Differential regulation of 5-hydroxytryptamine release by GABAA and GABAB receptors in midbrain raphe nuclei and forebrain of rats.

1. Extracellular 5-hydroxytryptamine (5-HT) was determined in dorsal raphe nucleus (DRN), median raphe nucleus (MRN) and nucleus accumbens by use of microdialysis in unanaesthetized rats. 2. Infusion of the gamma-aminobutyric acid (GABA)A receptor agonist muscimol into DRN and MRN resulted in decreased 5-HT in DRN and MRN, respectively. Muscimol infusion into nucleus accumbens had no effect on 5-HT. 3. Infusion of the GABAA receptor antagonist bicuculline into DRN resulted in increased DRN and nucleus accumbens 5-HT. Bicuculline infusion into MRN had no effect on 5-HT. This suggests that endogenous GABA had a tonic, GABAA receptor-mediated inhibitory effect on 5-HT in DRN, but not in MRN. 4. Infusion of the GABAB receptor agonist baclofen into DRN produced a decrease in DRN 5-HT. Baclofen infusion into nucleus accumbens resulted in decreased nucleus accumbens 5-HT. This suggests that GABAB receptors are present in the area of cell bodies and terminals of 5-hydroxytryptaminergic neurones. 5. Infusion of the GABAB receptor antagonists phaclofen and 2-hydroxysaclofen had no effect on midbrain raphe and forebrain 5-HT. This suggests that GABAB receptors did not contribute to tonic inhibition of 5-HT release. 6. In conclusion, 5-HT release is physiologically regulated by distinct subtypes of GABA receptors in presynaptic and postsynaptic sites.     

SDF-1α/CXCL12 enhances GABA and glutamate synaptic activity at serotonin neurons in the rat dorsal raphe nucleus

The serotonin (5-hydroxytryptamine; 5-HT) system has a well-characterized role in depression. Recent reports describe comorbidities of mood-immune disorders, suggesting an immunological component may contribute to the pathogenesis of depression as well. Chemokines, immune proteins which mediate leukocyte trafficking, and their receptors are widely distributed in the brain, mediate neuronal patterning, and modulate various neuropathologies. The purpose of this study was to investigate the neuroanatomical relationship and functional impact of the chemokine stromal cell-derived factor-1α/CXCL12 and its receptor, CXCR4, on the serotonin dorsal raphe nucleus (DRN) system in the rat using anatomical and electrophysiological techniques. Immunohistochemical analysis indicates that over 70% of 5-HT neurons colocalize with CXCL12 and CXCR4. At a subcellular level, CXCL12 localizes throughout the cytoplasm whereas CXCR4 concentrates to the outer membrane and processes of 5-HT neurons. CXCL12 and CXCR4 also colocalize on individual DRN cells. Furthermore, electrophysiological studies demonstrate CXCL12 depolarization of 5-HT neurons indirectly via glutamate synaptic inputs. CXCL12 also enhances the frequency of spontaneous inhibitory and excitatory postsynaptic currents (sIPSC and sEPSC). CXCL12 concentration-dependently increases evoked IPSC amplitude and decreases evoked IPSC paired-pulse ratio selectively in 5-HT neurons, effects blocked by the CXCR4 antagonist AMD3100. These data indicate presynaptic enhancement of GABA and glutamate release at 5-HT DRN neurons by CXCL12. Immunohistochemical analysis further shows CXCR4 localization to DRN GABA neurons, providing an anatomical basis for CXCL12 effects on GABA release. Thus, CXCL12 indirectly modulates 5-HT neurotransmission via GABA and glutamate synaptic afferents. Future therapies targeting CXCL12 and other chemokines may treat serotonin related mood disorders, particularly depression experienced by immune-compromised individuals.     

Allopregnanolone synthesis in cerebellar granule cells: roles in regulation of GABA(A) receptor expression and function during progesterone treatment and withdrawal

Rat cerebellar granule cells were cultured for 5 days with progesterone, resulting in the conversion of progesterone to allopregnanolone, a potent and efficacious modulator of gamma-aminobutyric acid (GABA) type-A receptors, as well as in decreases in the abundance of GABA(A) receptor alpha(1), alpha(3), alpha(5), and gamma(2) subunit mRNAs. These effects were accompanied by decreases in the efficacies of diazepam and the beta-carboline DMCM with regard to modulation of GABA-evoked Cl(-) currents. Withdrawal from such progesterone treatment resulted in a rapid and selective increase in the abundance of the GABA(A) alpha(4) subunit mRNA that was associated with a restoration of receptor sensitivity to the negative modulatory action of DMCM, a positive receptor response to flumazenil, and continued reduced responsiveness of receptors to diazepam. Prevention of allopregnanolone synthesis by the 5alpha-reductase inhibitor finasteride also prevented the changes in both GABA(A) receptor gene expression and receptor function elicited by progesterone treatment and withdrawal.     

In vivo evidence that 5-HT(2C) receptors inhibit 5-HT neuronal activity via a GABAergic mechanism

BACKGROUND AND PURPOSE: Recent evidence suggests that 5-HT(2C) receptor activation may inhibit midbrain 5-HT neurones by activating neighbouring GABA neurones. This hypothesis was tested using the putative selective 5-HT(2C) receptor agonist, WAY 161503. EXPERIMENTAL APPROACH: The effect of WAY 161503 on 5-HT cell firing in the dorsal raphe nucleus (DRN) was investigated in anaesthetised rats using single unit extracellular recordings. The effect of WAY 161503 on DRN GABA neurones was investigated using double label immunohistochemical measurements of Fos, glutamate decarboxylase (GAD) and 5-HT(2C) receptors. Finally, drug occupancy at 5-HT(2A) receptors was investigated using rat positron emission tomography and ex vivo binding studies with the 5-HT(2A) receptor radioligand [(11)C]MDL 100907. KEY RESULTS: WAY 161503 caused a dose-related inhibition of 5-HT cell firing which was reversed by the 5-HT(2) receptor antagonist ritanserin and the 5-HT(2C) receptor antagonist SB 242084 but not by the 5-HT(1A) receptor antagonist WAY 100635. SB 242084 pretreatment also prevented the response to WAY 161503. The blocking effects of SB 242084 likely involved 5-HT(2C) receptors because the drug did not demonstrate 5-HT(2A) receptor occupancy in vivo or ex vivo. The inhibition of 5-HT cell firing induced by WAY 161503 was partially reversed by the GABA(A) receptor antagonist picrotoxin. Also, WAY 161503 increased Fos expression in GAD positive DRN neurones and DRN GAD positive neurones expressed 5-HT(2C) receptor immunoreactivity. CONCLUSIONS AND IMPLICATIONS: These findings indicate that WAY 161503 inhibits 5-HT cell firing in the DRN in vivo, and support a mechanism involving 5-HT(2C) receptor-mediated activation of DRN GABA neurones.     

Multiple effects of allopregnanolone on GABAergic responses in single hippocampal CA3 pyramidal neurons

3α-Hydroxy, 5α-reduced pregnane steroids, such as allopregnanolone, are potent modulators of GABA(A) receptors and have many biological responses including sedative, anxiolytic, anticonvulsant and anesthetic actions. In the present study, we have investigated the effects of allopregnanolone on GABA(A) receptors in acutely isolated single hippocampal CA3 pyramidal neurons using the whole cell patch-clamp technique. Allopregnanolone induced membrane Cl(-) currents in a concentration-dependent manner, and the allopregnanolone-induced currents (I(AlloP)) were blocked by noncompetitive GABA(A) receptor antagonists. The I(AlloP) was not affected by the intracellular loading of γ-cyclodextrin (γ-CD), which efficiently sequesters several kinds of endogenous neurosteroids including allopregnanolone, suggesting that allopregnanolone accesses extracellular but not intracellular sites to activate GABA(A) receptors. Allopregnanolone prolonged the decay time constant of GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs), suggesting that allopregnanolone modulates the desensitization kinetics of postsynaptic GABA(A) receptors. The picrotoxin-sensitive tonic currents (I(tonic)), which were mediated by extrasynaptic GABA(A) receptors, were recorded from CA3 pyramidal neurons. The intracellular loading of γ-CD or allopregnanolone significantly decreased or increased the amplitude of picrotoxin-sensitive I(tonic), respectively, suggesting that endogenous neurosteroids might, at least in part, be involved in the generation of picrotoxin-sensitive I(tonic). Allopregnanolone also increased the frequency of GABAergic sIPSCs, in a manner dependent on the integrity of voltage-dependent Na(+) and Ca(2+) channels, suggesting that allopregnanolone activates presynaptic GABA(A) receptors to depolarize GABAergic nerve terminals. The present results suggest that allopregnanolone exerts its pharmacological and pathophysiological actions via the modulation of multiple types of GABA(A) receptor-mediated responses.     

GABAergic modulation of 5-HT7 receptor-mediated effects on 5-HT efflux in the guinea-pig dorsal raphe nucleus

5-HT(7) receptor mRNA and protein are localised in the dorsal raphe nucleus (DRN) on non-serotonergic neurones. The effect of 5-HT(7) receptor antagonism on 5-HT efflux was measured from guinea-pig DRN slices, using the technique of fast cyclic voltammetry. The 5-HT(7) receptor antagonist, SB-269970-A, significantly inhibited 5-HT efflux. The GABA(A) receptor agonist, muscimol, significantly inhibited 5-HT efflux, to a similar degree as SB-269970-A. In contrast, the GABA(A) receptor antagonist, bicuculline, significantly increased 5-HT efflux and attenuated the muscimol-induced inhibition. The muscimol and SB-269970-A effects were not additive and in the presence of bicuculline the SB-269970-A-induced inhibition of 5-HT efflux was attenuated. These data suggest that 5-HT(7) receptor antagonist-induced inhibition of 5-HT efflux occurs indirectly via activation of GABA(A) receptors. That is, 5-HT(7) receptors may be located on GABA interneurones and when activated decrease GABA release and hence decrease the inhibitory tone on 5-HT neurones, increasing 5-HT efflux in the DRN. Therefore, in the presence of GABAergic tone 5-HT(7) receptor antagonists would decrease 5-HT release from the DRN.     

Effects of repeated treatment with 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) on the autoregulatory control of dorsal raphe 5-HT neuronal firing and cortical 5-HT release.

These experiments were designed to examine the effects of repeated 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) treatment on the autoregulatory control of cortical 5-HT release and dorsal raphe nucleus (DRN) 5-HT neuronal cell firing. Repeated DOI treatment decreased the behavioural responsiveness (wet-dog shakes) of 5-HT2 receptors and attenuated the inhibitory effects of the 5-HT1A receptor agonist, 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)tetralin), on both cortical 5-HT release and DRN 5-HT neuronal firing. In contrast, the inhibitory effect of acute DOI on cortical 5-HT release and DRN 5-HT neuronal firing was unaffected by repeated DOI treatment. The results demonstrate that changes in the responsiveness of 5-HT2 receptor function may influence the responsiveness of presynaptic 5-HT1A receptors regulating 5-HT neuronal function. The results also provide further evidence that the inhibition of cortical 5-HT release and DRN 5-HT neuronal firing produced by DOI is not mediated by 5-HT2 receptor activation.     

Galanin is a potent in vivo modulator of mesencephalic serotonergic neurotransmission.

Neurochemical, molecular, immunohistochemical and behavioral methods were used to examine the in vivo effects of the neuropeptide galanin on central 5-HT neurotransmission and on 5-HT(1A) receptor-mediated responses. Intraventricularly infused galanin caused a long-lasting and dose-dependent reduction of basal extracellular 5-HT levels in the ventral hippocampus of awake rats as measured by microdialysis. Infusion of galanin into the dorsal raphe nucleus (DRN), but not intra-hippocampally, reduced 5-HT release. The effect of i.c.v. galanin on 5-HT release was blocked by the galanin receptor antagonist M35, acting most likely via galanin receptors at the level of the DRN. Galanin also reduced the levels of tryptophanhydroxylase mRNA in the DRN. Therefore, the effects of galanin on 5-HT(1A) receptor-mediated responses were further investigated. Surprisingly, galanin significantly attenuated the reduction of hippocampal 5-HT release induced by systemic injection of the 5-HT(1A) receptor agonist 8-OH-DPAT. Galanin also attenuated 8-OH-DPAT-induced hypothermia and locomotor activity in rats. These results indicate that galanin has important inhibitory actions on central 5-HT neurotransmission and on 5-HT(1A) receptor-mediated events.     

Allopregnanolone and ganaxolone increase the firing activity of dorsal raphe nucleus serotonergic neurons in female rats

Accumulating evidence suggest a reciprocal interaction between neurosteroids, especially 5alpha-pregnan-3alpha-ol,20-one (3alpha,5alpha-THP, allopregnanolone), and the serotonergic (5-HT) system. Both 5-HT and neurosteroids seem to play an important role in the pathophysiology of major depression. We have previously shown that a 7-d treatment with 3alpha,5alpha-THP drastically increases the spontaneous firing activity of dorsal raphe nucleus (DRN) 5-HT neurons in female rats. This study was thus undertaken to better characterize this modulation and to assess the effects of ganaxolone, a synthetic analogue of 3alpha,5alpha-THP. Female rats received 50 microg/kg.d of 3alpha,5alpha-THP or ganaxolone for 3 and 7 days. Others received 3alpha,5alpha-THP concomitantly with the antiprogestin RU486 (50 microg/kg.d, each), which was also administered alone. Acute experiments were also carried out with a single injection of 3alpha,5alpha-THP (1 microg/kg). Finally, both 3alpha,5alpha-THP and ganaxolone (50 microg/kg.d) were administered along with the selective serotonin reuptake inhibitor (SSRI) citalopram (10 mg/kg.d). In-vivo extracellular unitary recordings of 5-HT neurons from the DRN, revealed that 3alpha,5alpha-THP and ganaxolone increased their firing activity after 3 and 7 d of treatment. A 7-d treatment with RU486 had the same effect. Furthermore, an increase could be seen as soon as after 30-60 min following a single injection with 3alpha,5alpha-THP. Interestingly, both 3alpha,5alpha-THP and ganaxolone prevented the citalopram-induced reduction in firing activity after 3-d treatments. These data demonstrate the ability of 3alpha,5alpha-THP and ganaxolone to positively modulate the firing activity of DRN 5-HT neurons in female rats. Moreover, these results suggest that these neuroactive steroids might represent interesting adjuvants in the treatment of mood disorders in female patients.     

Effects of OPC-14523, a combined sigma and 5-HT1a ligand, on pre- and post-synaptic 5-HT1a receptors

OPC-14523 (OPC) is a novel compound with high affinity for sigma and 5-HT1A receptors that shows 'antidepressant-like' effects in animal models of depression. We have previously demonstrated that OPC produces an increase in 5-HT neurotransmission and a decreased response of 5-HT neurons to the acute administration of paroxetine in the DRN, an effect that appears to be mediated by OPC's 5-HT1A receptor affinity. The current study sets out to investigate more specifically the effects of OPC on 5-HT1A pre- and post-synaptic receptors, to assess whether it acts as an agonist or antagonist. Using an electrophysiological model of in vivo extracellular recordings in anaesthetized rats, the effects of OPC was assessed on pre-synaptic DRN 5-HT1A autoreceptors and post-synaptically on hippocampal 5-HT1A receptors of CA3 pyramidal neurons. OPC applied by microiontophoresis, produced a significant decrease in the firing activity of 5-HT neurons of the DRN and of quisqualate-activated CA3 pyramidal neurons of the dorsal hippocampus. The effects of OPC on 5-HT1A receptors were significantly reduced by the co-application of the 5-HT1A antagonist WAY-100635. In addition, the effects of OPC were not blocked by the injection of the sigma antagonists NE-100 or haloperidol. Therefore, OPC is acting as an agonist on both pre- and post-synaptic 5-HT1A receptors. The current findings combined with previous data on OPC suggest a pharmacological profile that warrants further investigation.     

3alpha,5beta-Reduced cortisol exhibits antagonist properties on cerebral cortical GABA(A) receptors

The tetrahydro-reduced derivatives of progesterone and deoxycorticosterone, allopregnanolone, and tetrahydrodeoxycorticosterone are potent positive modulators of GABA(A) receptors that are elevated by hypothalamic-pituitary-adrenal axis activation in rodents. In humans, 11-deoxycortisol and cortisol are important hypothalamic-pituitary-adrenal axis steroids. We hypothesized that C(3,5) reduction of 11-deoxycortisol and cortisol generates steroids with GABA(A) receptor activity. 3alpha,5beta-Reduced cortisol dose-dependently inhibited muscimol-stimulated chloride flux and tetrahydrodeoxycorticosterone potentiation of muscimol responses. Cortisol, 11-deoxycortisol, 5alpha-dihydrocortisol, 3alpha,5alpha-reduced cortisol, 3alpha,5alpha-reduced 11-deoxycortisol, and 3alpha,5beta-reduced 11-deoxycortisol had no activity at 1 muM and weaker negative modulatory activity at 10 muM. We conclude that cortisol metabolism may produce antagonistic GABAergic activity.     

5-HT1A receptor-mediated autoinhibition does not function at physiological firing rates: evidence from in vitro electrophysiological studies in the rat dorsal raphe nucleus

5-HT(1A)-mediated autoinhibition of neurones in the dorsal raphe nucleus (DRN) is considered to be the principal inhibitory regulator of 5-HT neuronal activity. The activation of this receptor by endogenous 5-HT was investigated using electrophysiological recordings from the rat DRN in vitro. At a concentration which blocked the inhibitory effect of exogenous 5-HT, the 5-HT(1A) antagonist WAY 100635 did not alter basal firing rate or modulate the excitatory response to the alpha(1)-agonist phenylephrine. Blockade of 5-HT reuptake by a concentration of fluoxetine, which enhanced the inhibitory effect of exogenous 5-HT, lowered phenylephrine-induced basal firing presumably due to potentiation of the effect of endogenous 5-HT. However, this effect was not firing rate dependent and neither the proportional increase nor the time-course of the response to a higher concentration of phenylephrine were altered in the presence of fluoxetine. These data suggest that the inhibitory 5-HT(1A) receptor on raphe neurones is neither tonically activated nor plays any role in modulating the response to excitatory transmitters. Thus, at physiological firing rates this receptor does not appear to function as an autoreceptor of serotonergic neurones of the DRN.     

Gender and gonadal status modulation of dorsal raphe nucleus serotonergic neurons. Part II. Regulatory mechanisms

In the companion paper, we showed that the spontaneous firing activity of DRN 5-HT neurons is significantly higher in male (M) than in freely cycling female (CF) rats. Moreover, during pregnancy, it increased in parallel to circulating levels of progesterone, peaking at day 17 of pregnancy (P17). In this second part, we assessed the role of three regulatory mechanisms potentially involved in these modifications of the 5-HT neurons firing activity. During pregnancy, the ED(50) for the response to LSD was decreased by about 70%, indicating a partial desensitization of 5-HT(1A) autoreceptors, which is consistent with the 5-HT neurons higher firing activity. The GABAergic tonic inhibition of 5-HT neurons was assessed using the responses to GABA, bicuculline and isoniazid. Together, they indicate a lower GABAergic tonic inhibition in males and P17 as compared to CF, which is in agreement with their greater 5-HT neurons firing rate. Finally, the efficacy of the long feedback loop, involving postsynaptic 5-HT(1A) receptors, did not seem affected by gender, ovariectomy or pregnancy since the response to systemic 8-OH-DPAT was similar. These results constitute strong evidence of mechanisms by which gender and hormonal fluctuations can modulate the 5-HT neurons function and influence vulnerability to mood disorders.     

Antimalarial drugs inhibit human 5-HT(3) and GABA(A) but not GABA(C) receptors

BACKGROUND AND PURPOSE: Antimalarial compounds have been previously shown to inhibit rodent nicotinic acetylcholine (nACh) and 5-HT(3) receptors. Here, we extend these studies to include human 5-HT(3A), 5-HT(3AB), GABA(A) alpha1beta2, GABA(A) alpha1beta2gamma2 and GABA(C) rho1 receptors.EXPERIMENTAL APPROACH: We examined the effects of quinine, chloroquine and mefloquine on the electrophysiological properties of receptors expressed in Xenopus oocytes.KEY RESULTS: 5-HT(3A) receptor responses were inhibited by mefloquine, quinine and chloroquine with IC(50) values of 0.66, 1.06 and 24.3 microM. At 5-HT(3AB) receptors, the potencies of mefloquine (IC(50)=2.7 microM) and quinine (15.8 microM), but not chloroquine (23.6 microM), were reduced. Mefloquine, quinine and chloroquine had higher IC(50) values at GABA(A) alpha1beta2 (98.7, 0.40 and 0.46 mM, respectively) and GABA(A) alpha1beta2gamma2 receptors (0.38, 1.69 and 0.67 mM, respectively). No effect was observed at GABA(C) rho1 receptors. At all 5-HT(3) and GABA(A) receptors, chloroquine displayed competitive behaviour and mefloquine was non-competitive. Quinine was competitive at 5-HT(3A) and GABA(A) receptors, but non-competitive at 5-HT(3AB) receptors. Homology modelling in combination with automated docking suggested orientations of quinine and chloroquine at the GABA(A) receptor binding site.CONCLUSIONS AND IMPLICATIONS: The effects of mefloquine, quinine and chloroquine are distinct at GABA(A) and GABA(C) receptors, whereas their effects on 5-HT(3AB) receptors are broadly similar to those at 5-HT(3A) receptors. IC(50) values for chloroquine and mefloquine at 5-HT(3) receptors are close to therapeutic blood concentrations required for malarial treatment, suggesting that their therapeutic use could be extended to include the treatment of 5-HT(3) receptor-related disorders.     

Conserved site for neurosteroid modulation of GABA A receptors

This study addresses whether the potentiation site for neurosteroids on GABA(A) receptors is conserved amongst different GABA(A) receptor isoforms. The neurosteroid potentiation site was previously identified in the alpha1beta2gamma2S receptor by mutation of Q241 to methionine or leucine, which reduced the potentiation of GABA currents by the naturally occurring neurosteroids, allopregnanolone or tetrahydrodeoxycorticosterone (THDOC). By using heterologous expression of GABA(A) receptors in HEK cells, in combination with whole-cell patch clamp recording methods, a relatively consistent potentiation by allopregnanolone of GABA-activated currents was evident for receptors composed of one alpha subunit isoform (alpha2-5) assembled with beta3 and gamma2S subunits. Using mutant alphabetagamma receptors, the neurosteroid potentiation was universally dependent on the conserved glutamine residue in M1 of the respective alpha subunit. Studying wild-type and mutant receptors composed of alpha4beta3delta subunits revealed that the delta subunit is unlikely to contribute to the neurosteroid potentiation binding site and probably affects the efficacy of potentiation. Thus, in keeping with the ability of neurosteroids to potentiate GABA currents via a broad variety of GABA(A) receptor isoforms in neurons, the potentiation site is structurally highly conserved on this important neurotransmitter receptor family.     

Effect of Mimosa pudica (Linn.) extract on anxiety behaviour and GABAergic regulation of 5-HT neuronal activity in the mouse

Mimosa pudica (Linn.) (M. pudica L.) is a plant used in some countries to treat anxiety and depression. In the present study we investigated the effects of an aqueous extract of M. pudica L. on mouse anxiety-like behaviour using the elevated T maze, and on regulation of dorsal raphe nucleus (DRN) 5-hydroxytryptamine (5-HT) neuronal activity using an in-vitro mouse brain slice preparation. Acute treatment with M. pudica L. extract had an anxiolytic effect on behaviour in the elevated T maze, specifically on inhibitory avoidance behaviour. Acute application of the extract alone had no effect on the activity of DRN 5-HT neurones. However, when co-applied with the GABA(A) receptor agonist THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol), the extract enhanced the inhibitory effect of the THIP on DRN 5-HT neurones. These observed effects of M. pudica L. on both behaviour and GABA modulation of 5-HT neuronal activity are similar to the effects of diazepam, the established anxiolytic and positive modulator of the GABA(A) receptor. This study suggests that the aqueous extract of M. pudica L. contains a positive modulator of GABA(A) receptor function and provides impetus for further investigation of the neuropharmacologically active constituents of the extract.     

Ovarian steroid regulation of 5-HT1A receptor binding and G protein activation in female monkeys.

Serotonin 5-HT(1A) receptors play an important role in serotonin neurotransmission and mental health. We previously demonstrated that estradiol (E) and progesterone (P) decrease 5-HT(1A) autoreceptor mRNA levels in macaques. In this study, we questioned whether E and P regulate 5-HT(1A) binding and function and G(alpha) subunit protein expression. Quantitative autoradiography for 5-HT(1A) receptors and G proteins using [3H]8-OH-DPAT and [35S]GTP-gamma-S, respectively, was performed on brain sections of rhesus macaques from four treatment groups: ovariectomized controls (OVX), E (28 d), P (28 d), and E (28 d) plus P (the last 14 d) treated. Western blot analysis for G(alpha) subunits was performed on raphe extracts from cynomolgus macaques that were OVX or OVX treated with equine estrogens (EE, 30 months). In the hypothalamus, E or E + P but not P alone decreased postsynaptic 5-HT(1A) binding sites. In the dorsal raphe nucleus (DRN), E, P, and E + P treatments decreased 5-HT(1A) autoreceptor binding. The Kd values for 8-OH-DPAT were the same for each treatment group. Both the basal and the R-(+)-8-OH-DPAT stimulated [35S]GTP-gamma-S binding were decreased during hormone replacement whereas the coupling efficiency between the receptor and G proteins was maintained. Finally, EE treatment reduced the level of G(alphai3), but not G(alphai1), G(alphao), and G(alphaz) in the DRN. In conclusion, these observations suggest that ovarian hormones may increase serotonin neurotransmission, in part, by decreasing 5-HT(1A) autoreceptors, 5-HT(1A) postsynaptic receptors, and the inhibitory G proteins for intracellular signal transduction.     

The interaction of cocaine with serotonin dorsal raphe neurons. Single-unit extracellular recording studies

Cocaine potently inhibits the spontaneous activity of dorsal raphe serotonin (5-hydroxytryptamine [5-HT] neurons which possess impulse-modulating receptors of the 5-HT1A subtype. In an investigation of the neuropharmacologic mechanisms underlying this electrophysiologic effect, we have compared cocaine with structurally and functionally similar compounds, attempted to reverse cocaine-induced suppression of 5-HT dorsal raphe nucleus (DRN) neuronal activity, and assessed the effects of 5-HT depletion on the response to cocaine. Extracellular recordings in chloral hydrate-anesthetized rats were obtained using single-unit recording techniques; drugs were infused intravenously IV) in a cumulative dose manner. The active isomer (-)-cocaine (ID50 = 0.5 +/- 0.15 mg/kg) and the phenyltropane analogue WIN 35428 (ID50 = 0.17 +/- 0.03 mg/kg) that share the ability of cocaine to block monoamine uptake also inhibit impulse activity in 5-HT neurons. In contrast, the inactive isomers (+)-cocaine, (+)-pseudococaine and the metabolite benzoylecgonine do not exhibit the same range of potency (maximal 20% to 30% inhibition at a cumulative dose of 8 to 16 mg/kg). A selective inhibitor of uptake for 5-HT (fluoxetine; ID50 = 1.8 +/- 0.5 mg/kg), but not norepinephrine (desipramine) or dopamine (GBR 12909), mimicked cocaine, as did the monoamine releaser amphetamine (ID50 = 2.86 +/- 0.46 mg/kg). The putative 5-HT1A autoreceptor antagonist spiperone reversed the cocaine-induced depression of firing rate in 64% of 5-HT neurons tested whereas receptor antagonists for dopamine D2 (haloperidol), 5-HT2 (ketanserin), gamma-aminobutyric acid (picrotoxin) and 5-HT1/beta-adrenergic (propranolol) were ineffective. Following treatment with the 5-HT synthesis inhibitor p-chlorophenylalanine (100 mg/kg/day of the base for 3 days), impulse depression induced by cocaine was significantly attenuated as compared to control, which suggests that the effects of cocaine may be dependent on endogenous 5-HT stores. In summary, these findings support the hypothesis that the inhibitory effects of cocaine on 5-HT DRN neurons are mediated by increased 5-HT available for interaction with 5-HT1A impulse-regulating autoreceptors in the DRN, as a consequence of cocaine-induced blockade of 5-HT reuptake processes. Further studies are required to clarify the relative contribution of cocaine-5-HT interactions to the behavioral and physiologic effects of this psychostimulant.     

Mechanisms of nicotine actions on dorsal raphe serotoninergic neurons

Nicotine, locally administered into the dorsal raphe nucleus (DRN) of rat midbrain slices, increased the discharge rate of 70% of serotoninergic neurons, decreased it in 30% and induced reciprocal oscillatory increases in serotonin (5-hydroxytryptamine, 5-HT) and gamma-aminobutyric acid (GABA) release. All of nicotine's stimulatory effects were maximal at 2.15 microM. Bicuculline, a GABA(A) receptor antagonist, increased the firing rate in 64% of serotoninergic neurons, decreased it in 36% and augmented serotonin and GABA release. Bicuculline increased nicotine's stimulatory effects on firing rate but did not reverse the inhibitory ones. N-[2-[4-(2-Methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinil-cyclohexanecarboxamide (WAY-100635), a 5-HT(1A) receptor antagonist, increased the firing rate of 88% of serotoninergic neurons, as well as serotonin and GABA release and reversed nicotine's inhibitory action on serotoninergic neurons. These data suggest that nicotine decreases the firing rate of one third of serotoninergic neurons through serotonin release and increases the firing rate of the remaining two thirds, due to stronger stimulatory than indirect inhibitory effects.