for DRN 5-HT neurons was defined as the occurrence of 2 spikes within 20 ms; end of a burst was defined as an ISI4 20 ms (Hajós et al., 2007). The following burst parameters were studied: spikes/minute, percent spikes in burst, spikes/burst, and interspike interval (ISI) in bursts. ...
... The small depth and low stability of modulation implies non-cell-autonomous processes possibly generated by a combination of top-down cortical influences and a fluctuating local activity. Indeed, low frequency LFP oscillations and the slow rhythmic modulation of discharge have been documented in the midbrain raphe nuclei (Hajos et al. 2007). ...
Key points:
•Median raphe is a key subcortical modulatory centre involved in several brain functions e.g. regulation of sleep-wake cycle, emotions and memory storage. •A large proportion of median raphe neurones are glutamatergic and implement a radically different mode of communication than serotonergic cells, but their in vivo activity is unknown. •We provide the first description of the in vivo, brain state-dependent firing properties of median raphe glutamatergic neurones identified by immunopositivity for the vesicular glutamate transporter type 3 (VGluT3) and serotonin (5HT). Glutamatergic populations (VGluT3+/5HT- and VGluT3+/5HT+)were compared to the purely serotonergic (VGluT3-/5HT+) and VGluT3-/5HT- neurones. •VGluT3+/5HT+ neurones fired similar to VGluT3-/5HT+ cells, whereas significantly diverged from the VGluT3+/5HT- population. Activity of the latter subgroup resembled the spiking of VGluT3-/5HT- cells, except their diverging response to sensory stimulation. •The VGluT3+ population of the median raphe may broadcast rapidly varying signals on top of a state-dependent, tonic modulation.
Abstract:
Subcortical modulation is crucial for information processing in the cerebral cortex. Besides the canonical neuromodulators, glutamate has recently been identified as a key cotransmitter of numerous monoaminergic projections. In the median raphe, a pure glutamatergic neurone population projecting to limbic areas was also discovered with a possibly novel, yet undetermined function. Here, we report the first functional description of the vesicular glutamate transporter type 3 (VGluT3)-expressing median raphe neurones. Since there is no appropriate genetic marker for the separation of serotonergic (5HT+) and non-serotonergic (5HT-) VGluT3+ neurones, we utilised immunohistochemistry after recording and juxtacellular labelling in anaesthetised rats. VGluT3+/5HT- neurones fired faster, more variably and were permanently activated during sensory stimulation, as opposed to the transient response of the slow firing VGluT3-/5HT+ subgroup. VGluT3+/5HT- cells were also more active during hippocampal theta. In addition, the VGluT3-/5HT- population - putative GABAergic cells - resembled the firing of VGluT3+/5HT- neurones, but without significant reaction to the sensory stimulus. Interestingly, the VGluT3+/5HT+ group - spiking slower than the VGluT3+/5HT- population - exhibited a mixed response i.e. the initial transient activation was followed by sustained elevation of firing. Phase coupling to hippocampal and prefrontal slow oscillations was found in VGluT3+/5HT- neurones, also differentiating them from the VGluT3+/5HT+ subpopulation. Taken together, glutamatergic neurones in the median raphe may implement multiple, highly divergent forms of modulation in parallel: a slow, tonic mode interrupted by sensory-evoked rapid transients and a fast one, capable of conveying complex patterns influenced by sensory inputs. This article is protected by copyright. All rights reserved.
... The response diversity may reflect the heterogeneity of DRN neurons in morphology, location and neurotransmitter phenotypes [34][35][36] . It is challenging to precisely identify neuron types using electrophysiological criteria in extracellular recordings 37,38 . Using optogenetic tagging, two recent recordings revealed that reward-predicting cues activate approximately half of 5-HT neurons 17,39 . ...
The dorsal raphe nucleus (DRN) is involved in organizing reward-related behaviours; however, it remains unclear how genetically defined neurons in the DRN of a freely behaving animal respond to various natural rewards. Here we addressed this question using fibre photometry and single-unit recording from serotonin (5-HT) neurons and GABA neurons in the DRN of behaving mice. Rewards including sucrose, food, sex and social interaction rapidly activate 5-HT neurons, but aversive stimuli including quinine and footshock do not. Both expected and unexpected rewards activate 5-HTneurons. After mice learn to wait for sucrose delivery, most 5-HT neurons fire tonically during waiting and then phasically on reward acquisition. Finally, GABA neurons are activated by aversive stimuli but inhibited when mice seek rewards. Thus, DRN 5-HT neurons positively encode a wide range of reward signals during anticipatory and consummatory phases of reward responses. Moreover, GABA neurons play a complementary role in reward processing.
Serotonin is synthesized from tryptophan in small groups of neurons within the central nervous system. These neurons, however, branch profusely and innervate all the nervous system, where, by releasing serotonin in different manners, they regulate a myriad of functions, including many behaviors. This chapter reviews the main functions of serotonin in the nervous system of invertebrates and vertebrates, showing that many of these have been conserved throughout evolution. It also summarizes the current knowledge about the mechanisms that control and regulate serotonin secretion from different compartments of the same neurons, evidencing their differences, which enable small numbers of neurons to display a wide variety of functions, including the regulation of our mood states.
Background: There is growing evidence that the treatment of several mental disorders can potentially benefit from activation of delta-opioid receptors. In the future, delta-agonists with a safe pharmacological profile can be used for the treatment of mood disorders in pregnant women. However, the data on prenatal exposure to delta-opioid agonists are missing. The present study is aimed to test the hypothesis that the activation of delta-opioid receptors during gravidity has positive effects on the behaviour accompanied by changes in glutamate and monoamine neurotransmission.
Methods: Gestating Wistar rats were chronically treated with a selective delta-agonist SNC80 or vehicle. Adult male and female offspring underwent novel object recognition (for the assessment of cognition) and open field (for the assessment of anxiety and habituation) tests, followed by in vivo electrophysiological examination of the activity of hippocampal glutamate and midbrain serotonin (5-HT) and dopamine neurons.
Results: We found that the maternal treatment with SNC80 did not affect the offspring’s anxiety, habituation, and 5-HT neuronal firing activity. Female offspring of SNC80-treated dams exhibited improved novelty recognition associated with decreased firing rate and burst activity of glutamate and dopamine neurons.
Conclusion: Maternal treatment with delta-opioid agonists during gestation may have a pro-cognitive effect on offspring without any negative effects on anxiety and habituation. The putative pro-cognitive effect might be mediated via mechanism(s) involving the firing activity of hippocampal glutamate and mesolimbic dopamine neurons.
When a neuron breaks silence, it can emit action potentials in a number of patterns. Some responses are so sudden and intense that electrophysiologists felt the need to single them out, labelling action potentials emitted at a particularly high frequency with a metonym – bursts. Is there more to bursts than a figure of speech? After all, sudden bouts of high‐frequency firing are expected to occur whenever inputs surge. The burst coding hypothesis advances that the neural code has three syllables: silences, spikes and bursts. We review evidence supporting this ternary code in terms of devoted mechanisms for burst generation, synaptic transmission and synaptic plasticity. We also review the learning and attention theories for which such a triad is beneficial. image
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.
Applying electrophysiological recording techniques as considerable technological progress in neurophysiological methods has provided the major advance in the neuroscience research and the study of brain–behavior relationships. This chapter presents the main electrophysiological methods currently used to study the effects of antidepressant treatments on neurotransmission. Electrophysiological studies combined with local or systemic administration of pharmacological agents will provide worthwhile strategy elucidating the pharmacological bases for reciprocal interactions of neurotransmission systems in vivo. The chapter provides authoritative review of commonly used methodologies in the field related to electrophysiological effects of antidepressant treatments on neurotransmission in the basic research level that has translational value for clinical settings.Key wordsAntidepressant
Electrophysiology
Extracellular recording
SSRIs
SNRIs
TCAs
MAOIs
NDRIs
TRIs
Degenerate neural circuits perform the same function despite being structurally different. However, it is unclear whether neural circuits with interacting neuromodulator sources can themselves be degenerate while maintaining the same neuromodulatory function. Here, we address this by computationally modelling the neural circuits of neuromodulators serotonin and dopamine, local glutamatergic and GABAergic interneurons, and their possible interactions, under reward/punishment-based conditioning tasks. We show that a single parsimonious, sparsely connected neural circuit model can recapitulate several separate experimental findings, but not all, suggesting potentially multiple circuits acting in parallel. Using computational simulations and dynamical systems analysis, we demonstrate that several different stable circuit architectures can produce the same observed network activity profile. Further, simulating dopamine (D2) receptor agonist can distinguish among sub-groups of these degenerate networks; a testable model prediction. Overall, this theoretical work suggests the plausibility of degeneracy within neuromodulator circuitry and has important implication for the stable and robust maintenance of neuromodulatory functions.
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.
Despite a wealth of clinical and preclinical data implicating the serotonin (5-HT) system in fear-related affective disorders, a precise definition of this neuromodulator's role in fear remains elusive. Using convergent anatomical and functional approaches, we interrogate the contribution to fear of basal amygdala (BA) 5-HT inputs from the dorsal raphe nucleus (DRN). We show the DRN→BA 5-HT pathway is engaged during fear memory formation and retrieval, and activity of these projections facilitates fear and impairs extinction. The DRN→BA 5-HT pathway amplifies fear-associated BA neuronal firing and theta power and phase-locking. Although fear recruits 5-HT and VGluT3 co-expressing DRN neurons, the fear-potentiating influence of the DRN→BA 5-HT pathway requires signaling at BA 5-HT1A/2A receptors. Input-output mapping illustrates how the DRN→BA 5-HT pathway is anatomically distinct and connected with other brain regions that mediate fear. These findings reveal how a discrete 5-HT circuit orchestrates a broader neural network to calibrate aversive memory.
Neurons that synthesize and release 5-hydroxytryptamine (5-HT; serotonin) express a core set of genes that establish and maintain this neurotransmitter phenotype and distinguish these neurons from other brain cells. Beyond a shared 5-HTergic phenotype, these neurons display divergent cellular properties in relation to anatomy, morphology, hodology, electrophysiology and gene expression, including differential expression of molecules supporting co-transmission of additional neurotransmitters. This diversity suggests that functionally heterogeneous subtypes of 5-HT neurons exist, but linking subsets of these neurons to particular functions has been technically challenging. We discuss recent data from molecular genetic, genomic and functional methods that, when coupled with classical findings, yield a reframing of the 5-HT neuronal system as a conglomeration of diverse subsystems with potential to inspire novel, more targeted therapies for clinically distinct 5-HT-related disorders.
Electrophysiological methods are commonly used in neuroscience and pharmacology to reveal the mechanisms of drug action. In vivo analysis of the mechanisms of drug action is a particularly important method in neuropharmacology. Here, we show the juxtacellular recording method to characterize the electrophysiological and neurochemical properties of neurons. Using juxtacellular recording, researchers can record the membrane potential from single neurons, and examine action potential parameters, such as the width and coefficient variance of inter-spike intervals. Additionally, recorded neurons can be labeled using neurobiotin, and neurochemical properties can be revealed by a combination of immunohistochemical staining and in situ hybridization. We introduce an experiment testing the effects of a phosphodiesterase 4 (PDE4) inhibitor on the fronto-striatal circuit using juxtacellular recording. The cerebral cortex-nucleus accumbens (NAcc)-external segment of globus pallidus (GPe)-subthalamic nucleus (STN)-substantia nigra pars reticulata (SNr) pathway is the neurobiological basis of many neuropsychiatric disorders. Several components of this pathway are particularly important for the regulation of motor action and cognitive function: 1) STN-SNr pathway (hyperdirect pathway), 2) NAcc-SNr pathway (direct pathway), and 3) GPe-STN-SNr pathway (indirect pathway). Researchers can record tri-phasic responses reflecting these pathways using electro-stimulation in cerebral cortex. A PDE4 inhibitor, roflumilast, affected the 2) direct pathway as well as the 3) indirect pathway, but not the 1) hyperdirect pathway. The current findings suggest that PDE4 inhibition could be considered as a possible treatment for cognitive deficits related to fronto-striatal disorders such as attention deficit/hyperactivity disorder, and Parkinson's disease.
Unlabelled:
Orexins (hypocretins) are neuropeptides that regulate multiple homeostatic processes, including reward and arousal, in part by exciting serotonergic dorsal raphe neurons, the major source of forebrain serotonin. Here, using mouse brain slices, we found that, instead of simply depolarizing these neurons, orexin-A altered the spike encoding process by increasing the postspike afterhyperpolarization (AHP) via two distinct mechanisms. This orexin-enhanced AHP (oeAHP) was mediated by both OX1 and OX2 receptors, required Ca(2+) influx, reversed near EK, and decayed with two components, the faster of which resulted from enhanced SK channel activation, whereas the slower component decayed like a slow AHP (sAHP), but was not blocked by UCL2077, an antagonist of sAHPs in some neurons. Intracellular phospholipase C inhibition (U73122) blocked the entire oeAHP, but neither component was sensitive to PKC inhibition or altered PKA signaling, unlike classical sAHPs. The enhanced SK current did not depend on IP3-mediated Ca(2+) release but resulted from A-current inhibition and the resultant spike broadening, which increased Ca(2+) influx and Ca(2+)-induced-Ca(2+) release, whereas the slower component was insensitive to these factors. Functionally, the oeAHP slowed and stabilized orexin-induced firing compared with firing produced by a virtual orexin conductance lacking the oeAHP. The oeAHP also reduced steady-state firing rate and firing fidelity in response to stimulation, without affecting the initial rate or fidelity. Collectively, these findings reveal a new orexin action in serotonergic raphe neurons and suggest that, when orexin is released during arousal and reward, it enhances the spike encoding of phasic over tonic inputs, such as those related to sensory, motor, and reward events.
Significance statement:
Orexin peptides are known to excite neurons via slow postsynaptic depolarizations. Here we elucidate a significant new orexin action that increases and prolongs the postspike afterhyperpolarization (AHP) in 5-HT dorsal raphe neurons and other arousal-system neurons. Our mechanistic studies establish involvement of two distinct Ca(2+)-dependent AHP currents dependent on phospholipase C signaling but independent of IP3 or PKC. Our functional studies establish that this action preserves responsiveness to phasic inputs while attenuating responsiveness to tonic inputs. Thus, our findings bring new insight into the actions of an important neuropeptide and indicate that, in addition to producing excitation, orexins can tune postsynaptic excitability to better encode the phasic sensory, motor, and reward signals expected during aroused states.
Our article in this journal some 15 years ago focussed on the role of serotonin (5-HT) autoreceptors in the mechanism of action of antidepressant drugs. Specifically in this regard, the results were summarised of rat microdialysis studies carried out to examine: (a) the relative importance of 5-HT1A and 5-HT1B autoreceptors, including (b) possible regional variation, and (c) potential changes in autoreceptor responsiveness following chronic selective serotonin reuptake inhibitor administration. In the present reflection piece, I recap some of the key findings against a brief background and provide an account of their bearing within the context of subsequent endeavours in the antidepressant drug research and development field. I conclude by shortly commenting on selected topics relevant to novel, interesting advances and avenues for future research.
The psychological feelings produced by cannabis have been described as fatuous euphoria, elation, and talkativeness. Alternatively, cannabis can induce low mood and depression especially after chronic use. Despite these clinical evidences, little was known about the capacity of cannabis to modulate serotonin (5-Hydroxytryptamine, 5-HT), the main neurotransmitter implicated in the regulation of mood and the pathology of mood disorders.
In the past 10 years, our laboratory has attempted to clarify how the cannabinoid type 1 receptor (CB1R) agonists, antagonists, and the fatty acid amide hydrolase (FAAH) inhibitors modulate the firing activity of 5-HT neurons located in the dorsal raphe nucleus.
While the CB1R agonist R-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolol[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate (WIN 55,212-2) produces a bell-shaped curve, increasing 5-HT firing at low doses (0.1–0.3 mg/kg) and decreasing firing at higher doses (>0.3 mg/kg), the FAAH inhibitor [3-(3-carbamoylphenyl)phenyl] N-cyclohexylcarbamate (URB597) produces a sigma-shaped curve, with a plateau at the highest doses tested (0.3 mg/kg). Δ9-Tetrahydrocannabinol (THC) produces a mixed response on 5-HT firing activity with 26 % of neurons showing an increase, 33 % showing a decrease, and 42 % showing no response. However, after 4 days, intraperitoneal (i.p.) injections of THC (1 mg/kg) produced a significant elevation of firing. The increase in firing following WIN 55,212-2 and THC was prevented by the CB1R antagonist rimonabant. Finally, both WIN 55,212-2 and THC evoked a robust decrease in 5-HT firing after long-term administration in adolescence.
These data show that CB1R agonists and FAAH inhibitors interact with the 5-HT system and that this effect may be related to emotional behaviors.
In combination, anxiety and depression afflict nearly one-third of the United States adult population. Mood and anxiety disorders have long been linked to dysregulation of the serotonin (5-HT) system. As one of the main sources of 5-HT to the forebrain, the dorsal raphe nucleus (DRN) plays a major role in both of these illnesses. The purpose of this chapter is to describe how in vitro and in vivo electrophysiological properties of DRN 5-HT cells, in relationship to their local and broad anatomical connectivity, relate to the pathophysiology of anxiety and depression. Specific DRN subregions will be discussed with regard to their afferent and efferent connections and how differences in regional discharge properties may contribute to these disorders. Lastly, the impact of two neuromodulators, GABA and nitric oxide, on 5-HT neurotransmission within the DRN and the putative contributions of these transmitters to the stress response will be addressed with the goal of providing insight into alternative treatments for stress-related psychopathology.
Serotonergic neurons are spontaneously active with slow regular discharge during alert behavior and decreased activity during sleep. Sleep-related inhibition of serotonergic neurons is mediated by GABAergic inputs that originate in hypothalamic and brainstem sleep centers. During alert behavior, tonic release of GABA contributes to feedback and feedforward inhibitory circuits that, together with serotonin autoreceptors, prevent excess stimulation of serotonergic neurons. Glutamatergic inputs to the raphe originate in the brainstem, several hypothalamic nuclei and cerebral cortex. Although glutamate receptor agonists strongly stimulate serotonergic neuronal discharge, the physiological significance of glutamatergic inputs is not well established. In the dorsal raphe nucleus (DRN), more glutamatergic fibers terminate on GABAergic than serotonergic neurons. Moreover, GABA normally restrains the excitatory influence of glutamatergic inputs to serotonergic neurons in the DRN. Peptidergic neurons modulate the activity of GABAergic and glutamatergic interneurons that synapse with serotonergic neurons in the DRN. These neuropeptides, for example CRF, endogenous opioids and Substance P, are implicated in responses to environmental challenges. Thus, stress can indirectly influence the activity of serotonergic neurons. In the raphe, GABAergic and glutamatergic interneurons may serve as a final common pathway for integrating information about environmental challenges and inputs from hypothalamic and brainstem centers that control the usual sleep-related inhibition of serotonergic neurons. Neuropeptides might thereby promote alert behavior to appropriately cope with stress. However, persistent peptidergic-induced changes in the strength of GABAergic and glutamatergic inputs to serotonergic neurons could contribute to insomnia, anxiety and major psychiatric disorders such as depression and schizophrenia.
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.
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.
We describe a novel and very effective single-cell labeling method with unique advantages for revealing the axonal and dendritic fields of any extracellularly recorded neuron. This procedure involves the use of fine glass micro-pipettes (tip diameter: approximately 1 micron), which contain biocytin or Neurobiotin dissolved in a salt solution, for the simultaneous juxtacellular recording and tracer iontophoresis. Once a neuron is well-isolated and identified, low intensity (< 10 nA) positive-current pulses are injected by way of the micro-electrode such as to modulate its firing. Juxtacellular tracer iontophoresis may last as long as the cell electrophysiologically remains in good health, while determining some of its physiological properties. Control experiments, including the selective killing of previously injected cells, provide convincing evidence that it is the stained unit that was recorded and 'tickled' by the juxtamembranous iontophoretic pulses. Electrophysiological and histochemical data further show that neuronal filling could occur during an electrically induced, transient, physical micro-damage of a somatic or dendritic membrane patch. This simple, single-cell staining method has been used to label several types of rat brain neurons, including projection neurons and interneurons. Its success rate ( > 86%) far exceeds that obtained by direct intracellular injections of tracers as shown by the labeling of a large sample of 100 individual cells (from 115 attempts) in the thalamic reticular (Rt) nucleus of 33 rats. We thereby demonstrate that Rt cells project to restricted regions of a single thalamic nucleus, including anterior thalamic nuclei, and that the thalamus and Rt complex have reciprocal connections. The juxtacellular procedure thus represents an ideal directed single-cell labeling tool for determination of functional properties, for subsequent identification, for delineation of overall neuronal architecture and for tracing neuronal pathways, provided care is taken to avoid the possible drawbacks and pitfalls that are illustrated and discussed in the present paper.
Substance P receptor [neurokinin 1 (NK1] antagonists (SPAs) represent a novel mechanistic approach to antidepressant therapy with comparable clinical efficacy to selective serotonin reuptake inhibitors (SSRIs). Because SSRIs are thought to exert their therapeutic effects by enhancing central serotonergic function, we have examined whether SPAs regulate neuronal activity in the dorsal raphe nucleus (DRN), the main source of serotonergic projections to the forebrain. Using in vivo electrophysiological techniques in the guinea pig, we found that administration of the highly selective NK1 receptor antagonist 1-(5-[[(2R,3S)-2-([(1R)-1-[3,5-bis(trifluoromethyl)phenyl]ethyl]oxy)-3-(4-phenyl)morpholin-4-yl]methyl]-2H-1,2,3-triazol-4-yl)-N,N-dimethylmethanamine (L-760735) caused an increase in DRN neuronal firing rate. However, unlike chronic treatment with fluoxetine, there was no detectable 5-HT1A autoreceptor desensitization. In vitro electrophysiological investigation showed that these effects were not mediated by a direct action in the DRN, an observation supported by immunocytochemical analysis that identified the lateral habenula (LHb) as a more likely site of action. Subsequently, we found that local application of L-760735 into the LHb increased firing in the DRN, which, together with our data showing that L-760735 increased metabolic activity in the cingulate cortex, amygdala, LHb, and DRN, indicates that the effects of L-760735 may be mediated by disinhibition of forebrain structures acting via a habenulo raphe projection. These findings support other evidence for an antidepressant profile of SPAs and suggest that regulation of DRN neuronal activity may contribute to their antidepressant mechanism of action but in a manner that is distinct from monoamine reuptake inhibitors.
The dorsal raphe nucleus (DR) has a topographic neuroanatomy consistent with the idea that different parts of this nucleus subserve different functions. Here we use dual in situ hybridization to describe the rostral-caudal neurochemical distribution of three major cell groups, serotonin (5-hydroxytryptamine; 5-HT), gamma-aminobutyric acid (GABA), and catecholamine, and their relative colocalization with each other and mRNA encoding four different receptor subtypes that have been described to influence DR responses, namely, 5HT-1A, alpha(1b) adrenergic (alpha(1b) ADR), and corticotropin-releasing factor type 1 (CRF-R1) and 2 (CRF-R2) receptors. Serotonergic and GABAergic neurons were distributed throughout the rostral-caudal extent of the DR, whereas catecholaminergic neurons were generally restricted to the rostral half of the nucleus. These phenotypes essentially represent distinct cell populations, because the neurochemical markers were rarely colocalized. Both 5HT-1A and alpha(1b) ADR mRNA were highly expressed throughout the DR, and the vast majority of serotonergic neurons expressed both receptors. A smaller percentage of GABAergic neurons also expressed 5HT-1A or alpha(1b) ADR mRNA. Very few catecholaminergic cells expressed either 5HT-1A or alpha(1b) ADR mRNA. CRF-R1 mRNA was detected only at very low levels within the DR, and quantitative colocalization studies were not technically feasible. CRF-R2 mRNA was mainly expressed at the middle and caudal levels of the DR. At midlevels, CRF-R2 mRNA was expressed exclusively in serotonin neurons, whereas, at caudal levels, approximately half the CRF-R2 mRNA was expressed in GABAergic neurons. The differential distribution of distinct neurochemical phenotypes lends support to the idea of functional differentiation of the DR.
The dorsal raphe nucleus (DRN) projects serotonergic axons throughout the brain and is involved in a variety of physiological functions. However, it also includes a large population of cells that contain other neurotransmitters. To clarify the physiological and pharmacological differences between the serotonergic and nonserotonergic neurons of the DRN, their postsynaptic responses to 5-hydroxytryptamine (5-HT, serotonin) and to selective activation of 5-HT1A or 5-HT2A/C receptors and their action potential characteristics were determined using in vitro patch-clamp recordings. The slices containing these neurons were then immunostained for tryptophan hydroxylase (TPH), a marker of serotonergic neurons. It was found that subpopulations of both serotonergic and nonserotonergic neurons responded to 5-HT with outward (i.e., inhibitory) and inward (i.e., excitatory) currents, responded to both 5-HT1A and 5-HT2A/C receptor activation with outward and inward currents, respectively, and displayed overlapping action potential characteristics. These findings suggest that serotonergic and nonserotonergic neurons in the DRN are both heterogeneous with respect to their individual pharmacological and electrophysiological characteristics. The findings also suggest that the activity of the different populations of DRN neurons will display heterogeneous changes when the serotonergic tone in the DRN is altered by neurological disorders or by drug treatment.
In the present study, changes of the neuronal activity of 5-hydroxytrypamine (5-HT) neurons of dorsal raphe nucleus(DRN) in a rat model of Parkinson's disease (PD) were investigated with glass microelectrode recording. The results showed that the discharge rates of 5-HT neurons in control and PD rats were (1.61+/-0.56) Hz and (2.61+/-1.97) Hz, respectively. The discharge rate of PD rats was significantly increased when compared to that of the control rats. In control rats, 79% of 5-HT neurons discharged regularly and 21% in bursts. In PD rats, however, 36% of 5-HT neurons discharged regularly, 16% irregularly and 47% in bursts. The percentage of 5-HT neurons discharging in bursts was obviously higher than that of the control rats (P<0.05). The data suggest that the discharge rate and bursting pattern of 5-HT neurons in DRN are increased in a rat model of Parkinson's disease.
Although anecdotal reports suggest that cannabis may be used to alleviate symptoms of depression, the psychotropic effects and abuse liability of this drug prevent its therapeutic application. The active constituent of cannabis, Δ⁹-tetrahydrocannabinol, acts by binding to brain CB1 cannabinoid receptors, but an alternative approach might be to develop agents that amplify the actions of endogenous cannabinoids by blocking their deactivation. Here, we show that URB597, a selective inhibitor of the enzyme fatty-acid amide hydrolase, which catalyzes the intracellular hydrolysis of the endocannabinoid anandamide, exerts potent antidepressant-like effects in the mouse tail-suspension test and the rat forced-swim test. Moreover, URB597 increases firing activity of serotonergic neurons in the dorsal raphe nucleus and noradrenergic neurons in the nucleus locus ceruleus. These actions are prevented by the CB1 antagonist rimonabant, are accompanied by increased brain anandamide levels, and are maintained upon repeated URB597 administration. Unlike direct CB1 agonists, URB597 does not exert rewarding effects in the conditioned place preference test or produce generalization to the discriminative effects of Δ⁹-tetrahydrocannabinol in rats. The findings support a role for anandamide in mood regulation and point to fatty-acid amide hydrolase as a previously uncharacterized target for antidepressant drugs.
• depression
• endocannabinoid
• fatty-acid amide hydrolase
• serotonin
• URB597
The serotonergic system plays a key role in the regulation of brain states, and many of the known features of serotonergic neurons appear to match this function. Midbrain raphe nuclei provide a diffuse projection to all regions of the forebrain, and raphe neurons exhibit a slow metronome-like activity that sets the ambient levels of serotonin across the sleep–wake cycle. Serotonergic cells have also been implicated, however, in a variety of more specific functions that can hardly be related to their low-rate monotonous patterns of discharges. The amazing variety of serotonergic receptors and their type-specific distribution on cortical neurons also raise the possibility of a more intimate coordination between the activity of serotonergic neurons and their target cortical circuits. Here we report an unexpected diversity in the behavior of immunohistochemically identified serotonergic neurons. Two outstanding subpopulations were identified by using the in vivo juxtacellular recording and labeling technique. The first subpopulation of serotonergic cells exhibited the classic clock-like activity with no apparent short timescale interaction with the hippocampal electroencephalogram. The other subpopulation discharged action potentials that were phase-locked to the hippocampal theta rhythm, the oscillatory pattern associated with acquisition of information and memory formation. These results indicate that the ascending serotonergic system comprises cells involved in complex information processing beyond the regulation of state transitions. The heterogeneity of serotonergic neuron behavior can also help to explain the complexity of symptoms associated with serotonergic dysfunction.
• hippocampus
• juxtacellular labeling
• midbrain raphe
• neuronal oscillations
Although anecdotal reports suggest that cannabis may be used to alleviate symptoms of depression, the psychotropic effects and abuse liability of this drug prevent its therapeutic application. The active constituent of cannabis, Delta(9)-tetrahydrocannabinol, acts by binding to brain CB, cannabinoid receptors, but an alternative approach might be to develop agents that amplify the actions of endogenous cannabinoids by blocking their deactivation. Here, we show that URB597, a selective inhibitor of the enzyme fatty-acid amide hydrolase, which catalyzes the intracellular hydrolysis of the endocannabinoid anandamide, exerts potent antidepressant-like effects in the mouse tail-suspension test and the rat forced-swim test. Moreover, URB597 increases firing activity of serotonergic neurons in the dorsal raphe nucleus and noradrenergic neurons in the nucleus locus ceruleus. These actions are prevented by the CB, antagonist rimonabant, are accompanied by increased brain anandamide levels, and are maintained upon repeated URB597 administration. Unlike direct CB, agonists, URB597 does not exert rewarding effects in the conditioned place preference test or produce generalization to the discriminative effects of Delta(9)-tetrahydrocannabinol in rats. The findings support a role for anandamide in mood regulation and point to fatty-acid amide hydrolase as a previously uncharacterized target for antidepressant drugs.
The differential projections from the dorsal raphe and median raphe nuclei of the midbrain were autoradiographically traced in the rat brain after 3H-proline micro-injections. Six ascending fiber tracts were identified, the dorsal raphe nucleus being the sole source of four tracts and sharing one with the median raphe nucleus. The tracts can be classified as those lying within the medial forebrain bundle (dorsal raphe forebrain tract and the median raphe forebrain tract) and those lying entirely outside (dorsal raphe arcuate tract, dorsal raphe periventricular tract, dorsal raphe cortical tract, and raphe medial tract). The dorsal raphe forebrain tract lies in the ventrolateral aspect of the medial forebrain bundle (MFB) and projects mainly to lateral forebrain areas (e.g., basal ganglion, amygdala, and the pyriform cortex). The median raphe forebrain tract lies in the ventromedial aspect of the MFB and projects to medial forebrain areas (e.g., cingulate cortex, medial septum, and hippocampus). The dorsal raphe cortical tract lies ventrolaterally to the medial longitudinal fasciculus and projects to the caudate-putamen and the parieto-temporal cortex. The dorsal raphe periventricular tract lies immediately below the midbrain aqueduct and projects rostrally to the periventricular region of the thalamus and hypothalamus. The dorsal raphe arcuate tract curves laterally from the dorsal raphe nucleus to reach the ventrolateral edge of the midbrain and projects to ventrolateral geniculate body nuclei and the hypothalamic suprachiasmatic nuclei. Finally, the raphe medial tract receives fibers from both the median and dorsal raphe nuclei and runs ventrally between the fasciculus retroflexus and projects to the interpeduncular nucleus and the midline mammillary body.
Further studies were done to test whether the fiber tracts travelling in the MFB contained 5-HT. Unilateral (left) injections of 5,7-dihydroxytryptamine (5 μgm/400 nl) 18 days before midbrain raphe microinjections of 3H-proline produced a reduction in the grain concentrations in all the ascending fibers within the MFB. Furthermore, pharmacological and behavioural evidence was obtained to show that the 5-HT system had been unilaterally damaged; these animals displayed preferential ipsilateral turning in a rotameter which was strongly reversed to contralateral turning after 5-hydroxytryptophan administration.
The results show that DR and MR nuclei have numerous ascending projections whose axons contain the transmitter 5-HT. The results agree with the neuroanatomical distribution of the 5-HT system previously determined biochemically, histochemically, and neurophysiologically. The midbrain serotonin system seems to be organized by a series of fiber pathways. The fast transport rate in these fibers was found to be about 108 mm/day.
Background: Selectively-bred alcohol-preferring (P) rats have fewer serotonin (5-HT) neurons in the dorsal raphe nucleus (DRN) than do alcohol-nonpreferring (NP) rats. The present study was designed to test the hypothesis that the remaining 5-HT neurons in P rats compensated for their reduced number by increasing neuronal activity.
Methods: Spontaneous activity was recorded from single-spiking and bursting 5-HT neurons in the DRN of unanesthetized paralyzed, alcohol-naive P, NP, and Wistar rats. Firing frequencies, the percentages of action potentials in bursts, and the percentages of bursting neurons were evaluated.
Results: There were no significant differences among the three groups of rats in any of the parameters measured. Power analyses were performed on preliminary data to determine the sample sizes necessary for detection of significant differences. The mean firing frequencies of single-spiking 5-HT neurons averaged 1.8 (37 neurons), 1.7 (17 neurons), and 1.8 (41 neurons) spikes per second in P, NP, and Wistar rats, respectively. For bursting 5-HT neurons, the percentages of action potentials in bursts for P, NP, and Wistar rats were 55.0% (24 neurons), 49.7% (18 neurons), and 55.1% (21 neurons). The mean percentages of bursting 5-HT neurons encountered per electrode penetration were 44% for P rats (n= 28), 44% for NP rats (n= 14), and 34% for Wistar rats (n= 26).
Conclusions: The results indicate that the sample of 5-HT neurons recorded in the DRN of P rats had not compensated for a reduced number by altering neuronal activity.
The excitability of various neurones in the mammalian central nervous system (CNS), ranging from motoneurones to serotonergic neurones, is enhanced by alpha 1-adrenoceptor agonists. Excitations mediated via alpha 1-adrenoceptors are associated with a slow depolarization and an increase in input resistance, probably resulting from a decrease in resting potassium conductance. However, the involvement of voltage-dependent transient currents in mediating alpha 1 excitatory effects has not been evaluated. An early transient outward current has been described which is important in regulating the frequency of repetitive firing; it is activated by depolarizing voltage steps from potentials more negative than rest and blocked by 4-aminopyridine. This current, which has been termed 'IA', was found originally in invertebrates and subsequently in various vertebrate neurones. The present single-electrode voltage-clamp study demonstrates an early transient outward current (IA) in serotonergic neurones which is suppressed by noradrenaline and the alpha 1-agonist phenylephrine; a suppression of IA may account in part for the acceleration of pacemaker activity induced by alpha 1-agonists in serotonergic neurones.
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.
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).
Several lines of evidence indicate that brief (< 25 ms) bursts of high-frequency firing have special importance in brain function. Recent work shows that many central synapses are surprisingly unreliable at signaling the arrival of single presynaptic action potentials to the postsynaptic neuron. However, bursts are reliably signaled because transmitter release is facilitated. Thus, these synapses can be viewed as filters that transmit bursts, but filter out single spikes. Bursts appear to have a special role in synaptic plasticity and information processing. In the hippocampus, a single burst can produce long-term synaptic modifications. In brain structures whose computational role is known, action potentials that arrive in bursts provide more-precise information than action potentials that arrive singly. These results, and the requirement for multiple inputs to fire a cell suggest that the best stimulus for exciting a cell (that is, a neural code) is coincident bursts.
Improved clinical antidepressant efficacy may result if the acute inhibition of 5-HT cell firing induced by antidepressants is prevented. Here we examined whether inhibition of 5-HT cell firing by non-selective 5-HT uptake inhibiting antidepressant drugs is reversed by a selective 5-HT1A receptor antagonist. In addition, we examined whether concomitant blockade of NA uptake offsets the inhibition of 5-HT cell firing resulting from 5-HT uptake blockade. Antidepressants which block 5-HT uptake (paroxetine, clomipramine, amitriptyline, venlafaxine), all caused dose-dependent and complete inhibition of 5-HT cell firing. Desipramine, a selective NA uptake blocker, caused a slight reduction in firing. The selective 5-HT1A receptor antagonist, WAY 100635, reversed the inhibition of 5-HT cell firing induced by clomipramine, amitriptyline, venlafaxine, and paroxetine, but not that induced by the alpha 1 adrenoceptor antagonist, prazosin. Desipramine, at a dose which increased extracellular NA in the DRN, reversed the effect of prazosin but did not alter the ability of paroxetine to inhibit 5-HT cell firing. Our data indicate that antidepressant drugs with 5-HT uptake blocking properties inhibit 5-HT cell firing via activation of 5-HT1A autoreceptors, and do so irrespective of their effects on NA uptake. These data are discussed in relation to the application of 5-HT1A receptor antagonists to enhance the clinical efficacy of antidepressant drugs.
The effects of risperidone on brain 5‐hydroxytryptamine (5‐HT) neuronal functions were investigated and compared with other antipsychotic drugs and selective receptor antagonists by use of single cell recording and microdialysis in the dorsal raphe nucleus (DRN).
Administration of risperidone (25–400 μg kg ⁻¹ , i.v.) dose‐dependently decreased 5‐HT cell firing in the DRN, similar to the antipsychotic drug clozapine (0.25–4.0 mg kg ⁻¹ , i.v.), the putative antipsychotic drug amperozide (0.5–8.0 mg kg ⁻¹ , i.v.) and the selective α 1 ‐adrenoceptor antagonist prazosin (50–400 μg kg ⁻¹ , i.v.).
The selective α 2 ‐adrenoceptor antagonist idazoxan (10–80 μg kg ⁻¹ , i.v.), in contrast, increased the firing rate of 5‐HT neurones in the DRN, whereas the D 2 and 5‐HT 2A receptor antagonists raclopride (25–200 μg kg ⁻¹ , i.v.) and MDL 100,907 (50–400 μg kg ⁻¹ , i.v.), respectively, were without effect. Thus, the α 1 ‐adrenoceptor antagonistic action of the antipsychotic drugs might, at least partly, cause the decrease in DRN 5‐HT cell firing.
Pretreatment with the selective 5‐HT 1A receptor antagonist WAY 100,635 (5.0 μg kg ⁻¹ , i.v.), a drug previously shown to antagonize effectively the inhibition of 5‐HT cells induced by risperidone, failed to prevent the prazosin‐induced decrease in 5‐HT cell firing. This finding argues against the notion that α 1 ‐adrenoceptor antagonism is the sole mechanism underlying the inhibitory effect of risperidone on the DRN cells.
The inhibitory effect of risperidone on 5‐HT cell firing in the DRN was significantly attenuated in rats pretreated with the 5‐HT depletor PCPA ( p ‐chlorophenylalanine; 300 mg kg ⁻¹ , i.p., day ⁻¹ for 3 consecutive days) in comparison with drug naive animals.
Administration of risperidone (2.0 mg kg ⁻¹ , s.c.) significantly enhanced 5‐HT output in the DRN.
Consequently, the reduction in 5‐HT cell firing by risperidone appears to be related to increased availability of 5‐HT in the somatodendritic region of the neurones leading to an enhanced 5‐HT 1A autoreceptor activation and, in turn, to inhibition of firing, and is probably only to a minor extent caused by its α 1 ‐adrenoceptor antagonistic action.
We examined the involvement of the frontal cortex in the 5-HT1A receptor-induced inhibition of 5-HT neurones in the dorsal raphe nucleus (DRN) of the anaesthetized rat using single-unit recordings complemented by Fos-immunocytochemistry.
Both transection of the frontal cortex as well as ablation of the medial region of the prefrontal cortex (mPFC) significantly attenuated the inhibition of 5-HT neurones induced by systemic administration of the 5-HT1A receptor agonist, 8-OH-DPAT (0.5–16 μg kg−1, i.v.). In comparison, the response to 8-OH-DPAT was not altered by ablation of the parietal cortex. The inhibitory effect of 8-OH-DPAT was reversed by the 5-HT1A receptor antagonist, WAY 100635 (0.1 mg kg−1, i.v.) in all neurones tested.
In contrast, cortical transection did not alter the sensitivity of 5-HT neurones to iontophoretic application of 8-OH-DPAT into the DRN. Similarly, cortical transection did not alter the sensitivity of 5-HT neurones to systemic administration of the selective 5-HT reuptake inhibitor, paroxetine (0.1–0.8 mg kg−1, i.v.).
8-OH-DPAT evoked excitation of mPFC neurones at doses (0.5–32 μg kg−1, i.v.) in the range of those which inhibited 5-HT cell firing. At higher doses (32–512 μg kg−1, i.v.) 8-OH-DPAT inhibited mPFC neurones. 8-OH-DPAT (0.1 mg kg−1, s.c.) also induced Fos expression in the mPFC. The neuronal excitation and inhibition, as well as the Fos expression, were antagonized by WAY 100635.
These data add further support to the view that the inhibitory effect of 5-HT1A receptor agonists on the firing activity of DRN 5-HT neurones involves, in part, activation of a 5-HT1A receptor-mediated postsynaptic feedback loop centred on the mPFC.
British Journal of Pharmacology (1999) 126, 1741–1750; doi:10.1038/sj.bjp.0702510
It is now nearly 5 years since the last of the currently recognised 5-HT receptors was identified in terms of its cDNA sequence. Over this period, much effort has been directed towards understanding the function attributable to individual 5-HT receptors in the brain. This has been helped, in part, by the synthesis of a number of compounds that selectively interact with individual 5-HT receptor subtypes--although some 5-HT receptors still lack any selective ligands (e.g. 5-ht1E, 5-ht5A and 5-ht5B receptors). The present review provides background information for each 5-HT receptor subtype and subsequently reviews in more detail the functional responses attributed to each receptor in the brain. Clearly this latter area has moved forward in recent years and this progression is likely to continue given the level of interest associated with the actions of 5-HT. This interest is stimulated by the belief that pharmacological manipulation of the central 5-HT system will have therapeutic potential. In support of which, a number of 5-HT receptor ligands are currently utilised, or are in clinical development, to reduce the symptoms of CNS dysfunction.
Selectively-bred alcohol-preferring (P) rats have fewer serotonin (5-HT) neurons in the dorsal raphe nucleus (DRN) than do alcohol-nonpreferring (NP) rats. The present study was designed to test the hypothesis that the remaining 5-HT neurons in P rats compensated for their reduced number by increasing neuronal activity.
Spontaneous activity was recorded from single-spiking and bursting 5-HT neurons in the DRN of unanesthetized paralyzed, alcohol-naive P, NP, and Wistar rats. Firing frequencies, the percentages of action potentials in bursts, and the percentages of bursting neurons were evaluated.
There were no significant differences among the three groups of rats in any of the parameters measured. Power analyses were performed on preliminary data to determine the sample sizes necessary for detection of significant differences. The mean firing frequencies of single-spiking 5-HT neurons averaged 1.8 (37 neurons), 1.7 (17 neurons), and 1.8 (41 neurons) spikes per second in P, NP, and Wistar rats, respectively. For bursting 5-HT neurons, the percentages of action potentials in bursts for P, NP, and Wistar rats were 55.0% (24 neurons), 49.7% (18 neurons), and 55.1% (21 neurons). The mean percentages of bursting 5-HT neurons encountered per electrode penetration were 44% for P rats (n = 28), 44% for NP rats (n = 14), and 34% for Wistar rats (n = 26).
The results indicate that the sample of 5-HT neurons recorded in the DRN of P rats had not compensated for a reduced number by altering neuronal activity.
We have previously described a population of 5-hydroxytryptamine neurons which repetitively fires bursts of usually two (but occasionally three or four) action potentials, with a short (<20 ms) interspike interval within a regular low-frequency firing pattern. Here we used a paradigm of electrical stimulation comprising twin pulses (with 7- or 10-ms inter-pulse intervals) to mimic this burst firing pattern, and compared the effects of single- and twin-pulse electrical stimulations in models of pre- and postsynaptic 5-hydroxytryptamine function. Firstly, we measured the effect of direct electrical stimulation (2 Hz for 2 min) of rat brain slices on efflux of preloaded [3H]5-hydroxytryptamine. In this in vitro model, twin-pulse stimulation increased the efflux of tritium by about twice as much as did single-pulse stimulation. This effect was evident in the medial prefrontal cortex (area under the curve: 2. 59+/-0.34 vs 1.28+/-0.22% relative fractional release), as well as in the caudate-putamen (3.93+/-0.65 vs 2.17+/-0.51%) and midbrain raphe nuclei (5.42+/-1.05 vs 2.51+/-0.75%). Secondly, we used in vivo microdialysis to monitor changes in endogenous extracellular 5-hydroxytryptamine in rat medial prefrontal cortex in response to electrical stimulation (3 Hz for 10 min) of the dorsal raphe nucleus. In this model, twin-pulse stimulation of the dorsal raphe nucleus increased 5-hydroxytryptamine by approximately twice as much as did single-pulse stimulation at the same frequency (area under the curve: 50.4+/-9.0 vs 24.2+/-4.4 fmol). Finally, we used in vivo extracellular recording to follow the response of postsynaptic neurons in the rat medial prefrontal cortex to 5-hydroxytryptamine released by dorsal raphe stimulation. Electrical stimulation of the dorsal raphe nucleus (1 Hz) induced a clear-cut poststimulus inhibition in the majority of cortical neurons tested. In these experiments, the duration of poststimulus inhibition following twin-pulse stimulation was markedly longer than that induced by single-pulse stimulation (200+/-21 vs 77+/-18.5 ms). Taken together, the present in vitro and in vivo data suggest that in 5-hydroxytryptamine neurons, short bursts of action potentials will propagate along the axon to the nerve terminal and will enhance both the release of 5-hydroxytryptamine and its postsynaptic effect.
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 dorsal (DR) and median raphe (MR) nuclei contain 5-hydroxytryptamine (serotonin, 5-HT) cell bodies that give rise to the majority of the ascending 5-HT projections to the forebrain limbic areas that control emotional behavior. In the past, the electrophysiological identification of neurochemically identified 5-HT neurons has been limited. Recent technical developments have made it possible to re-examine the electrophysiological characteristics of identified 5-HT- and non-5-HT-containing neurons. Visualized whole cell electrophysiological techniques in combination with fluorescence immunohistochemistry for 5-HT were used. In the DR, both 5-HT- and non-5-HT-containing neurons exhibited similar characteristics that have historically been attributed to putative 5-HT neurons. In contrast, in the MR, the 5-HT-and non-5-HT-containing neurons had very different characteristics. Interestingly, the MR 5-HT-containing neurons had a shorter time constant and larger afterhyperpolarization (AHP) amplitude than DR 5-HT-containing neurons. The 5-HT(1A) receptor-mediated response was also measured. The efficacy of the response elicited by 5-HT(1A) receptor activation was greater in 5-HT-containing neurons in the DR than the MR, whereas the potency was similar, implicating greater autoinhibition in the DR. Non-5-HT-containing neurons in the DR were responsive to 5-HT(1A) receptor activation, whereas the non-5-HT-containing neurons in the MR were not. These differences in the cellular characteristics and 5-HT(1A) receptor-mediated responses between the MR and DR neurons may be extremely important in understanding the role of these two 5-HT circuits in normal physiological processes and in the etiology and treatment of pathophysiological states.
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.
Early life adversity is associated with an increased incidence of psychiatric illness in adulthood. Although the mechanisms underlying this association are unclear, one possible substrate is brain 5-hydroxytryptamine neurotransmission, which is reportedly abnormal in several psychiatric disorders. This study examined the effect of a rat model of early life adversity, early maternal separation, on 5-hydroxytryptamine neurotransmission in adulthood. In vitro electrophysiological experiments revealed that, in early maternal separation rats compared with controls, the sensitivity of α1-adrenoceptors on 5-hydroxytryptamine neurons in the dorsal raphe nucleus was significantly reduced, whilst the sensitivity of 5-hydroxytryptamine1A receptors showed a nonsignificant trend to reduction. In in vivo microdialysis experiments, the 5-hydroxytryptamine1A receptor agonist-induced suppression of 5-hydroxytryptamine release in the frontal cortex was reduced in early maternal separation animals, suggesting desensitization of 5-hydroxytryptamine1A autoreceptors. There was no increase in basal 5-hydroxytryptamine in the frontal cortex as measured by microdialysis and a nonsignificant trend towards increased basal firing activity of classical (non-bursting) 5-hydroxytryptamine neurons in the dorsal raphe nucleus measured by in vivo electrophysiology. Finally, early maternal separation failed to alter expression of messenger ribonucleic acids coding for 5-hydroxytryptamine1A or α1B receptors in the dorsal raphe nucleus as measured by in situ hybridization histochemistry, suggesting that functional changes in receptor sensitivity observed are not due to changes in receptor gene transcription. The findings demonstrate that early life adversity programs changes in sensitivity of the two principal regulators of 5-hydroxytryptamine neuronal activity. Similar effects in humans may contribute to the increased incidence of psychiatric illness in individuals exposed to early life adversity.
Neurochemical and electrophysiological studies on the functional significance of burst firing in serotonergic neurons
- S E Gartside
- E Hajó S-Korcsok
- E Bagdy
- L G Harsing
- Jr
- T Sharp
- M Hajó S
Gartside, S.E., Hajó s-Korcsok, E., Bagdy, E., Harsing, L.G. Jr,Sharp, T. &
Hajó s, M. (2000) Neurochemical and electrophysiological studies on the
functional significance of burst firing in serotonergic neurons. Neuroscience,
98, 295-300.
Early life adversity programs changes in central 5-HT neuronal function in adulthood
- S E Gartside
- D A Johnson
- M M Leitch
- C Troakes
- C D Ingram
Gartside, S.E., Johnson, D.A., Leitch, M.M., Troakes, C. & Ingram, C.D.
(2003) Early life adversity programs changes in central 5-HT neuronal
function in adulthood. Eur. J. Neurosci., 17, 2401–2408.
Neurochemical and electrophysiological studies on the functional significance of burst firing in serotonergic neurons
- Gartside