Stress-related serotonergic systems: implications for symptomatology of anxiety and affective disorders

Previous studies have suggested that serotonergic neurons in the midbrain raphe complex have a functional topographic organization. Recent studies suggest that stimulation of a bed nucleus of the stria terminalis-dorsal raphe nucleus pathway by stress- and anxiety-related stimuli modulates a subpopulation of serotonergic neurons in the dorsal part of the dorsal raphe nucleus (DRD) and caudal part of the dorsal raphe nucleus (DRC) that participates in facilitation of anxiety-like responses. In contrast, recent studies suggest that activation of a spinoparabrachial pathway by peripheral thermal or immune stimuli excites subpopulations of serotonergic neurons in the ventrolateral part of the dorsal raphe nucleus/ventrolateral periaqueducal gray (DRVL/VLPAG) region and interfascicular part of the dorsal raphe nucleus (DRI). Studies support a role for serotonergic neurons in the DRVL/VLPAG in inhibition of panic-like responses, and serotonergic neurons in the DRI in antidepressant-like effects. Thus, data suggest that while some subpopulations of serotonergic neurons in the dorsal raphe nucleus play a role in facilitation of anxiety-like responses, others play a role in inhibition of anxiety- or panic-like responses, while others play a role in antidepressant-like effects. Understanding the anatomical and functional properties of these distinct serotonergic systems may lead to novel therapeutic strategies for the prevention and/or treatment of affective and anxiety disorders. In this review, we describe the anatomical and functional properties of subpopulations of serotonergic neurons in the dorsal raphe nucleus, with a focus on those implicated in symptoms of anxiety and affective disorders, the DRD/DRC, DRVL/VLPAG, and DRI.     

Impaired Chemosensitivity of Mouse Dorsal Raphe Serotonergic Neurons Overexpressing Serotonin 1A (Htr1a) Receptors

Background

Serotonergic system participates in a wide range of physiological processes and behaviors, but its role is generally considered as modulatory and noncrucial, especially concerning life-sustaining functions. We recently created a transgenic mouse line in which a functional deficit in serotonin homeostasis due to excessive serotonin autoinhibition was produced by inducing serotonin 1A receptor (Htr1a) overexpression selectively in serotonergic neurons (Htr1a raphe-overexpressing or Htr1a^RO mice). Htr1a^RO mice exhibit episodes of autonomic dysregulation, cardiovascular crises and death, resembling those of sudden infant death syndrome (SIDS) and revealing a life-supporting role of serotonergic system in autonomic control. Since midbrain serotonergic neurons are chemosensitive and are implicated in arousal we hypothesized that their chemosensitivity might be impaired in Htr1a^RO mice.

Principal findings

Loose-seal cell-attached recordings in brainstem slices revealed that serotonergic neurons in dorsal raphe nucleus of Htr1a^RO mice have dramatically reduced responses to hypercapnic challenge as compared with control littermates. In control mice, application of 9% CO₂ produced an increase in firing rate of serotonergic neurons (0.260±0.041 Hz, n = 20, p = 0.0001) and application of 3% CO₂ decreased their firing rate (−0.142±0.025 Hz, n = 17, p = 0.0008). In contrast, in Htr1a^RO mice, firing rate of serotonergic neurons was not significantly changed by 9% CO₂ (0.021±0.034 Hz, n = 16, p = 0.49) and by 3% CO₂ (0.012±0.046 Hz, n = 12, p = 0.97).

Conclusions

Our findings support the hypothesis that chemosensitivity of midbrain serotonergic neurons provides a physiological mechanism for arousal responses to life-threatening episodes of hypercapnia and that functional impairment, such as excessive autoinhibition, of midbrain serotonergic neuron responses to hypercapnia may contribute to sudden death.     

Fibroblast Growth Factor 8 Deficiency Compromises the Functional Response of the Serotonergic System to Stress

Functionally heterogeneous populations of serotonergic neurons, located within the dorsal raphe nucleus (DR), play a role in stress-related behaviors and neuropsychiatric illnesses such as anxiety and depression. Abnormal development of these neurons may permanently alter their structure and connections, making the organism more susceptible to anxiety-related disorders. A factor that critically regulates the development of serotonergic neurons is fibroblast growth factor 8 (Fgf8). In this study, we used acute restraint stress followed by behavioral testing to examine whether Fgf8 signaling during development is important for establishing functional stress- and anxiety-related DR neurocircuits in adulthood. Wild-type and heterozygous male mice globally hypomorphic for Fgf8 were exposed to acute restraint stress and then tested for anxiety-like behavior on the elevated plus-maze. Further, we measured c-Fos immunostaining as a marker of serotonergic neuronal activation and tissue 5-hydroxyindoleacetic acid concentrations as a marker of serotonin functional output. Results showed that Fgf8 hypomorphs exhibited 1) an exaggerated response of DR anxiety-promoting circuits and 2) a blunted response of a DR panic-inhibiting circuit to stress, effects that together were associated with increased baseline anxiety-like behavior. Overall, our results provide a neural substrate upon which Fgf8 deficiency could affect stress response and support the hypothesis that developmental disruptions of serotonergic neurons affect their postnatal functional integrity.     

Effect of specific serotonergic lesions on cholinergic neurons in the hippocampus,cortex and striatum

Local injection of 5, 7-dihydroxytryptamine into the median raphe nucleus of rats pretreated with desipramine decreases the serotonin content of the hippocampus and cortex. The turnover of acetylcholine, as measured by the rate of decline of acetylcholine content after hemicholinium-3, the rate of decline of acetylcholine content after hemicholinium-3, is not affected in the hippocampus or the striatum, but is increased in the cortex by such treatment. Local injection of 5, 7-dihydroxytryptamine into the dorsal raphe nucleus of desipramine-treated rats decreases the serotonin content of the hippocampus, cortex, and striatum. The turnover of acetylcholine is increased in the hippocampus and cortex, but not affected in the striatum. Thus, serotonergic neurons from the median raphe nucleus appear to tonically inhibit cholinergic neurons in the cortex, and serotonergic neurons from the dorsal raphe nucleus appear to tonically inhibit cholinergic neurons in the hippocampus and cortex. These serotonergic neurons do not appear to act tonically on striatal cholinergic neurons.     

The Distribution of Serotonergic Nerves in Microencephalic Rats Treated Prenatally with Methylazoxymethanol

Prenatal exposure of pregnant rats to methylazoxymethanol acetate (MAM) induces microencephaly in the offspring. In the present study of these microencephalic rats (MAM rats) we used quantitative autoradiography to investigate [³H] paroxetine binding sites, which are a selective marker of serotonin (5-HT) transporters (5-HTT). The binding in the accumbens, cortex, hippocampus, and dorsolateral thalamus was significantly increased in MAM rats, compared to the control rats, while there was a significant decrease in the dorsal raphe nucleus of the MAM rats. The levels of 5-HTT mRNA in the dorsal raphe nuclei were analyzed by in situ hybridization, which revealed a significant decrease in 5-HTT mRNA-positive neurons in the MAM rats compared to the control rats. The results imply serotonergic hyperinnervation in the cerebral hemispheres of MAM rats, while a target-dependent secondary degeneration of 5-HT neurons might be induced in the dorsal raphe nuclei of MAM rats.     

Chemically defined collateral projections from the pons to the central nucleus of the amygdala and hypothalamic paraventricular nucleus in the rat

Triple fluorescence labelling was employed to reveal the distribution of chemically identified neurons within the pontine laterodorsal tegmental nucleus and dorsal raphe nucleus which supply branching collateral input to the central nucleus of the amygdala and hypothalamic paraventricular nucleus. The chemical identity of neurons in the laterodorsal tegmental nucleus was revealed by immunocytochemical detection of choline-acetyltransferase or substance P; in the dorsal raphe nucleus, the chemical content of the neurons was revealed with antibody recognizing serotonin. The projections were defined by injections of two retrograde tracers, rhodamine-and fluorescein-labelled latex microspheres, in the central nucleus of the amygdala and paraventricular nucleus, respectively. Neurons projecting to both the central nucleus of the amygdala and the paraventricular nucleus were distributed primarily within the caudal extensions of the laterodorsal tegmental nucleus and dorsal raphe nucleus. Approximately 11% and 7% of the labelled cells in the laterodorsal tegmental nucleus and dorsal raphe nucleus projected via branching collaterals to the paraventricular nucleus and central nucleus of the amygdala. About half of these neurons in the laterodorsal tegmental nucleus were cholinergic, and one-third were substance-P-ergic; in the dorsal raphe nucleus, approximately half of the neurons containing both retrograde tracers were serotonergic. These results indicate that pontine neurons may simultaneously transmit signals to the central nucleus of the amygdala and paraventricular nucleus and that several different neuroactive substances are found in the neurons participating in these pathways. This coordinated signalling may lead to synchronized responses of the central nucleus of the amygdala and paraventricular nucleus for the maintenance of homeostasis. Interactions between different neuroactive substances at the target site may serve to modulate the responses of individual neurons.     

Evidence for projections from medullary nuclei onto serotonergic and dopaminergic neurons in the midbrain dorsal raphe nucleus of the rat

Summary The anterograde tracer Phaseolus vulgaris-leucoagglutinin was injected into the medial nucleus of the solitary tract and into the rostral dorsomedial medulla. A sequential two-color immunoperoxidase staining was accomplished in order to demonstrate the co-distribution of presumed terminal axons with chemically distinct neurons in the dorsal raphe nucleus of the midbrain central gray, i.e., B7 serotonergic and A10dc dopaminergic neurons. Black-stained efferent fibers from the medial nucleus of the solitary tract and the rostral dorsomedial medulla intermingled with brown-stained serotonergic (5-hydroxytryptamine-immunoreactive) or dopaminergic (tyrosine hydroxylase-immunoreactive) neurons. Light microscopy revealed that the black-stained efferent axons exhibited numerous en passant and terminal varicosities that were often found in close apposition to brown-stained serotonergic and dopaminergic somata, and to proximal and distal dendrites and dendritic processes. The close association of immunoreactive elements suggests the presence of axo-somatic and axodendritic synaptic contacts of medullary fibers with serotonergic and dopaminergic neurons in the dorsal raphe nucleus. These projections could be involved in the modulation of dorsal raphe neurons, depending on the autonomic status of an animal.     

Serotonin receptor activation reduces calcium current in an acutely dissociated adult central neuron

The release of serotonin (5-HT) from the terminals of serotonergic (raphe) neurons is under inhibitory feed-back control. 5-HT, acting on raphe cell body autoreceptors, also mediates inhibitory postsynaptic potentials as a result of release from collaterals from neighboring raphe neurons. This may involve a ligand (5-HT)-gated increase in the membrane potassium conductance, leading to a decrease in action potential frequency, which could indirectly reduce calcium influx into nerve terminals. In this report we demonstrate that 5-HT can also directly reduce calcium influx at potentials including and bracketing the peak of calcium current activation. Using acutely isolated, patch-clamped dorsal raphe neurons, we found that low concentrations of 5-HT and the 5-HT1A-selective agonist 8-OH-DPAT reversibly decrease whole-cell calcium current. This effect is antagonized by the putative 5-HT1A-selective antagonist NAN 190. Hence, the inhibition of calcium current may serve a physiological role in these cells and elsewhere in the brain.     

Development of serotonergic neurons of dorsal raphe nuclei in mice with knockout of monoamine oxidase a and 5-HT1A and 5-HT1B autoreceptor

The morphological changes in the development of serotonergic neurons of the dorsal raphe nuclei in the medulla oblongata was studied by immunocytochemistry in mice with knockout of 1A and 1B serotonin autoreceptors as well as monoamine oxidase A. Serotonin autoreceptors regulate electric activity of serotonergic neurons as well as the synthesis and release of the neurotransmitter, while monoamine oxidase A catalyzes its degradation. These genetic modifications proved to have no effect on the number of serotonergic neurons in the medulla oblongata but induced morphofunctional changes. Decreased cell size and increased intracellular serotonin level were observed in the case of monoamine oxidase A deficiency, while excessive cell size and decreased intracellular serotonin level were observed in the case of autoreceptor deficiency. The data obtained confirm the hypothesis of autoregulation of serotonergic neurons in development.     

Inhibition of serotonergic medullary raphe obscurus neurons suppresses genioglossus and diaphragm activities in anesthetized but not conscious rats.

Although exogenous serotonin at the hypoglossal motor nucleus (HMN) activates the genioglossus muscle, endogenous serotonin plays a minimal role in modulating genioglossus activity in awake and sleeping rats (Sood S, Morrison JL, Liu H, and Horner RL. Am J Respir Crit Care Med 172: 1338-1347, 2005). This result therefore implies that medullary raphe neurons also play a minimal role in the normal physiological control of the HMN, but this has not yet been established because raphe neurons release other excitatory neurotransmitters onto respiratory motoneurons in addition to serotonin. This study tests the hypothesis that inhibition of medullary raphe serotonergic neurons with 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) suppresses genioglossus and diaphragm activities in awake and sleeping rats. Ten rats were implanted with electrodes to record sleep-wake states and genioglossus and diaphragm activities. Microdialysis probes were also implanted into the nucleus raphe obscurus (NRO). Experiments in 10 anesthetized and vagotomized rats were also performed using the same methodology. In anesthetized rats, microdialysis perfusion of 0.1 mM 8-OH-DPAT into the NRO decreased genioglossus activity by 60.7+/-9.0% and diaphragm activity by 13.3+/-3.4%. Diaphragm responses to 7.5% CO2 were also significantly reduced by 8-OH-DPAT. However, despite the robust effects observed in anesthetized and vagotomized rats, there was no effect of 0.1 mM 8-OH-DPAT on genioglossus or diaphragm activities in conscious rats awake or asleep. The results support the concept that endogenously active serotonergic medullary raphe neurons play a minimal role in modulating respiratory motor activity across natural sleep-wake states in freely behaving rodents. This result has implications for pharmacological strategies aiming to manipulate raphe neurons and endogenous serotonin in obstructive sleep apnea.     

GABAB receptor mRNA in the raphe nuclei: co-expression with serotonin transporter and glutamic acid decarboxylase

We have used double-label in situ hybridization techniques to examine the cellular localization of GABAB receptor mRNA in relation to serotonin transporter mRNA and glutamic acid decarboxylase mRNA in the rat dorsal raphe, median raphe and raphe magnus nuclei. The degree of cellular co-localization of these markers notably varied among the different nuclei. In the dorsal raphe, cell bodies showing GABAB receptor mRNA were very abundant, the 85% being also labelled for serotonin transporter mRNA, and a low proportion (5%) showing glutamic acid decarboxylase mRNA. In the median raphe, the level of co-expression of GABAB receptor mRNA with serotonin transporter mRNA was significantly lower. Some cells were also identified that contained GABAB receptor mRNA in the absence of either one of the other mRNA species studied. Our results support the presence of GABAB receptors in serotonergic as well as GABAergic neurones in the dorsal and median raphe, providing the anatomical basis for the reported dual inhibitory/disinhibitory effect of the GABAB agonist baclofen on serotonergic function.     

Effects of benzodiazepines on central serotonergic mechanisms.

If the rat conflict test were a valid animal model of anxiety neurosis, evidence which implicates serotonin systems in the anxiety-reducing actions of benzodiazepine tranquilizers could be summarized as follows: (1) The punishment-lessening effects of benzodiazepines in the conflict test are mimicked by serotonin antagonists (methysergide, cinanserin, bromolysergic acid), serotonin synthesis inhibition (PCPA), and serotonin nerve terminal damage (5,6-dihydroxytryptamine). (2) Punishment effects may be intensified by the serotonin precursor, 5-hydroxytryptophan (in combination with a monoamine oxidase inhibitor), serotonin agonists (alpha-methyltryptamine), or intraventricular injections of serotonin itself. Intraventricularly administered serotonin also antagonizes the punishment-lessening effects of benzodiazepines. (3) Stimulation of the serotonergic cell bodies in the dorsal raphe nucleus by local application of crystalline carbachol causes intense suppression of behavior. The suppressive effects of raphe stimulation are antagonized by systemic administration of benzodiazepines. (4) In biochemical experiments, the decrease in norepinephrine turnover induced by oxazepam rapidly undergoes tolerance, whereas the decrease induced in serotonin turnover is maintained over repeated doses. These results parallel findings in the conflict test which indicate that the depressant action of oxazepam rapidly undergoes tolerance, whereas the anxiety-reducing action is maintained over repeated doses. Although central serotonin neurons are thus implicated in the therapeutic actions of benzodiazepine tranquilizers, it is quite possible that the drugs actually act indirectly to reduce serotonin activity. The concept that benzodiazepines may exert a primary action on GABA-containing neurons, which in turn regulate serotonergic transmission, was supported by preliminary psychopharmacological evidence. The GABA-antagonist picrotoxin, at doses that do not disrupt unpunished behavior, fully antagonizes the punishment-lessening effects of benzodiazepines in the conflict test.     

Raphe magnus nucleus is involved in ventilatory but not hypothermic response to CO2.

There is evidence that serotonin [5-hydroxytryptamine (5-HT)] is involved in the physiological responses to hypercapnia. Serotonergic neurons represent the major cell type (comprising 15-20% of the neurons) in raphe magnus nucleus (RMg), which is a medullary raphe nucleus. In the present study, we tested the hypothesis 1) that RMg plays a role in the ventilatory and thermal responses to hypercapnia, and 2) that RMg serotonergic neurons are involved in these responses. To this end, we microinjected 1) ibotenic acid to promote nonspecific lesioning of neurons in the RMg, or 2) anti-SERT-SAP (an immunotoxin that utilizes a monoclonal antibody to the third extracellular domain of the serotonin reuptake transporter) to specifically kill the serotonergic neurons in the RMg. Hypercapnia caused hyperventilation and hypothermia in all groups. RMg nonspecific lesions elicited a significant reduction of the ventilatory response to hypercapnia due to lower tidal volume (Vt) and respiratory frequency. Rats submitted to specific killing of RMg serotonergic neurons showed no consistent difference in ventilation during air breathing but had a decreased ventilatory response to CO(2) due to lower Vt. The hypercapnia-induced hypothermia was not affected by specific or nonspecific lesions of RMg serotonergic neurons. These data suggest that RMg serotonergic neurons do not participate in the tonic maintenance of ventilation during air breathing but contribute to the ventilatory response to CO(2). Ultimately, this nucleus may not be involved in the thermal responses to CO(2).     

Nonexocytotic serotonin release tonically suppresses serotonergic neuron activity

The firing activity of serotonergic neurons in raphe nuclei is regulated by negative feedback exerted by extracellular serotonin (5-HT)_o acting through somatodendritic 5-HT1A autoreceptors. The steady-state [5-HT]_o, sensed by 5-HT1A autoreceptors, is determined by the balance between the rates of 5-HT release and reuptake. Although it is well established that reuptake of 5-HT_o is mediated by 5-HT transporters (SERT), the release mechanism has remained unclear. It is also unclear how selective 5-HT reuptake inhibitor (SSRI) antidepressants increase the [5-HT]_o in raphe nuclei and suppress serotonergic neuron activity, thereby potentially diminishing their own therapeutic effect. Using an electrophysiological approach in a slice preparation, we show that, in the dorsal raphe nucleus (DRN), continuous nonexocytotic 5-HT release is responsible for suppression of phenylephrine-facilitated serotonergic neuron firing under basal conditions as well as for autoinhibition induced by SSRI application. By using 5-HT1A autoreceptor-activated G protein–gated inwardly rectifying potassium channels of patched serotonergic neurons as 5-HT_o sensors, we show substantial nonexocytotic 5-HT release under conditions of abolished firing activity, Ca²⁺ influx, vesicular monoamine transporter 2–mediated vesicular accumulation of 5-HT, and SERT-mediated 5-HT transport. Our results reveal a cytosolic origin of 5-HT_o in the DRN and suggest that 5-HT_o may be supplied by simple diffusion across the plasma membrane, primarily from the dense network of neurites of serotonergic neurons surrounding the cell bodies. These findings indicate that the serotonergic system does not function as a sum of independently acting neurons but as a highly interdependent neuronal network, characterized by a shared neurotransmitter pool and the regulation of firing activity by an interneuronal, yet activity-independent, nonexocytotic mechanism.     

Brain serotonin and blood pressure regulation: studies using in vivo electrochemistry and direct tissue assay

Hypotensive responses to tryptophan and 5-hydroxytryptophan infusions were studied in normotensive male Sprague-Dawley rats. Results showed that 5-hydroxytryptophan but not tryptophan lowered pressure in a dose dependent way in direct relation to the production of brain serotonin and 5-HIAA. Intrinsic release of serotonin from brain was also studied during periods of induced hypertension and hypotension. Brain monoamine responses to blood pressure changes induced by intravenous phenylephrine and nitroprusside were measured in dorsal raphe nucleus and nucleus tractus solitarius by in vivo electrochemistry. Results showed that 5-HIAA was increased during drug induced hypertension and during reflex hypertension which followed a period of hypotension. These changes were blocked by sinoaortic denervation indicating that these central serotonergic neurons are responding to increased pressure sensed by baroreceptors. Therefore, serotonin has a role in blood pressure regulation as a pharmacologic agent and as a neurotransmitter in homeostatic control of pressure.     

The orexinergic synaptic innervation of serotonin- and orexin 1-receptor-containing neurons in the dorsal raphe nucleus

Orexin/hypocretin has been well demonstrated to excite the serotonergic neurons in the dorsal raphe nucleus (DRN). We studied the morphological relationships between orexin-containing axon terminals and serotonin- as well as orexin-receptor-containing neurons in the dorsal raphe nucleus. Using immunohistochemical techniques at the light microscopic level, orexin A (OXA)-like immunoreactive neuronal fibers in the DRN were found to make close contact with serotonergic neurons, while some of the serotonergic neurons also expressed the orexin 1 receptor (OX1R). At the electron microscopic level, double-immunostaining experiments showed that the orexin A-like immunoreactive fibers were present mostly as axon terminals that made synapses on the serotonin- and orexin 1-receptor-containing neurons. While only axodendritic synapses between orexin A-containing axon terminals and serotonergic neurons were detected, the synapses made by orexin A-containing axon terminals on the orexin 1-receptor-containing neurons were both axodendritic and axosomatic. The present study suggests that excitation effect of orexin A on dorsal raphe serotonergic neurons is via synaptic communication through orexin 1 receptor.     

Role of norepinephrine in regulating the activity of serotonin-containing dorsal raphe neurons

Previous studies have yielded conflicting results concerning the role of noradrenergic afferents to the dorsal raphe nucleus in regulating the activity of serotonergic neurons. In the present study, we recorded the activity of serotonin-containing dorsal raphe neurons in mouse brain slices $\text{in vitro}$ under the following conditions: (a) no treatment, (b) phenylephrine added to the incubation medium, (c) in tissue obtained from mice that were anesthetized with halothane, (d) same condition as c, with phenylephrine added to the incubation medium, and (e) same as condition c, with the addition of bicuculline to the incubation medium. The data revealed that the neurons recorded with no treatment exhibited a spontaneous discharge rate of 3.40 ± 0.29 spikes/sec and a cell/tract ratio of 1.15, while cells recorded from tissue slices obtained from halothane anesthetized mice exhibited a discharge rate of 2.01 ± 0.27 spikes/sec and a cell/track ratio of 0.58. Addition of phenylephrine to the incubation media in slices obtained from anesthetized mice increased both the discharge rate (4.23 ± 0.30 spikes/sec) and cell/tract ratio (1.28). Similarly, addition of bicuculline to the incubation media increased both the discharge rate (4.09 ± 0.46 spikes/sec) and cell/tract ratio (1.21) in mouse brain slices obtained from anesthetized animals. Thus, we conclude that a noradrenergic input (which is removed in the tissue slice preparation) is not necessary to maintain the spontaneous activity of serotonergic dorsal raphe units. Halothane anesthesia depressed the activity of these neurons, presumably by releasing GABA from interneurons. Finally, while dorsal raphe neurons are not dependent upon an excitatory noradrenergic input to maintain their spontaneous activity, these neurons can be excited by noradrenergic a fferents under certain conditions.     

Synthesis of Serotonin in Traumatized Rat Brain

Abstract: Previous studies have demonstrated that focal freezing lesions in rats cause a widespread decrease of cortical glucose use in the lesioned hemisphere and this was interpreted as a reflection of depression of cortical activity. The serotonergic neurotransmitter system was implicated in these alterations when it was shown that (1) cortical serotonin metabolism was increased widely in focally injured brain and (2) inhibition of serotonin synthesis prevented the development of cortical hypometabolism. In the present studies we applied an autoradiographic method that uses the accumulation of the ¹⁴C-labeled analogue of serotonin α-methylserotonin to assess changes in the rate of serotonin synthesis in injured brain. The results confirmed that 3 days after the lesion was made, at the time of greatest depression of glucose use, serotonin synthesis was significantly increased in cortical areas throughout the injured hemisphere. The increase was also seen in the dorsal hippocampus and area CA3, as well as in the medial geniculate and dorsal raphe, but not in any other subcortical structures including median raphe. Present results suggest that the functional changes in the cortex of the lesioned hemisphere are associated with an increased rate of serotonin synthesis mediated by activation of the dorsal raphe. We also documented by α-[¹⁴C]aminoisobutyric acid autoradiography that there was increased permeability of the blood-brain barrier, but this was restricted to the rim of the lesion.     

The role of various calcium and potassium channels in the regulation of somatodendritic serotonin release

We prepared slices from midbrain containing the raphe nuclei and from hippocampus of rats. The brain slices were loaded with [³H]serotonin and superfused in order to measure the release of radioactivity at rest and in response to electrical stimulation. No difference was observed in the resting and stimulated fractional release of tritium in the somatodendritic and axon terminal parts of serotonergic neurons. The selective 5-HT_1A receptor agonist 8-OH-DPAT decreased the electrically induced tritium effux from raphe nuclei slices preloaded with [³H]serotonin, and this inhibition was reversed by 5-HT_1A receptor antagonist (+)WAY-100135. The 5-HT_1B receptor agonist CGS-12066B but not 8-OH-DPAT, inhibited the stimulation-evoked tritium efflux from hippocampal slices after labeling with [³H]serotonin. The electrical stimulation-evoked tritium efflux in raphe nuclei slices incubate with [³H]serotonin was completely external Ca²⁺-dependent, and omega-conotoxin GVIA and Cd²⁺, but not diltiazem, inhibited the tritium overflow. In raphe nuclei slices 4-aminopyridine enhanced the electrical stimulation-induced trititum release in a concentration-dependent manner. The inhibition of tritium efflux by 8-OH-DPAT was abolished with 4-aminopyridine. Glibenclamide or tolbutamide proved to be ineffective. These data indicate that (1) different 5-HT receptor subtypes (5-HT_1A and 5-HT_1B) regulate dendritic and axon terminal 5-HT release; (2) serotonin release from the dendrites may be regulated by the voltage-sensitive N-type Ca²⁺ channels; (3) the 5-HT_1A receptor-mediated inhibition of serotonin release may be due to opening of voltage-sensitive K⁺ channels.