Short- and long-term consequences of prenatal exposure to the cannabinoid agonist WIN55,212-2 on rat glutamate transmission and cognitive functions.

The aim of the present review is to summarize integrated neurochemical, morphological and neurobehavioral evidence, in particular from our laboratory, which emphasize the short- and long-term consequences of prenatal exposure to the cannabinoid receptor agonist WIN55,212-2 on rat glutamate transmission and cognitive functions. The results obtained provide evidence that maternal exposure to WIN55,212-2 induces an impairment of cognitive capacities in the offspring. This impairment is associated with alterations of cortical and hippocampal glutamate outflow, cortical neuron morphology and hippocampal long-term potentiation. These findings are in line with clinical data showing that the consumption of marijuana by women during pregnancy has negative consequences on the cognitive functions of their children. Thus, although it is difficult and sometimes misleading to extrapolate findings obtained from animal models to humans, the possibility that an alteration of glutamate transmission might underlie, at least in part, some of the cognitive deficits affecting the offspring of marijuana users, is supported.

Neurotensin receptor mechanisms and its modulation of glutamate transmission in the brain: relevance for neurodegenerative diseases and their treatment.

The extracellular accumulation of glutamate and the excessive activation of glutamate receptors, in particular N-methyl-D-aspartate (NMDA) receptors, have been postulated to contribute to the neuronal cell death associated with chronic neurodegenerative disorders such as Parkinson's disease. Findings are reviewed indicating that the tridecaptide neurotensin (NT) via activation of NT receptor subtype 1 (NTS1) promotes and reinforces endogenous glutamate signalling in discrete brain regions. The increase of striatal, nigral and cortical glutamate outflow by NT and the enhancement of NMDA receptor function by a NTS1/NMDA interaction that involves the activation of protein kinase C may favour the depolarization of NTS1 containing neurons and the entry of calcium. These results strengthen the hypothesis that NT may be involved in the amplification of glutamate-induced neurotoxicity in mesencephalic dopamine and cortical neurons. The mechanisms involved may include also antagonistic NTS1/D2 interactions in the cortico-striatal glutamate terminals and in the nigral DA cell bodies and dendrites as well as in the nigro-striatal DA terminals. The possible increase in NT levels in the basal ganglia under pathological conditions leading to the NTS1 enhancement of glutamate signalling may contribute to the neurodegeneration of the nigro-striatal dopaminergic neurons found in Parkinson's disease, especially in view of the high density of NTS1 receptors in these neurons. The use of selective NTS1 antagonists together with conventional drug treatments could provide a novel therapeutic approach for treatment of Parkinson's disease.

Receptor-receptor interactions within receptor mosaics. Impact on neuropsychopharmacology.

Future therapies for diseases associated with altered dopaminergic signaling, including Parkinson's disease, schizophrenia and drug addiction or drug dependence may substantially build on the existence of intramembrane receptor-receptor interactions within dopamine receptor containing receptor mosaics (RM; dimeric or high-order receptor oligomers) where it is believed that the dopamine D(2) receptor may operate as the 'hub receptor' within these complexes. The constitutive adenosine A(2A)/dopamine D(2) RM, located in the dorsal striato-pallidal GABA neurons, are of particular interest in view of the demonstrated antagonistic A(2A)/D(2) interaction within these heteromers; an interaction that led to the suggestion and later demonstration that A(2A) antagonists could be used as novel anti-Parkinsonian drugs. Based on the likely existence of A(2A)/D(2)/mGluR5 RM located both extrasynaptically on striato-pallidal GABA neurons and on cortico-striatal glutamate terminals, multiple receptor-receptor interactions within this RM involving synergism between A(2A)/mGluR5 to counteract D(2) signaling, has led to the proposal of using combined mGluR5 and A(2A) antagonists as a future anti-Parkinsonian treatment. Based on the same RM in the ventral striato-pallidal GABA pathways, novel strategies for the treatment of schizophrenia, building on the idea that A(2A) agonists and/or mGluR5 agonists will help reduce the increased dopaminergic signaling associated with this disease, have been suggested. Such treatment may ensure the proper glutamatergic drive from the mediodorsal thalamic nucleus to the prefrontal cortex, one which is believed to be reduced in schizophrenia due to a dominance of D(2)-like signaling in the ventral striatum. Recently, A(2A) receptors also have been shown to counteract the locomotor and sensitizing actions of cocaine and increases in A(2A) receptors have also been observed in the nucleus accumbens after extended cocaine self-administration, probably representing a compensatory up-regulation to counteract the cocaine-induced increases in dopamine D(2) and D(3) signaling. Therefore, A(2A) agonists, through antagonizing D(2) and D(3) signaling within A(2A)/D(2) and A(2A)/D(3) RM heteromers in the nucleus accumbens, may be found useful as a treatment for cocaine dependence. Furthermore, antagonistic cannabinoid CB(1)/D(2) interactions requiring A(2A) receptors have also been discovered and possibly operate in CB(1)/D(2)/A(2A) RM located principally on striatal glutamate terminals but also on some ventral striato-pallidal GABA neurons, thereby opening up a new mechanism for the integration of endocannabinoid, DA and adenosine mediated signals. Thus, A(2A), mGluR5 and/or CB(1) receptors can form integrative units with D(2) receptors within RM displaying different compositions, topography and localization. Also galaninR/5-HT(1A) RM probably participates in the transmission of the ascending 5-hydroxytryptamine neurons, where galanin receptors antagonize 5-HT(1A) recognition and signaling. Subtype specific galanin receptor antagonists may therefore represent novel antidepressant drugs. These results suggest the importance of a complete understanding of the function of these RM with regard to disease. Ultimately receptor-receptor interactions within RM that modify dopaminergic and serotonergic signaling may give new strategies for treatment of a wide range of diseases associated with altered dopaminergic and serotonergic signaling.

Prenatal exposure to the cannabinoid receptor agonist WIN 55,212-2 and carbon monoxide reduces extracellular glutamate levels in primary rat cerebral cortex cell cultures.

The effects of prenatal exposure to the cannabinoid receptor agonist WIN 55,212-2 (0.5 mg/kg s.c.), alone or in combination with carbon monoxide, on extracellular glutamate levels in primary rat cerebral cortical neuronal cultures, were investigated. Dam weight gain, pregnancy length and litter size at birth were not affected by prenatal treatment with WIN 55,212-2 and carbon monoxide alone or in combination. Basal and K(+)-evoked extracellular glutamate levels were reduced in cortical cultures from pups born to mothers exposed to WIN 55,212-2 and carbon monoxide alone or in combination compared to cultures from rats born to vehicle-treated mothers. In cultures obtained from rats exposed to vehicle or carbon monoxide alone during gestation, WIN 55,212-2 (0.01-100 nM) increased extracellular glutamate levels, displaying a bell-shaped concentration-response curve. In cultures from rats born to mothers exposed to WIN 55,212-2 alone or in combination with carbon monoxide the WIN 55,212-2 ( 1 nM)-induced increase in extracellular glutamate levels was lower than that observed in cultures from rats born to vehicle-treated mothers and similar at those observed at 10 and 100 nM concentrations. The selective CB1 receptor antagonist SR141716A (10 nM) counteracted the WIN 55,212-2-induced increase in extracellular glutamate levels in cultures exposed to vehicle or carbon monoxide during gestation, but failed to antagonise it in cultures from rats born to mothers exposed to WIN 55,212-2 alone or in combination with carbon monoxide. These findings provide evidence that prenatal exposure to the cannabinoid receptor agonist WIN 55,212-2 and carbon monoxide, alone or in combination, is associated with an impairment in cortical glutamatergic transmission. It could be speculated that such detrimental effects might be involved in the reported deficit in learning and memory associated with prenatal marijuana exposure.

The vigilance promoting drug modafinil increases extracellular glutamate levels in the medial preoptic area and the posterior hypothalamus of the conscious rat: prevention by local GABAA receptor blockade.

The effects of modafinil on glutamatergic and GABAergic transmission in the rat medial preoptic area (MPA) and posterior hypothalamus (PH), are analysed. Modafinil (30-300 mg/kg) increased glutamate and decreased GABA levels in the MPA and PH. Local perfusion with the GABAA agonist muscimol (10 microM), reduced, while the GABAA antagonist bicuculline (1 microM and 10 microM) increased glutamate levels. The modafinil (100 mg/kg)-induced increase of glutamate levels was antagonized by local perfusion with bicuculline (1 microM). When glutamate levels were increased by the local perfusion with the glutamate uptake inhibitor L-trans-PDC (0.5 mM), modafinil produced an additional enhancement of glutamate levels. Modafinil (1-33 microM) failed to affect [3H]glutamate uptake in hypothalamic synaptosomes and slices. These findings show that modafinil increases glutamate and decreases GABA levels in MPA and PH. The evidence that bicuculline counteracts the modafinil-induced increase of glutamate levels strengthens the evidence for an inhibitory GABA/glutamate interaction in the above regions controlling the sleep-wakefulness cycle.

The modulation of cortical acetylcholine release by GABA, GABA-like drugs and benzodiazepines in freely moving guinea-pigs.

In order to define the modulatory role played by gamma-aminobutyric acid (GABA) in corticopetal cholinergic projections, the effect of this amino acid and related drugs on gross behaviour, the EEG and the release of acetylcholine (ACh) from the cerebral cortex in freely moving guinea-pigs was studied. gamma Aminobutyric acid, injected intracerebroventricularly (20-50 mumol) induced a three-phase picture: first (5-15 min) behavioural activation and increased release of ACh, then (30-90 min) depression, EEG synchronization and reduced release of ACh, and finally "rebound" stimulation. Ethanolamine-O-sulphate (EOS) injected intraventricularly (28 mumol/kg) or intraperitoneally (14 mmol/kg) reproduced the first two phases of the effects of GABA (i.e. stimulation followed by inhibition), while diazepam (0.7 and 3.5 mumol/kg, i.p.) and flurazepam (32 mumol/kg, i.p.) caused, at first, only depression. Muscimol and 4,5,6,7-tetrahydroisoxazolo(4,5-c)pyridine-3-ol (THIP) injected intraventricularly (in the nmol range) or intraperitoneally (in the mumol range) produced behavioural activation and increased release of ACh; the depressant signs appeared only after very large, toxic doses. Picrotoxin and bicuculline, at sub-convulsive doses, reduced the symptomatology caused by GABA and antagonized the sedation produced by diazepam. Methysergide (8-16 mumol/kg, i.p.) prevented the behavioural activation and the increased release of ACh by GABA, unmasked the depression due to subthreshold doses of diazepam (i.c.v., 7-70 nmol) and reversed the stimulation induced by muscimol into sedation and reduced the outflow of ACh. Pretreatment with 5,7-HT also dampened and shortened the stimulation by muscimol.(ABSTRACT TRUNCATED AT 250 WORDS)

Amplification of cortical serotonin release: a further neurochemical action of the vigilance-promoting drug modafinil.

The present in vitro and in vivo studies examined the effects of modafinil on serotonergic transmission in the rat frontal cortex. In the in vitro study modafinil (0.3-30 microM) increased electrically-evoked, but not spontaneous, serotonin ([(3)H]5-HT) efflux from cortical slices in a concentration-dependent manner while the indirect serotonin agonist dl-fenfluramine (1-15 microM) enhanced both spontaneous and evoked [(3)H]5-HT efflux. The effects of modafinil were more pronounced when the 5-HT reuptake was blocked by paroxetine. Contrary to paroxetine (0.3-3 microM) and dl-fenfluramine (1-5 microM), modafinil failed to influence the [(3)H]5-HT uptake. In the in vivo study modafinil (3-100 mg/kg i.p.) increased 5-HT dialysate levels, the maximal effect being already reached at the 30 mg/kg dose. dl-fenfluramine (5 mg/kg) induced an increase in 5-HT levels which was significantly higher than that displayed by modafinil at 30 mg/kg. In the presence of paroxetine (3 microM), the effect of modafinil at 30 mg/kg was higher than that observed in the absence of 5-HT reuptake inhibition. Finally, in the presence of the selective 5-HT(1A) receptor agonist 8-OH-DPAT, modafinil at 100 mg/kg failed to affect 5-HT dialysate levels. These results demonstrate that modafinil regulates cortical serotonergic transmission and suggest that the drug preferentially acts by amplifying the electro-neurosecretory coupling mechanisms and via mechanisms which do not involve the reuptake process.

Facilitation of GABA release by neurotensin is associated with a reduction of dopamine release in rat nucleus accumbens.

The main aim of the present study was to investigate the effects of local perfusion with the tridecapeptide neurotensin on extracellular GABA and dopamine levels in the nucleus accumbens of the halothane-anaesthetized rat, using in vivo microdialysis. In an initial set of characterization studies we examined the Na+ dependence of neurotransmitter release by local perfusion with ouabain, veratridine and tetrodotoxin. Local perfusion with the Na+ ATPase inhibitor ouabain (10 microM) or the Na+ channel agonist veratridine (20 microM) perfused into the nucleus accumbens increased both extracellular GABA and dopamine levels. The Na+ channel antagonist tetrodotoxin (1 microM) consistently decreased (24% of basal) dopamine levels, while even at 10 microM it did not affect GABA. However, tetrodotoxin (10 microM) abolished the veratridine-induced increase in both GABA and dopamine, demonstrating that Na(+)-dependent neuronal activity is involved in this release mechanism. In a second set of experiments a hypothesis for a functional link between neurotensin, dopamine and GABA in the medial nucleus accumbens was tested. Towards this aim, the effects of local perfusion with a high 1 microM concentration of neurotensin into the nucleus accumbens increased both GABA (210% of basal value) and dopamine (145% of basal) release. However, a low (10 nM) concentration of neurotensin again increased GABA release (160% of basal), but decreased that of dopamine (75% of basal value). Furthermore, the local perfusion with the GABAA receptor antagonist bicuculline abolished the neurotensin (10 nM) induced inhibition of dopamine release without affecting the increase in GABA release. These findings suggest that neurotensin modulates both GABA and dopamine neurotransmission in the nucleus accumbens.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term effects on cortical glutamate release induced by prenatal exposure to the cannabinoid receptor agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinyl-methyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone: an in vivo microdialysis study in the awake rat.

The aim of the present in vivo microdialysis study was to investigate whether prenatal exposure to the CB(1) receptor agonist WIN55,212-2 mesylate (WIN; (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinyl-methyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone), at a dose of 0.5 mg/kg (s.c. from the fifth to the 20th day of gestation), that causes neither malformations nor overt signs of toxicity, influences cortical glutamate extracellular levels in adult (90-day old) rats. Dam weight gain, pregnancy length and litter size at birth were not significantly affected by prenatal treatment with WIN. Basal and K(+)-evoked dialysate glutamate levels were lower in the cerebral cortex of adult rats exposed to WIN during gestation than in those born from vehicle-treated mothers. In both group of animals WIN (0.1 mg/kg, i.p.) increased dialysate glutamate levels. However, while the blockade of the CB1 receptors with the selective receptor antagonist SR141716A completely counteracted the WIN-induced increase in those rats exposed to vehicle during gestation, it failed to antagonise the increase in those born from WIN-treated dams. These findings suggest that prenatal exposure to the CB1 receptor agonist WIN, at a concentration which is not associated with gross malformations and/or overt signs of toxicity, induces permanent alterations in cortical glutamatergic function. The possibility that these effects might underlie, at least in part, some of the cognitive deficits affecting the offspring of marijuana users is discussed.

Evidence for a differential cholecystokinin-B and -A receptor regulation of GABA release in the rat nucleus accumbens mediated via dopaminergic and cholinergic mechanisms.

In the present study we characterized the cholecystokinin receptor regulation of (i) the dopamine D2 agonist binding sites in striatal sections including the nucleus accumbens and (ii) GABA and dopamine release in the central part of the rat nucleus accumbens, by combining the in vitro filter wipe-off and the in vivo microdialysis techniques. In the binding study we demonstrate that sulphated cholecystokinin octapeptide (1 nM) increased (219 +/- 30%) the KD value of the D2 agonist [3H]N-propylnorapomorphine binding sites in sections from the striatum including the accumbens. This effect was counteracted by the cholecystokinin-B antagonist PD134308 (50 nM). In a parallel study using microdialysis in the central nucleus accumbens, we found that local perfusion with sulphated cholecystokinin octapeptide (1 microM) induced an increase in GABA (135 +/- 7%) and dopamine (146 +/- 8%) release which was unaffected by the cholecystokinin-A antagonist L-364,718 (10 nM). In contrast, when the cholecystokinin-B antagonist PD134308 (10 nM) was co-perfused with the peptide it prevented the increase in dopamine and decreased GABA release (-24 +/- 2%). This reduction was counteracted by the addition to the perfusate medium of the cholecystokinin-A antagonist or the cholinergic muscarinic M2 receptor antagonist AF-DX 116 (0.1 microM). Taken together, these data demonstrate that the facilitation by sulphated cholecystokinin octapeptide of GABA and dopamine release in the central accumbens probably reflects an inhibitory effect of the peptide on both pre- and postsynaptic D2 receptors, mediated via cholecystokinin-B receptor activation. In addition, for the first time we provide evidence for a differential cholecystokinin-A and -B receptor-mediated regulation of GABA transmission in the central accumbens, where the cholecystokinin-B receptor exerts a dominant excitatory influence while the cholecystokinin-A receptor mediates an inhibition of GABA release via a local muscarinic M2 receptor.

Nigral neurotensin receptor regulation of nigral glutamate and nigroventral thalamic GABA transmission: a dual-probe microdialysis study in intact conscious rat brain.

Dual-probe microdialysis in the awake rat was employed to investigate the effects of intranigral perfusion with the tridecapeptide neurotensin on local dialysate glutamate and GABA levels in the substantia nigra pars reticulata and on dialysate GABA levels in the ventral thalamus. Intranigral neurotensin (10-300nM, 60min) dose-dependently increased (+29+/-3% and +46+/-3% vs basal for the 100 and 300nM concentrations, respectively) local dialysate glutamate levels, while the highest 300nM concentration of the peptide exerted a long-lasting and prolonged reduction in both local and ventral thalamic (-20+/-4% and -22+/-2%, respectively) GABA levels. Intranigral perfusion with the inactive neurotensin fragment neurotensin(1-7) (10-300nM, 60min) was without effect. Furthermore, the non-peptide neurotensin receptor antagonist SR 48692 (0.2mg/kg) and tetrodotoxin (1microM) fully counteracted the intranigral neurotensin (300nM)-induced increase in local glutamate. SR 48692 (0.2mg/kg) also counteracted the decreases in nigral and ventral thalamic GABA release induced by the peptide. In addition, intranigral perfusion with the dopamine D(2) receptor antagonist raclopride (1microM) fully antagonized the neurotensin (300nM)-induced decreases in nigral and ventral thalamic GABA levels. The ability of nigral neurotensin receptor activation to differently influence glutamate and GABA levels, whereby it increases nigral glutamate and decreases both nigral and ventral thalamic GABA levels, suggests the involvement of neurotensin receptor in the regulation of basal ganglia output at the level of the nigra.

Glycine-induced changes in acetylcholine release from guinea-pig brain slices.

The effect of glycine (Gly) on acetylcholine (ACh) release from superfused, resting or electrically-stimulated slices of guinea-pig caudate nucleus (CN), brain stem (BS) and cerebral cortex (CC) was studied. The amino acid 1 X 10(-4)-6 X 10(-3)mol 1(-1) reduced the electrically-induced release and increased the spontaneous and KCl-evoked transmitter outflow, mostly in CN but also in BS, whereas it was ineffective in CC. Taurine, chosen as a structurally related compound, moderately affected only the spontaneous release in CN. Strychnine 2 X 10(-7) mol 1(-1) was per se ineffective, but prevented most Gly effects. The Gly-induced increase of ACh outflow in resting CN slices, however, could be completely antagonized only by administering strychnine and picrotoxin together. These findings suggest that: (i) the overall pattern of Gly influence on cholinergic function is similar to that previously described for gamma-aminobutyric acid (GABA); (ii) specific receptors seem to be present in BS and, above all, in CN; (iii) a positive cooperation between endogenous GABA and Gly is evident in resting CN slices; (iv) the absence of any apparent endogenous glycinergic control on the cholinergic neurones casts doubt on but does not exclude the existence of glycinergic neurones in CN.

Cortical acetylcholine release is increased and gamma-aminobutyric acid outflow is reduced during morphine withdrawal.

The effects of naloxone on acetylcholine (ACh) and gamma-aminobutyric acid (GABA) outflow from the cerebral cortex of freely moving, morphine-dependent guinea-pigs was studied. The cortical efflux of ACh in chronically-treated guinea-pigs was about half of that of normal animals. GABA efflux was unaffected. During opioid withdrawal precipitated by naloxone (0.1-10 mg kg-1, i.p.) the guinea-pigs showed jumping, hyperactivity and wet dog shakes, the intensity of which was directly related to the dose of naloxone. The withdrawal syndrome was accompanied by a dose-dependent increase in ACh release and reduction in GABA outflow; ACh release was increased by naloxone at doses lower (0.1-3 mg kg-1) than those acting on GABA efflux (1-10 mg kg-1). Atropine (10 mg kg-1) and diazepam (5 mg kg-1) did not prevent GABA and ACh changes.

5-Hydroxytryptamine-mediated effects of nicotine on endogenous GABA efflux from guinea-pig cortical slices.

1. The effect of nicotine on endogenous basal GABA outflow was studied in guinea-pig cerebral cortex slices. 2. Nicotine 1.86-18.6 mumol l-1 significantly decreased the basal, tetrodotoxin-sensitive GABA efflux, whereas at higher concentrations (186-620 mumol l-1) nicotine increased it. The inhibition was prevented by mecamylamine while the facilitation was blocked by mecamylamine, (+)-tubocurarine and tetrodotoxin. 3. The effect of nicotine was due to an indirect 5-hydroxytryptaminergic action. In fact, MDL 72222 (1 mumol l-1) completely prevented the alkaloid inhibition and methysergide (1 mumol l-1) reversed the facilitation into inhibition; concomitant treatment with methysergide and MDL 72222 antagonized the effect of nicotine at 186 mumol l-1 4. Lower concentrations of 5-HT (3-10 mumol l-1) decreased, whereas higher concentrations (30-100 mumol l-1) increased, spontaneous GABA outflow. The inhibition of GABA efflux was prevented by MDL 72222 whereas the facilitation was reversed by methysergide (1 mumol l-1) into inhibition, and prevented by MDL 72222 1 mumol l-11. 5. These results suggest that, by activating nicotinic receptors present on 5-hydroxytryptaminergic terminals, nicotine releases 5-HT which, in turn, inhibits or increases the secretory activity of cortical GABA interneurones via 5-HT3 and methysergide-sensitive receptors, respectively.

Effect of acute and subchronic nicotine treatment on cortical efflux of [3H]-D-aspartate and endogenous GABA in freely moving guinea-pigs.

1. The [3H]-D-aspartate preloading of the parietal cortex of freely moving guinea-pigs equipped with epidural cups makes it possible to investigate drug effects on the efflux of this radiolabel, assumed as a marker of the glutamatergic structures underlying the cup. In the same model, the efflux of [3H]-gamma-aminobutyric acid ([3H]-GABA) and endogenous GABA can be measured. 2. Nicotine, 0.9-3.6 mg kg-1, s.c., or 3-5 micrograms, i.c.v., increased the efflux of [3H]-D-aspartate but reduced that of GABA. 3. These effects were mediated through mecamylamine-sensitive receptors but the ganglionic blocking agent was devoid of any primary activity. 4. The inhibition of GABA efflux induced by nicotine 3.6 mg kg-1, s.c., was abolished by methysergide 2 mg kg-1, i.p. and was reduced by naloxone 3 mg kg-1, i.p. pretreatment, suggesting the involvement of tryptaminergic and opioid systems. In contrast, muscarinic and catecholamine antagonists were ineffective. 5. Chronic treatment with nicotine (3.6 mg kg-1, twice daily for 16 days) reduced the facilitatory effect of [3H]-D-aspartate and abolished the inhibition of endogenous GABA efflux. 6. A slight increase in the number of nicotinic binding sites (by use of [3H]-nicotine as ligand) was found in the neocortex of chronically treated guinea-pigs. 7. The higher degree of tolerance to chronic nicotine treatment shown by GABA as compared with [3H]-D-aspartate efflux suggests that adaptative changes of the inhibitory neuronal pools prevail. This may contribute to the reinforcing and addictive properties of nicotine.

Changes in gamma-aminobutyric acid release induced by topical administration of drugs affecting its metabolism and receptors: studies in freely moving guinea pigs with epidural cups.

The effect of local application of drugs affecting gamma-aminobutyric acid metabolism and receptors on cortical aminoacid release has been investigated in freely-moving guinea pigs equipped with epidural cups. Topical treatment with gamma-aminobutyric acid reuptake and/or metabolism inhibitors (alone and in combination) produced a slow and progressive increase in cortical aminoacid release. The inhibition of gamma-aminobutyric acid-transaminase with ethanolamino-O-sulphate seemed to be a suitable procedure for enhancing the gamma-aminobutyric acid efflux without interfering with its autoreceptor-mediated negative feedback, tested with the gamma-aminobutyric acid agonist (+/-)baclofen and antagonist phaclofen. A substantial part of the gamma-aminobutyric acid outflowing from the cortex was of neuronal origin since tetrodotoxin halved the basal efflux in the presence of gamma-aminobutyric acid reuptake and/or metabolism inhibitors. These results, considered together, indicate that the epidural cup technique may be a useful approach to study changes in cortical gamma-aminobutyric acid release induced by drugs acting on gabaergic transmission and directly applied on the surface of the cortex.

Inhibitory cholinergic control of endogenous GABA release from electrically stimulated cortical slices and K(+)-depolarized synaptosomes.

In the present study we characterize the optimal experimental conditions under which to investigate the cholinergic regulation of endogenous electrically evoked gamma-aminobutyric acid (GABA) release from guinea pig cortical slices. Superfusion with the neuronal GABA reuptake inhibitor, SKF89976A (10 microM) caused cortical GABA release to be linearly correlated with the frequency of electrical stimulation (5, 10, 20 Hz). Electrically evoked GABA release (10 Hz) was tetrodotoxin-sensitive and Ca(2+)-dependent and was under GABAB autoreceptor control. Under these experimental conditions, acetylcholine (0.1-10 microM) and physostigmine (30 microM) decreased the electrically evoked GABA release while the M2 receptor antagonist AFDX-116 (0.01-0.1 microM) counteracted these effects. Similar results were also observed in a cortical synaptosomal preparation stimulated with K+ (10 mM). These findings demonstrate an inhibitory cholinergic regulation of electrically evoked GABA release via M2 receptors located on cortical GABAergic terminals.

Changes in pituitary-adrenal activity affect the binding properties of striatal dopamine D-2 receptors but not their modulation by neurotensin and cholecystokinin-8.

The effects of adrenalectomy were investigated on the binding properties of [ (3)H] N- propylnorapomorphine ([(3)H]NPA) binding sites in membrane preparations from the rat neostriatum. Adrenalectomy (1 week) was found to increase the K(D) value of [(3)H]NPA binding sites by 30 +/- 3%, without affecting the B(max) values. These effects were fully counteracted by replacement treatment with corticosterone (5 mg/kg, twice daily, 1 week, last injection given 2 h before killing). The ability of neurotensin and cholecystokinin-8 (sulphated) in vitro to increase the K(D) value of [(3)H]NPA binding sites was maintained also following adrenalectomy. The average increases were 16+/-7 and 21+/-6%, respectively. When added together the two peptides failed to increase the K(D) value any further. The B(max) values of [(3)H]NPA binding sites were not affected by either adrenalectomy or by the peptides. These results indicate a glucocorticoid regulation of the binding characteristics of dopamine D-2 receptors in the rat striatum. The mechanism mediating the effects of adrenalectomy may involve an altered expression or regulation of the D-2 receptor or of its connected G-proteins. Probably, it is different from the mechanism whereby the neuropeptides modulate the D-2 receptor, since the neuropeptide-induced modulation was also maintained following the changes induced by adrenalectomy.

Receptor-receptor interactions and their relevance for receptor diversity. Focus on neuropeptide/dopamine interactions.

Receptor diversity in combination with receptor-receptor subtype specific interactions, which can be antagonistic or synergistic in character, markedly increase plasticity in WT and VT in the nervous system. In this way switching among transmission lines for the various DA receptor subtypes becomes possible. Some of these aspects are supported by our work on selective modulation of D2 receptors by CCK and NT. Selective regulation of D2 receptors via CCK-8 receptor subtypes and NT receptors may underlie CCK/DA interactions and NT/DA interactions in the basal ganglia. These studies underline the importance of receptor-receptor interactions exerted at the membrane level between neuropeptide receptors and D2 receptors, which are determined at least in part by the ongoing activity at D1 receptors. In the case of both CCK/D2 and NT/D2 receptor interactions, it has been possible, by means of intrastriatal and intraaccumbens microdialysis, to obtain a functional correlate to the receptor interactions found in the membrane preparations from the striatum. Schizophrenia may be in part related to reduced release of CCK and/or NT peptides or to alterations in their receptor interactions with the D2 receptor. This view may lead to new therapeutic approaches.

Intramembrane interactions between neurotensin receptors and dopamine D2 receptors as a major mechanism for the neuroleptic-like action of neurotensin.

Evidence has been presented that behavioral actions of NT, inducing its neuroleptic-like action, can be explained on the basis of NT-D2 intramembrane receptor-receptor interactions in the basal ganglia, unrelated to the coexistence phenomenon, leading to reduced affinity and transduction of the D2 agonist binding site. By reducing selectively D2 receptor transduction at the pre- and postsynaptic level, the NT receptor appears capable of switching the DA synapses towards a D1 receptor-mediated transduction, illustrating how receptor-receptor interactions can increase the functional plasticity of central synapses (FIG. 12).