Met- and Leu-enkephalin immunoreactivity in separate neurones.

A pair of pentapeptides, Met- and Leu-enkephalin were recently isolated from brain tissue. The two peptides seem to represent endogenous opiate receptor ligands and have by immunocytochemical and radioimmunoassay studies been shown to occur in an extensive system of cerebral and peripheral nerves. The relative proportions between Met- and Leu-enkephalin varies between different brain regions and also between different species, suggesting the existence of separate populations of Met- and Leu-enkephalin nerves. Until now, however, immunocytochemistry has given no support for this notion. We report here evidence of separate populations of Met- and Leu-enkephalin nerves.     

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.     

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.     

Localization of dystrophin to postsynaptic regions of central nervous system cortical neurons

Moderate non-progressive cognitive impairment is a consistent feature of Duchenne muscular dystrophy (DMD), although no central nervous system (CNS) abnormality has been identified. Recent studies have elucidated the molecular defect in DMD, including the absence of the protein dystrophin in affected individuals. Normal brain tissue contains dystrophin messenger RNA and dystrophin is present in low abundance in the brain and seems to be regulated in this tissue, at least in part, by a promoter that differs from that in muscle. Until now, antibodies and immunocytochemical methods used to demonstrate dystrophin at the plasma membrane of mouse and human muscle have proven inadequate to localize precisely dystrophin in the mammalian CNS. We have now made an antibody (anti 6-10) which is much more sensitive than those previously available to immunolabel dystrophin in the CNS. Using this antibody, we found that in the mouse, dystrophin is particularly abundant in the neurons of the cerebral and cerebellar cortices, and that it is localized at postsynaptic membrane specializations. Dystrophin may have a different role in neurons than in muscle, and an alteration at the synaptic level may be the basis of the cognitive impairment in DMD.     

Neurotransmitter turnover in rat striatum is correlated with morphine self-administration

Drugs of abuse probably exert their reinforcing effects through 'reward' pathways in the central nervous system (CNS). Neuronal systems mediating opiate reinforcement have been investigated using pharmacological and electrolytic lesion procedures. Drugs that interfere with catecholaminergic and cholinergic neuronal activity decrease intravenous (i.v.) morphine self-administration in monkeys and rats. Electrolytic lesion procedures in rats have demonstrated that the medial forebrain bundle and caudate nucleus are important in maintaining i.v. morphine self-administration. We have now carried out a direct investigation of striatal (caudate nucleus, putamen and globus pallidus) neuronal systems. We show here that striatal catecholaminergic systems are important in mediating opiate reinforcement, and present direct evidence for the involvement of neurotransmitter systems in morphine reward.     

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).     

Naloxone augments electrophysiological signs of selective attention in man

Previous research on the behavioural functions of endogenous opioid systems in rodents suggested a possible opioid role in the regulation of attention. This proposal was consistent with reports that opiate administration in man impairs the ability to concentrate while opiate antagonists augment behavioural and electrophysiological indices of arousal and attention. We examined the effects of the opiate antagonist naloxone on electrophysiological measures of attention in normal human subjects, using a paradigm which dissociates selective information processing from concurrent processes of general arousal or alertness that may be present. We now report electrophysiological evidence that naloxone improves the selectivity of auditory attention in the presence of competing sources of stimuli. These findings indicate a role for the endogenous opioid systems in the regulation of selective attention in man.     

Coffee contains potent opiate receptor binding activity

Opiate receptor-active peptide fragments (exorphins) have been identified recently in casein and gluten hydrolysates, and morphine has been found in bovine and human milk. To determine whether similar peptides or alkaloids occur in other foodstuffs, we have screened potential sources using a rat brain homogenate assay to detect opiate receptor activity. We report here that instant coffee powders from a variety of manufacturers compete with tritiated naloxone for binding to opiate receptors in the rat brain membrane preparations, with no significant difference between normal and decaffeinated coffee. The receptor binding activity resembles that seen with opiate antagonists, in that there was no change in the half-maximal effective dose (ED50) in the presence of 100 mM Na+; on bioassay, the activity was similarly shown to be antagonistic and specific for opiate-induced inhibition of twitch. Preliminary characterization of the activity reveals that it has a molecular weight (MW) in the range 1,000-3,500, is heat-stable, ether-extractable, not modified by enzymatic digestion with papain, and clearly separable from caffeine and morphine on TLC. As its concentration in an average cup of coffee is five times the ED50, these data suggest that drinking coffee may be followed by effects mediated via opiate receptors, as well as effects of caffeine.     

Enkephalins presynaptically inhibit cholinergic transmission in sympathetic ganglia.

Recent biochemical and immunohistochemical studies have shown that the opioid peptides, enkephalins, occur in nerve terminals and cell bodies in mammalian sympathetic ganglia1-3. Opiates and enkephalins are thought to inhibit synaptic transmission in the peripheral nervous tissues as well as in the central nervous system4-12. The mechanisms of the opiate actions, however, are not entirely clear; both pre- and postsynaptic sites of action have been proposed7-9,11,12. As acetylcholine is known to be the major neurotransmitter in the autonomic ganglia and as the mechanism of synaptic transmission is well clarified13, analysis of the peptide action could be more easily but equally usefully carried out in the peripheral synapses than in central synapses. We now report that enkephalins presynaptically inhibit cholinergic transmission in sympathetic ganglia.     

Benzodiazepines antagonize cholecystokinin-induced activation of rat hippocampal neurones

Cholecystokinin (CCK) is a neuropeptide present in the mammalian central nervous system (CNS). In all species studied so far, the highest concentrations of this neuropeptide have been found in the cerebral cortex, the amygdala and the hippocampus. Five molecular forms of CCK having 39, 33, 13, 8 and 4 amino acid residues have been identified in the CNS, the sulphated octapeptide (CCK8) being the most abundant form detected. Specific CCK binding sites have been demonstrated in the rat, guinea pig and human brain. CCK8, applied by microiontophoresis to deep cortical neurones and hippocampal pyramidal neurones, has a powerful excitatory effect, whereas the non-sulphated CCK octapeptide has no such effect on these neurones. Low doses of benzodiazepines depress the spontaneous activity of hippocampal pyramidal neurones. We report here that benzodiazepines at very low doses antagonize selectively the CCK8-induced activation of rat hippocampal pyramidal neurones. This antagonistic action might be involved in the anxiolytic effect of these drugs.     

Opposite motivational effects of endogenous opioids in brain and periphery

Many psychoactive drugs, including the opiates, have been shown to have paradoxical reinforcing effects. Opiates produce positive reinforcing effects when they are paired with visual and textural environmental stimuli in rats, yet, at similar doses and over the same routes of administration, produce aversive effects, as shown when they are paired with taste stimuli. Similarly, in human, the positive reinforcing effects of opiates are well known to addicts and recreational drug users, yet patients receiving opiates as analgesics often report nauseous reactions. At present there is no evidence to differentiate between the neural substrates that mediate these opposite motivational effects. We now report an initial step in the resolution of this paradox by demonstrating that endogenous and exogenous opioids produce positive reinforcing effects through an action on brain opiate receptors, and aversive effects through an action on peripheral opiate receptors (especially in the gut).     

Noradrenaline in the ventral forebrain is critical for opiate withdrawal-induced aversion

Cessation of drug use in chronic opiate abusers produces a severe withdrawal syndrome that is highly aversive, and avoidance of withdrawal or associated stimuli is a major factor contributing to opiate abuse. Increased noradrenaline in the brain has long been implicated in opiate withdrawal, but it has not been clear which noradrenergic systems are involved. Here we show that microinjection of beta-noradrenergic-receptor antagonists, or of an alpha2-receptor agonist, into the bed nucleus of the stria terminalis (BNST) in rats markedly attenuates opiate-withdrawal-induced conditioned place aversion. Immunohistochemical studies revealed that numerous BNST-projecting cells in the A1 and A2 noradrenergic cell groups of the caudal medulla were activated during withdrawal. Lesion of these ascending medullary projections also greatly reduced opiate-withdrawal-induced place aversion, whereas lesion of locus coeruleus noradrenergic projections had no effect on opiate-withdrawal behaviour. We conclude that noradrenergic inputs to the BNST from the caudal medulla are critically involved in the aversiveness of opiate withdrawal.     

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.     

Synaptosomes possess an exocytotic pool of glutamate

There is increasing evidence that L-glutamate is a major excitatory neurotransmitter in the central nervous system. Immunocytochemical studies indicate that glutamate within nerve terminals may be concentrated in vesicles and glutamate-accumulating vesicles have recently been isolated. Exocytotic release of glutamate from synaptosomes (isolated nerve terminals) has not been convincingly demonstrated, however, and remains highly controversial. In order to study the kinetics of release of endogenous L-glutamate from guinea pig cerebral cortical synaptosomes we have devised a continuous enzymatic assay. This has enabled us to identify a pool, equivalent to 15-20% of the total synaptosomal glutamate, which is capable of rapid Ca2+-dependent exocytotic release.     

Aspartate and glutamate as possible neurotransmitters of cells in layer 6 of the visual cortex

Earlier work has suggested that aspartate, glutamate and gamma-aminobutyric acid (GABA) act as transmitters in the cerebral cortex. There is reasonable evidence for the identity of the cell population responsible for GABA release but until now there has been little evidence concerning the sources for release of aspartate and glutamate. Here we have used two approaches to identify possible neurotransmitters used by cells in the visual cortex: measurement of the efflux of endogenous compounds in conditions of synaptic release and localization of these compounds to particular cell classes using neurotransmitter-specific histochemical techniques. Our results suggest that the acidic amino acids aspartate and glutamate may be cortical neurotransmitters, as shown by calcium-dependent release from endogenous stores and by uptake specific to pyramidal cells in layer 6 of the cortex. These substances may therefore have a role in the function of layer 6 cells, which are responsible for the recurrent projection from the cortex to the lateral geniculate nucleus and for the projection within the cortex from layer 6 to layer 4.     

Met-enkephalin circulates in human plasma

The physiological roles of Met-enkephalin and Leu-enkephalin are still unknown. They may act as neurotransmitters in the central and peripheral nervous systems. Met-enkephalin has been detected in several species in a variety of tissues including brain, spinal cord and gut using bioassays, opiate receptor assays and radioimmunoassays (RIA). It has also been detected in human gut immunocytochemically and in human brain and cerebrospinal fluid by opiate receptor assay and RIA. However, all reported assays show some degree of cross-reaction with Leu-enkephalin and unequivocal differentiation between the two enkephalins and the larger endorphins has not always been possible. Thus the existence of Met-enkephalin in human tissues and fluids remains in doubt. Using a highly specific RIA, we have now obtained evidence that Met-enkephalin-like material circulates in the plasma of normal subjects and may be secreted by the adrenal gland. Chromatographically the material exists in plasma mainly as the intact pentapeptide and not as the biologically inactive degradation product Gly-Gly-Phe-Met as would be expected from metabolic studies.     

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.     

Pericytes are required for blood-brain barrier integrity during embryogenesis

Vascular endothelial cells in the central nervous system (CNS) form a barrier that restricts the movement of molecules and ions between the blood and the brain. This blood-brain barrier (BBB) is crucial to ensure proper neuronal function and protect the CNS from injury and disease. Transplantation studies have demonstrated that the BBB is not intrinsic to the endothelial cells, but is induced by interactions with the neural cells. Owing to the close spatial relationship between astrocytes and endothelial cells, it has been hypothesized that astrocytes induce this critical barrier postnatally, but the timing of BBB formation has been controversial. Here we demonstrate that the barrier is formed during embryogenesis as endothelial cells invade the CNS and pericytes are recruited to the nascent vessels, over a week before astrocyte generation. Analysing mice with null and hypomorphic alleles of Pdgfrb, which have defects in pericyte generation, we demonstrate that pericytes are necessary for the formation of the BBB, and that absolute pericyte coverage determines relative vascular permeability. We demonstrate that pericytes regulate functional aspects of the BBB, including the formation of tight junctions and vesicle trafficking in CNS endothelial cells. Pericytes do not induce BBB-specific gene expression in CNS endothelial cells, but inhibit the expression of molecules that increase vascular permeability and CNS immune cell infiltration. These data indicate that pericyte-endothelial cell interactions are critical to regulate the BBB during development, and disruption of these interactions may lead to BBB dysfunction and neuroinflammation during CNS injury and disease.