Opioid inhibition of rat medial vestibular nucleus neurones in vitro and its dependence on age

Extracellular and whole-cell patch clamp intracellular recordings were made from rat medial vestibular nucleus (MVN) neurones in vitro, and their responses to selective μ-, κ- and δ-opioid receptor agonists and antagonists were examined. Of 127 neurones tested, the large majority were inhibited in a dose-dependent manner by the δ-opioid receptor agonists [d-Ala², d-Leu⁵]-enkephalin (DADLE) and [d-Pen², Pen⁵]-enkephalin (DPLPE). The μ-opioid receptor agonist morphine and the κ-receptor agonist U50,488 did not affect the tonic discharge rate of any of the 63 MVN cells tested. The δ-receptor antagonist naltrindole effectively antagonised the inhibitory effects of DADLE and DPLPE. Weak excitatory responses to high doses of DADLE were seen in only two MVN cells. These results demonstrate the presence of δ- but not μ- or κ-opioid receptors on tonically active MVN neurones. Whole-cell intracellular recordings from MVN cells in a current clamp showed that the DADLE-induced inhibition was accompanied by membrane hyperpolarisation and decrease in input resistance, while voltage clamp experiments showed that DADLE induced an outward membrane current that was reduced but not abolished by 20 mM tetraethylammonium bromide. Thus the mechanisms of action of DADLE in inhibiting MVN cells involve the potentiation of outward K currents, in a similar way to the effects of opioids in other areas of brain. The inhibitory effects of DADLE increased linearly with age, so that the responses to DADLE in the youngest animals used here (60–80 g, approx. 3 weeks of age) were relatively small, increasing significantly over the following 2–3 weeks. This age-dependence may be due to post-natal changes in the density of δ-opiate receptors or the efficacy of the signalling pathways activated by them in the MVN cells over this time. Received: 17 November 1997 / Accepted: 26 March 1998     

Excitation and inhibition of rat medial vestibular nucleus neurones by 5-hydroxytryptamine

The effects of 5-hydroxytryptamine (5-HT) and related compounds on the discharge rate of tonically active medial vestibular nucleus (MVN) neurones were studied in an in vitro slice preparation of the dorsal brainstem of the rat. The majority (87 of 107, 82%) of MVN neurones were excited by 5-HT. Nine cells (8%) showed a biphasic response to 5-HT, which consisted of a brief inhibition followed by excitation. Eleven cells (10%) were inhibited by 5-HT. The excitatory effects of 5-HT were mimicked by alpha-methyl-5-HT and antagonised by ketanserin and ritanserin, indicating the involvement of the 5-HT₂ subtype of 5-HT receptor. In biphasic cells, blockade of 5-HT₂ receptors by ketanserin reduced the excitatory component of the response and revealed an enhanced initial inhibition. The inhibitory effects in biphasic cells, and in cells that showed a pure inhibition in response to 5-HT, were blocked by pindobind-5-HT and mimicked by 8-hydroxy-2-(di-n-propylamino)-tetralin indicating the involvement of 5-HT1A receptors. The significance of these findings in relation to the effects of 5-HT on vestibular reflex function is discussed.     

Tonic activity of rat medial vestibular nucleus neurones in vitro and its inhibition by GABA

Summary The spontaneous discharge of 48 medial vestibular nucleus (MVN) neurones was recorded extracellularly in horizontal slices of the rat brainstem in vitro. The mean tonic rate of discharge was 17.1±8.2 imp/s, similar to that observed by others in transverse (coronal) slices of the rat and guinea pig MVN. The tonic rate of discharge of individual MVN cells either increased or decreased after synaptic blockade in low Ca²⁺ media, suggesting that ongoing synaptic activity has an important influence on the spontaneous activity of MVN cells in vitro. However the persistence of tonic activity after synaptic blockade indicates that an intrinsic, pacemaker-like mechanism is involved in the generation of the tonic activity. GABA, muscimol, baclofen and 3-APA inhibited the tonic activity of all MVN cells tested. Bicuculline antagonised, and picrotoxin blocked, the inhibitory responses to muscimol, but the effects of GABA were only partially blocked in 50 M picrotoxin. The effects of baclofen and 3-APA persisted in low Ca²⁺ media, and were antagonised by saclofen and phaclofen. Picrotoxin-resistant responses to GABA persisted in low Ca²⁺ media, and were also antagonised by saclofen. These results suggest that the inhibitory control of MVN neurones by GABA involves both the GABA_A and GABA_B subtypes of GABA receptor. GABA_B receptors appear to be distributed both pre- and post-synaptically in the rat MVN. The possible significance of the intrinsic, tonic activity of MVN cells in normal vestibular function and in vestibular compensation, and the effects of GABA, are discussed.     

Muscarinic inhibition of endogenous glutamate release from rat hippocampus synaptosomes

The effects of acetylcholine (ACh) on the depolarization-evoked release of endogenous glutamic acid (Glu) have been studied using synaptosomes prepared from rat hippocampus and depolarized in superfusion with 15 mM KCl. Acetylcholine inhibited Glu release in a concentration-dependent way. The natural agonist was particularly effective causing 50% inhibition of Glu release at 10 microM in the absence of acetylcholinesterase (AChE) inhibitors. The inhibitory effect of ACh on the K+-evoked release of Glu was antagonized by the selective muscarinic receptor antagonist atropine but not by the nicotinic receptor antagonist mecamylamine. The data represent the first demonstration that muscarinic receptors located on Glu axon terminals in rat hippocampus may modulate the release of Glu.     

Feeding-induced decrease in extracellular glutamate level in the rat nucleus accumbens: dependence on glutamate uptake

In vivo microdialysis combined with high-performance liquid chromatography and electrochemical detection was used to monitor extracellular glutamate levels in the medial nucleus accumbens of Sprague-Dawley rats during their feeding behaviour. Consumption of a palatable new diet or a diet to which rats were previously exposed caused a decrease in extracellular level of glutamate in the nucleus accumbens during and after feeding. The presentation of an inedible object (a piece of rubber) instead of the expected food caused a marked increase in extracellular glutamate levels. In contrast, if the piece of rubber was presented to rats that did not expect food delivery, the extracellular level of glutamate remained unchanged during the rubber presentation. The feeding-induced decrease in the extracellular glutamate level did not depend on food deprivation and was completely prevented by intraaccumbal infusions through the dialysis probe of 10 mM D,L-threo-beta-hydroxyaspartate (a glutamate uptake inhibitor). Intraaccumbal infusions of 10 microM S-(-)-raclopride L-tartrate (a D2/D3 dopamine receptor antagonist) or 1 microM tetrodotoxin (a voltage-dependent Na(+) channel blocker) also completely reversed the decrease in extracellular glutamate level in response to food intake. The D1/D5 dopamine receptor antagonist SCH-23390 (10 microM) administered into the nucleus accumbens had no significant effect on the feeding-induced decrease in extracellular glutamate level.From the data obtained we suggest that the decrease in the extracellular level of glutamate in the medial nucleus accumbens in response to feeding appears to arise from a temporal increase in glutamate uptake that is probably operated by dopamine inputs to the nucleus accumbens via D2/D3 receptors. Our findings also suggest that the dissociation between the expected biological value of a presented object and the reality might be an important determinant for regulation of glutamate release in this brain area during feeding behaviour.     

Dopamine neurones of the ventral tegmentum project to both medial and lateral habenula

Using the techniques of microdissection and microradiochemical assay for catecholamines, it has been shown that a specific subgroup of ventral tegmental area (VTA) dopamine neurones project to the medial sector of lateral habenula. In addition a new dopamine pathway, arising from midline VTA neurons, has been shown to project to the medial sector of medial habenular. These findings are discussed and some implications for habenular functions are stated.     

Dual GABAergic synaptic response of fast excitation and slow inhibition in the medial habenula of rat epithalamus

We report here a novel action of GABAergic synapses in regulating tonic firing in the mammalian brain. By using gramicidin-perforated patch recording in rat brain slices, we show that cells of the medial habenula of the epithalamus generate tonic firing in basal conditions. The GABAergic input onto these cells at postnatal days 18-25 generates a combinatorial activation of fast excitation and slow inhibition. The fast excitation, mediated by gamma-aminobutyric acid type A receptors (GABA A Rs), is alone capable of triggering robust action potentials to increase cell firing. This excitatory influence of GABAergic input results from the Cl(-) homeostasis that maintains intracellular Cl(-) at high levels. The GABA A excitation is often followed by a slow inhibition mediated by GABA B Rs that suppresses tonic firing. Interestingly, in a subpopulation of the cells, the GABA B inhibition exhibits a remarkably low threshold for synaptic activation in that low-strength GABAergic input often activates selectively the GABA B slow inhibition, whereas the GABA A excitation requires further increases in stimulus strength. Our study demonstrates that the dual activation of GABAergic excitation and inhibition through GABA A Rs and GABA B Rs generates distinct temporal patterns of cell firing that alter the cellular output in an activity-dependent manner.     

Group II metabotropic glutamate receptors are differentially expressed in the medial nucleus of the trapezoid body in the developing and adult rat

The existence of a neuronal-glial signalling through the activation of neurotransmitter receptors expressed in glia is well-documented. In excitatory synapses, glutamate released from presynaptic terminals activates not only postsynaptic receptors, but also ionotropic and metabotropic glutamate receptors localized in the glia ensheathing the synapses. The medial nucleus of the trapezoid body of the auditory system is involved in the localization of sounds in the space. In this nucleus, the large excitatory synaptic terminals formed by the calyces of Held on the principal globular cell bodies are wrapped by astrocytic processes. Since these synapses are functional from early postnatal days, glia receiving excitatory synaptic signals from the calyces may participate in modulating the maturation and development of the system.Groups I and II of metabotropic glutamate receptors (mGluRs) have been localized in glial cells in different brain regions. To investigate whether group II mGluRs are present in the medial nucleus of the trapezoid body, we have studied the pattern of expression of mGluR2/3 in the developing and mature nucleus by means of immunocytochemichal methods. The most remarkable finding was the switch in the occurrence of mGluR2/3 from glial to neuronal compartments. Thus, a preferential localization of mGluR2/3 immunoreactivity was observed in astrocytic processes surrounding the calyces of Held during the early postnatal development. In contrast, the main feature in adult rats was the presence of the group II mGluRs in presynaptic calyces of Held and postsynaptic principal globular cells.From these observations we suggest a role for group II mGluRs in neuronal-glial signalling in the calyx of Held-principal globular neuron synapses. Activation of these receptors might be relevant to the maturation and modulation of synaptic transmission in the medial nucleus of the trapezoid body.     

Immunocytochemical evidence for in vitro release of glutamate and GABA from separate nerve terminal populations in the rat pontine nuclei

Summary A quantitative electron microscopic immunocytochemical method was used to study the synaptic handling of glutamate and GABA in slice preparations from the rat pontine nuclei. Slices were subjected to a depolarizing stimulus (55 mM K⁺, 20 min) in the presence of a physiological or low Ca²⁺concentration. Depolarization at physiological [Ca²⁺] evoked a depletion of glutamate-like immunoreactivity from nerve terminals that contain round vesicles and establish asymmetric synaptic contacts. When depolarization was induced in the presence of only 0.1 mM Ca²⁺ (10 mM Mg²⁺ added), the loss of glutamate was significantly reduced or abolished, indicative of a Ca²⁺dependent component of glutamate release. By means of a double labeling immunocytochemical method we could identify a population of nerve terminals that displayed strong GABA-like immunoreactivity, and a level of glutamate like immunoreactivity that was low but yet clearly above background level. This type of terminal contains elongated or pleomorphic vesicles and establishes symmetric synaptic contacts. In these terminals, depolarization evoked a Ca²⁺-dependent depletion of GABA like immunoreactivity, but failed to change the level of glutamate like immunoreactivity. The present study demonstrates that two different types of nerve terminal in the rat pontine nuclei contain releasable pools of glutamate and GABA, respectively, and that the GABA releasing terminals also contain a non releasable pool of glutamate. The glutamate of the latter pool could act as precursor of GABA.     

Group II metabotropic glutamate receptor-mediated regulation of dopamine release from slices of rat nucleus accumbens

The role of metabotropic glutamate receptors (mGluRs) in the regulation of dopamine release in the rat nucleus accumbens was investigated. Fifteen millimolar of KCl stimulated the release of [(3)H]dopamine from the slices of the rat nucleus accumbens. Both an mGluR agonist 1S,3R-1-amino-cyclopentane-1,3-dicarboxylate (ACPD) and a preferential group II mGluR agonist, (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (L-CCG-1), significantly inhibited the KCl-evoked [(3)H]dopamine release in the nucleus accumbens. This inhibitory effect of L-CCG-1 on the KCl-evoked dopamine release was significantly attenuated by preferential group II mGluR antagonists such as (2S,3S,4S)-2-methyl-2-(carboxypropyl)glycine (MCCG) and (RS)-alpha-methyl-4-tetrazolylphenylglycine (MTPG); in contrast, the preferential group III mGluR agonist L-2-amino-4-phosphonobutylate (L-AP4), failed to show any effect on the KCl-evoked [(3)H]dopamine release in the nucleus accumbens. Moreover, the inhibitory effect of L-CCG-1 on the KCl-evoked [(3)H]dopamine release from the slices of the rat nucleus accumbens was preserved in the presence of tetrodotoxin. These results show that group II mGluRs may play a more significant role in regulating dopamine release than group III mGluRs, and that the group II mGluRs may negatively regulate dopamine release, presumably through those expressed at the dopaminergic nerve terminals or through those expressed at glutamatergic nerve terminals in the nucleus accumbens.     

Metabolic inhibition and low internal ATP activate K-ATP channels in rat dopaminergic substantia nigra neurones

The patch-clamp technique was used to study whole-cell currents of acutely dissociated rat substantia nigra (SN) neurones. In perforated-patch current-clamp recordings, inhibition of mitochondrial metabolism by rotenone (5 M) produced a hyperpolarisation and inhibited electrical activity. These effects were reversed by the sulphonylureas tolbutamide (0.5 mM) or glibenclamide (0.5 M). Under voltageclamp conditions, rotenone induced a timeand voltage-independent K⁺ current which was selectively blocked by sulphonylureas. The glibenclamide-sensitive current reversed at –81.7±2.7 mV (n=5) and showed marked inward rectification. Intracellular dialysis with 0.3 mM adenosine 5-triphosphate (ATP), but not 2 mM or 5 mM ATP, in standard whole-cell recordings also resulted in activation of a sulphonylurea-sensitive K⁺ current with similar properties (reversal potential, –81.9±2.5 mV, n=5). The close similarity in the properties of the ATP-sensitive K⁺ current observed in whole-cell recordings and the K⁺ current activated by metabolic inhibition in perforated-patch recordings suggest that they both result from activation of the same type of ATP-sensitive K⁺ channel. Sulphonylureas had no effect on electrical activity or membrane currents in the absence of rotenone in perforated-patch recordings, or in cells dialysed with 5 mM ATP, indicating that in SN neurones these drugs are selective for the ATP-sensitive K⁺ current.     

Measurement of 5-hydroxytryptamine release in the rat medial vestibular nucleus using in vivo microdialysis

The function of the 5-hydroxytryptamine (5-HT) system in the medial vestibular nucleus (MVN) was evaluated using KCl-evoked 5-HT release assessed by in vivo microdialysis. The release of 5-HT in the MVN was increased by the local application of KCl (100 mM) in a calcium-dependent manner. Likewise, perfusion of a 5-HT reuptake blocking agent, clomipramine (1 microM), increased extracellular levels of 5-HT in the MVN. Finally, pretreatment with the selective 5-HT(1A) agonist, 8-hydroxy-2-(di-N-propylamino) tetralin (1 mg/kg, intraperitoneally), attenuated the KCl-evoked 5-HT release. These findings suggest that 5-HT stored in vesicles is released from nerve terminals in the MVN. In addition, its regulation may be mediated by control of somatodendritic 5-HT(1A) receptor activation.     

GABA release from suprachiasmatic nucleus terminals is necessary for the light-induced inhibition of nocturnal melatonin release in the rat.

The daily rhythm of melatonin production in the mammalian pineal is driven by the endogenous circadian pacemaker in the suprachiasmatic nuclei. The major release period of melatonin is closely linked to the dark phase of the 24-h day/night cycle. Environmental light will affect melatonin release in two ways: (i) it entrains the rhythm of the circadian oscillator; and (ii) it causes an acute suppression of nocturnal melatonin release. These two effects of light are both mediated by the suprachiasmatic nucleus and enable the pineal gland to convey information about day length to the reproductive system through changes in melatonin levels. Glutamate is currently believed to be the major transmitter in the retinal ganglion cell fibers reaching the suprachiasmatic nucleus. At present no information is available, however, about the transmitter(s) implicated in the further propagation, i.e. from the suprachiasmatic nucleus onwards, of the light information. In the present study we provide evidence that the endogenous release of GABA from suprachiasmatic nucleus terminals is implicated in the further transmission of light information to the pineal gland. Bilateral administration of the GABA-antagonist bicuculline to hypothalamic target areas of the suprachiasmatic nucleus completely prevents the inhibitory effect of nocturnal light on melatonin secretion and the present study thus documents that retina-mediated photic activation of suprachiasmatic nucleus neurons induces the release of GABA from efferent suprachiasmatic nucleus nerve terminals, resulting in an inhibition of melatonin release by the pineal gland. Together with our previous (electro)physiological data these results identify GABA as an important mediator of rapid synaptic transmission of suprachiasmatic nucleus output to its target areas.     

Dendritic trees of neurones in the rat supraoptic nucleus

The cytoarchitecture of the rat supraoptic nucleus was studied using the Golgi-Cox method with a neutral red or Cresyl violet counterstain to allow the limits of the nucleus to be determined accurately. A small number of stained neurones were seen in each of the brains sectioned but they were unevenly distributed in the supraoptic nucleus. There were more cells posteriorly and close to the optic tract. A minority of cells studied (a total of 4) were round (diameter approximately 20 μm) and multipolar and were interpreted as interneurones. The majority of stained cells studied (60) had oval cell bodies (mean long diameter 22.7 μm in coronal sections) with a single fine axon directed dorsally and medially and 1–3 heavier dendrites passing ventrally. Both cell bodies and dendrites of the major cell type showed spines and both dendrites and axons had some varicosities.The anatomy of the larger cell group (the presumed neurosecretory cells) is thus consistent with their having a substantial synaptic input and the presence of long dendrites (up to 205 μm) implies that caution is necessary in the interpretation of experiments where cell activity is monitored while active materials are placed near the cell bodies.