Immunocytochemical localization of nuclear estrogen receptors and progestin receptors within the rat dorsal raphe nucleus

Estradiol and progesterone modulate central serotonergic activity; however, the mechanism(s) of action remain unclear. Recently, estradiol-induced progestin receptors (PRs) have been localized within the majority of serotonin (5-HT) neurons in the female macaque dorsal raphe nucleus (DRN; Bethea [1994] Neuroendocrinology 60:50–61). In the present study, we investigated whether estrogen receptors (ERs) and/or PRs exist within 5-HT and/or non-5-HT cells in the female and male rat DRN and whether estradiol treatment alters the expression of these receptors. Young adult female and male Sprague-Dawley rats were gonadectomized, and 1 week later, half of the animals received a subcutaneous Silastic implant of estradiol-17β. Animals were transcardially perfused 2 days later with acrolein and paraformaldehyde, and sequential dual-label immunocytochemistry was performed on adjacent sections by using either a PR antibody or an ERα antibody. This was followed by an antibody to either the 5-HT-synthesizing enzyme, tryptophan hydroxylase (TPH), or to the astrocytic marker, glial fibrillary acidic protein (GFAP). Cells containing immunoreactivity (ir) for nuclear ERs or PRs were identified within the rat DRN in a region-specific distribution in both sexes. No colocalization of nuclear ER-ir or PR-ir with cytoplasmic TPH-ir or GFAP-ir was observed in either sex or treatment, indicating that the steroid target cells are neither 5-HT neurons nor astrocytes. Females were found to have approximately 30% more PR-labeled cells compared with males throughout the DRN (P < 0.05), but no sex difference was detected in the number of neurons demonstrating ER-ir. In both sexes, 2 days of estradiol exposure decreased the number of cells with ER-ir, whereas it greatly increased the number of cells containing PR-ir in several DRN regions (P < 0.005). Collectively, these findings demonstrate the existence of nonserotonergic cells that contain nuclear ERs or PRs within the female and male rat DRN, including estradiol-inducible PRs. These findings point to a species difference in ovarian steroid regulation of 5-HT activity between the macaque and the rat, perhaps transsynaptically via local neurons in the rat brain. J. Comp. Neurol. 391:322–334, 1998. © 1998 Wiley-Liss, Inc.     

Differential galanin receptor-1 and galanin expression by 5-HT neurons in dorsal raphé nucleus of rat and mouse: evidence for species-dependent modulation of serotonin transmission

Galanin and galanin receptors are widely expressed by neurons in rat brain that either synthesize/release and/or are responsive to, classical transmitters such as gamma-aminobutyric acid, acetylcholine, noradrenaline, histamine, dopamine and serotonin (5-hydroxytryptamine, 5-HT). The dorsal raphé nucleus (DRN) contains approximately 50% of the 5-HT neurons in the rat brain and a high percentage of these cells coexpress galanin and are responsive to exogenous galanin in vitro. However, the precise identity of the galanin receptor(s) present on these 5-HT neurons has not been previously established. Thus, the current study used a polyclonal antibody for the galanin receptor-1 (GalR1) to examine the possible expression of this receptor within the DRN of the rat and for comparative purposes also in the mouse. In the rat, intense GalR1-immunoreactivity (IR) was detected in a substantial population of 5-HT-immunoreactive neurons in the DRN, with prominent receptor immunostaining associated with soma and proximal dendrites. GalR1-IR was also observed in many cells within the adjacent median raphé nucleus. In mouse DRN, neurons exhibited similar levels and distribution of 5-HT-IR to that in the rat, but GalR1-IR was undetectable. Consistent with this, galanin and GalR1 mRNA were also undetectable in mouse DRN by in situ hybridization histochemistry, despite the detection of GalR1 mRNA (and GalR1-IR) in adjacent cells in the periaqueductal grey and other midbrain areas. 5-HT neuron activity in the DRN is primarily regulated via 5-HT1A autoreceptors, via inhibition of adenylate cyclase and activation of inward-rectifying K+ channels. Notably, the GalR1 receptor subtype signals via identical mechanisms and our findings establish that galanin modulates 5-HT neuron activity in the DRN of the rat via GalR1 (auto)receptors. However, these studies also identify important species differences in the relationship between midbrain galanin and 5-HT systems, which should prompt further investigations in relation to comparative human neurochemistry and which have implications for studies of animal models of relevant neurological conditions such as stress, anxiety and depression.     

Estrogen-regulated progestin receptors are found in the midbrain raphe but not hippocampus of estrogen receptor alpha (ER alpha) gene-disrupted mice

Estrogen and progesterone may modulate serotonergic function through intracellular receptors, alpha (ER alpha) and/or beta (ER beta), and the progestin receptor (PR). Studies in macaque and rat suggest species differences in steroid action. Presently, we examined the mouse. To identify whether ER alpha is involved in estrogen induction of PR in midbrain raphe, we studied the ER alpha gene-disrupted (alpha ERKO) mouse. The hippocampus was examined as another estrogen/progestin-sensitive brain area reported to express ER alpha, ER beta, and PR. Female and male homozygous alpha ERKO and wildtype mice were gonadectomized and given estradiol benzoate or vehicle. Dual-label immunocytochemistry was performed for PR or ER alpha and the serotonin-synthesizing enzyme, tryptophan hydroxylase (TPH). Cells exhibiting PR immunoreactivity (PR-ir) or ER alpha-ir were observed in dorsal and median raphe and hippocampus in both sexes. No ER alpha-ir cells were observed in alpha ERKO brains. In raphe, PR-ir or ER alpha-ir often colocalized with TPH-ir. Thus, estrogen and progesterone may directly modulate gene expression in select serotonergic neurons via ER alpha and PR in female and male mice. Estrogen significantly increased the number of PR-ir cells, and the percentage of PR-ir cells colocalizing TPH-ir in both raphe nuclei, regardless of sex and genotype. Although less among alpha ERKO mice, the significant estrogen induction of PRs implicates the involvement of another ER, perhaps ER beta. In hippocampus, distinct estrogen-induced PR-ir cells were observed only in wildtype animals, demonstrating an ER alpha-mediated event in this forebrain region. Collectively, these findings suggest that estrogen can regulate the expression of one gene (the PR) via multiple mechanisms, based upon brain region.     

Serotonin 5-HT(2) receptors activate local GABA inhibitory inputs to serotonergic neurons of the dorsal raphe nucleus

The purpose of the present study was to characterize the synaptic currents induced by bath-applied serotonin (5-HT) in 5-HT cells of the dorsal raphe nucleus (DRN) and to determine which 5-HT receptor subtypes mediate these effects. In rat brain slices, 5-HT induced a concentration-dependent increase in the frequency of inhibitory postsynaptic currents (IPSCs) in 5-HT neurons recorded intracellularly in the ventral part of the DRN (EC(50): 86 microM); 5-HT also increased IPSC amplitude. These effects were blocked by the GABA(A) receptor antagonist, bicuculline (10 microM) and by the fast sodium channel blocker, TTX, suggesting that 5-HT had increased impulse flow in local GABAergic neurons. DAMGO (300 nM), a selective mu-agonist, markedly suppressed the increase in IPSC frequency induced by 5-HT (100 microM) in the DRN. A near maximal concentration of the selective 5-HT(2A) antagonist, MDL100,907 (30 nM), produced a large reduction ( approximately 70%) in the increase in IPSC frequency induced by 100 microM 5-HT; SB242,084 (30 nM), a selective 5-HT(2C) antagonist, was less effective ( approximately 24% reduction). Combined drug application suppressed the increase in 5-HT-induced IPSC frequency almost completely, suggesting involvement of both 5-HT(2A) and 5-HT(2C) receptors. Unexpectedly, the phenethylamine hallucinogen, DOI, a partial agonist at 5-HT(2A/2C) receptors, caused a greater increase (+334%) in IPSC frequency than did 5-HT 100 microM (+80%). This result may be explained by an opposing 5-HT(1A) inhibitory effect since the selective 5-HT(1A) antagonist, WAY-100635, enhanced the 5-HT-induced increase in IPSCs. These results indicate that within the DRN-PAG area there may be a negative feedback loop in which 5-HT induces an increase in IPSC frequency in 5-HT cells by exciting GABAergic interneurons in the DRN via 5-HT(2A) and, to a lesser extent, 5-HT(2C) receptors. Increased GABA tone may explain the previous observation of an indirect suppression of firing of a subpopulation of 5-HT cells in the DRN induced by phenethylamine hallucinogens in vivo.     

Colocalization of Natriuretic Peptide and Estrogen Receptor Immunoreactivities in Preoptic Nuclei in the Female Rat

Estrogen is known to play an important role in regulating reproductive function in female rats through actions exerted at the preoptic area, a part of the brain that is markedly sexually dimorphic and which contains abundant estrogen receptors. A critical question to our understanding of estrogen's action on the brain is to identify the types of neurons that contain estrogen receptors (ER). Previous studies have shown that atrial natriuretic peptide (ANP) is in abundance in the preoptic area, and that ANP and other natriuretic peptides are capable of regulating gonadotropin secretion. In an effort to determine whether ERs are present in natriuretic peptide-immunoreactive (NP-ir) neurons in the preoptic area of the rat, double label immunocytochemistry was performed. Since ER-ir, as demonstrated with antibody H222 is known to be localized predominantly in cell nuclei, while NP-ir is present in the cytoplasm, single cells can be double labeled. Diaminobenzidine tetrahydrochloride was used for localization of NP-ir neurons, while nickel-enhanced diaminobenzidine tetrahydrochloride was used for localization of ER-ir. The results revealed that many nuclei throughout the preoptic area contained neurons that were ER-ir or NP-ir and that a substantial number were double labeled. Cell counts in selected preoptic nuclei and components, including the anteroventral periventricular nucleus, periventricular preoptic nucleus, medial part of the medial preoptic nucleus, and central part of the medial preoptic nucleus revealed that 13.6%, 11.1%, 13.5%, and 24.4%, respectively, of the NP-ir neurons in these nuclei also contained ER-ir. Collectively, a total of 14.9% of the NP-ir neurons in these nuclei also contained ER-ir. These results support the hypothesis that estrogen may regulate the activity of NP-ir cells in the preoptic area and thereby may be capable of altering functions associated with NPs in this region of the brain, including not only regulation of gonadotropin secretion, but also control of blood pressure and fluid balance.     

Frontocortical 5-HT4 receptors exert positive feedback on serotonergic activity: viral transfections, subacute and chronic treatments with 5-HT4 agonists.

BACKGROUND: We recently identified a facilitory control exerted by serotonin4 (5-HT4) receptors on the in vivo firing activity of dorsal raphe nucleus (DRN) serotonergic (5-HT) neurons. However, these findings were based on acute administrations of 5-HT4 receptor agonists and antagonists, which were active only in a subpopulation of 5-HT neurons. We had no evidence that this influence was significant when considering the entire DRN, nor if it was persistent after chronic treatments. In addition, the poor distribution of 5-HT4 receptors within the DRN raised the question of the neuroanatomical bases underlying this control. METHODS AND RESULTS: Here we show that the subacute intraperitoneal (IP) injection of the 5-HT4 receptor agonists prucalopride (2.5 mg/kg) and RS 67333 (1.5 mg/kg) 30 minutes before the beginning of recordings augment the mean firing rate of DRN neurons by 40% and 66%, respectively. These increases remain stable when the compounds are administered continuously during 3 and 21 days; the effects of the 3-day treatment are blocked by the 5-HT4 receptor antagonist GR 125487 (1000 microg/kg, intravenous [i.v.]). In addition, stereotaxic microinjections of herpes simplex viruses, transformed to overexpress 5-HT4 receptors, increase DRN 5-HT neuronal mean activity when performed in the medial prefrontal cortex (mPFC) but not in the striatum or in the hippocampus. CONCLUSIONS: This finding suggests the existence of a 5-HT(4)-dependent activation of DRN that may involve the mPFC, unveiling the 5-HT4 receptor as a putative player in the physiopathology of several disorders related to central 5-HT dysfunction.     

Serotonin 5-HT2 receptors activate local GABA inhibitory inputs to serotonergic neurons of the dorsal raphe nucleus

The purpose of the present study was to characterize the synaptic currents induced by bath-applied serotonin (5-HT) in 5-HT cells of the dorsal raphe nucleus (DRN) and to determine which 5-HT receptor subtypes mediate these effects. In rat brain slices, 5-HT induced a concentration-dependent increase in the frequency of inhibitory postsynaptic currents (IPSCs) in 5-HT neurons recorded intracellularly in the ventral part of the DRN (EC₅₀: 86 μM); 5-HT also increased IPSC amplitude. These effects were blocked by the GABA_A receptor antagonist, bicuculline (10 μM) and by the fast sodium channel blocker, TTX, suggesting that 5-HT had increased impulse flow in local GABAergic neurons. DAMGO (300 nM), a selective μ-agonist, markedly suppressed the increase in IPSC frequency induced by 5-HT (100 μM) in the DRN. A near maximal concentration of the selective 5-HT_2A antagonist, MDL100,907 (30 nM), produced a large reduction (70%) in the increase in IPSC frequency induced by 100 μM 5-HT; SB242,084 (30 nM), a selective 5-HT_2C antagonist, was less effective (24% reduction). Combined drug application suppressed the increase in 5-HT-induced IPSC frequency almost completely, suggesting involvement of both 5-HT_2A and 5-HT_2C receptors. Unexpectedly, the phenethylamine hallucinogen, DOI, a partial agonist at 5-HT_2A/2C receptors, caused a greater increase (+334%) in IPSC frequency than did 5-HT 100 μM (+80%). This result may be explained by an opposing 5-HT_1A inhibitory effect since the selective 5-HT_1A antagonist, WAY-100635, enhanced the 5-HT-induced increase in IPSCs. These results indicate that within the DRN–PAG area there may be a negative feedback loop in which 5-HT induces an increase in IPSC frequency in 5-HT cells by exciting GABAergic interneurons in the DRN via 5-HT_2A and, to a lesser extent, 5-HT_2C receptors. Increased GABA tone may explain the previous observation of an indirect suppression of firing of a subpopulation of 5-HT cells in the DRN induced by phenethylamine hallucinogens in vivo.     

Dual control of dorsal raphe serotonergic neurons by GABA(B) receptors. Electrophysiological and microdialysis studies

We assessed the role of GABA(B) receptors in the control of serotonergic (5-HT) neurons of the dorsal raphe nucleus (DRN) by using microdialysis in vivo and intra- and extracellular recording in vitro in the rat. The GABA(B) agonist R(+)baclofen (but not the inactive S(-)enantiomer) enhanced the 5-HT output in the DRN (4. 7-fold at 15 mg/kg s.c.) and, to a much lesser extent, striatum of unanesthetized rats. Phaclofen (2 mg/kg s.c.) antagonized the effects of 6 mg/kg R(+)baclofen in dorsal striatum. Using dual-probe microdialysis, R(+)baclofen (0.1-100 microM) applied in the DRN enhanced the local 5-HT output (4.5-fold at 100 microM) but decreased that in striatum at 100 microM. At concentrations higher than 100 microM there was a moderate decrement in the elevation of 5-HT in the DRN. In midbrain slices, bath R(+)baclofen exerted a biphasic effect on DRN 5-HT neurons. Consistent with a reduced striatal 5-HT release when infused in the DRN, R(+)baclofen (0.1-30 microM) induced an outward current in 5-HT neurons (IC(50) = 1.4 microM). Lower R(+)baclofen concentrations (0.01-1 microM) preferentially reduced GABAergic inhibitory postsynaptic currents induced by N-methyl-D-aspartate (20 microM) in 5-HT neurons (IC(50) = 72 nM). Using extracellular recordings, R(+)baclofen (300 nM) enhanced the ability of NMDA to induce firing in a subpopulation of serotonergic neurons. These results are consistent with a preferential activation by a low concentration of R(+)baclofen of presynaptic GABA(B) receptors on GABAergic afferents that could disinhibit 5-HT neurons and increase 5-HT release.     

Serotoninergic dorsal raphe neurons possess functional postsynaptic nicotinic acetylcholine receptors

Very few neurons in the telencephalon have been shown to express functional postsynaptic nicotinic acetylcholine receptors (nAChRs), among them, the noradrenergic and dopaminergic neurons. However, there is no evidence for postsynaptic nAChRs on serotonergic neurons. In this study, we asked if functional nAChRs are present in serotonergic (5-HT) and nonserotonergic (non-5-HT) neurons of the dorsal raphe nucleus (DRN). In rat midbrain slices, field stimulation at the tegmental pedunculopontine (PPT) nucleus evoked postsynaptic currents (eEPSCs) with different components in DRN neurons. After blocking the glutamatergic and GABAergic components, the remaining eEPSCs were blocked by mecamylamine and reduced by either the selective alpha7 nAChR antagonist methyllycaconitine (MLA) or the selective alpha4beta2 nAChR antagonist dihydro-beta-eritroidine (DHbetaE). Simultaneous addition of MLA and DHbetaE blocked all eEPSCs. Integrity of the PPT-DRN pathway was assessed by both anterograde biocytin tracing and antidromic stimulation from the DRN. Inward currents evoked by the direct application of acetylcholine (ACh), in the presence of atropine and tetrodotoxin, consisted of two kinetically different currents: one was blocked by MLA and the other by DHbetaE; in both 5-HT and non-5-HT DR neurons. Analysis of spontaneous (sEPSCs) and evoked (eEPSCs) synaptic events led to the conclusion that nAChRs were located at the postsynaptic membrane. The possible implications of these newly described nAChRs in various physiological processes and behavioral events, such as the wake-sleep cycle, are discussed.     

Expression of 5-HT1A receptor mRNA in rat dorsal raphe nucleus and ventrolateral periaqueductal gray neurons after peripheral inflammation

In the present study we observed the expression of 5-hydroxytryptamine (5-HT)1A receptor mRNA in the dorsal raphe nucleus (DRN) and ventrolateral periaqueductal gray (vlPAG) neurons, especially in 5-HT immunoreactive neurons (5-HT-IR), using in situ hybridization (ISH) and double staining with fluorescent ISH (FISH) and immunohistochemical (FIH) techniques. The findings of this study demonstrated that 5-HT1A receptor mRNA was expressed with moderate to high level in the DRN and vlPAG neurons. Following carrageenan inflammation, the expression of 5-HT1A receptor mRNA in the DRN and bilateral vlPAG neurons was significantly increased. The peak occurred at 3-8h followed by a clear decrease at 24 h, which basically corresponded to the time-course of behavioral hyperalgesia. Moderate 5-HT1A receptor mRNA and 5-HT immunoreactive (5-HT-IR) double-labeled cells were observed in the DRN and vlPAG, suggesting that some of 5-HT1A receptors in the DRN and vlPAG may be autoreceptors. Eight hours after carrageenan injection, the number of the double labeled cells was significantly increased. These results suggest that the synthesis of 5-HT1A receptors, including autoreceptors, is increased in the DRN and vlPAG during peripheral inflammation.     

A role for midbrain raphe gamma aminobutyric acid neurons in 5-hydroxytryptamine feedback control

New data show that 5-hydroxytryptamine (5-HT) neurons of the dorsal raphe nucleus (DRN) are subject to feedback control from 5-HT2 receptors, but the circuitry involved is uncertain. This study investigated whether 5-HT2 receptor agonism activates DRN gamma-aminobutyric acid (GABA) neurons, which are known to inhibit neighbouring 5-HT neurons. Systemic administration of the 5-HT2 receptor agonist, DOI, caused a striking increase in Fos-immunoreactivity in the DRN. This effect was abolished by the 5-HT2 antagonists ritanserin and MDL 100907, but not SB 206553, indicating the involvement of 5-HT2A receptors. Importantly, DOI-induced Fos-immunoreactivity in the DRN was extensively colocalized in GAD67-immunoreactive neurons. These findings suggest that activated local GABA neurons may play an important role in 5-HT2 receptor-mediated feedback control of DRN 5-HT neurons.     

Immunocytochemical evidence for the existence of substance P receptor (NK1) in serotonin neurons of rat and mouse dorsal raphe nucleus

In addition to its neurotransmitter/modulator role in pain perception, substance P (SP) is involved in a regulation of mood, as antagonists of its neurokinin-1 receptor (NK1r) have been found to have antidepressant-like effects in humans. In rodents, treatment with NK1r antagonists has been shown to increase the firing of dorsal raphe nucleus (DRN) serotonin (5-hydroxytryptamine, 5-HT) neurons and to induce a desensitization of their 5-HT1A autoreceptors, suggesting local interactions between the SP and 5-HT systems. To search for the presence of NK1r on 5-HT neurons of the DRN, we used light and electron microscopic immunocytochemistry, as well as confocal microscopy, after single- and double-labelling of NK1r and of the biosynthetic enzyme of 5-HT, tryptophan hydroxylase (TpOH). A significant number of 5-HT (TpOH-positive) cell bodies and dendrites endowed with NK1r were thus demonstrated in the caudal part of rat and mouse DRN. As visualized by electron microscopy after gold immunolabelling, NK1r was mostly cytoplasmic in 5-HT neurons, while predominating on the plasma membrane in the case of TpOH-negative dendrites. The proportion of NK1r observed on the plasma membrane of 5-HT neurons was, however, slightly higher in mouse than rat. Thus, in both rat and mouse DRN, a subpopulation of 5-HT neurons is endowed with NK1r receptors and may be directly involved in the antidepressant-like effects of NK1r antagonists. These 5-HT neurons represent a new element in the neuronal circuitry currently proposed to account for the role of SP in mood regulation.     

Uncontrollable, but not controllable, stress desensitizes 5-HT1A receptors in the dorsal raphe nucleus

Uncontrollable stressors produce behavioral changes that do not occur if the organism can exercise behavioral control over the stressor. Previous studies suggest that the behavioral consequences of uncontrollable stress depend on hypersensitivity of serotonergic neurons in the dorsal raphe nucleus (DRN), but the mechanisms involved have not been determined. We used ex vivo single-unit recording in rats to test the hypothesis that the effects of uncontrollable stress are produced by desensitization of DRN 5-HT(1A) autoreceptors. These studies revealed that uncontrollable, but not controllable, tail shock impaired 5-HT(1A) receptor-mediated inhibition of DRN neuronal firing. Moreover, this effect was observed only at time points when the behavioral effects of uncontrollable stress are present. Furthermore, temporary inactivation of the medial prefrontal cortex with the GABA(A) receptor agonist muscimol, which eliminates the protective effects of control on behavior, led even controllable stress to now produce functional desensitization of DRN 5-HT(1A) receptors. Additionally, behavioral immunization, an experience with controllable stress before uncontrollable stress that prevents the behavioral outcomes of uncontrollable stress, also blocked functional desensitization of DRN 5-HT(1A) receptors by uncontrollable stress. Last, Western blot analysis revealed that uncontrollable stress leads to desensitization rather than downregulation of DRN 5-HT(1A) receptors. Thus, treatments that prevent controllable stress from being protective led to desensitization of 5-HT(1A) receptors, whereas treatments that block the behavioral effects of uncontrollable stress also blocked 5-HT(1A) receptor desensitization. These data suggest that uncontrollable stressors produce a desensitization of DRN 5-HT(1A) autoreceptors and that this desensitization is responsible for the behavioral consequences of uncontrollable stress.     

Cellular effects of swim stress in the dorsal raphe nucleus

Swim stress regulates forebrain 5-hydroxytryptamine (5-HT) release in a complex manner and its effects are initiated in the serotonergic dorsal raphe nucleus (DRN). The purpose of this study was to examine the effects of swim stress on the physiology of DRN neurons in conjunction with 5-HT immunohistochemistry. Basic membrane properties, 5-HT(1A) and 5-HT(1B) receptor-mediated responses and glutamatergic excitatory postsynaptic currents (EPSCs) were measured using whole-cell patch clamp techniques. Rats were forced to swim for 15min and 24h later DRN brain slices were prepared for electrophysiology. Swim stress altered the resting membrane potential, input resistance and action potential duration of DRN neurons in a neurochemical-specific manner. Swim stress selectively elevated glutamate EPSC frequency in 5-HT DRN neurons. Swim stress non-selectively reduced EPSC amplitude in all DRN cells. Swim stress elevated the 5-HT(1B) receptor-mediated inhibition of glutamatergic synaptic activity that selectively targeted 5-HT cells. Non-5-HT DRN neurons appeared to be particularly responsive to the effects of a milder handling stress. Handling elevated EPSC frequency, reduced EPSC decay time and enhanced a 5-HT(1B) receptor-mediated inhibition of mEPSC frequency selectively in non-5-HT DRN cells. These results indicate that swim stress has both direct, i.e., changes in membrane characteristics, and indirect effects, i.e., via glutamatergic afferents, on DRN neurons. These results also indicate that there are distinct local glutamatergic afferents to neurochemically specific populations of DRN neurons, and furthermore that these distinct afferents are differentially regulated by swim stress. These cellular changes may contribute to the complex effects of swim stress on 5-HT neurotransmission and/or the behavioral changes underlying the forced swimming test model of depression.     

Neurochemical identification of stereotypic burst-firing neurons in the rat dorsal raphe nucleus using juxtacellular labelling methods

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.     

AMPA and NMDA receptor regulation of firing activity in 5-HT neurons of the dorsal and median raphe nuclei

The glutamatergic regulation of 5-hydroxytryptamine (5-HT) neuronal activity has not been extensively studied. Here, we used extracellular single unit recording in midbrain slices to examine glutamate receptor mediated effects on 5-HT neuronal activity in the dorsal raphe nucleus (DRN) and the median raphe nucleus (MRN). Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA; 1 and 3 microm) concentration-dependently increased firing in 5-HT neurons in both the DRN and the MRN. The response to AMPA was blocked by the AMPA receptor antagonist, 6,7-dinitroquinoxaline-2,3(1H-4H)-dione (DNQX; 10 microm) but not the N-methyl-d-aspartate (NMDA) receptor antagonist, 2-amino-5-phosphonopentanoic acid (AP-5; 50 microm). NMDA (10-100 microm) also increased 5-HT neuronal firing in a concentration-dependent manner in both the DRN and MRN; a response that was blocked by AP-5 (50 microm). In some DRN neurons the NMDA response was partially antagonized by DNQX (10 microm) suggesting that NMDA, as well as directly activating 5-HT neurons, evokes local release of glutamate, which indirectly activates AMPA receptors on 5-HT neurons. Responses of DRN 5-HT neurons to AMPA and NMDA were enhanced by the gamma-amino-butyric acid (GABA)(A) receptor antagonist, bicuculline (50 microm), suggesting that both AMPA and NMDA increase local release of GABA. Finally in the DRN the 5-HT(1A) receptor antagonist, WAY100635 (100 nm), failed to enhance the response of 5-HT neurons to AMPA and caused only a small increase in the excitatory response to NMDA suggesting a low degree of tonic activation of 5-HT(1A) autoreceptors even when 5-HT neuronal firing rate is high.     

Distribution of 5-hydroxytriptamine2C receptor mRNA in rat retina

There are several factors that suggest serotonin [5-hydroxytryptamine (5-HT)] plays a role as a neurotransmitter/neuromodulator within the retina. The presence of mRNAs encoding 5-HT receptors (5-HTR) of the types 5-HT2CR and 5-HT5AR within the rat retina was investigated using in situ hybridization of digoxigenin-labeled probes. The 5HT5AR probe produced no labeling, whereas the 5HT2CR probe hybridized in cells scattered in the inner nuclear and ganglion cell layers. Thus, the 5HT2CR gene is expressed by retinal neurons, some of which represent third-order neurons, either amacrine or ganglion cells. This suggests that 5-HT may modulate the outgoing signal from the retina.     

Tetraethylammonium ions block the nicotinic cholinergic receptors of cochlear outer hair cells

An involvement of serotonin (5-HT) 1A receptors in the etiology of psychiatric disorders has been suggested. Hypo-responsiveness of the 5-HT1A receptor is linked to anxiety and constitutive deletion of the 5-HT1A receptor produces anxiety-like behaviors in the mouse. Evidence that 5-HT1A receptor inactivation increases the therapeutic effects of antidepressants has also been presented. The present studies used in vivo microdialysis and homologous recombination techniques to examine the contribution of 5-HT1A autoreceptors to these effects. Basal and fluoxetine-evoked extracellular concentrations of 5-HT were quantified in the striatum, a projection area of dorsal raphe neurons (DRN), of wild-type (WT) and 5-HT1A receptor knock out (KO) mice. The density of 5-HT transporters was also determined. Basal 5-HT concentrations did not differ in WT and KO mice. Fluoxetine (10 mg/kg) increased 5-HT concentrations in both genotypes. This increase was, however, 2-fold greater in KO mice. In contrast, no differences in K(+)-evoked 5-HT concentrations were seen. Similarly, neither basal nor stimulation-evoked DA differed across genotype. Autoradiography revealed no differences between genotype in the density of 5-HT transporters or post-synaptic 5-HT2A receptors, an index of 5-HT neuronal activity. These experiments demonstrate that, under basal and KCl stimulated conditions, adaptive mechanisms in the 5-HT system compensate for the lack of 5-HT1A autoreceptor regulation of DRN. Furthermore, they suggest that the absence of release-regulating 5-HT1A autoreceptors in the DRN can not account for the anxiety phenotype of KO mice. The enhanced response to fluoxetine in KO mice is consistent with pharmacological studies and suggests that adaptive mechanisms that occur in response to 5-HT1A receptor deletion are insufficient to oppose increases in 5-HT concentrations produced by acute inhibition of the 5-HT transporter.     

Flattening plasma corticosterone levels increases the prevalence of serotonergic dorsal raphe neurons inhibitory responses to nicotine in adrenalectomised rats

Major depression is characterized by a diminished activity of the brain serotonergic system as well as by the flattening of plasma cortisol levels. Nicotine improves mood in patients with major depression and in experimentally depressed animals by increasing brain serotonin (5-HT), noradrenaline and dopamine levels. The present study was directed to determine if flattening plasma glucocorticoid levels changes nicotine's stimulatory effects upon 5-HT DRN neurons. The experiments were performed in brain slices obtained from rats previously (14 days) adrenalectomised and implanted subcutaneously with one pellet containing 75 mg of corticosterone (Adx + CSR rats). Whole cell voltage and current clamp techniques were used to study the activity of immunocitochemically identified 5-HT DRN neurons. Administration of nicotine (1 μM) in sham-operated animals produced stimulatory effects in all 5-HT DRN neurons studied. In Adx + CSR rats however, nicotine inhibited 75% of 5-HT DRN neurons and increased the potassium-dependent inward rectifying current. The inhibitory effect of nicotine upon 5-HT DRN neurons was dependent on serotonin release inside the DRN, since it was converted into a stimulatory response by the selective antagonist of 5-HT_1A receptors N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridyl)cyclohexanecarboxamide (WAY100635, 25 nM). Adx + CSR rats also presented an increased function of 5-HT_1A autoreceptors, since, in these rats, serotonin (1–10 μM) produced a higher increase in the potassium dependent inward rectifying current in comparison with sham-operated animals. Serotonin release inside DRN was mediated by α4β2 nicotinic acetylcholine receptors since the selective antagonist of these receptors dihydro-β-erytroidine hydrobromide (DHβE, 100 nM) blocked the inhibitory effects of nicotine 5-HT DRN neurons. These data indicate that, in the experimental model of adrenalectomised rats implanted with corticosterone pellets, nicotine increases the function of 5-HT_1A receptors of 5-HT DRN neurons.     

Segregation of serotonin 5-HT2A and 5-HT3 receptors in inhibitory circuits of the primate cerebral cortex

An emerging concept of cortical network organization is that distinct segments of the pyramidal neuron tree are controlled by functionally diverse inhibitory microcircuits. We compared the expression of two serotonin receptor subtypes, the G-protein-coupled 5-hydroxytryptamine2A receptors and the ion-channel gating 5-HT3 receptors, in cortical neuron types, which control these microcircuits. Here we show, using light and electron microscopic immunocytochemical techniques, that 5-HT2A receptors are segregated from 5-HT3 receptors in the macaque cerebral cortex. 5-HT2A receptor immunolabel was found in pyramidal cells and also in GABAergic interneurons known to specialize in the perisomatic inhibition of pyramidal cells: large and medium-size parvalbumin- and calbindin-containing interneurons. In contrast, 5-HT3 label was only present in small GABA-, substance P receptor-, and calbindin-containing neurons and in medium-size calretinin-containing neurons: interneurons known to preferentially target the dendrites of pyramidal cells. This cellular segregation indicates a serotonin-receptor-specific segmentation of the GABAergic inhibitory actions along the pyramidal neuron tree.