A functional role for vasoactive intestinal polypeptide in anterior cingulate cortex

Vasoactive intestinal polypeptide (VIP) is present in high concentrations in the cerebral cortex, where it is the putative neurotransmitter of a major intracortical neuronal system. Homogenates of cortical tissue contain high-affinity, specific binding sites for VIP as well as an adenylate cyclase system which is sensitive to this peptide. As with many of the other peptidergic systems which have been identified in the central nervous system (CNS), it has proved extremely difficult to elucidate the nature and extent of the functional role of VIP in specific brain areas. Here, using the quantitative autoradiographic 14C-deoxyglucose technique in rats to provide insight into functional processes, we describe the increases in glucose utilization which occur locally in anterior cingulate cortex following the unilateral injection of VIP (20 pmol) into this key brain area and, additionally, the focal alterations in glucose use in CNS regions having known neuronal connections with the injected region (for example, ipsilateral mediodorsal thalamus, ventral tegmental area, nucleus accumbens, caudate nucleus and contralateral cingulate cortex). These data provide evidence that VIP may modify the processing of afferent and efferent information within the anterior cingulate cortex in the conscious rat.     

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).     

Specific tricyclic antidepressant binding sites in rat brain.

The discovery of high-affinity binding sites for psychoactive drugs such as benzodiazepines, opiates and neuroleptics has opened up new approaches to the study of these drugs and their mechanisms of action. Although most tricyclic antidepressants inhibit neuronal uptake of noradrenaline and serotonin, their mechanism of action remains unclear. Changes in the sensitivity of the beta-receptor after chronic tricyclic antidepressant treatment suggest that they modulate noradrenergic neurotransmission. Tricyclic antidepressants also act directly on cholinergic, histaminergic, alpha-adrenergic and serotonergic receptors. It is not clear, however, which, if any, of these effects are related to the primary antidepressant effect or whether they are simply responsible for some of the side effects. We have thus investigated the possibility that specific binding sites for tricyclic antidepressants exist in the central nervous system. So far, binding studies using 3H-labelled tricyclic antidepressant drugs have only detected binding to histaminergic H2 and cholinergic muscarinic receptors and low-affinity binding. We demonstrate here a population of specific high-affinity binding sites for 3H-imipramine on brain membranes which may be responsible for the antidepressant effects of these drugs.     

beta-Carboline-3-carboxylic acid ethyl ester antagonizes diazepam activity

Analogous to the progression of events in the opiate receptor-enkaphalin area, the first reports that benzodiazepines have selective and specific high-affinity binding sites in brain have stimulated a search for the endogenous 'ligand' or substance that might normally act at these sites. Braestrup and co-workers have extracted from human urine a gamma-fraction (ref. 10) which they have recently identified as beta-carboline-3-carboxylic acid ethyl ester (beta CEE). They reported that this substance is extremely potent in displacing 3H-diazepam from brain binding sites and proposed that a beta-carboline-3-carboxylic acid derivative might, in part, be the endogenous ligand for the brain benzodiazepine receptor. We have examined several synthetically derived beta-carboline-3-carboxylic acid analogues and now present data obtained from testing only the beta CEE described by Braestrup et al. In addition to confirming these workers' observation that this compound is a potent displacer of 3H-diazepam from brain tissue, our pharmacological data indicate that beta CEE has activity that is opposite to, rather than similar to, that of diazepam.     

Co-localization of GABA receptors and benzodiazepine receptors in the brain shown by monoclonal antibodies

The most abundant inhibitory neurotransmitter in the central nervous system, gamma-aminobutyric acid (GABA), exerts its main effects via a GABAA receptor that gates a chloride channel in the subsynaptic membrane. These receptors can contain a modulatory unit, the benzodiazepine receptor, through which ligands of different chemical classes can increase or decrease GABAA receptor function. We have now visualized a GABAA receptor in mammalian brain using monoclonal antibodies. The protein complex recognized by the antibodies contained high- and low-affinity binding sites for GABA as well as binding sites for benzodiazepines, indicative of a GABAA receptor functionally associated with benzodiazepine receptors. As the pattern of brain immunoreactivity corresponds to the autoradiographical distribution of benzodiazepine binding sites, most benzodiazepine receptors seem to be part of GABAA receptors. Two constituent proteins were identified immunologically. Because the monoclonal antibodies cross-react with human brain, they provide a means for elucidating those CNS disorders which may be linked to a dysfunction of a GABAA receptor.     

Synaptic localization of kainic acid binding sites

The heterocyclic compound kainic acid (KA) is a potent excitant when applied to mammalian neurones. Lesions caused by injections of KA into the rat striatum and hippocampus cause similar patterns of damage to those seen in Huntington's chorea and status epilepticus, respectively. Although it was originally thought to be a glutamate agonist, it is now clear that KA does not act on the majority of the receptors for glutamate, and in fact seems to act on a class of receptors which are distinct from those which mediate responses to other excitatory amino acids. The potent and selective neurotoxic effects of this compound may be mediated by these same receptors. At present, the relative distribution of junctional and extrajunctional (non-synaptic) receptors is unknown and resolution of this issue would provide important insights into the action of KA on the central nervous system (CNS). We show here that KA binding sites are greatly enriched in isolated synaptic junctions from rat brain and, using an in vitro autoradiographic technique, we have found that these binding sites are concentrated specifically in terminal fields where KA acts as a potent neurotoxin.     

Inositol trisphosphate receptor localization in brain: variable stoichiometry with protein kinase C

Many neurotransmitters, hormones and growth factors act at membrane receptors to stimulate the phosphodiesteratic hydrolysis of phosphatidyl-inositol 4,5-bisphosphate generating the comessengers inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and diacylglycerol. Diacylglycerol stimulates protein kinase C3 while Ins(1,4,5)P3 is postulated to activate specific receptors leading to release of intracellular calcium, probably from the endoplasmic reticulum. In recent preliminary reports, Rubin and associates detected 32P-Ins(1,4,5)P3 binding to liver and adrenal microsomes and to permeabilized neutrophils and liver cells. We now report the biochemical and autoradiographic demonstration in brain of high affinity, selective binding sites for 3H- and 32P-labelled Ins(1,4,5)P3 at levels 100-300 times higher than those observed in peripheral tissues. The potencies of various myoinositol analogues at the Ins(1,4,5)P3 binding site correspond to their potencies in releasing calcium from microsomes, supporting the physiological relevance of this receptor. Brain autoradiograms demonstrate discrete, heterogeneous localization of Ins(1,4,5)P3 receptors. In some regions localizations of Ins(1,4,5)P3 receptors resemble those of protein kinase C14, while in others they differ markedly, suggesting a novel mechanism whereby the relative activity of the two limbs of the PI cycle can be differently regulated.     

Anatomical distributions of four pharmacologically distinct 3H-L-glutamate binding sites

Glutamate is thought to serve as a major excitatory neurotransmitter throughout the central nervous system (CNS); electrophysiological studies indicate that its action is mediated by multiple receptors. Four receptors have been characterized by their selective sensitivity to N-methyl-D-aspartate (NMDA), kainic acid (KA), quisqualic acid (QA) and 2-amino-4-phosphonobutyric acid (APB). Electrophysiological evidence indicates that these receptors are all present in the rat hippocampus and that the anatomically discrete synaptic fields within the hippocampus exhibit differential sensitivity to the selective excitatory amino acid agents. Thus, we have used the hippocampus as a model system to investigate possible subpopulations of 3H-L-glutamate binding sites. By using quantitative autoradiography, the pharmacological specificity of 3H-L-glutamate binding in discrete terminal fields was determined. We report here that there are at least four distinct classes of 3H-L-glutamate binding sites which differ in their anatomical distribution, pharmacological profile and regulation by ions. Two of these sites seem to correspond to the KA and NMDA receptor classes, and a third site may represent the QA receptor. The fourth binding site does not conform to present receptor classifications. None of these binding sites corresponds to the major glutamate binding site observed in biochemical studies.     

Differential localization of type I and type II benzodiazepine binding sites in substantia nigra

A number of studies have suggested the existence of multiple benzodiazepine binding sites in the brain. We have recently reported the physical separation of two apparent benzodiazepine binding site subtypes, the pharmacological properties, and distribution in tissue sections of which correspond to the putative type I and type II sites. Benzodiazepine and gamma-aminobutyric acid (GABA) receptors have been shown to interact, and lesions of the GABAergic striatonigral pathway, which lead to GABA supersensitivity, both increase the numbers of GABA binding sites and enhance GABA-stimulated benzodiazepine binding. We demonstrate here that degeneration of striatonigral fibres increases the density of putative type I benzodiazepine binding sites in the substantia nigra and decreases the density of the putative type II sites. This suggests that type I sites that increase after denervation are postsynaptic, whereas the type II sites reduced by the lesion may be localized to axons or terminals of the striatonigral pathways.     

Localization of D-2 dopamine receptors to intrinsic striatal neurones by quantitative autoradiography

Recent work with positron emission and single photon emission computed tomography has demonstrated the feasibility of studying striatal dopamine receptors in the living human brain. For the proper interpretation of these studies in normal and diseased states, the cellular localization of these receptors must be definitively established. It has been claimed, on the basis of receptor binding studies with tissue homogenates in rats, that 30-50% of striatal D-2 dopamine receptors are located on axons or terminals of the corticostriatal pathway. This finding has been incorporated into major reviews and classifications of dopamine receptors. The recent development of quantitative autoradiographic methods for diffusible ligands has facilitated the study of neurotransmitter receptors in cytoarchitechtonically intact tissue. Because this technique provides the necessary anatomic resolution that is lacking in homogenate binding studies, we have used it to re-examine the localization of striatal dopamine receptors. Here we present evidence that D-2 receptors are located exclusively on kainic acid-sensitive intrinsic neuronal elements in the striatum. We report that discrete cortical ablation does not alter 3H-spiperone binding to rat striatum and thus our results do not support the existence of D-2 dopamine receptors on the terminals of the corticostriatal pathway.     

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.     

A novel analgesic dipeptide from bovine brain is a possible Met-enkephalin releaser.

It is generally accepted that morphine exerts its analgesic effect by binding to specific opiate receptors in the brain and spinal cord. Since Hughes et al. isolated and identified two endogenous pentapeptides, Met- and Leu-enkephalin, from the brain and found that they acted as agonists at opiate receptors, alpha-, beta- and gamma-endorphins, larger peptides than enkephalins and having morphine-like activity, have been identified in either the brain or pituitary of various species. Several studies have demonstrated that enkephalins possess analgesic properties and that they are distributed in the pain-mediated pathways in the central nervous system. These findings suggest that enkephalins are important neurotransmitters or neuromodulators regulating pain transmission. We now report the isolation of a novel substance which has a Met-enkephalin releasing action. Our findings suggest the possibility of a regulating mechanism for the release of endogenous opioid peptides, especially Met-enkephalin.     

Evidence for opiate receptors on pituicytes

A hypothalamo-neurohypophyseal enkephalinergic pathway has been described and the pars nervosa of the rat pituitary contains enkephalin-like material which may coexist in vasopressin and oxytocin terminals. At the level of the pars nervosa itself, stereospecific opiate receptors with properties very similar to those of brain receptors have been described, and opiates have been shown to inhibit the release of both vasopressin and oxytocin. The location of the opiate receptors involved has been presumed to be pre-terminal on the neurosecretory fibres. Using an autoradiographic technique to visualize opiate receptors, however, we now report that destruction of the neurosecretory fibres following pituitary stalk section does not result in a significant change in the neural lobe opiate receptor population. This suggests that the opiate receptors within the neural lobe may be present on pituicytes rather than on neurosecretory fibres.     

Molecular heterogeneity of benzodiazepine receptors

Benzodiazepines exhibit reversible, stereospecific high affinity binding to mammalian brain membranes, and the respective binding sites for 3H-flunitrazepam represent pharmacologically and clinically relevant receptors for benzodiazepines. Recently it has been demonstrated that reversibly bound 3H-flunitrazepam becomes irreversibly attached to a specific membrane protein with apparent molecular weight of 50,000 when incubations are performed in the presence of UV light. Irreversible binding of 3H-flunitrazepam to this protein had pharmacological properties similar to reversible benzodiazepine receptor binding, indicating that 3H-flunitrazepam is a photoaffinity label for the benzodiazepine receptor. Using irreversible binding of 3H-flunitrazepam and subsequent electrophoretic separation of the labelled proteins in SDS-gels followed by fluorography, we found that in hippocampus and several other brain regions at least two different types of benzodiazepine receptors exist. Each seems to be associated with a gamma-aminobutyric acid (GABA) receptor.     

Identification and distribution of 5-HT3 receptors in rat brain using radioligand binding

Functional serotonin (5-hydroxytryptamine, 5-HT) receptors have been divided into three subtypes: 5-HT1-like, 5-HT2 and 5-HT3 (ref. 1). Brain binding sites have been identified for both the 5-HT1 and 5-HT2 subtypes. Receptors of the 5-HT3 type have been characterized on isolated peripheral tissue models such as the rat vagus nerve, guinea-pig ileum and isolated rabbit heart. Using these models, selective 5-HT3 receptor antagonists such as MDL 72222 (ref. 5), ICS 205-930 (ref. 6), GR38032F (ref. 7) and BRL 43694 (ref. 8) have been developed. Recently, GR38032F, MDL 72222 and ICS 205-930 have been shown to have behavioural effects in rodents and primates that undoubtedly reflect an action in the central nervous system (refs 9-11 and unpublished observations), suggesting the existence of 5-HT3 receptors in the brain. Here we report direct evidence for the existence of 5-HT3 receptors in rat brain tissue and their distribution, based on high affinity binding of the potent 5-HT3 receptor antagonist 3H-GR65630 to homogenates of rat entorhinal cortex. Selective 5-HT3 receptor antagonists and agonists inhibited binding of 3H-GR65630 with high affinities which correlated well with their actions on the rat isolated vagus nerve. Binding was differentially distributed throughout the brain with high concentrations in cortical and limbic areas.     

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).     

Identification of presynaptic serotonin autoreceptors using a new ligand: 3H-PAT

Binding studies with appropriate labelled ligands have revealed the existence of two types of serotonin (5-HT) receptor, 5-HT1 and 5-HT2, in the central nervous system of mammals. The 5-HT1 type is characterized by a higher affinity for agonists than for antagonists, whereas the 5-HT2 type binds preferentially to antagonists. However, neither of these receptor types apparently corresponds to the presynaptic autoreceptor controlling 5-HT release. In an attempt to identify the presynaptic autoreceptor directly, we synthesized the tritiated derivative of 8-hydroxy-2-(di-n-propylamino) tetralin (PAT), a new tetralin derivative with potent 5-HT agonist properties and carried out binding studies with rat brain membranes. As we report here, in the hippocampus, the properties of 3H-PAT binding sites correspond closely to those of 5-HT1 sites. In contrast, in the striatum, 3H-PAT binding sites exhibit a subcellular distribution and pharmacological characteristics usually associated with presynaptic autoreceptors. Furthermore, a marked loss of 3H-PAT binding sites occurs in the striatum (but not in the hippocampus) after the selective degeneration of serotoninergic fibres in 5,7-hydroxytryptamine (5,7-HT)-treated rats. Conversely, the sprouting of additional 5-HT terminals in the brain stem of adult rats treated at birth with 5,7-HT is associated with an increased density of 3H-PAT binding sites in this region. 3H-PAT thus seems to be a useful ligand for studying the biochemical and pharmacological characteristics of presynaptic autoreceptors in selected regions of rat brain.     

Substance P-immunoreactive retinal ganglion cells and their central axon terminals in the rabbit

Retinal ganglion cells are the projection neurons that link the retina to the brain. Peptide immunoreactive cells in the ganglion cell layer (GCL) of the mammalian retina have been noted but their identity has not been determined. We now report that, in the rabbit, 25-35% of all retinal ganglion cells contain substance P-like (SP) immunoreactivity. They were identified by either retrograde transport of fluorescent tracers injected into the superior colliculus, or by retrograde degeneration after optic nerve section. SP immunoreactive cells are present in all parts of the retina and have medium to large cell bodies with dendrites that ramify extensively in the proximal inner plexiform layer. Their axons terminate in the dorsal lateral geniculate nucleus, superior colliculus and accessory optic nuclei, and these terminals disappear completely after contralateral optic nerve section and/or eye enucleation. In the dorsal lateral geniculate nucleus large, beaded, immunoreactive axons and varicosities make up a narrow plexus just below the optic tract, where they define a new geniculate lamina. The varicosities make multiple synaptic contacts with dendrites of dorsal lateral geniculate nucleus projection neurons and presumptive interneurons in complex glomerular neuropil. This is direct evidence that some mammalian retinal ganglion cells contain substance P-like peptides and strongly suggests that, in the rabbit, substance P (or related tachykinins) may be a transmitter or modulator in a specific population or populations of retinal ganglion cells.     

Neuropeptides modulate the beta-adrenergic response of purified astrocytes in vitro

Neuropeptides may have functions in the central nervous system (CNS) other than altering neuronal excitability. For example, they may act as regulators of brain metabolism by affecting glycogenolysis. Since it has been suggested that glial cells might provide metabolic support for neuronal activity, they may well be one of the targets for neuropeptide regulation of metabolism. Consistent with this view are reports that peptide-containing nerve terminals have been seen apposed to astrocytes, but it is also quite possible that peptides could act at sites lacking morphological specialization. Primary cultures containing CNS glial cells have been shown to respond to beta-adrenergic agonists with an increase in cyclic AMP and, as a result, with an increase in glycogenolysis and have also been shown to respond to a variety of peptides with changes in cyclic AMP. In the study reported here, we have examined the effects of several peptides on relatively pure cultures of rat astrocytes. We demonstrate that the increase in intracellular cyclic AMP induced by noradrenaline is markedly enhanced by somatostatin and substance P and is inhibited by enkephalin, even though these peptides on their own have little or no effect on the basal levels of cyclic AMP. Vasoactive intestinal peptide (VIP) on the other hand increases cyclic AMP in the absence of noradrenaline. These results suggest that neuropeptides influence glial cells as well as neurones in the CNS and, in the case of somatostatin and substance P, provide further examples of neuropeptides modulating the response to another chemical signal without having a detectable action on their own.