A novel class (H3) of histamine receptors on perivascular nerve terminals

Two types of histamine receptor, the H1- and H2-receptors, are found not only on vascular smooth muscle cells but on the perivascular autonomic nerve terminals. Activation of the prejunctional histamine receptors modifies transmitter release from the nerve terminals. Recently, histamine was shown to inhibit its own release from depolarized slices of rat cerebral cortex. This phenomenon was found to be mediated by a novel class of histamine receptor, the H3-receptor, that was pharmacologically distinct from the H1- and H2-receptors. Up to now, there has been no indication whether this third class of histamine receptor is present in any tissue other than the brain. We report here that histamine depresses sympathetic neurotransmission in the guinea-pig mesenteric artery by interacting with histamine H3-receptors on the perivascular nerve terminals. The pharmacological properties of these receptors are similar to those reported for the H3-receptors in the brain. Our data provide evidence for the existence of H3-receptors in the autonomic nervous system.     

Mediation of thalamic sensory input by both NMDA receptors and non-NMDA receptors

Excitatory amino acids such as L-glutamate and L-aspartate are well established as neurotransmitter candidates in the mammalian central nervous system, and three types of receptor for these substances have been proposed, characterized by the agonists N-methyl-D-aspartate (NMDA), kainate and quisqualate. All these receptors have been suggested to have synaptic roles in excitatory transmission in the brain. Here I demonstrate that NMDA receptors play a crucial role in the observed response of ventrobasal thalamus (VB) neurones to natural stimulation of somatosensory afferents, but do not appear to be responsible for the short-latency excitation seen on electrical stimulation of the afferents which is apparently mediated by excitatory amino-acid receptors of the non-NMDA type. This result indicates an involvement of NMDA and non-NMDA receptors in the responses of VB neurones to stimulation of somatosensory somatosensory afferents, depending on the mode of stimulation of the pathway.     

Activation of multiple-conductance state chloride channels in spinal neurones by glycine and GABA

In the mammalian central nervous system, glycine and gamma-aminobutyric acid (GABA) bind to specific and distinct receptors and cause an increase in membrane conductance to CI- (refs 5-7). Neurones in various regions of the nervous system show differential sensitivity to glycine and GABA; thus GABA and glycine receptors are spatially distinct from one another. However, on the basis of desensitization experiments on spinal cord neurones, it was suggested that the receptors for glycine and GABA may share the same CI- channel. We now report that in small membrane patches, isolated from the soma of spinal neurones, both receptor channels display several (multiple) conductance states. Two of the states are common to both receptor channels. However, the most frequently observed 'main conductance states' of the GABA and glycine receptor channels are different. Both channels display the same anion selectivity. We propose that one class of multistate CI- channel is coupled to either GABA or glycine receptors. The main conductance state adopted by this channel is determined by the receptor to which it is coupled.     

Pertussis toxin reverses adenosine inhibition of neuronal glutamate release

Adenosine and its analogues are potent inhibitors of synaptic activity in the central and peripheral nervous system. In the central nervous system (CNS), this appears to arise primarily by inhibition of presynaptic release of transmitters, including glutamate, which is possibly the major excitatory transmitter in the brain. In addition, postsynaptic effects of adenosine have been reported which would also serve to reduce neurotransmission. The mechanism by which adenosine inhibits CNS neurotransmission is unknown, although it appears to exert its effect via an A1 receptor which in some systems is negatively coupled to adenylate cyclase. In an attempt to elucidate the mechanism of inhibition, we have examined the effect of pertussis toxin (PTX) on the ability of the stable adenosine analogue (-)phenylisopropyladenosine (PIA) to inhibit glutamate release from cerebellar neurones maintained in primary culture. PTX, by ADP-ribosylating the nucleotide-binding protein Ni, prevents coupling of inhibitory receptors such as the A1 receptor to adenylate cyclase. As reported here, we found that PTX, as well as preventing inhibition of adenylate cyclase by PIA, also converts the PIA-induced inhibition of glutamate release to a stimulation. Our results suggest strongly that purinergic inhibitory modulation of transmitter release occurs by inhibition of adenylate cyclase.     

GABA affects the release of gastrin and somatostatin from rat antral mucosa

gamma-Aminobutyric acid (GABA) is regarded as the major inhibitory neurotransmitter in the central nervous system of vertebrates. GABA exerts its inhibitory actions by interacting with specific receptors on pre- and postsynaptic membranes and has been shown to inhibit somatostatin release from hypothalamic neurones in vitro. Concepts of innervation of the gastrointestinal tract have been expanded by recent studies which suggest that GABAergic neurones are not confined solely to the central nervous system but may also exist in the vertebrate peripheral autonomic nervous system. Jessen and coworkers have demonstrated the presence, synthesis and uptake of GABA by the myenteric plexus of the guinea pig taenia coli, and have documented the presence of glutamic acid decarboxylase (GAD) in isolated myenteric plexus. This enzyme is responsible for the conversion of glutamic acid to GABA in GABAergic neurones. The possibility that GABA may have a role in neurotransmission or neuromodulation in the enteric nervous system of the vertebrate gut has been suggested by several investigators. Furthermore, GABA receptors have been demonstrated on elements of the enteric nervous system. The effects of GABA on gastrointestinal endocrine cell function have not been examined. We report here the effects of GABA on gastrin and somatostatin release from isolated rat antral mucosa in short-term in vitro incubations.     

Evidence that substance P is a neurotransmitter in the myenteric plexus

Substance P (SP) is an undecapeptide originally isolated from the gut and since shown to occur within neurones in several parts of the peripheral and central nervous systems. Immunohistochemical studies indicate an exceedingly dense network of SP-containing nerves within the myenteric plexus of the guinea pig ileum. These nerves are intrinsic to the gut wall and can release SP to contract the longitudinal muscle layer. We have previously shown that SP directly depolarizes myenteric neurones and that this depolarization has a time course and ionic mechanism similar to the slow excitatory postsynaptic potential (e.p.s.p.) which can be produced by electrical stimulation of presynaptic nerves within the myenteric ganglia. We wondered whether SP might mediate this slow synaptic potential. We report here that the SP depolarization and the slow e.p.s.p. are reversibly depressed by chymotrypsin, an enzyme which degrades SP, although the responses to acetylcholine, serotonin and an unknown hyperpolarizing transmitter are unaffected. The results provide direct evidence that a peptide can mediate chemical transmission between neurones in the mammalian nervous system.     

Excitatory effect of histamine on neuronal activity of rat cerebellar fastigial nucleus in vitro

The cerebellar fastigial nucleus (FN) holds an important role in motor control and body balance. Previous studies have revealed that the nucleus is innervated by direct hypothalamocerebellar histaminergic fibers. However, the functional role of histaminergic projection in cerebellar FN has never been established. In this study, we investigated the effect of histamine on neuronal firing of cerebellar FN by using slice preparations. Sixty-five FN cells were recorded from 47 cerebellar slices, and a vast majority of the cells responded to histamine stimulation with an excitatory response (58/65, 89.2%). Perfusing slices with low-Ca2 /high-Mg2 medium did not block the histamine-induced excitation (n=10), supporting a direct postsynaptic action of histamine on the cells. Furthermore, the excitatory effect of histamine on FN neurons was not blocked by selective histamine H1 receptor antagonist triprolidine (n=15) or chlorpheniramine (n=10), but was effectively suppressed by ranitidine (n=15), a highly selective histamine H2 receptor antagonist. On the other hand, highly selective histamine H2 receptor agonist dimaprit (n=20) instead of histamine H1 receptor agonist 2-pyridylethylamine (n=16) mimicked the ex- citatory effect of histamine on FN neurons. The dimaprit-induced FN neuronal excitation was effectively antagonized by selective histamine H2 receptor antagonist ranitidine (n=13) but not influenced by se- lective histamine H1 receptor antagonist triprolidine (n=15). These results demonstrate that histamine excites cerebellar FN cells via the histamine H2 receptor mechanism and suggest that the hypotha- lamocerebellar histaminergic fibers may modulate cerebellar FN-mediated sensorimotor integration through their excitatory innervations on FN neurons.     

Glutamate stimulates inositol phosphate formation in striatal neurones

The major excitatory amino acids, glutamate (Glu) and aspartate (Asp), are thought to act at three receptor subtypes in the mammalian central nervous system (CNS). These are termed quisqualate (QA), N-methyl-D-aspartate (NMDA) and kainate (KA) receptors according to the specific agonist properties of these compounds revealed by electrophysiological studies. Although Glu has been shown to stimulate cyclic GMP formation in brain slices, direct regulation of second messenger systems (cyclic AMP, Ca2+ or inositol phosphates) subsequent to activation of excitatory amino-acid receptors, has not been extensively studied. Here we demonstrate that in striatal neurones, excitatory amino acids, but not inhibitory or non-neuroactive amino acids, induce a three- to fourfold increase in inositol mono-, di- and triphosphate (IP, IP, IP) formation with the relative potency QA greater than Glu greater than NMDA, KA. The Glu-evoked formation of inositol phosphates appears to result principally from actions at QA as well as NMDA receptors on striatal neurones. Our results suggest that excitatory amino acids stimulate inositol phosphate formation directly, rather than indirectly by the evoked release and subsequent actions of adenosine or acetylcholine.     

Glycogenolysis induced by serotonin in brain: identification of a new class of receptor

Serotonin-containing neurones in brain have been proposed to have a role in the control of physiological mechanisms such as sleep, thermoregulation, pain perception and endocrine secretions as well as in the physiopathology of migraine or depressive illness. One difficulty in testing these possibilities lies in the scarcity of pharmacological agents able to interact selectively with the probably multiple classes of serotonin receptors in the central nervous system. Development of such agents would be facilitated by simple in vitro models in which biological responses to serotonin in mammalian brain could be quantified. Thus a serotonin-sensitive adenylate cyclase has been characterized in rat brain, but the response to serotonin is weak in newborn and practically absent in adult animals. In addition, two pharmacologically distinct classes of serotoninergic binding site have been identified using 3H-serotonin and 3H-spiperone as ligands, but their identification as receptors remains to be established. More recently, serotonin has been shown to stimulate phosphorylation of a neuronal protein in slices from the facial motor nucleus, although the receptors mediating this action were not characterized. We now report that serotonin stimulates glycogen hydrolysis in slices of cerebral cortex, that this action is mediated by a novel class of receptors and that tricyclic antidepressants are among the best competitive antagonists of the indolamine.     

Regulation of glutamate receptors by cations.

Current evidence suggests that glutamate is a major excitatory neurotransmitter in the mammalian central nervous system (CNS); particularly, glutamate excites most neurones in the CNS. Until recently this effect was widely used to study glutamate receptors and to distinguish them from those of other excitatory amino acids. The development of ligand binding studies for many neurotransmitters has facilitated the study of receptors at the molecular level and using these methods we recently reported the existence in hippocampal membranes of pharmacologically distinct sodium-dependent and sodium-independent glutamate binding sites, the former related to high-affinity uptake and the latter exhibiting several characteristics of postsynaptic receptor sites. We now report that, as with other neurotransmitters, several ions regulate the Na-independent binding of glutamate; the monovalent cations induce a decreased binding while certain divalent cations enhance this Na-independent binding. Additionally, since some of these effects appear to be irreversible, we propose that the regulation of glutamate binding by cations might account for the extremely long-lasting potentiation of synaptic responses found in the hippocampus following bursts of repetitive electrical stimulation (see ref. 9 for a review).     

NMDA-receptor activation increases cytoplasmic calcium concentration in cultured spinal cord neurones

Excitatory amino acids act via receptor subtypes in the mammalian central nervous system (CNS). The receptor selectively activated by N-methyl-D-aspartic acid (NMDA) has been best characterized using voltage-clamp and single-channel recording; the results suggest that NMDA receptors gate channels that are permeable to Na+, K+ and other monovalent cations. Various experiments suggest that Ca2+ flux is also associated with the activation of excitatory amino-acid receptors on vertebrate neurones. Whether Ca2+ enters through voltage-dependent Ca2+ channels or through excitatory amino-acid-activated channels of one or more subtype is unclear. Mg2+ can be used to distinguish NMDA-receptor-activated channels from voltage-dependent Ca2+ channels, because at micromolar concentrations Mg2+ has little effect on voltage-dependent Ca2+ channels while it enters and blocks NMDA receptor channels. Marked differences in the potency of other divalent cations acting as Ca2+ channel blockers compared with their action as NMDA antagonists also distinguish the NMDA channel from voltage-sensitive Ca2+ channels. However, we now directly demonstrate that excitatory amino acids acting at NMDA receptors on spinal cord neurones increase the intracellular Ca2+ activity, measured using the indicator dye arsenazo III, and that this is the result of Ca2+ influx through NMDA receptor channels. Kainic acid (KA), which acts at another subtype of excitatory amino-acid receptor, was much less effective in triggering increases in intracellular free Ca2+.     

Oxytocin induces morphological plasticity in the adult hypothalamo-neurohypophysial system

The hypothalamo-neurohypophysial system offers a unique example in the adult mammalian central nervous system (CNS) of a functional and structural plasticity related to a physiological state. During lactation, oxytocin neurones evolve a synchronized electrical activation which permits pulsatile hormone release at milk ejection. At the same time, in the supraoptic (SON) and paraventricular nuclei, glial coverage of neurones diminishes, so that large portions of their surface membrane become directly juxtaposed; synaptic remodelling also associates pairs of neurones through the formation of common presynaptic terminals. These structural changes, reversible after weaning, affect exclusively oxytocinergic neurones and could facilitate their synchronized electrical activity. As several observations suggest that oxytocin itself is released centrally, we have examined the effect of prolonged intracerebroventricular infusions of oxytocin on the structure of the SON of non-lactating animals. We report here that the peptide indeed engenders the structural reorganization characteristic of the oxytocin system when it is physiologically activated. Similar infusion of vasopressin has no effect. Our observations thus demonstrate that a central neuropeptide can induce anatomical changes in the adult CNS, and suggest that oxytocin can regulate its own release by contributing to the dramatic restructuring of the nuclei containing the neurones responsible for its secretion.     

An opiate system in the goldfish retina

Recently, in addition to conventional neurotransmitters such as acetylcholine, dopamine, glycine and gamma-aminobutyric acid (GABA), putative neuroactive peptide transmitters have been localized to specific retinal amacrine cells. In particular, opiate receptors 2,3, assayable enkephalin immunoreactivity and enkephalin-immunoreactive neurones 1,5 have been described in avian and mammalian retinae. However, little physiological evidence has been obtained for the involvement of neuropeptides in retinal function. Here we report that exogenous opiates affect both the release of GABA from GABAergic amacrine cells and the firing patterns of ganglion cells in the goldfish retina. Our results show that the output of the retina is modulated by an opiate system whose neural organization and pharmacological properties resemble those described elsewhere in the vertebrate central nervous system.     

Presence of a low molecular weight endogenous inhibitor on 3H-muscimol binding in synaptic membranes

The specific binding of 3H-muscimol to synaptic membrane preparations obtained from the rate brain has been though to reflect the association of gamma-aminobutyric acid (GABA), a potential candidate as an inhibitory neurotransmitter in the mammalian central nervous system (CNS), with its synaptic receptors. Treatment of synaptic membranes with Triton X-100 significantly increases the specific binding of 3H-muscimol. Several reports also indicate the presence of endogenous substances, such as GABA, acidic protein and phosphatidylethanolamine, which inhibit Na-independent binding of 3H-GABA in the synaptic membranous fractions from the rat brain. We report here that in the supernatant obtained from Triton-treated synaptic membranes there exists a new type of endogenous inhibitor of 3H-muscimol binding which is apparently different from the inhibitory substances described previously. The new inhibitor has a low molecular weight (MW) and probably originated from neurones rather than glial cells. We have termed this endogenous inhibitor the GABA receptor binding inhibitory factor (GRIF).     

A physiological role for endogenous zinc in rat hippocampal synaptic neurotransmission.

The mammalian central nervous system (CNS) contains an abundance of the transition metal zinc, which is highly localized in the neuronal parenchyma. Zinc is actively taken up and stored in synaptic vesicles in nerve terminals, and stimulation of nerve fibre tracts that contain large amounts of zinc, such as the hippocampal mossy fibre system, can induce its release, suggesting that it may act as a neuromodulator. The known interaction of zinc with the major excitatory and inhibitory amino-acid neurotransmitter receptors in the CNS supports this notion. That zinc has a role in CNS synaptic transmission, however, has so far not been shown. Here we report a physiological role for zinc in the young rat hippocampus (postnatal, P3-P14 days). Our results indicate that naturally occurring spontaneous giant depolarizing synaptic potentials (GDPs) in young CA3 pyramidal neurones, mediated by the release of GABA (gamma-aminobutyric acid), are induced by endogenously released zinc. These synaptic potentials are inhibited by specific zinc-chelating agents. GDPs are apparently generated by an inhibitory action of zinc on both pre- and postsynaptic GABAB receptors in the hippocampus. Our study implies that zinc modulates synaptic transmission in the immature hippocampus, a finding that may have implications for understanding benign postnatal seizures in young children suffering with acute zinc deficiency.     

Kappa- and delta-opioid receptor agonists differentially inhibit striatal dopamine and acetylcholine release

At least three different families of endogenous opioid peptides, the enkephalins, endorphins and dynorphins, are present in the mammalian central nervous system (CNS). Immunocytochemical studies have demonstrated their localization in neurones, which supports the view that these peptides may have a role as neurotransmitter or neuromodulators. However, the target cells and cellular processes acted upon by the opioid peptides are still largely unknown. One possible function of neuropeptides, including the opioid peptides, may be presynaptic modulation of neurotransmission in certain neuronal pathways, for example, by inhibition or promotion of neurotransmitter release from the nerve terminals. Here we report that dynorphin and some benzomorphans potently and selectively inhibit the release of (radiolabelled) dopamine from slices of rat corpus striatum, by activating kappa-opioid receptors. In contrast, [Leu5]enkephalin and [D-Ala2, D-Leu5]enkephalin selectively inhibit acetylcholine release by activating delta-opioid receptors.     

Acetylcholine hyperpolarizes central neurones by acting on an M2 muscarinic receptor

Acetylcholine (ACh) is considered to act as a neurotransmitter in the mammalian brain by binding to membrane receptors and bringing about a change in neurone excitability. In the case of muscarinic receptors, cell excitability is usually increased; this effect results from a closure of membrane potassium channels in cortical cells. However, some central neurones are inhibited by ACh, and we hypothesized that these two opposite effects of ACh resulted from interactions with different subtypes of muscarinic receptor. We made intracellular recordings from neurones in the rat nucleus parabrachialis, a group of neurones in the upper pons some of which themselves synthesize ACh. ACh and muscarine caused a membrane hyperpolarization which resulted from an increase in the membrane conductance to potassium ions. The muscarinic receptor subtype was characterized by determining the dissociation equilibrium constant (KD) for pirenzepine during the intracellular recording; the value of approximately 600 nM indicates a receptor in the M2 class. This muscarinic receptor is quite different from that which brings about a decrease in potassium conductance in other neurones, which has a pirenzepine KD of approximately 10 nM (M1 receptors). It is possible that antagonists selective for this kind of M2 receptor would be useful in the management of conditions, such as Alzheimer's disease, which are associated with a reduced effectiveness of cholinergic neurones.     

Cloning of a putative high-affinity kainate receptor expressed predominantly in hippocampal CA3 cells.

Kainic acid is a potent neurotoxin for certain neurons. Its neurotoxicity is thought to be mediated by an excitatory amino-acid-gated ion channel (ionotropic receptor) possessing nanomolar affinity for kainate. Here we describe a new member of the rat excitatory amino-acid receptor gene family, KA-1, that has a 30% sequence similarity with the previously characterized alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunits GluR-A to -D. The pharmacological profile of expressed recombinant KA-1 determined in binding experiments with [3H]kainate is different from that of the cloned AMPA receptors and similar to the mammalian high-affinity kainate receptor (kainate greater than quisqualate greater than glutamate much greater than AMPA) with a dissociation constant of about 5 nM for kainate. The selectively high expression of KA-1 messenger RNA in the CA3 region of the hippocampus closely corresponds to autoradiographically located high-affinity kainate binding sites. This correlation, as well as the particular in vivo pattern of neurodegeneration observed on kainate-induced neurotoxicity, suggests that KA-1 participates in receptors mediating the kainate sensitivity of neurons in the central nervous system.     

Multiple classes of glutamate receptor on depolarizing bipolar cells in retina

Multiple subtypes of excitatory amino acid receptor have been found on individual dissociated neurones. These findings were obtained from cells without intact synaptic connections, so the functional roles for such receptor subtypes are unknown. We have recorded intracellular responses from depolarizing bipolar cells (DBC) that receive direct synaptic input from two distinct populations of neurones: rods and cones. We report here that 2-amino-4-phosphonobutyrate (APB), a glutamate analogue, reveals two subtypes of glutamate receptors on DBCs. APB acts on the same receptor that mediates synaptic transmission from rods but has no action on the second subtype of glutamate receptor. These results show that the rod and cone inputs to DBCs are mediated by pharmacologically distinct receptors and that subtypes of glutamate receptor existing on single neurones can subserve separate, functionally defined synaptic inputs.     

Highly potent and selective ligands for histamine H3-receptors

New drugs selective for histamine H3-receptors can be used to establish that these receptors are involved in the feedback control of histamine synthesis and release, and to demonstrate their distribution in the brain and peripheral tissues. These drugs provide new tools for affecting physiological and possibly pathological conditions in which histamine is involved.