The pharmacology of the neurochemical transmission in the midbrain raphe nuclei of the rat

Midbrain slices containing the dorsal and medial raphe nuclei were prepared from rat brain, loaded with [(3)H]serotonin ([(3)H]5-HT), superfused and the release of [(3)H]5-HT was determined at rest and in response to electrical stimulation. Compartmental analysis of [(3)H]5-HT taken up by raphe tissue indicated various pools where the neurotransmitter release may originate from these stores differed both in size and rate constant. 5-HT release originates not only from vesicles but also from cytoplasmic stores via a transporter-dependent exchange process establishing synaptic and non-synaptic neurochemical transmission in the serotonergic somatodendritic area. Manipulation of 5-HT transporter function modulates extracellular 5-HT concentrations in the raphe nuclei: of the SSRIs, fluoxetine was found 5-HT releaser, whereas citalopram did not exhibit this effect. Serotonergic projection neurons in the raphe nuclei possess inhibitory 5-HT(1A) and 5-HT(1B/1D) receptors and facilitatory 5-HT(3) receptors, which regulate 5-HT release in an opposing fashion. This observation indicates that somatodendritic 5-HT release in the raphe nuclei is under the control of several 5-HT homoreceptors. 5-HT(7) receptors located on glutamatergic axon terminals indirectly inhibit 5-HT release by reducing glutamatergic facilitation of serotonergic projection neurons. An opposite regulation of glutamatergic axon terminals was also found by involvement of the inhibitory 5-HT(7) and the stimulatory 5-HT(2) receptors as these receptors inhibit and stimulate glutamate release in raphe slice preparation, respectively, Furthermore, postsynaptic 5-HT(1B/1D) heteroreceptors interact with release of GABA in inhibitory fashion in raphe GABAergic interneurons. Serotonergic projection neurons also possess glutamate and GABA heteroreceptors; NMDA and AMPA receptors release 5-HT, whereas both GABAA and GABAB receptors inhibit somatodendritic 5-HT release. Evidence was found for reciprocal interactions between serotonergic and glutamatergic as well as serotonergic and GABAergic innervations in the raphe nuclei. Serotonergic neurons in the raphe nuclei also receive noradrenergic innervation arising from the locus coeruleus and alpha-1 and alpha-2 adrenoceptors inhibited [(3)H]5-HT release in our experimental conditions. The close relation between 5-HT transporter and release-mediating 5-HT autoreceptors was also shown by addition of L-deprenyl, a drug possessing inhibition of type B monoamine oxidase and 5-HT reuptake. L-Deprenyl selectively desensitizes 5-HT(1B) but not 5-HT(1A) receptors and these effects are not related to inhibition of 5-HT metabolism but rather to inhibition of 5-HT transporter.     

Adult AMPA GLUA1 Receptor Subunit Loss in 5-HT Neurons Results in a Specific Anxiety-Phenotype with Evidence for Dysregulation of 5-HT Neuronal Activity

Both the glutamatergic and serotonergic (5-HT) systems are implicated in the modulation of mood and anxiety. Descending cortical glutamatergic neurons regulate 5-HT neuronal activity in the midbrain raphe nuclei through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors. To analyze the functional role of GLUA1-containing AMPA receptors in serotonergic neurons, we used the Cre-ERT2/loxP-system for the conditional inactivation of the GLUA1-encoding Gria1 gene selectively in 5-HT neurons of adult mice. These Gria1^5-HT−/− mice exhibited a distinct anxiety phenotype but showed no alterations in locomotion, depression-like behavior, or learning and memory. Increased anxiety-related behavior was associated with significant decreases in tryptophan hydroxylase 2 (TPH2) expression and activity, and subsequent reductions in tissue levels of 5-HT, its metabolite 5-hydroxyindoleacetic acid (5-HIAA), and norepinephrine in the raphe nuclei. However, TPH2 expression and activity as well as monoamine levels were unchanged in the projection areas of 5-HT neurons. Extracellular electrophysiological recordings of 5-HT neurons revealed that, while α1-adrenoceptor-mediated excitation was unchanged, excitatory responses to AMPA were enhanced and the 5-HT_1A autoreceptor-mediated inhibitory response to 5-HT was attenuated in Gria1^5-HT−/− mice. Our data show that a loss of GLUA1 protein in 5-HT neurons enhances AMPA receptor function and leads to multiple local molecular and neurochemical changes in the raphe nuclei that dysregulate 5-HT neuronal activity and induce anxiety-like behavior.     

Distinct modulatory effects of 5-HT on excitatory synaptic transmissions in the nucleus tractus solitarius of the rat

The second-order relay neurons in the nucleus tractus solitarius (NTS) receive numerous peripheral afferent inputs mainly from the vagus nerve. Their activity is modified by several neuromodulators and hence autonomic responses are properly regulated. Serotonin (5-HT) is an important candidate for such neuromodulators, since serotonergic inputs and distribution of 5-HT receptors are provided in the NTS. However, its mechanism of action remains unclear. To evaluate the serotonergic modulation of synaptic transmission, we examined the effects of 5-HT (1.0-10.0 μM) on the solitary tract-evoked excitatory postsynaptic currents (eEPSCs) and spontaneously occurring EPSCs (sEPSCs) in the preselected second-order neurons of the rat NTS. 5-HT concentration-dependently decreased the amplitude of eEPSCs, which was accompanied by an increase in paired-pulse ratio. The inhibitory effect of 5-HT was mimicked by α-methylserotonin and blocked by ketanserin. 5-HT had no effect on the inward current induced in the NTS neurons by topically applied α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA). On the other hand, 5-HT increased the frequency of sEPCSs without effect on their amplitude. This excitatory effect of 5-HT was mimicked by 2-methylserotonin and antagonized by ondansetron. The results suggest a dual modulation of the excitatory synaptic transmission by 5-HT in the NTS; presynaptic inhibition of the peripheral inputs synapsing to the relay neurons via 5-HT(2) receptors and presynaptic excitation of inputs from the intrinsic local network via 5-HT(3) receptors. These effects of 5-HT may provide important means of optimizing the autonomic responses mediated by the NTS network.     

Effect of acute and repeated versus sustained administration of the 5-HT1A receptor agonist ipsapirone: electrophysiological studies in the rat hippocampus and dorsal raphe

The present study was aimed at examining the adaptation of presynaptic 5-HT_1A autoreceptors in the dorsal raphe and of postsynaptic 5-HT_1A receptors in the dorsal hippocampus during long-term administration of the 5-HT_1A receptor agonist ipsapirone given either repeatedly or in a sustained fashion. Concurrent microiontophoretic application of ipsapirone did not attentuate the suppressant effect of 5-hydroxytyptamine (5-HT) on 5-HT neurons, but markedly decreased it when co-applied on CA₃ pyramidal neurons in the dorsal hippocampus. Thus, ipsapirone acted as a full agonist in the dorsal raphe and as a partial agonist in the dorsal hippocampus. Ipsapirone (15 mg/kg/day, s.c. × 2 days) delivered by osmotic minipumps markedly decreased the firing activity of the dorsal raphe 5-HT neurons. After 14 days of treatment, there was a complete recovery of their firing activity and a desensitization of their somatodendritic 5-HT_1A autoreceptors, as assessed using microiontophoretic applications of 5-HT and 8-hydroxy-2(di-n-propylamino)tetraline (8-OH-DPAT) onto 5-HT neurons. The same degree of desensitization was obtained when ipsapirone was administered with repeated injections (7.5 mg/kg b.i.d., s.c. × 14 days). In contrast, the two modalities of ipsapirone adminsitration left unaltered the responsiveness of CA₃ pyramidal neurons to microiontophoretic applications of 5-HT and 8-OH-DPAT. In conclusion, long-term administration of ipsapirone most likely increases 5-HT neurotransmission by enhancing the tonic activation of postsynaptic 5-HT_1A receptors. Therefore, the use of sustained release preparation of 5-HT_1A receptor agonists should not alter their therapeutic effectiveness in anxiety and affective disorders since the same effects on 5-HT_1A receptor functions were produced in this rat model by the sustained and the repeated modes of administration of ipsapirone. Received: 24 September 1996 / Accepted: 28 April 1997     

Review article: roles played by 5-hydroxytryptamine in the physiology of the bowel

The application of 5-HT to the gut elicits a wide variety of effects because of the expression and wide distribution in the bowel of many subtypes of 5-HT. There is, however, no reason to believe that all of these receptors are stimulated by endogenous 5-HT. 5-HT has been found to be the neurotransmitter of a subset of myenteric interneurons, which evoke a slow excitatory postsynaptic response mediated by 5-HT_1P receptors. The major enteric depot of 5-HT is found in mucosal enterochromaffin cells, which are sensory transducers that utilize 5-HT to activate both intrinsic (via 5-HT_1P and 5-HT₄ receptors) and extrinsic (via 5-HT₃ receptors) primary afferent nerves. Mucosal 5-HT is inactivated by uptake into epithelial cells mediated by the same 5-HT transporter utilized by serotonergic neurons. Antagonism of 5-HT₃ receptors by compounds such as alosetron should be useful in treating functional bowel disease because they can inhibit excitation of extrinsic sensory nerves by 5-HT without interfering with intrinsic enteric reflexes.     

Effect of prolonged administration of tianeptine on 5-HT neurotransmission: an electrophysiological study in the rat hippocampus and dorsal raphe

Extracellular unitary recordings of dorsal hippocampus CA₃ pyramidal neurons and of dorsal raphe 5-hydroxytryptamine (5-HT) neurons were used to assess the effect of tianeptine, a putative antidepressant, on the efficacy of 5-HT neurotransmission. Sustained tianeptine administration (20 mg/kg/day, s.c. × 14 days) did not modify the firing activity of 5-HT neurons in the dorsal raphe. Their responsiveness to the intravenous injection of LSD, an agonist of the somatodendritic 5-HT autoreceptor, and of 8-OH-DPAT, a selective 5-HT_1A agonist, was also unaffected by this treatment. The responsiveness of CA₃ pyramidal neurons to microiontophoretic application of 5-HT remained unchanged after sustained tianeptine administration, but it was markedly enhanced in rats treated with repeated electroconvulsive shocks. Finally, the duration of suppression of firing activity of CA₃ pyramidal neurons produced by electrical stimulation of the ascending 5-HT pathway, delivered at 1 Hz and 5 Hz, was not modified in rats treated with tianeptine. Methiothepin, an antagonist of the terminal autoreceptor enhanced the effectiveness of 5-HT pathway stimulation to the same extent in control and tianeptinetreated rats. The present results indicate that, administered at a dose known to stimulate 5-HT reuptake (20 mg/kg/day, s.c.; by minipump), and for a period of time (14 days) for which other antidepressant treatments have been shown to enhance 5-HT function, tianeptine does not modify the efficacy of 5-HT synaptic transmission in the rat hippocampus.     

Raphe serotonin neurons are not homogenous: electrophysiological, morphological and neurochemical evidence

The median (MR) and dorsal raphe (DR) nuclei contain the majority of the 5-hydroxytryptamine (5-HT, serotonin) neurons that project to limbic forebrain regions, are important in regulating homeostatic functions and are implicated in the etiology and treatment of mood disorders and schizophrenia. The primary synaptic inputs within and to the raphe are glutamatergic and GABAergic. The DR is divided into three subfields, i.e., ventromedial (vmDR), lateral wings (lwDR) and dorsomedial (dmDR). Our previous work shows that cell characteristics of 5-HT neurons and the magnitude of the 5-HT_1A and 5-HT_1B receptor-mediated responses in the vmDR and MR are not the same. We extend these observations to examine the electrophysiological properties across all four raphe subfields in both 5-HT and non-5-HT neurons. The neurochemical topography of glutamatergic and GABAergic cell bodies and nerve terminals were identified using immunohistochemistry and the morphology of the 5-HT neurons was measured. Although 5-HT neurons possessed similar physiological properties, important differences existed between subfields. Non-5-HT neurons were indistinguishable from 5-HT neurons. GABA neurons were distributed throughout the raphe, usually in areas devoid of 5-HT neurons. Although GABAergic synaptic innervation was dense throughout the raphe (immunohistochemical analysis of the GABA transporters GAT1 and GAT3), their distributions differed. Glutamate neurons, as defined by vGlut3 anti-bodies, were intermixed and co-localized with 5-HT neurons within all raphe subfields. Finally, the dendritic arbor of the 5-HT neurons was distinct between subfields. Previous studies regard 5-HT neurons as a homogenous population. Our data support a model of the raphe as an area composed of functionally distinct subpopulations of 5-HT and non-5-HT neurons, in part delineated by subfield. Understanding the interaction of the cell properties of the neurons in concert with their morphology, local distribution of GABA and glutamate neurons and their synaptic input, reveals a more complicated and heterogeneous raphe. These results provide an important foundation for understanding how specific subfields modulate behavior and for defining which aspects of the circuitry are altered during the etiology of psychological disorders.This article is part of a Special Issue entitled ‘Serotonin’.     

Potentiation of excitatory serotonergic responses by MK-801 in the medial prefrontal cortex

New atypical antipsychotics show a greater affinity to serotonergic rather than to dopamine receptors, suggesting that serotonin (5-HT) has a major role in the pathophysiology and treatment of schizophrenia. The goal of this study was to characterise the response of pyramidal neurons in the medial prefrontal cortex (mPFC) to 5-HT and NMDA before and after administration of the NMDA receptor antagonist, MK-801 (dizocilpine), a well-validated pharmacological model of psychosis. mPFC pyramidal (glutamatergic) neurons were recorded in urethane-anaesthetised rats. The responses to NMDA and 5-HT were assessed using in vivo electrophysiology and microiontophoresis. The 5-HT_2A/2C antagonist ritanserin and the 5-HT_1A antagonist WAY100635 were used to block 5-HT responses. MK-801 decreased the NMDA-induced excitatory responses and increased NMDA-evoked burst activity among mPFC pyramidal neurons. Three subpopulations of pyramidal cells were identified according to their responses to 5-HT: excitation (33%), inhibition (40%) and non-response (27%). The inhibitory responses were blocked by WAY100635 in 100% of cases, but not by ritanserin; the excitatory responses were blocked by ritanserin in 75% of cases, but not by WAY100635. The administration of MK-801 potentiated the firing rate of excitatory responses but did not modify the inhibitory responses induced by microiontophoretic application of 5-HT. These results suggest that MK-801 modifies 5-HT synapses in the mPFC by potentiating the excitatory 5-HT_2A/2C responses and attenuating NMDA excitations. These data indicate that 5-HT excitatory transmission is selectively impaired at the mPFC level in this pharmacological model of schizophrenia.     

8-OH-DPAT disruption of prepulse inhibition in rats: reversal with (+)WAY 100,135 and localization of site of action

Recent studies have implicated central serotonergic systems in the modulation of prepulse inhibition (PPI), an operational measure of sensorimotor gating, which has been used to identify gating deficits in psychiatric disorders, such as schizophrenia, Huntington's disease, and obsessive compulsive disorder. Both serotonin (5-HT) releasers and agonists at 5-HT_1A, 5-HT_1B, and 5-HT₂ receptors reduce PPI in the rat. The present experiments demonstrate that the disruption of PPI in rats induced by the systemic administration of the 5-HT_1A agonist, 8-OH-DPAT (8-hydroxy-2(di-n-propylamino)tetralin; 0.2 mg/kg), can be attenuated by the novel, selective 5-HT_1A antagonist (+)WAY 100,135, (20.0 mg/kg),N-tert-butyl-3-(4-(2-methoxyphenyl)-piperazin-1-yl)-2-phenyl-propanamide. Further experiments addressing the central site of action of 8-OH-DPAT revealed that the microinjection of 8-OH-DPAT (5.0 µg/0.5 l) into either the median raphe nucleus (MR) or dorsal raphe nucleus (DR) disrupts PPI. The reduction in PPI produced by intra-raphe microinjections of 8-OH-DPAT was prevented by a systemic injection of (+)WAY 100,135. These results support the hypothesis that somatodendritic 5-HT_1A autoreceptors within the midbrain raphe subserve the PPI-disruptive effects of systemically administered 8-OH-DPAT. The decrement in PPI after intra-raphe infusions of a high dose of 8-OH-DPAT, however, was substantially less than the decrement in PPI after systemic administration of the drug. Hence, sites in addition to the somatodendritic autoreceptors may also play an important role in 8-OH-DPAT-induced disruption of PPI. Together with previous reports that 5-HT releasers and other 5-HT agonists also disrupt PPI, the results support the hypothesis that the serotonergic system modulates PPI through multiple receptor and anatomical systems.     

Presynaptic control of serotonin on striatal dopamine function

Rationale

The influences of the serotonergic system on dopamine (DA) neuron activity have received considerable attention during the last three decades due to the real opportunity to improve disorders related to central DA neuron dysfunctions such as Parkinson??s disease, schizophrenia, or drug abuse with serotonergic drugs. Numerous biochemical and behavioral data indicate that serotonin (5-HT) affects dopaminergic terminal function in the striatum.

Objective

The authors propose a thorough examination of data showing controversial effects induced by striatal 5-HT on dopaminergic activity.

Results

Inhibitory and excitatory effects of exogenous 5-HT have been reported on DA release and synthesis, involving various striatal 5-HT receptors. 5-HT also promotes an efflux of DA through reversal of the direction of DA transport. By analogy with the mechanism of action described for amphetamine, the consequences of 5-HT entering DA terminals might explain both the excitatory and inhibitory effects of 5-HT on presynaptic DA terminal activity, but the physiological relevance of this mechanism is far from clear. The recent data suggest that the endogenous 5-HT system affects striatal DA release in a state-dependent manner associated with the conditional involvement of various 5-HT receptors such as 5-HT_2A, 5-HT_2C, 5-HT₃, and 5-HT₄ receptors.

Conclusion

Methodological and pharmacological issues have prevented a comprehensive overview of the influence of 5-HT on striatal DA activity. The distribution of striatal 5-HT receptors and their restricted influence on DA neuron activity suggest that the endogenous 5-HT system exerts multiple and subtle influences on DA-mediated behaviors.     

Blockade of pre-and post-synaptic 5-HT1A receptors does not modify the effect of fluoxetine or 5-hydroxytryptophan on ethanol and food intake in rats

Selective serotonin reuptake inhibitors (SSRIs) or serotonin precursors inhibit ethanol and food intake by increasing the synaptic availability of 5-HT in the central nervous system. However, these agents can also increase 5-HT levels at somatodendritic 5-HT_1A autoreceptors, with negative effects on serotonergic transmission. (+)WAY100135 [N-ter-butyl 3-4-(2-methoxy-phenyl) piperazin-1-yl-2-phenylpropa-namide dihydrochloride] is a selective antagonist both at pre-and post-synaptic 5-HT_1A receptors. The present study investigated the effect on ethanol and food intake of (+)WAY100135, given alone or coadministered with the SSRI fluoxetine or the 5-HT precursor 5-hydroxytryptophan (5-HTP) in genetically selected alcohol-preferring rats. Blockade of presynaptic 5-HT_1A receptors after injection of (+)WAY100135, 0.1 or 1 μg/rat, into the dorsal raphe did not significantly modify ethanol, food or total fluid intake. The same doses of (+)WAY100135 did not modify the inhibition of ethanol and food intake induced by intraperitoneal (IP) injection of fluoxetine, 5 mg/kg. Subcutaneous (SC) administration of (+)WAY100135 (1 or 10 mg/kg) did not affect the 3-h, or the overnight intake of ethanol, food or total fluids. Given together with IP fluoxetine (5 mg/kg) or SC 5-HTP (100 mg/kg plus carbidopa, 12.5 mg/kg), the same SC doses of (+)WAY100135 did not modify their inhibitory effect on ethanol and food consumption. Present findings suggest that blockade either of pre-or of pre-and postsynaptic 5-HT_1A receptors does not potentiate the inhibitory effect of fluoxetine or 5-HTP on ethanol and food intake. Received: 2 November 1996/Final version: 23 April 1997     

In vivo control of 5-hydroxytryptamine release by terminal autoreceptors in rat brain areas differentially innervated by the dorsal and median raphe nuclei

Selective serotonin reuptake inhibitors (SSRIs) reduce the 5-HT release in vivo. This effect is due to the activation of somatodendritic 5-HT_1A receptors and it displays a regional pattern comparable to that of selective 5-HT_1A agonists, i.e., preferentially in forebrain areas innervated by the dorsal raphe nucleus (DRN). However, despite a comparatively lower 5-HT_1A-mediated inhibition of 5-HT release and a greater density of serotonergic uptake sites in hippocampus, the net elevation produced by the systemic administration of SSRIs is similar in various forebrain areas, regardless of the origin of serotonergic fibres. As terminal autoreceptors may also limit the SSRI-induced elevations of 5-HT in the extracellular brain space, we reasoned that a differential control of 5-HT release by terminal autoreceptors in DRN- and median raphe-innervated areas might be accountable. To examine this possibility, we have conducted a regional microdialysis study in the DRN, MRN and four forebrain regions preferentially innervated either by the DRN (frontal cortex, striatum) or the median raphe nucleus (MRN; dorsal and ventral hippocampus) using freely moving rats. Dialysis probes were perfused with 1 μM of the SSRI citalopram to augment the endogenous tone on terminal 5-HT autoreceptors. The non-selective 5-HT₁ antagonist methiothepin (10 and 100 μM, dissolved in the dialysis fluid) increased extracellular 5-HT in frontal cortex and dorsal hippocampus in a concentration-dependent manner. The 5-HT_1B/1D antagonist GR 127935 was ineffective at 10 μM and tended to reduce 5-HT in dorsal hippocampus at 100 μM. The local infusion of 100 μM methiothepin significantly elevated the extracellular 5-HT concentration to 142–173% of baseline (mean values of 260 min post-administration) in the DRN, MRN, frontal cortex, striatum and hippocampus (dorsal and ventral). Comparable elevations were noted in the four forebrain regions examined. As observed in frontal cortex and dorsal hippocampus, the perfusion of 10 μM GR 127935 did not elevate 5-HT in DRN, MRN, striatum or ventral hippocampus. Because the stimulated 5-HT release in the DRN has been suggested to be under control of 5-HT_1B/1D receptors, we examined the possible contribution of these receptor subtypes to the effects of methiothepin in the DRN. The perfusion of sumatriptan (0.01–10 μM) or GR 127935 (0.01–10 μM) did not significantly modify the 5-HT concentration in dialysates from the DRN. Thus, the present data suggest that the comparable effects of SSRIs in DRN- and MRN-innervated forebrain regions are not explained by a preferential attenuation of 5-HT release by terminal 5-HT_1B autoreceptors in hippocampus, an area with a low inhibitory influence of somatodendritic 5-HT_1A receptors. Methiothepin-sensitive autoreceptors (possibly 5-HT_1B) appear to play an important role not only in the projection areas but also with respect to the control of 5-HT release in the DRN and MRN. In addition, our findings indicate that GR 127935 is not an effective antagonist of the actions of 5-HT at rat terminal autoreceptors. Received: 27 February 1998 / Accepted: 12 June 1998     

Modulation of the subthalamic nucleus activity by serotonergic agents and fluoxetine administration

Rationale

Within the basal ganglia, the subthalamic nucleus (STN) is the only glutamatergic structure and occupies a central position in the indirect pathway. In rat, the STN receives serotonergic input from the dorsal raphe nucleus and expresses serotonergic receptors.

Objective

This study examined the consequences of serotonergic neurotransmission modulation on STN neuron activity.

Methods

In vivo single-unit extracellular recordings, HPLC determination, and rotarod and bar test were performed in control, 4-chloro-DL-phenylalanine methyl ester hydrochloride- (pCPA, a serotonin synthesis inhibitor) and chronically fluoxetine-treated rats.

Results

The pCPA treatment and the administration of serotonin (5-HT) receptor antagonists increased number of bursting neurons in the STN. The systemic administration of the 5-HT_1A agonist, 8-OH-DPAT, decreased the firing rate and increased the coefficient of variation of STN neurons in pCPA-treated rats but not in control animals. Additionally, microinjection of 8-OH-DPAT into the STN reduced the firing rate of STN neurons, while microinjection of the 5-HT_2C agonist, Ro 60-0175, increased the firing rate in both control and fluoxetine-treated animals. Finally, the fluoxetine challenge increased the firing rate of STN neurons in fluoxetine-treated rats and induced catalepsy.

Conclusions

Our results indicate that the depletion and the blockage of 5-HT modify STN neuron firing pattern. STN neuron activity is under the control of 5-HT_1A and 5-HT_2C receptors located both inside and outside the STN. Finally, fluoxetine increases STN neuron activity in chronically fluoxetine-treated rats, which may explain the role of this nucleus in fluoxetine-induced extrapyramidal side effects.     

Acute and chronic tryptophan depletion differentially regulate central 5-HT1A and 5-HT2A receptor binding in the rat

Rationale Tryptophan depletion is used to reduce central serotonergic function and to investigate its role in psychiatric illness. Despite widespread clinical use, its effects on serotonin (5-HT) receptors have not been well characterized. Objective The aim of this study was to examine the effect of acute (ATD) and chronic tryptophan depletion (CTD) on free-plasma tryptophan (TRP), central TRP and 5-HT and brain 5-HT_1A and 5-HT_2A receptor binding in the rat. Methods TRP and 5-HT were measured by high-performance liquid chromatography and receptor levels determined by homogenate radioligand binding and in-vitro receptor autoradiography. Results Free-plasma TRP, central TRP and central 5-HT levels were significantly and similarly reduced by ATD and 1- and 3-week CTD compared to controls. ATD significantly reduced 5-HT_1A binding in the dorsal raphe (14%) but did not significantly alter postsynaptic 5-HT_1A binding (frontal cortex, remaining cortex and hippocampus) or 5-HT_2A binding (cortex and striatum). One-week CTD did not significantly alter cortical 5-HT_2A binding or postsynaptic 5-HT_1A binding. Furthermore, 3-week CTD did not significantly alter 5-HT_1A binding but significantly increased cortical 5-HT_2A binding without affecting striatal or hippocampal levels. In the CTD 1 and 3-week groups, rat body weight was significantly decreased as compared to controls. However, weight loss was not a confounding factor for decreased cortical 5-HT_2A-receptor binding. Conclusion ATD-induced reduction in somatodendritic 5-HT_1A autoreceptor binding may represent an intrinsic ‘homeostatic response’ reducing serotonergic feedback in dorsal raphe projection areas. In contrast, the increase in 5-HT_2A receptor after CTD may be a compensatory response to a long-term reduction in 5-HT.     

Serotonergic regulation of neuronal excitability in the prefrontal cortex

The cerebral cortex receives a dense serotonergic innervation originating predominantly from the dorsal raphe nucleus. This innervation regulates cortical functioning by activating multiple serotonin receptors that are differentially expressed by pyramidal cells and interneurons. Electrophysiological studies in the prefrontal cortex indicate that receptors of the 5-HT_1A and 5-HT_2A subtypes are the main serotonin receptors regulating membrane excitability in pyramidal cells. Most pyramidal cells in layer V coexpress 5-HT_1A and 5-HT_2A receptors that together regulate how these neurons encode excitatory input into neuronal firing. In contrast, a subset of large pyramidal cells of deep layer V appears to express exclusively 5-HT_2A receptors that depolarize and excite these cells. Serotonin also depolarizes and excites at least two classes of GABAergic interneurons by acting on 5-HT₃ and 5-HT_2A receptors. The differential expression of serotonin receptors in different pyramidal cells and interneurons is consistent with a growing appreciation of the anatomical, molecular and functional heterogeneity of pyramidal cells and interneurons of the cerebral cortex. These findings begin to lay the ground for a cellular-level understanding of the serotonergic regulation of the prefrontal cortex.     

Stress- and antidepressant treatment-induced modifications of 5-HT7 receptor functions in the rat brain

This paper summarizes a series of electrophysiological studies aimed at finding the effects of the activation of 5-HT₇ receptors on neuronal excitability as well as on excitatory and inhibitory synaptic transmission in the hippocampus and in the frontal cortex of the rat. These studies demonstrated that 5-HT₇ receptors play an important role in the modulation of the activity of the hippocampal network by regulating the excitability of pyramidal cells of the CA1 area, as well as via their effect on GABA and glutamatergic transmission. The reactivity of 5-HT₇ receptors in the hippocampus is decreased by repeated administration of antidepressant drugs and increased by a prolonged high level of corticosterone. More importantly, administration of antidepressant drug, imipramine, prevents the occurrence of corticosterone-induced changes in the function of hippocampal 5-HT₇ receptors. It has also been found that the blockade of 5-HT₇ receptors by the selective antagonist SB 269970, lasting for a few days, causes similar changes to those observed after long-term administration of antidepressants. Thus, it seems that the pharmacological blockade of 5-HT₇ receptors produces faster effects compared to classic antidepressant drugs. A similarity between the changes in the glutamatergic transmission induced by the blockade of 5 HT₇ receptors and those caused by repeated administration of the antidepressant drug, imipramine, has also been found in the frontal cortex. It has also been shown that the changes in glutamatergic transmission and the impairment of long-term synaptic plasticity in the frontal cortex of animals subjected to repeated restraint stress are reversed by the blockade of 5-HT₇ receptors.Overall, these studies, together with the data provided by other investigators, support the hypothesis that 5-HT₇ receptor antagonists may become a prototype of a new class of antidepressant drugs. Such compounds will not function by blocking 5-HT reuptake, as many of the currently used drugs, but through a direct interaction with the 5-HT₇ receptor. This type of action is highly selective and usually does not require the occurrence of adaptive changes in neuronal functions, thus allowing for a much quicker therapeutic effect.     

Early desensitization of somato-dendritic 5-HT1A autoreceptors in rats treated with fluoxetine or paroxetine

Electrophysiological and autoradiographic approaches were used to assess possible changes in 5-hydroxytryptamine (serotonin) 5-HT_1A receptors in the rat dorsal raphe nucleus after a subchronic treatment with fluoxetine or paroxetine, two specific serotonin reuptake inhibitors with antidepressant properties. Fluoxetine or paroxetine were injected daily (5 mg/kg, i.p.) for various time periods up to 21 days. Electrophysiological recordings performed 24 h after the last injection showed that the potency of the 5HT_1A receptor agonist, 8-OH-DPAT, to depress the firing of serotoninergic neurons in the dorsal raphe nucleus within brain stem slices was significantly reduced as early as after a 3-day treatment with either drug. The proportion of recorded neurons showing desensitization of somatodendritic 5-HT_1A autoreceptors increased along the treatment from 40% on the 3rd day to 60–80% on the 21st day. At no time during the treatment, was the specific binding of [³H]8-OHDPAT (agonist radioligand) or [³H] WAY-100 635 (antagonist radioligand) to 5-HT_1A receptors modified in the dorsal raphe nucleus or in other brain areas, suggesting that neither the density nor the coupling of these receptors to G-proteins were probably altered in rats injected with fluoxetine or paroxetine for up to 21 days.These results show that adaptive desensitization of somatodendritic 5-HT_1A autoreceptors within the dorsal raphe nucleus can already be detected after a 3-day treatment with selective serotonin reuptake inhibitors. Rather than the desensitization per se, it may be the progressive increase in the number of serotoninergic neurons with desensitized 5-HT_1A autoreceptors which plays a critical role in the (slowly developing) antidepressant action of these drugs.     

Acute 5-HT1A autoreceptor knockdown increases antidepressant responses and serotonin release in stressful conditions

Rationale

Identifying the etiological factors in anxiety and depression is critical to develop more efficacious therapies. The inhibitory serotonin_1A receptors (5-HT_1AR) located on 5-HT neurons (autoreceptors) limit antidepressant responses and their expression may be increased in treatment-resistant depressed patients.

Objectives

Recently, we reported that intranasal administration of modified small interference RNA (siRNA) molecules targeting 5-HT_1AR in serotonergic neurons evoked antidepressant-like effects. Here we extended this finding using marketed siRNAs against 5-HT_1AR (1A-siRNA) to reduce directly the 5-HT_1A autoreceptor expression and evaluate its biological consequences under basal conditions and in response to stressful situations.

Methods

Adult mice were locally infused with vehicle, nonsense siRNA, and 1A-siRNA into dorsal raphe nucleus (DR). 5-HT_1AR knockout mice (1A-KO) were also used. Histological approaches, in vivo microdialysis, and stress-related behaviors were performed to assess the effects of 5-HT_1A autoreceptor knockdown.

Results

Intra-DR 1A-siRNA infusion selectively reduced 5-HT_1AR mRNA and binding levels and canceled 8-OH-DPAT-induced hypothermia. Basal extracellular 5-HT in medial prefrontal cortex (mPFC) did not differ among treatments. However, 1A-siRNA-treated mice displayed less immobility in the tail suspension and forced swim tests, as did 1A-KO mice. This was accompanied by a greater increase in prefrontal 5-HT release during tail suspension test. Moreover, intra-DR 1A-siRNA infusion augmented the increase of extracellular 5-HT in mPFC evoked by fluoxetine, up to the level in 1A-KO mice.

Conclusion

Together with our previous report, the present results indicate that acute suppression of 5-HT_1A autoreceptor expression evokes robust antidepressant-like effects, likely mediated by an increased capacity of serotonergic neurons to release 5-HT in stressful conditions.     

Regulation of central synaptic transmission by 5-HT(1B) auto- and heteroreceptors

Although 5-HT(1B) receptors are believed to be expressed on nerve terminals, their precise mode of action is not fully understood because of the lack of selective antagonists. The 5-HT(1B) receptor knockout mouse was used in the present investigation to assess the function of 5-HT(1B) receptors in the modulation of synaptic transmission in three areas of the central nervous system: the dorsal raphe, the ventral midbrain, and the nucleus accumbens. N-(3-Trifluoromethylphenyl)piperazine, a 5-HT(1B) receptor agonist, potently inhibited 5-HT(1A) receptor-mediated slow inhibitory postsynaptic potentials (IPSPs) in the dorsal raphe of wild-type but not knockout mice. Both synaptically released 5-HT and exogenous 5-HT caused a presynaptic inhibition that outlasted the postsynaptic hyperpolarization only in wild-type mice. In the ventral midbrain, 5-HT(1B) receptor-dependent inhibition of gamma-aminobutyric acid(B) IPSPs in dopamine neurons was present in wild-type animals and absent in knockout animals. Similar results were obtained in the nucleus accumbens measuring glutamate-mediated excitatory postsynaptic currents in medium spiny neurons. Finally, cocaine, which blocks 5-HT uptake, inhibited IPSPs in the dorsal raphe and the ventral midbrain of wild-type but not knockout mice, whereas cocaine produced comparable inhibition of excitatory postsynaptic currents in the nucleus accumbens of both types of animals. These results indicate that 5-HT(1B) receptors function as autoreceptors and heteroreceptors to exert presynaptic inhibition of transmitter release in the central nervous system. Furthermore, this study underscores the role played by presynaptic 5-HT(1B) receptors in mediating the effects of cocaine on synaptic transmission.     

5-HT1A receptor agonists: recent developments and controversial issues

During the last decade, serotonin (5-HT)_1A receptors have been a major target for neurobiological research and drug development. 5-HT_1A receptors have been cloned and a variety of selective agonists, such as the aminotetraline 8-OH-DPAT and the pyrimidinylpiperazine ipsapirone, have become available. Demonstrations of apparent intrinsic activity of these ligands at 5-HT_1A receptors, however, depend highly on the particular assay system. This may be due to the possible existence of receptor subtypes and to assay (or brain region)-dependent differences in receptor reserve and the nature of receptor-effector coupling. Nevertheless, the apparent intrinsic activity of 8-OH-DPAT seems to be higher (although possibly not yet maximal) than that of the pyrimidinylpiperazines. In the brain, 5-HT_1A receptors are located presynaptically as somatodendritic receptors on 5-HT neurons and postsynaptically in particular limbic and cortical regions. Although it is generally accepted that presynaptic 5-HT_1A receptors control 5-HT neuronal activity, recent evidence suggests an additional role of postsynaptic 5-HT_1A receptors in cortex as part of a negative feedback loop. Anxiolytic and antidepressive properties of selective 5-HT_1A receptor agonists have now been confirmed by clinical studies. Although it is well established that the latter properties depend on theagonistic activity of these compounds, theoptimal level of intrinsic activity is still a matter of debate and may be dependent on the clinical indication. Such compounds may also have antiaggressive effects, and possibly anticraving effects (manifested by their alcohol intake-reducing effects in dependent animals), but the specificity of these so-called anti-impulsivity effects is still controversial and not yet tested clinically. Anticataleptic, antiemetic and neuroprotective properties have been demonstrated in different species. Behavioral studies on the mechanisms underlying the anxiolytic and antidepressive effects have examined the relative contribution of pre-and postsynaptic 5-HT_1A receptors by means of local cerebral application and lesion techniques. Most evidence points towards a critical involvement of presynaptic receptors in the anxiolytic effects of 5-HT_1A receptor agonists (although a possible contribution of postsynaptic receptors cannot be excluded). With regard to the antidepressive properties, a case can be made for the reverse; i.e., a strong involvement of postsynaptic receptors and a questionable contribution of presynaptic receptors. However, as the therapeutic effects of those 5-HT_1A receptor (partial) agonists which have been tested clinically require repeated administration, attention has been directed increasingly towards chronic studies. These studies have shown that a number of electrophysiological, biochemical, behavioral and endocrinological 5-HT_1A receptor-related events adapt differentially to repeated or sustained administration. Thus, several hypotheses accounting for the delayed onset of action have been advanced. Among these, time-dependent downregulation /desensitization of eitherpre- orpostsynaptic 5-HT_1A receptors, or cortical 5-HT₂ receptors have received much attention. However, these hypotheses have their weaknesses, and it is argued thatfunctional sensitization of particular postsynaptic 5-HT_1A receptor-mediated events remains a valuable alternate hypothesis. Basic research on the role of 5-HT_1A receptors in psychopathology and in the therapeutic effects of clinically effective therapeutics, as well as on the mechanism of action of 5-HT_1A receptor ligands, will enable rational design of ligands with particular profiles of intrinsic activity at different 5-HT_1A receptor populations, and may contribute to a more efficient treatment of a multiplicity of brain disorders.