Studies on the respiratory control mechanism of medullary raphe nuclei and their serotonergic system

Respiratory control mechanism of the medullary raphe nuclei were studied with some references to their serotonergic mechanisms. Anesthetized, paralyzed and artificially ventilated cats were used and their phrenic nerve efferent activity was always observed as an indicator of central respiratory activity. Following results were obtained. 1) Electrical stimulation of medullary raphe nuclei, namely, nucleus raphe magnus, obscurus and pallidus, produced dominantly inhibitory responses in the phrenic nerve activity, while raphe stimulation in the pons and more rostral portion did not produce any respiratory responses. The blood pressure was depressed by raphe stimulation, too, almost in parallel to the respiratory inhibition. These inhibitory responses in respiration and blood pressure were partially antagonized by cyproheptadine (0.3-0.5 mg/kg i.v.) and methysergide (0.3-0.5 mg/kg i.v.). 2) Raphe stimulation inhibited remarkably activities of the medullary inspiratory and expiratory neurons, similarly. 3) In the experiment, where single shot stimulus was added to the raphe nuclei at the various time point in the respiratory phase, raphe stimulation showed the retardative effect of inspiratory switching, in addition to the inhibitory effect of phrenic burst activity. 4) The mechanism of respiratory inhibition produced by raphe stimulation was analyzed by evoked potentials in the averaged phrenic nerve activity. The post-stimulus averaged potentials of the phrenic nerve consist of the depolarizing potentials of about 10 msec duration and the subsequent hyperpolarizing potentials of several 10 msec duration, the duration time depending on the stimulus intensity. When stimulation was given in high frequency, the post-stimulus averaged potential became flattened, and the phrenic burst activity was inhibited almost completely. But in the case of stimulation in ventral parts of the raphe nuclei, the initial depolarizing potential was comparatively more prominent, and when high frequency stimulation was given, continuous firing was observed in the phrenic nerve activity. At the time of the continuous firing, respiratory rhythmicity was disappeared completely. 5) Propranolol (0.3-1.0 mg/kg i.v.), which have been recognized to have 5-HT1 antagonistic activity, reduced the hyperpolarizing potentials of the post-stimulus averaged potentials, and methysergide (0.3-1.0 mg/kg i.v.), 5-HT1 and 5-HT2 antagonist, reduced both depolarizing and hyperpolarizing potentials. These phenomena would suggest strongly that hyperpolarizing and depolarizing potentials are related to the 5-HT1 and 5-HT2 receptors, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for a serotonergically mediated sympathoexcitatory response to stimulation of medullary raphe nuclei

The cardiovascular role of serotonin (5-HT) containing neurons in the midline medullary raphe nuclei was studied in anesthetized cats. High frequency electrical stimulation of nucleus (n.) raphe (r.) pallidus, n.r. obscurus and n.r. magnus produced both pressor and depressor responses. Single shock stimulation of pressor sites produced an excitatory evoked potential of sympathetic nervous discharge (SND) recorded from the inferior cardiac nerve. Conversely, single shock stimulation of vasodepressor sites resulted in a computer-summed inhibition of SND. The mean conduction velocity in the sympathoexcitatory medullo-spinal pathway to sympathetic preganglionic neurons was calculated to be 1.24 m/s. The 5-HT antagonists methysergide and metergoline blocked the excitation of sympathetic activity evoked from medullary raphe nuclei. In contrast, these agents failed to alter the sympathoexcitatory response to electrical stimulation of lateral medulla pressor sites or the sympathoinhibitory response elicited by raphe stimulation. The 5-HT uptake inhibitor chlorimipramine increased the duration of the sympathoexcitatory response evoked from the raphe but not from the lateral medulla. Finally, mid-collicular transection did not effect the excitation of sympathetic activity elicited by stimulation of medullary raphe nuclei. These data suggest that serotonergic neurons in the midline medullary raphe nuclei provide an excitatory input to sympathetic neurons in the spinal cord.     

Excitation of hypothalamic paraventricular neurons by stimulation of the raphe nuclei

Extracellular recordings were made from 467 anti-dromically identified neurosecretory neurons and 148 non-neurosecretory neurons in the paraventricular nucleus of the hypothalamus of hemispherectomized cats under pentobarbital anesthesia. Stimulation of the dorsal, median, and pontine raphe nuclei excited 31%, 26%, and 12% of neurosecretory neurons tested, respectively, and inhibited 9%, 7%, and 8%. The excitatory responses in 13 of 14 neurons tested were blocked by either of two intravenously administered 5-HT2 antagonists, cyproheptadine or methysergide. The 5-HT1A antagonist, (-)pindolol, partially blocked the excitatory responses elicited by raphe stimulation in three of five neurons tested. The inhibitory responses to raphe stimulation were not affected by application of these antagonists. More non-neurosecretory neurons than neurosecretory neurons were excited in response to raphe stimulation and these excitatory responses were also blocked by these antagonists. We conclude that most electrically stimulated synaptic inputs from the midbrain raphe nuclei to the hypothalamic paraventricular nucleus are excitatory and are mainly mediated by 5-HT2 receptors.     

Ascending afferent regulation of rat midbrain dopamine neurons

Standard, extracellular single-unit recording techniques were used to examine the electrophysiological and pharmacological responsiveness of midbrain dopamine (DA) neurons to selected, ascending afferent inputs. Sciatic nerve stimulation-induced inhibition of nigrostriatal DA (NSDA) neurons was blocked by both PCPA (5-HT synthesis inhibitor) and 5,7-DHT (5-HT neurotoxin), suggesting mediation by a serotonergic (5-HT) system. Direct stimulation of the dorsal raphe (which utilizes 5-HT as a neurotransmitter and inhibits slowly firing NSDA neurons) inhibited all mesoaccumbens DA (MADA) neurons tested. Paradoxically, DPAT, a 5-HT_1a agonist which inhibits 5-HT cell firing, enhanced sciatic nerve stimulation-induced inhibition of NSDA neurons. MADA neurons were not inhibited by sciatic nerve stimulation and, therefore, could not be tested in this paradigm. In contrast to the dorsal raphe, electrical stimulation of the pedunculopontine tegmental nucleus preferentially excited slowly firing NSDA and MADA neurons. Thus, both excitatory and inhibitory ascending afferents influence the activity of midbrain DA neurons, and intact 5-HT systems are necessary for sciatic nerve stimulation to alter DA cell activity. However, the role that 5-HT plays in mediating peripheral sensory input remains unclear.     

A role for nitric oxide in serotonin neurons of the midbrain raphe nuclei

Neuronal nitric oxide synthase (nNOS) catalyses the production of the neurotransmitter nitric oxide. nNOS is expressed in the dorsal raphe nucleus (DRN), a source of ascending serotonergic projections. In this study, we examined the distribution nNOS and the function of nitric oxide in the DRN and adjacent median raphe nucleus (MRN) of the rat. We hypothesized that nNOS is differentially expressed across the raphe nuclei and that nitric oxide influences the firing activity of a subgroup of 5‐HT neurons. Immunohistochemistry revealed that, nNOS is present in around 40% of 5‐HT neurons, throughout the DRN and MRN, as well as in some non‐5‐HT neurons immediately adjacent to the DRN and MRN. The nitric oxide receptor, soluble guanylyl cyclase, was present in all 5‐HT neurons examined in the DRN and MRN. In vitro extracellular electrophysiology revealed that application of the nitric oxide donor, diethylamine NONOate (30–300 µM) inhibited 60%–70% of putative 5‐HT neurons, excited approximately 10% of putative 5‐HT neurons and had no effect on the rest. The inhibitory response to nitric oxide was blocked by [1H‐[1,2,4]oxadiazolo‐[4, 3‐a]quinoxalin‐1‐one (ODQ, 30 or 100 µM), indicating mediation by soluble guanylyl cyclase. Juxtacellular labelling revealed that nitric oxide inhibits firing in both putative 5‐HT neurons which express nNOS and those which do not express nNOS. Our data are consistent with the notion that nitric oxide acts as both a trans‐synaptic and autocrine signaller in 5‐HT neurons in the DRN and MRN and that its effects are widespread and primarily inhibitory.     

Influence of inhibitory and excitatory inputs on serotonin efflux differs in the dorsal and median raphe nuclei

The dorsal (DRN) and median raphe nuclei (MRN) are two major sources of serotonergic projections to forebrain that are involved in regulation of behavioral state and motor activity, and implicated in affective disorders such as depression and schizophrenia. To investigate afferent influences on serotonergic neurons, this study compared the role of endogenous GABA and glutamate in the DRN and MRN using microdialysis and measurement of locomotor activity in freely behaving rats. Local infusion of the GABA(A) receptor antagonist bicuculline increased serotonin (5-HT) efflux in the DRN but not the MRN. In contrast, infusion of glutamate receptor antagonists produced larger decreases in 5-HT efflux in the MRN compared with the DRN. Moreover, glutamate receptor antagonists attenuated the increase in 5-HT efflux produced by GABA receptor blockade in the DRN. Thus, the disinhibitory effect of GABA blockers could be ascribed in part to an enhanced influence of glutamate. Measurements of locomotor activity indicate that changes in 5-HT were not simply correlated with behavioral activity induced by drug infusion. In summary, the role of inhibitory and excitatory afferents was strikingly different in the DRN and MRN. GABA afferents were the predominant tonic influence on serotonergic neurons in the DRN. In contrast, glutamatergic but not GABAergic afferents had a strong tonic influence on serotonergic neurons in the MRN.     

Nociceptive responses in nucleus parafascicularis thalami are modulated by dorsal raphe stimulation and microiontophoretic application of morphine and serotonin

Single-cell experiments were undertaken to examine the hypothesis that serotonin (5-HT) and morphine participate in ascending pain suppression phenomena. The observations demonstrate that: 1) dorsal raphe stimulation (DRS) modulates the spontaneous activity and the noxious-evoked responses of parafasciculus (PF) neurons, and the modulating effects of DRS are altered by either naloxone or methysergide; 2) morphine ejection into the PF alters the spontaneous activity and the noxious-evoked responses of PF neurons, and naloxone prevents morphine effects; and 3) serotonin ejection into the PF alters the spontaneous activity and the noxious-evoked responses of PF neurons and methysergide prevents the serotonin effects. These findings support the hypothesis that opioid and serotonin participate, at least in part, in the control of ascending pain mechanisms.     

Inhibition of lateral vestibular nucleus neurons by 5-hydroxytryptamine derived from the dorsal raphe nucleus

Electrophysiological studies were performed to elucidate the effect of 5-hydroxytryptamine (5-HT) originating in the dorsal raphe nucleus (DR) on neuronal activity in the lateral vestibular nucleus (LVN) neurons, using cats anesthetized with alpha-chloralose. LVN neurons were classified into monosynaptic and polysynaptic neurons according to their responses to vestibular nerve stimulation. Conditioning stimuli applied to the DR inhibited orthodromic spikes elicited by vestibular nerve stimulation predominantly in polysynaptic neurons of the LVN. The iontophoretic application of 5-HT also inhibited orthodromic spikes of the LVN neurons. A close correlation was observed between the effects of DR conditioning stimulation and iontophoretically applied 5-HT in the same neurons. These inhibitions with both treatments were antagonized during the application of methysergide, a 5-HT antagonist. In the majority of LVN polysynaptic neurons that responded to antidromic stimulation of the ipsilateral or contralateral abducens nucleus, orthodromic spikes elicited by vestibular nerve stimulation were inhibited by DR conditioning stimulation and the iontophoretic application of 5-HT. In contrast, LVN neurons that responded to antidromic stimulation of the vestibulospinal tract were rarely affected by these treatments. These results indicate that 5-HT derived from the DR inhibits the synaptic transmission of LVN polysynaptic neurons ascending to the abducens nucleus, and suggest that 5-HT derived from the DR is involved in the regulation of the vestibulo-ocular reflex.     

The dorsal and medial raphe projections to the substantia nigra in the rat: Electrophysiological, biochemical and behavioural observations

Electrophysiological experiments have been performed in urethane anaesthetized rats to investigate the projections from the dorsal (DRN) and medial raphe nuclei (MRN) to the substantia nigra. The biochemical and behavioural effects following discrete electrolytic lesions in the dorsal and medial raphe have also been investigated.Stimulation of the DRN produced predominantly inhibition of spontaneous activity of single neurones in the substantia nigra though some neurones were also excited. Bilateral stimulation of the substantia nigra produced antidromic spikes in DRN and MRN neurones.Lesions of the DRN and MRN produced a significant reduction in substantia nigra 5-HT concentration. Additionally, DRN lesions reduced striatal 5-HT, while MRN lesions reduced hippocampal 5-HT. Both lesions increased substantia nigra HVA concentration but did not affect DA concentration. Neither DRN nor MRN lesions affected striatal HVA, although DA levels were significantly elevated after 14 days.Animals with DRN lesions explored more than controls or MRN-lesioned animals. However, this behaviour was transient and was not observed after 14 days. On the other hand, MRN-lesioned animals were significantly hyperactive.These observations suggest that the substantia nigra receives a direct monosynaptic inhibitory input from the DRN and MRN and that these pathways use 5-HT as a neurotransmitter serving to tonically inhibit dopaminergic neurones. While 5-HT and dopamine appear to be involved in the control of motor behaviour, the precise relationship between these serotoninergic and dopaminergic systems in this respect is unclear.     

Dorsal raphe nuclear stimulation of SCN serotonin release and circadian phase-resetting

Serotonin (5-HT) is strongly implicated in the regulation of mammalian circadian rhythms. However, little is known of the functional relationship between the circadian clock located in the suprachiasmatic nucleus (SCN) and its source of serotonergic innervation, the midbrain raphe nuclei. In previous studies, we reported that electrical stimulation of the dorsal or median raphe nuclei (DRN and MRN, respectively) induced 5-HT release in the SCN. Notably, DRN- but not MRN-stimulated 5-HT release was blocked by the 5-HT(1,2,7) antagonist, metergoline, suggesting that the DRN signals to the SCN indirectly via the activation of a 5-HT-responsive multisynaptic pathway. In the present study, pretreatment with the 5-HT(2,7) antagonist, ritanserin, also significantly inhibited DRN-electrically stimulated SCN 5-HT release. However, pretreatment with the 5-HT(1A) antagonist, NAN-190, or the 5-HT(2) antagonists ketanserin and cinanserin had little suppressive effect on this DRN-stimulated 5-HT release. In complementary behavioral trials, electrical stimulation of the DRN during subjective midday caused a 1.3-h advance in the free-running circadian activity rhythm under constant darkness, which was inhibited by metergoline. Collectively, these results are evidence that: (1) DRN-stimulated 5-HT release in the SCN requires the activation of an intermediate target with receptors having 5-HT(7) pharmacological characteristics; (2) electrical stimulation of the DRN induces phase-resetting of the circadian activity rhythm; and (3) activation of 5-HT receptors is necessary for this DRN-stimulated circadian phase-resetting. In view of the dynamic changes in DRN neuronal activity incumbent with the daily sleep-activity cycle, and its functional linkages to the SCN and intergeniculate leaflet, the DRN could serve to provide behavioral/arousal state information to various sites comprising the brain circadian system.     

Inhibition of neurons in the amygdala by dorsal raphe stimulation: Mediation through a direct serotonergic pathway

This study presents data showing that the dorsal raphe nucleus (DRN) has a marked inhibitory influence upon neurons in the amygdala and that this inhibitory effect is mediated by a direct DRN-amygdala serotonergic pathway. The evidence may be briefly summarized as follows: (1) on the same amygdaloid cells, both iontophoresis of serotonin (5-HT) and electrical stimulation of the DRN markedly inhibited spontaneous single unit activities; (2) the latency of DRN-induced inhibition was relatively short and is compatible with the conduction velocities (which were determined by antidromic activation of the 5-HT pathway) of unmyelinated 5-HT fibers; (3) destruction of 5-HT projections by 5, 7-dihydroxytryptamine (5, 7-DHT) or pharmacological depletion of 5-HT by parachlorophenylalanine (PCPA) prevented the inhibitory responses to DRN stimulation in the great majority of cells studied; (4) in PCPA-pretreated animals, injection of 5-hydroxytryptophan (5-HTP) reversed the PCPA effect, restoring the responses of amygdaloid cells to DRN stimulation. In the amygdala, the presumptive 5-HT antagonists which we tested did not block the inhibitory effects of 5-HT except that intravenously administered LSD blocked the inhibitory responses produced by submaximal DRN stimulation. The implications of these results for the possible functions of 5-HT in the amygdala is discussed.     

Selective 5-HT receptor inhibition of glutamatergic and GABAergic synaptic activity in the rat dorsal and median raphe

The dorsal (DR) and median (MR) raphe nuclei contain 5-hydroxytryptamine (5-HT) cell bodies that give rise to the majority of the ascending 5-HT projections to the forebrain. The DR and MR have differential roles in mediating stress, anxiety and depression. Glutamate and GABA activity sculpt putative 5-HT neuronal firing and 5-HT release in a seemingly differential manner in the MR and DR, yet isolated glutamate and GABA activity within the DR and MR has not been systematically characterized. Visualized whole-cell voltage-clamp techniques were used to record excitatory and inhibitory postsynaptic currents (EPSC and IPSC) in 5-HT-containing neurons. There was a regional variation in action potential-dependent (spontaneous) and basal [miniature (m)] glutamate and GABAergic activity. mEPSC activity was greater than mIPSC activity in the DR, whereas in the MR the mIPSC activity was greater. These differences in EPSC and IPSC frequency indicate that glutamatergic and GABAergic input have distinct cytoarchitectures in the DR and MR. 5-HT(1B) receptor activation decreased mEPSC frequency in the DR and the MR, but selectively inhibited mIPSC activity only in the MR. This finding, in concert with its previously described function as an autoreceptor, suggests that 5-HT(1B) receptors influence the ascending 5-HT system through multiple mechanisms. The disparity in organization and integration of glutamatergic and GABAergic input to DR and MR neurons and their regulation by 5-HT(1B) receptors may contribute to the distinction in MR and DR regulation of forebrain regions and their differential function in the aetiology and pharmacological treatment of psychiatric disease states.     

Inhibitory influence of the mesocortical dopaminergic system on spontaneous activity or excitatory response induced from the thalamic mediodorsal nucleus in the rat medial prefrontal cortex

Since the medial prefrontal cortex receives converging projections from the mediodorsal nucleus of the thalamus (MD) and the dopaminergic neurons located in the ventromedial mesencephalic tegmentum (VMT) the responses of cortical neurons to ipsilateral VMT and MD stimulation (50–150 μA; 0.2–0.5 ms duration) were analyzed in ketamine anaesthetized rats. MD stimulation at 1 Hz blocked the firing of 90% of the spontaneously active cortical units tested (mean latency, 15 ms; mean duration, 182 ms). MD stimulation at 5–10Hz evoked single spike responses (mean latency, 16 ms) in 80% of the units tested. Ten to 15 days after kainic acid injection into the MD the number of cortical neurons inhibited (1 Hz) or excitated (5–10 Hz) was reduced to 57 and 18%, respectively. Following stimulation of the VMT (at a frequency of 1–5 Hz), 85% of cortical neurons showed an arrest of spontaneous firing occurring after a mean latency of 17 ms and lasting 109 ms on the average. Most of the cells displaying the VMT inhibitory effect were excitated by MD stimulation. Moreover VMT stimulation, applied 3–45 ms before that of MD, blocked the excitation induced by MD in 75% of the units tested. After injection of 6-hydroxydopamine into the medial forebrain bundle or intraperitoneal administration of α-methyl-paratyrosine (α-MpT), the number of units tested responding to VMT stimulation was of 19 and 35%, respectively. Moreover in these treated rats, the proportion of excitatory responses to MD blocked by VMT stimulation was reduced to 5 and 6%. On the other hand, the effects induced by VMT stimulation were not affected after specific destruction of the noradrenergic ascending system. These results suggest that the mesocortical dopaminergic neurons modulate the influence of the main thalamic afferent on the prefrontal cortical cells.     

Contribution of decreased serotonin release to the antidyskinetic effects of deep brain stimulation in a rodent model of tardive dyskinesia: comparison of the subthalamic and entopeduncular nuclei

Mechanisms whereby deep brain stimulation (DBS) of the subthalamic nucleus (STN) or internal globus pallidus (GPi) reduces dyskinesias remain largely unknown. Using vacuous chewing movements (VCMs) induced by chronic haloperidol as a model of tardive dyskinesia (TD) in rats, we confirmed the antidyskinetic effects of DBS applied to the STN or entopeduncular nucleus (EPN, the rodent homolog of the GPi). We conducted a series of experiments to investigate the role of serotonin (5-HT) in these effects. We found that neurotoxic lesions of the dorsal raphe nuclei (DRN) significantly decreased HAL-induced VCMs. Acute 8-OH-DPAT administration, under conditions known to suppress raphe neuronal firing, also reduced VCMs. Immediate early gene mapping using zif268 in situ hybridization revealed that STN-DBS inhibited activity of DRN and MRN neurons. Microdialysis experiments indicated that STN-DBS decreased 5-HT release in the dorsolateral caudate-putamen, an area implicated in the etiology of HAL-induced VCMs. DBS applied to the EPN also suppressed VCMs but did not alter 5-HT release or raphe neuron activation. While these findings suggested a role for decreased 5-HT release in the mechanisms of STN DBS, further microdialysis experiments showed that when the 5-HT lowering effects of STN DBS were prevented by pretreatment with fluoxetine or fenfluramine, the ability of DBS to suppress VCMs remained unaltered. These results suggest that EPN- and STN-DBS have different effects on the 5-HT system. While decreasing 5-HT function is sufficient to suppress HAL-induced VCMs, 5-HT decrease is not necessary for the beneficial motor effects of DBS in this model.     

Effects of electrical and chemical stimulation of nucleus raphe magnus on responses to renal nerve stimulation

Electrical stimulation of the nucleus raphe magnus (NRM) inhibits some somatic and visceral input at the spinal level. This study was designed to examine the effects of electrical and chemical stimulation of NRM on neuronal responses to afferent renal nerve (ARN) stimulation. In chloralose-anesthetized rats, electrical stimulation of ARN elicited predominantly excitatory responses in spinal gray neurons. In 10 neurons studied, electrical stimulation of the NRM elicited an inhibition of spontaneous activity of 8 neurons and inhibited evoked responses to ARN stimulation in 6 neurons. Microinjection of glutamate (5-10 nmol in 0.5-1 microliter) into the NRM elicited an inhibition of spontaneous activity in 9 neurons, a facilitation in 6 neurons and no response in 8 neurons receiving ARN input. Responses evoked by ARN stimulation were inhibited in 12 neurons, facilitated in 4 neurons and not affected in 8 neurons. We conclude that renal input can be modulated at the spinal level by activation of the NRM and adjacent tissue. Furthermore, the inhibition of spinal gray neuronal responses elicited by stimulation of the NRM appears to be due, at least in part, to activation of fibers of passage since non-selective electrical stimulation is more efficacious than selective chemical stimulation of neuronal soma and dendrites.     

Norepinephrine and serotonin: Opposite effects on the activity of lateral geniculate neurons evoked by optic pathway stimulation

This study compares the responses of single units in the rat dorsal lateral geniculate nucleus (LGNd) to microiontophoretically applied norepinephrine (NE) and serotonin (5-HT). Most of the cells were identified physiologically as P-type (geniculocortical relay) neurons. At low iontophoretic currents (1 to 20 nA), NE caused a delayed increase in the spontaneous firing rate of these units, whereas 5-HT invariably slowed the discharge frequency. To compare the effects of the two monoamines on evoked activity, P-cells were driven by electrical stimulation of the afferent visual pathway at the level of the optic chiasm. NE caused a marked facilitation of both the short-latency (2 to 4 ms) and the delayed (70 to 230 ms) responses to such stimulation. The α-adrenoceptor antagonist phentolamine (10 nA), which by itself had no consistent effect on evoked activity, strongly diminished the response to NE. In contrast to NE, 5-HT was a powerful depressant of electrically evoked activity; neither phentolamine nor the 5-HT antagonist methysergide antagonized this response. Firing of LGNd units evoked by flashes of light was also facilitated by NE and depressed by 5-HT. We conclude that LGNd relay neurons exhibit the following unique features in their responsiveness to monoamines: (i) microiontophoretically applied NE facilitates, but 5-HT depresses, the spontaneous or synaptically evoked activity of virtually every cell; (ii) there is no dissociation between the actions of NE on spontaneous and evoked activity, as is the case in other brain regions.     

Interleukin-1 inhibits firing of serotonergic neurons in the dorsal raphe nucleus and enhances GABAergic inhibitory post-synaptic potentials

In vitro electrophysiological data suggest that interleukin-1 may promote non-rapid eye movement sleep by inhibiting spontaneous firing of wake-active serotonergic neurons in the dorsal raphe nucleus (DRN). Interleukin-1 enhances GABA inhibitory effects. DRN neurons are under an inhibitory GABAergic control. This study aimed to test the hypothesis that interleukin-1 inhibits DRN serotonergic neurons by potentiating GABAergic inhibitory effects. In vitro intracellular recordings were performed to assess the responses of physiologically and pharmacologically identified DRN serotonergic neurons to rat recombinant interleukin-1beta. Coronal slices containing DRN were obtained from male Sprague-Dawley rats. The impact of interleukin-1 on firing rate and on evoked post-synaptic potentials was determined. Evoked post-synaptic potentials were induced by stimulation with a bipolar electrode placed on the surface of the slice ventrolateral to DRN. Addition of interleukin-1 (25 ng/mL) to the bath perfusate significantly decreased firing rates of DRN serotonergic neurons from 1.3 +/- 0.2 Hz (before administration) to 0.7 +/- 0.2 Hz. Electrical stimulation induced depolarizing evoked post-synaptic potentials in DRN serotonergic neurons. The application of glutamatergic and GABAergic antagonists unmasked two different post-synaptic potential components: a GABAergic evoked inhibitory post-synaptic potentials and a glutamatergic evoked excitatory post-synaptic potentials, respectively. Interleukin-1 increased GABAergic evoked inhibitory post-synaptic potentials amplitudes by 30.3 +/- 3.8% (n = 6) without affecting glutamatergic evoked excitatory post-synaptic potentials. These results support the hypothesis that interleukin-1 inhibitory effects on DRN serotonergic neurons are mediated by an interleukin-1-induced potentiation of evoked GABAergic inhibitory responses.     

Blockade of 5-HT(2) receptors in the periaqueductal grey matter (PAG) abolishes the anxiolytic-like effect of 5-HT(1A) receptor antagonism in the median raphe nucleus in mice

Several lines of evidence support the involvement of serotonergic (5-HT) neurons of the median raphe nucleus (MRN) in anxiety-like behaviour. In this context, it is known that blockade of 5-HT_1A somatodendritic autoreceptors in the midbrain raphe nuclei increases the firing rate of these neurons, disinhibiting 5-HT release in postsynaptic target areas such as amygdala, hippocampus and periaqueductal grey matter (PAG). However, while activation of 5-HT_1A or 5-HT₂ receptors in forebrain targets such as the amygdala or hippocampus enhances anxiety-like behaviours in rodents, stimulation of both receptor subtypes in the midbrain PAG markedly reduces anxiety-like behaviour. In view of these findings, the present study investigated whether the anti-anxiety effects induced by pharmacological disinhibition of 5-HT neurons in the MRN are attenuated by the blockade of 5-HT₂ receptors within the PAG. Mice received combined intra-PAG injection with ketanserin (10 nmol/0.1 μl), a 5-HT₂ receptor antagonist, followed by intra-MRN injection of WAY-100635 (5.6 nmol/0.1 μl), a highly selective 5-HT_1A receptor antagonist. They were then individually exposed to the elevated plus-maze (EPM), with the videotaped behavioural sessions subsequently scored for both conventional and ethological measures. The results confirmed that intra-MRN infusion of WAY100635 reduces behavioural indices of anxiety without significantly altering general activity measures, and further showed that this effect was completely blocked by intra-PAG pretreatment with an intrinsically-inactive dose of ketanserin. Together, these results suggest that 5HT₂ receptor populations located within the midbrain PAG play a significant role in the reduction of anxiety observed following disinhibition of 5-HT neurons in the MRN.     

The effect of morphine on responses of mediodorsal thalamic nuclei and nucleus submedius neurons to colorectal distension in the rat

In halothane-anesthetized rats, we characterized the responses of single neurons in the nuclei of medial thalamus (MT), specifically the mediodorsal thalamic nucleus (MD) and the nucleus submedius (Sm), to a noxious visceral stimulus (colorectal balloon distension, CRD), and studied the effects of intravenous morphine (Mor) on these responses using standard extracellular microelectrode recording techniques. 62 MD and 46 Sm neurons were isolated on the basis of spontaneous activity. 47 of the MD neurons (76%) responded to CRD, of which 70% had excitatory and 30% had inhibitory responses. 38 of the Sm neurons (83%) responded to CRD, of which 89% had excitatory and 11% had inhibitory responses. Responses of MD and Sm neurons excited by CRD were related significantly to distension pressure (20–100 mmHg), with maximum excitation occurring at 60 and 100 mmHg, respectively. MD neurons inhibited by CRD also had graded responses to graded CRD, with maximum inhibition occurring at 80 mmHg. The responses to noxious (pinch, heat) and nonnoxious (tap, brush) cutaneous stimuli were studied in 59 of the MD and 44 of the Sm neurons isolated. 22 of the MD neurons (37%) studied had cutaneous receptive fields, of which 59% were large and bilateral, 41% were small and usually contralateral receptive fields. 55% of these neurons were nociceptive-specific, 45% responded to both noxious and nonnoxious cutaneous stimulation. 29 of the Sm neurons (66%) studied had cutaneous receptive fields, of which 72% were large and usually bilateral, 14% were small and bilateral, 14% were small and contralateral receptive fields. 90% of these neurons were nociceptive-specific, 10% responded to both noxious and nonnoxious stimulation. No MD or Sm neurons responded exclusively to nonnoxious cutaneous stimulation. Mor (0.125, 0.25, 0.5 and 1 mg/kg IV) attenuated MD and Sm neuronal excitatory responses to CRD in a dose-dependent fashion, abolishing evoked activity with a dose of 0.5 mg/kg (p<0.05) and 1 mg/kg (p<0.05), respectively. Naloxone (0.4 mg/kg IV) reversed the effects of Mor. Mor and naloxone had no effects on spontaneous activity. These data support the involvement of MD and Sm neurons in visceral nociception, and are consistent with a role of Sm in affective-motivational, and MD in both sensory-discriminative and affective-motivational aspects of nociception.     

An electrophysiological study of amygdalohypothalamic projections to the ventromedial nucleus of the rat.

The influence of the amygdala on the activity of single neurons within the hypothalamic ventromedial nucleus (HVM) was studied in pentobarbital or urethane anesthetized rats. The results are summarized as follows: (1) Stimulation of different amygdaloid nuclei or of the stria terminalis (ST) evoked a prominent field potential within HVM and altered the spike discharge patterns of the majority of HVM neurons. (2) More than 80% of 428 HVM neurons tested with single amygdala shocks exhibited excitation or excitation-inhibition sequences; the remainder displayed inhibitory responses of 100-150 msec duration at latencies slightly longer than for most of the observed excitatory responses. ST stimulation also evoked excitation or excitation-inhibition sequences from 85% of 240 HVM neurons tested; of the remainder, those with spontaneous activity displayed inhibitory responses with durations of 100-150 msec at latencies slightly longer than for most observed excitatory responses. (3) Evoked potential interaction studies suggested that stimulation of either ST or the amygdala activated the same population of HVM neurons. Single cells tested with both amygdala and ST stimulation displayed similar patterns of response. HVM field potentials and single unit responses to amygdala stimulation were markedly diminished by lesions of ST. Thus, in the rat, only one pathway, i.e., the stria terminalis, contains amygdalofugal fibres to the ventromedial hypothalamic nucleus. (4) The orthodromic responses of HVM neurons were dependent on the frequency of amygdala stimulation. Less than 50% of HVM neurons responded to amygdala stimuli at frequencies greater than 33Hz. Many cells could not be activated at stimulation frequencies greater than 10 Hz, and the spontaneous discharges from certain HVM neurons were effectively abolished at this stimulation frequency. (5) Evidence of prominent postsynaptic inhibition was present throughout HVM. Seventeen HVM neurons displayed amygdala evoked unitary activity different from that of the majority of HVM neurons, and these cells were considered to represent possible inhibitory neurons. In contrast to most HVM neurons activated via probable monosynaptic amygdalohypothalamic pathways, these putative inhibitory neurons were apparently activated via polysynaptic pathways. (6) In summary, these results suggest that the amygdala exerts a prominent monosynaptic influence on the activity of many HVM neurons, coupled with polysynaptic activation of powerful local postsynaptic inhibitory mechanism. In the rat, these amygdala evoked events depend on the integrity of the stria terminalis.