Effects of serotonin on the hypothalamic-pituitary GABAergic system

The effects of serotonin (5-HT) and its precursor, 5-hydroxytryptophan (5-HTP) on the GABAergic system in the mediobasal hypothalamus (MBH) and the anterior pituitary were studied. The IP administration of 5-HTP produced a transient increase (only at 45 min after the injection) in glutamate decarboxylase activity (GAD) of MBH and in GABA concentration in anterior pituitary. Besides, 5-HTP administration increased the in vitro evoked GABA release from MBH. The administration of 5-HTP to hypophysectomized rats partially reversed the inhibitory effects of hypophysectomy on GABA concentration in MBH. We also examined the direct effect of 5-HT on some parameters on the hypothalamic GABAergic system. The presence of 5-HT in the incubation medium increased GAD activity and evoked GABA release from MBH. These observations indicate that the serotoninergic stimulation of the hypothalamic GABAergic system could be a direct effect which may, at least partially, be independent of the feedback mechanism induced by prolactin on the GABAergic neurons. The serotoninergic increase of prolactin secretion could be accomplished through stimulation of the hypothalamic GABAergic transmission.     

Effect of oestradiol on the responses of regional brain serotonin to stress in the ovariectomized rat

Summary The effect of oestradiol on the response of regional brain 5-hydroxytryptamine (serotonin, 5-HT) to 24 hours electrical footshock was investigated in ovariectomized rats. The stress applied four weeks after ovariectomy induced significant decreases in 5-HT concentrations in the fronto-parietal brain cortex (Bc), hippocampus (Hipp), striatum (CP), medial-basal hypothalamus (MBH) and the preoptic-anterior hypothalamic area (PAHA). Pretreatment with oestradiol prevented the footshock-induced decreases in 5-HT in the Hipp, CP, MBH and the PAHA. It was concluded that oestradiol may prevent stress-induced response of regional brain 5-HT in ovariectomized rats. An interaction between oestradiol and brain 5-HT is proposed in the control of the course of the oestrous cycles in stressful situations.     

Interactions between GABAergic and serotoninergic systems with excitatory amino acid neurotransmission in the hypothalamic control of gonadotropin secretion in prepubertal female rats

The present studies were designed to study the interrelationships between GABAergic, serotoninergic and excitatory amino acids systems (EAAs) in the control of gonadotropin secretion in prepubertal female rats. For this purpose we determined the effects of N-methyl-d-aspartate (NMDA), an exogenous agonist of EAAs receptors, on LH and FSH secretion in 16-day-old female rats in which the GABA-A and GABA-B receptors were blocked by bicuculline and baclofen or serotonin (5-HT) depleted by p-choloroamphetamine (PCA). In addition the effects of the GABAergic and serotoninergic systems on LH and FSH secretion were evaluated in animals treated with dibenzocycloalkenimine (diocilpine MK-801), an antagonist of NMDA neurotransmission. While muscimol, a GABA- A agonist, induced a significant increase in LH and FSH levels (P<0.01), baclofen, a GABA-B agonist, had an inhibitory effect on these hormones (P<0.01). MK 801, a NMDA receptor antagonist, not only suppressed the stimulatory effect of NMDA on LH and FSH but also blocked the stimulatory effect of muscimol without modifying the inhibitory action of baclofen on both gonadotropins. Bicuculline, a GABA-A receptor antagonist, did not modify the release effect of NMDA on LH and FSH. 5-HTP, a precursor of 5-HT that increases the levels of this neurotransmitter in the central nervous system significantly increased (P<0.01) the plasma levels of LH and FSH, and this effect was blocked by the NMDA receptor antagonist MK-801. We conclude that the stimulatory effects of GABAergic and serotoninergic systems in prepubertal female rats are connected with the activation of EAA neurotransmission, while the stimulatory effects of NMDA appear to be independent of serotoninergic and GABAergic actions on LH and FSH secretion. Since both GABA and serotonin systems change their effects on LH and FSH during sexual maturation from a stimulatory action in prepubertal to an inhibitory action in adult rats and since NMDA neurotransmission has a stimulatory effect on gonadotropin secretion both in prepubertal and adult rats, it is clear that the interrelationships between GABAergic and serotoninergic systems with EAAs in the gonadotropin control are different in prepubertal and in adult rats.     

Regulation of striatal serotonin release by the lateral habenula-dorsal raphe pathway in the rat as demonstrated by in vivo microdialysis: role of excitatory amino acids and GABA

Striatal extracellular levels of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were monitored with the microdialysis technique during electrical stimulation of the lateral habenula-dorsal raphe (LHb-NRD) pathway in halothane anaesthetized rats. A new double-loop probe, with an improved recovery factor, was implanted into the head of the caudate-putamen and perfused with Ringer solution containing 1 microM of the 5-HT uptake blocker indalpine. Samples were collected every 15 min and analyzed with HPLC coupled to fluorimetric detection. Low frequency stimulation of the LHb (1.5 and 3 Hz, 0.5 mA) produced no detectable changes in striatal indole levels, whereas 15 Hz stimulation induced a 70% increase in 5-HT release. This effect was most likely mediated by a direct LHb-NRD link, since it persisted after ibotenic acid lesions of the interpeduncular nucleus (which is the major projection area for the medial habenular nucleus), but was completely abolished after transection of the fasciculus retroflexus, which carries the axons of the LHb-NRD pathway. The possible identity of the transmitter operating in the LHb-NRD pathway was investigated by NRD injections of kynurenic acid, a potent blocker of excitatory amino acid transmission, and by NRD injections of the GABA antagonist bicuculline. Kynurenic acid (300 nl, 50 mM) did not by itself induce any detectable changes in spontaneous indole output, but completely blocked the effect of LHb stimulation. Injection of bicuculline (300 nl, 2 mM) increased the striatal 5-HT output by about 70%, and potentiated the effect of LHb stimulation by a further 50%. In none of the experiments performed in this study were there any significant changes in striatal 5-HIAA output. These data are compatible with the idea that excitatory amino acids in the LHb-NRD pathway are involved in the regulation of striatal 5-HT release, and that this influence is modulated by GABAergic synaptic activity at the level of the NRD.     

Electrophysiological evidence for convergence of inputs from the medial prefrontal cortex and lateral habenula on single neurons in the dorsal raphe nucleus

Neuronal projections to the dorsal raphe nucleus (DRN) from the medial prefrontal cortex (mPFC) and lateral habenula nucleus (LHb) provide the two key routes by which information processed by mood regulatory, cortico-limbic-striatal circuits input into the 5-HT system. These two projections may converge as it appears that both activate local GABAergic neurons to inhibit 5-HT neurons in the DRN. Here we have tested this hypothesis by measuring the effect of stimulation of the mPFC and LHb on the activity of 5-HT and non-5-HT, putative gamma-amino butyric acid (GABA) neurons in the DRN using extracellular recordings in anaesthetized rats. A total of 119 5-HT neurons (regular, slow firing, broad spike width) and 21 non-5-HT, putative GABA neurons (fast-firing, narrow spike width) were tested. Electrical stimulation of the mPFC or LHb caused a poststimulus inhibition (30 ms latency) of 101/119 5-HT neurons, of which 61 (60%) were inhibited by both the mPFC and LHb. Electrical stimulation of the mPFC or LHb also caused a short latency (12-20 ms) poststimulus facilitation of 10/21 non-5-HT neurons, of which 5 (50%) were activated by both the mPFC and LHb. These data indicate that a significant number of 5-HT neurons and non-5-HT neurons in the DRN are influenced by both the mPFC and LHb. Moreover, the data are compatible with the hypothesis and that there is a convergence of mPFC and LHb inputs on local circuit GABAergic neurons in the DRN which in turn inhibit the activity of 5-HT neurons.     

Long-term oestradiol treatment enhances hippocampal synaptic plasticity that is dependent on muscarinic acetylcholine receptors in ovariectomised female rats

Short-term oestradiol treatment modulates hippocampus-dependent memory and synaptic plasticity in the hippocampus. Long-term oestradiol treatment can also enhance hippocampus- dependent memory, although the effects of long-term oestradiol treatment on synaptic plasticity are unknown. We investigated the effects of long-term oestradiol treatment on synaptic plasticity at the Schaeffer Collateral/CA1 synapse in 8-month-old female rats. In addition, we determined the role of endogenous activation of muscarinic acetylcholine receptors (mAChRs) in synaptic transmission and plasticity using scopolamine (1 μm), an antagonist of mAChRs. Hippocampus slices from ovariectomised rats that were treated with oestradiol-containing capsules for 5 months were compared with slices from ovariectomised rats that received cholesterol-containing capsules. Unexpectedly, scopolamine application significantly increased the baseline field excitatory postsynaptic potentials (fEPSP) and decreased paired pulse facilitation (PPF) in slices from cholesterol-treated rats. Baseline fEPSPs and PPF were not significantly modulated in slices from oestradiol-treated rats by scopolamine. Slices from oestradiol-treated rats showed enhanced long-term potentiation relative to slices from cholesterol-treated rats. Scopolamine significantly reduced the magnitude of plasticity in slices from oestradiol-treated rats. Taken together, these results suggest that mAChRs have a significant effect on baseline synaptic transmission through a decrease in the probability of glutamate release in slices from cholesterol-treated rats. Long-term oestradiol treatment blocks this effect and enhances theta-burst stimulation-induced synaptic plasticity in the middle-aged female rat, and this effect is mediated by activation of mAChRs.     

Electrical stimulation of the nucleus raphe magnus in the rat. Effects on 5-HT metabolism in the spinal cord

The direct electrical stimulation (with biphasic pulses of 1 msec, 10 pulses/sec, 200 microA, for 30 min) of the nucleus raphe magnus in chloral hydrate anaesthesized rats produced a significant acceleration (+50%) of 5-HT synthesis in the spinal cord as revealed by the increased rate of 5-HTP accumulation occurring at this level after the blockade of central 5-HTP decarboxylase with benserazid. In contrast, no change was detected in 5-HT metabolism in the forebrain of stimulated rats. The acceleration of 5-HT synthesis was likely not due to an increased availability of tryptophan for the rate-limiting enzyme, tryptophan hydroxylase, since the concentration of this amino acid was changed neither in the spinal cord, nor in the forebrain of stimulated rats. The measurement of tryptophan hydroxylase activity in soluble extracts from the spinal cord of control and stimulated rats revealed that the acceleration in 5-HT synthesis produced by the electrical stimulation of the nucleus raphe magnus was not associated with a persisting activation of this enzyme. Although one cannot completely exclude that a short-lasting activation of tryptophan hydroxylase, no longer detectable in soluble extracts, has occurred in the spinal cord of stimulated rats, the present findings rather suggest that the rate of 5-HT synthesis can be controlled by factors other than only the concentration of tryptophan and the intrinsic activity of tryptophan hydroxylase in serotoninergic neurons. The demonstration of an acceleration of 5-HT synthesis in bulbospinal serotoninergic neurons under stimulating conditions close to those producing analgesia in rats further supports the role of these neuronal systems in the physiological mechanisms of pain control.     

Neuronal control of [3H]GABA uptake in the ependymocytes of the subcommissural organ: an in vivo model of neuron-glia interaction

The rat subcommissural organ (SCO) is a particular but adequate paradigm for the approach, in vivo, to some aspects of neuron-glia interaction in gamma-aminobutyric acid (GABA) uptake. The rat SCO ependymocytes (the main component of this structure lying at the junction of the aqueduct and the third ventricle) accumulate [3H]GABA by a highly specific uptake mechanism and receive a serotoninergic input forming typical synaptic contacts. It seems that there is a correlation between the capacity of the rat SCO ependymocytes to take up [3H]GABA and the presence of a serotonin (5-HT) innervation. Indeed, in the newborn rat, no uptake of [3H]GABA was observed before the onset of this innervation and the increased [3H]GABA accumulation in the SCO was correlated with the appearance of the 5-HT terminals in the SCO. Moreover, in the mouse, whose SCO is devoid of a 5-HT innervation, no accumulation of [3H]GABA was observed in the SCO ependymocytes. Thus, the 5-HT innervation could be involved directly or indirectly in the onset of the GABA uptake carriers. On the other hand, in adult rats parachlorophenylalanine (pCPA) treatment decreased the 5-HT content of the SCO, and increased [3H]GABA accumulation; such an augmentation was not observed when rats were treated with pCPA plus 5-hydroxytryptophan to restore the 5-HT content. However, an increase in 5-HT content of the SCO by pargyline treatment appeared to have no effect on [3H]GABA uptake. Control of GABA uptake activity by 5-HT in the SCO ependymocytes could be an interesting model for the study of a possible interaction between amino-acids and other neurotransmitters by terminating their action in the extracellular space.     

A cortical GABA-5HT interaction in the mechanism of action of the antidepressant trazodone

The aim of the study was to investigate whether the antidepressant trazodone (TRZ), a serotonin-2 receptor antagonist/reuptake inhibitor, modifies gamma-amino-butyric acid (GABA) extracellular levels in the cerebral cortex, by acting on 5-HT(2A) receptors, and through this mechanism increases 5-HT levels. For this purpose the effect of TRZ on the release of GABA was studied in adult male rats in synaptosomes, cortical slices, and "in vivo" by microdialysis. In cortical slices, the release of both GABA and 5-HT was determined. GABA and 5-HT were identified and their levels quantified by HPLC. The inhibition of 5-HT uptake by TRZ was also measured. In synaptosomes, TRZ antagonized dose-dependently, at concentrations from 10(-10) to 10(-6) M, the increase in GABA release induced by (+/-)DOI, a 5-HT(2A/2C) agonist, and the alpha receptor agonist phenylephrine, both 10(-6) M. The pIC50 values were 8.31+/-0.24, and 5.99+/-0.52, respectively. In the same preparation, [3H]5-HT accumulation was inhibited by citalopram and TRZ with pIC(50) of 7.8+/-0.44 and 5.9+/-0.09, respectively, a finding confirming the weak activity of TRZ in comparison with a SSRI. In cortical slices, TRZ exerted a biphasic effect on GABA release. At concentrations from 10(-10) to 10(-7) M it inhibited and from 10(-6) to 10(-4) M increased GABA release. 5-HT release was enhanced by TRZ throughout the entire range of concentrations tested. However, the increase was delayed after low and rapid after high concentrations. AMI-193, a 5-HT(2A) antagonist (10(-10) to 10(-5) M), reduced GABA release in a dose-response manner, while it induced an increase of 5-HT outflow. On the contrary, (+/-)DOI (10(-10) to 10(-5) M) increased GABA release and inhibited 5-HT levels. Perfusion with the GABA(A) receptor antagonist bicuculline was also followed by an increase in 5-HT release. In microdialysis experiments, TRZ 1.25 mg kg(-1) s.c. brought about a decrease in GABA extracellular levels, while an increase was found after the dose of 2.5 mg kg(-1). These findings demonstrate that TRZ, at concentrations which do not inhibit 5-HT uptake, reduces the cortical GABAergic tone by decreasing GABA extracellular levels, through the blockade of 5-HT(2A) receptors. The attenuation of GABAergic tone is responsible for an increase in 5-HT levels. A further increase also results from 5-HT uptake inhibition caused by higher doses of TRZ. The ensuing high 5-HT levels enhance GABA release, which in turn inhibits 5-HT release.     

Modulation of the brain aversive system by GABAergic and serotonergic mechanisms

Experiments performed in our laboratory, using electrical stimulation combined with microinjection of drugs in the dorsal midbrain central grey (CG) of the rat, evidenced that direct stimulation of GABA receptors with locally administered gamma-aminobutyric acid (GABA) or the GABAA receptor agonists 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol, isoguvacine and muscimol raised the aversive threshold, defined as the lowest electrical current intensity inducing flight or escape behaviour when applied to the dorsal CG. The GABAB receptor agonist baclofen was ineffective. Also, enhancement of endogenous GABA action through local injection of the benzodiazepines chlordiazepoxide and midazolam or of pentobarbital resulted in anti-aversive effects. Ro 15-1788 antagonized both chlordiazepoxide and midazolam, suggesting benzodiazepine receptor mediation. In contrast to pro-GABAergic drugs, microinjection of the GABA antagonists bicuculline and picrotoxin into the CG elicited flight behaviour, like the electrical stimulation. Similar experiments with drugs influencing serotonergic neurotransmission evidenced that intra-CG microinjection of serotonin (5-HT) or of the direct 5-HT receptor agonist 5-methoxy-N,N-dimethyltryptamine increased the aversive threshold. The anti-aversive effect of 5-HT was potentiated by the selective inhibitor of 5-HT neuronal uptake, zimelidine. Also, the latter drug increased the aversive threshold when given alone. The anti-aversive effect of 5-HT was antagonized by local pretreatment with either metergoline or ketanserin, the latter being a selective blocker of 5-HT2 receptors. In contrast to the GABA antagonists mentioned above, the 5-HT receptor blockers did not evoke aversive behaviour per se. Therefore, both GABAergic and serotonergic mechanisms are likely to play an inhibitory role in the dorsal CG integrating aversive behaviour.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of stress on regional brain serotonin metabolism after progesterone treatment and upon plasma progesterone in the rat

Summary The effect of progesterone upon stress altered serotonin (5-HT) metabolism in various regions of the rat brain was investigated with regard to a possible connection with premenstrual and post-partum depression. When electric footshock was administered to ovariectomized rats pretreated with progesterone or its vehicle, there were generally higher 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) concentrations after progesterone. 5-HT levels were significantly higher in thalamus, hippocampus, raphe, and frontal cortex, 5-HIAA rose significantly in hippocampus, raphe, and frontal cortex. Whereas after electric footshock alone the septum showed highest increases of 5-HT and 5-HIAA and hippocampus ranged last, after pretreatment with progesterone increases of 5-HT and 5-HIAA were least pronounced in septum but rather high in hippocampus. Electric footshock administered to ovariectomized rats also resulted in an increase of plasma progesterone concentration.     

Serotonin mediates oestrogen stimulation of cell proliferation in the adult dentate gyrus

Characterizing the mechanisms by which endogenous factors stimulate neurogenesis is of special interest in view of the possible implication of newly generated cells in hippocampal functions or disorders. The aim of this study was to determine whether serotonin (5-HT) and oestradiol (E2) act through a common pathway to increase cell proliferation in the adult dentate gyrus (DG). We also investigated the effects of long-lasting changes in oestrogen levels on cell proliferation. Combining ovariectomy with inhibition of 5-HT synthesis using p-chlorophenylalanine (PCPA) treatment produced approximately the same decreases in the number of bromodeoxyuridine (BrdU) and PSA-NCAM immunolabelled cells in the subgranular layer as ovariectomy alone. Administration of 5-hydroxytryptophan (5-HTP) restored cell proliferation primarily decreased by ovariectomy, whereas oestradiol was unable to reverse this change in ovariectomized rats treated with PCPA. These findings demonstrate that 5-HT mediates oestrogen stimulation of cell proliferation in adult dentate gyrus. However, increase in ovarian hormones during pregnancy has no effect on dentate cell proliferation. This finding suggests that concomitant changes in other factors, such as glucocorticoids, may counterbalance the positive regulation of cell proliferation by 5-HT and oestradiol. Finally, oestrogen may regulate structural plasticity by stimulating PSA-NCAM expression independently of neurogenesis, as shown for instance by the increases in the number of PSA-NCAM labelled cells in pregnants. As 5-HT and oestrogen are involved in mood disorders, our data suggest that the positive regulation of cell proliferation and neuroplasticity by these two factors may contribute to restore hippocampal connectivity in depressive patients.     

Estradiol benzoate decreases nigral GABAergic activity in male rats

Repeated doses of estradiol benzoate (10 micrograms/kg, s.c., once a day for 2, 5 or 8 days) to male rats decreased gamma-aminobutyric acid (GABA) content and glutamate decarboxylase (GAD) activity in substantia nigra (SN) but failed to change these parameters in hippocampus, cerebral cortex, cerebellum, lateral septum and olfactory tubercle. In the caudate nucleus, estradiol benzoate decreased GABA concentration but did not modify GAD activity. A decrease in nigral GABA concentration and GAD activity was also observed 24 and 48 but not 3 h after a single injection of estradiol benzoate. These data are consistent with results on GAD activity reported by McGinnis et al. in ovariectomized rats. Kinetic analysis of nigral GAD activity revealed that repeated estradiol benzoate injection reduced the Vmax without affecting the Km of GAD. Estradiol benzoate also reduced the rate of nigral GABA accumulation resulting from local infusion of gabaculine, suggesting that the steroid decreases GABA turnover in male rat SN. Hypophysectomy decreased GABA content and GAD activity in SN and GABA content in striatum. Administration of estradiol benzoate for 8 days to hypophysectomized rats failed to decrease further these parameters. Taken together, these data suggest that estradiol benzoate decreases SN GABAergic activity and that the integrity of the pituitary gland is required for this effect.     

Reduction of GABAB inhibitory postsynaptic potentials by serotonin via pre- and postsynaptic mechanisms in CA3 pyramidal cells of rat hippocampus in vitro.

The action of serotonin (5-HT) on GABAergic synaptic transmission was investigated with intracellular recordings in CA3 pyramidal cells of rat hippocampal slices. Local application of 5-HT (500 microM) hyperpolarized CA3 pyramidal cells, decreased cellular input resistance, and reduced slow afterhyperpolarizations. Serotonin attenuated the late (GABAB) component of polysynaptic inhibitory postsynaptic potentials (IPSPs; 47% of control) without affecting the early (GABAA) component. During bath application of the excitatory amino acid antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (20 microM) and 2-amino-5-phosphonovalerate (AP-5) (40 microM), 5-HT similarly decreased the amplitude of the late (GABAB) component (17% of control) of monosynaptic IPSPs but did not affect the early (GABAA) component. The mean reversal potentials of poly- and monosynaptic IPSPs were unaffected by 5-HT. The conductance increases associated with the late component of poly- and monosynaptic IPSPs were reduced by 5-HT. Hyperpolarizing responses evoked in CA3 pyramidal cells by somatic applications of gamma-aminobutyric acid (GABA) were unaffected by 5-HT. During bath application of bicuculline (20-50 microM), hyperpolarizing responses elicited by dendritic GABA application were reduced by 5-HT (71% of control). The effect of 5-HT on these direct GABAB hyperpolarizations (29% decrease in response) does not appear sufficient to fully account for the effect of 5-HT on late GABAB IPSPs (53-83% decrease in response). Therefore, 5-HT may reduce GABAB IPSPs in CA3 pyramidal cells 1) by a postsynaptic action on pyramidal cells and 2) by a selective presynaptic action on GABAergic interneurons mediating the GABAB IPSP. This presynaptic action of 5-HT does not appear to involve excitatory afferents onto inhibitory interneurons.     

mu-Opioids disinhibit and kappa-opioids inhibit serotonin efflux in the dorsal raphe nucleus

The relative importance of GABAergic and glutamatergic afferents in mediating the effects of mu- and kappa-opioids on serotonin (5-HT) efflux in vivo has not been firmly established. Thus, we used microdialysis in the dorsal raphe nucleus (DRN) of freely behaving rats to study the effect of GABA and glutamate receptor antagonists on opioid-induced changes in 5-HT efflux. Infusing the mu-opioid agonist DAMGO (300 microM) increased extracellular 5-HT in the DRN by approximately 70%. During infusion of the GABA(A) receptor blocker bicuculline (100 microM), extracellular 5-HT increased by approximately 250%, and subsequent infusion of DAMGO decreased 5-HT to approximately 70% above the pre-bicuculline baseline. These data are consistent with the hypothesis that mu-opioids disinhibit 5-HT neurons, an effect attenuated by direct inhibition of 5-HT efflux or inhibition of excitatory influences on 5-HT efflux. To further test this hypothesis, glutamate receptor blockers, AP-5 (1 mM) and DNQX (300 microM), were co-infused with DAMGO. The glutamate receptor antagonists prevented decreases in 5-HT elicited by DAMGO in the presence of bicuculline. This indicates that DAMGO inhibits glutamatergic afferents, which partly offsets the disinhibitory influence of mu-opioids on 5-HT efflux. In contrast, the kappa-opioid agonist, U-50,488 (300 microM), decreased 5-HT by approximately 30% in the DRN. Glutamate and GABA receptor antagonists did not block this effect. In conclusion, mu-opioids inhibit GABAergic and glutamatergic afferents, thereby indirectly affecting 5-HT efflux in the DRN. In contrast, kappa-opioids inhibit 5-HT efflux independent of effects on glutamatergic and GABAergic afferents.     

Valproate Effect on γ-Aminobutyric Acid Release in Pars Reticulata of Substantia Nigra: Combination of Push-pull Perfusion and Fluorescence Histochemistry

Summary: To study further the previously demonstrated suppressive in vivo effect of sodium valproate (VPA) on y-aminobutyric acid (GABA) release in the preoptic area, we examined GABA neurotransmission in substantia nigra (SN), using the push-pull cannula technique in freely moving ovariectomized rats. To clarify whether the area in the substantia nigra that is actually perfused is pars reticulata, known to receive rich GABAergic input from striatum, we used the retrograde fluorescence tracer fast blue (FB) after each perfusion experiment, applying the tracer through the push-pull cannula. Nigral perfusion with VPA caused significant suppression of local GABA release. This effect was more marked in a subgroup of animals showing retrograde labeled cell bodies in striatum, i.e., animals with a tracer application site and therefore also a perfusion site precisely in pars reticulata. Our data suggest that VPA inhibits GABA release in rat SN as it does in the preoptic area, which may be in agreement with our hypothesis of enhanced GABAergic neurotransmission by VPA, causing suppression of presynaptic GABA release through negative feedback actions on the GABA autoreceptor complex. Furthermore, the combination of push-pull cannula technique and retrograde fluorescence tracer application appears to be an important tool to prove afferent connections of the area actually perfused in neuronal networks.     

Tooth-pulp-evoked rostral spinal trigeminal nucleus neuron activity is inhibited by conditioning sciatic nerve stimulation in the rat: possible role of 5-HT3 receptor mediated GABAergic inhibition

The purpose of the present study was to determine whether modulation of the trigeminal spinal nucleus oralis (TSNO) neurons related to tooth-pulp (TP)-evoked jaw-opening reflex (JOR) after electrical stimulation of the sciatic nerve (SN) is mediated by the descending serotonergic (5-HT(3)) inhibitory system activated by inhibitory GABAergic interneurons. In 30 anesthetized rats, the activity of TSNO neurons (87.5%, 35/40) and all digastric muscle electromyograms (dEMG, n=30) in response to TP stimulation (at an intensity of 3.5 times the threshold for JOR) were inhibited by conditioning stimulation of the SN (5.0 mA x 0.5 ms, 1 Hz, conditioning-test intervals; 50 ms). The inhibitory effects were significantly attenuated after intravenous administration of the 5-HT(3) receptor antagonist ICS 205-930 (n=6). Using multibarrel electrodes, iontophoretic application of ICS 205-930 into the TSNO significantly reduced the SN stimulation-induced inhibition of TP-evoked TSNO neuronal excitation (n=6), and in the same neurons, iontophoretic application of the GABA(A) receptor antagonist bicuculline into the TSNO greatly inhibited their effect. On the other hand, we found the expression of 5-HT(3) receptor immunoreactive neurons in the TSNO. These results suggest that SN stimulation may activate the descending serotonergic (5-HT(3)) inhibitory system through activation of inhibitory GABAergic interneurons, which inhibit excitatory responses of the TSNO neurons to TP stimulation.     

Modulation of striatal dopamine metabolism by the activity of dorsal raphe serotonergic afferences

Serotonin (5-HT)-dopamine (DA) interaction was studied in the caudate nucleus after electrical stimulation of the dorsal raphe (DR), an area containing 5-HT cell bodies and sending afferences to nigrostriatal dopaminergic neurons. The DR was stimulated by means of a bipolar stainless steel electrode for 16 min (10 Hz, 0.6 ms, 200 microA). 5-HT and DA metabolism were monitored before, during and after stimulation by in vivo differential pulse voltammetry. This electrochemical technique uses carbon fiber electrodes implanted in brain areas to record oxidation peaks corresponding to extracellular 5-hydroxyindolacetic acid (5-HIAA) and dihydroxyphenylacetic acid (DOPAC). Changes in the concentrations of the metabolites were recorded every 2 min in freely moving rats. Both 5-HIAA and DOPAC increased in the first minutes after the beginning of stimulation, the rise lasting 30 min after the end. That DR was closely involved was borne out by the fact that stimulation in the surrounding areas had no effect on either metabolite. Classical biochemical determinations in tissue samples were also used to study the effect on DA release: 3-methoxytyramine (3-MT) levels, measured in basal conditions and after blockade of its degradation by pargyline, were not changed, indicating that DR stimulation, though increasing DA metabolism, does not affect release. However, modulation of DA transmission by 5-HT afferences seems possible in certain circumstances. This 5-HT-DA interaction appears to be presynaptic (on dopaminergic terminals or cell bodies) since it is not prevented by kainic acid degeneration of striatal neurons.     

Castration rapidly decreases hypothalamic γ-aminobutyric acidergic neuronal activity in both male and female rats

The postcastration LH response is greater and somewhat more rapid in male than female rats. We have previously demonstrated that hypothalamic γ-aminobutyric acid (GABA)ergic neuronal activity decreases following gonadectomy in male rats. To investigate whether these same hypothalamic GABA neurons decrease their activity postcastration in female rats, and whether more rapid and or greater postcastration decreases occur in male rats, we determined the timing and magnitude of the postcastration decreases in GABA turnover which are associated with the sexually dimorphic postcastration LH response. Adult male and 4-day cycling female rats were castrated between 0800 and 1000 h (females ovariectomized on diestrus day 1). Serum LH levels increased significantly by 12 h postcastration in both males and females with the magnitude of the increases being 6.2-fold in males and 2.8-fold in females. GABA turnover was determined in 16 microdissected brain structures by the GABA transaminase inhibition method at 0 h (sham-operated controls), 6 h, 12 h and 1, 2, 4 and 6 days postcastration. In male rats, in the diagonal band of Broca at the level of the organum vasculosum of the lamina terminalis [DBB(ovlt)], the rate of GABA turnover decreased significantly already by 6 h postcastration compared with the 0 h controls, and remained suppressed through 6 days. This rapid down regulation of DBB(ovlt) GABAergic neurons also occurred in female rats, however, the duration of the decrease was not as prolonged as in male rats. Similar changes occurred in the tuberoinfundibular GABAergic (TIGA) neurons projecting to the median eminence in both males and females. Down regulation of these GABAergic neurons precedes or is coincident with increased postcastration LH secretion in both sexes, and the duration of the decreases is consistent with the less robust postcastration LH response in female rats. In addition, the rate of GABA turnover decreased after castration in the interstitial (bed) nucleus of the stria terminalis, ventral aspect (INSTv), the medial preoptic nucleus, dorsomedial aspect (MPNdm) and the ventromedial nucleus, ventrolateral aspect (VMNvl) in male rats, and in the INSTv and VMNvl of female rats, while there was no effect of castration in other hypothalamic regions or control structures. The result in the female VMNvl is consistent with reports that GABA facilitates lordosis behavior in this hypothalamic structure. These findings are consistent with the hypothesis that discrete hypothalamic populations of sex steroid-sensitive GABAergic neurons mediate the postcastration LH responses in both male and female rats, and may underlie other sexually dimorphic adult phenotypes such as sex behavior.