Effects of nicotine in the dopaminergic system of mice lacking the alpha4 subunit of neuronal nicotinic acetylcholine receptors

The mesostriatal dopaminergic system influences locomotor activity and the reinforcing properties of many drugs of abuse including nicotine. Here we investigate the role of the alpha4 nicotinic acetylcholine receptor (nAChR) subunit in mediating the effects of nicotine in the mesolimbic dopamine system in mice lacking the alpha4 subunit. We show that there are two distinct populations of receptors in the substantia nigra and striatum by using autoradiographic labelling with 125I alpha-conotoxin MII. These receptors are comprised of the alpha4, beta2 and alpha6 nAChR subunits and non-alpha4, beta2, and alpha6 nAChR subunits. Non-alpha4 subunit-containing nAChRs are located on dopaminergic neurons, are functional and respond to nicotine as demonstrated by patch clamp recordings. In vivo microdialysis performed in awake, freely moving mice reveal that mutant mice have basal striatal dopamine levels which are twice as high as those observed in wild-type mice. Despite the fact that both wild-type and alpha4 null mutant mice show a similar increase in dopamine release in response to intrastriatal KCl perfusion, a nicotine-elicited increase in dopamine levels is not observed in mutant mice. Locomotor activity experiments show that there is no difference between wild-type and mutant mice in basal activity in both habituated and non-habituated environments. Interestingly, mutant mice sustain an increase in cocaine-elicited locomotor activity longer than wild-type mice. In addition, mutant mice recover from depressant locomotor activity in response to nicotine at a faster rate. Our results indicate that alpha4-containing nAChRs exert a tonic control on striatal basal dopamine release, which is mediated by a heterogeneous population of nAChRs.     

Sex differences in anxiety,sensorimotor gating and expression of the alpha4 subunit of the GABAA receptor in the amygdala after progesterone withdrawal

In a progesterone withdrawal (PWD) model of premenstrual anxiety, we have previously demonstrated that increased hippocampal expression of the alpha4 subunit of the GABAA receptor (GABAA-R) is closely associated with higher anxiety levels in the elevated plus maze. However, several studies indicate that sex differences in regulation of the GABAA-R in specific brain regions may be an important factor in the observed gender differences in mood disorders. Thus, we investigated possible sex differences in GABAA-R subunit expression and anxiety during PWD. To this end, we utilized the acoustic startle response (ASR) to assess anxiety levels in male and female rats undergoing PWD as the ASR is also applicable to the assessment of human anxiety responses. We also investigated GABAA-R alpha4 subunit expression in the amygdala, as the amygdala directly regulates the primary startle circuit. Female rats exhibited a greater ASR during PWD than controls, indicating higher levels of anxiety and arousal. In contrast, male rats undergoing PWD did not demonstrate an increased ASR. The sex differences in the ASR were paralleled by sex differences in the expression of the GABAA-R alpha4 subunit in the amygdala such that alpha4 subunit expression was up-regulated in females during PWD whereas alpha4 levels in males undergoing PWD were not altered relative to controls. These findings might have implications regarding gender differences in human mood disorders and the aetiology of premenstrual anxiety.     

Diazepam-induced changes on sleep and the EEG spectrum in mice: role of the alpha3-GABA(A) receptor subtype

Benzodiazepines reduce EEG slow-wave activity in non-REM sleep by potentiating GABAergic neurotransmission at GABAA receptors via a modulatory binding site. However, the mechanisms of action underlying the effects of benzodiazepines on sleep and the sleep EEG are still unknown. Slow waves during sleep are generated by the corticothalamic system and synchronized by the inhibitory GABAergic neurons of the reticular thalamic nucleus. This region contains exclusively alpha3-containing GABAA receptors. We investigated the role of these receptors in the mediation of diazepam effects on the sleep EEG by studying point-mutated mice in which the alpha3-GABAA receptor is diazepam-insensitive [alpha3(H126R)]. Sleep was recorded for 12 h after i.p. injection of 3 mg/kg diazepam or vehicle at light onset in alpha3(H126R) and wild-type controls (n = 13-17 per genotype). The main effect was a marked reduction of slow-wave activity (EEG power density in 0.75-4.00 Hz) in non-REM sleep and a concomitant increase in frequencies above 15.00 Hz in non-REM sleep and waking in both genotypes. Neither effect of diazepam differed significantly between the genotypes. Despite the exclusive expression of alpha3-containing GABAA receptors in the reticular thalamic nucleus, these receptors do not seem to be critical for the mediation of the effects of diazepam on the sleep EEG.     

Cannabinoid mechanism in reinstatement of heroin-seeking after a long period of abstinence in rats

Because opioid and cannabinoid systems have been reported to interact in the modulation of addictive behaviour, this study was aimed at investigating the ability of cannabinoid agents to reinstate or prevent heroin-seeking behaviour after a prolonged period of extinction. In rats previously trained to self-administer heroin intravenously, non-contingent non-reinforced priming administrations of heroin and cannabinoids were presented after long-term extinction, and lever pressing following injections was observed. Results showed that: (i) intravenous priming infusions of heroin (0.1 and 0.2 mg/kg) lead to reinstatement of drug-seeking behaviour; (ii) intraperitoneal priming injections of the central cannabinoid receptor agonists R-(+)-(2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrol[1,2,3-de]-1,4-benzoxazinyl) (1-naphthalenyl)methanonemesylate (WIN 55,212-2, 0.15 and 0.3 mg/kg) and (-)-cis-3-[2-hydroxy-4(1,1-dimethyl-heptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol (CP 55,940, 0.05 and 0.1 mg/kg), but not delta9-tetrahydrocannabinol (delta9-THC, 0.1-1.0 mg/kg), effectively restored heroin-seeking behaviour; (iii) intraperitoneal priming injection of the central cannabinoid receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)4-methyl-1H-pyrazole-3-carboxamide (SR 141716A, 0.3 mg/kg) did not reinstate responding, but (iv) completely prevented heroin-induced reinstatement of drug-seeking behaviour. Moreover, heroin-seeking behaviour was still present for a few days following cannabinoid primings, indicating a long-lasting effect of cannabinoids on responding for heroin. These findings indicate that relapse to heroin after an extended drug-free period is triggered by cannabinoid agonists and that SR 141716A prevents drug-seeking behaviour, suggesting that the use of the cannabinoid antagonist could have some therapeutic benefits in heroin-induced relapse.     

High level of mGluR7 in the presynaptic active zones of select populations of GABAergic terminals innervating interneurons in the rat hippocampus

The release of neurotransmitters is modulated by presynaptic metabotropic glutamate receptors (mGluRs), which show a highly selective expression and subcellular location in glutamatergic terminals in the hippocampus. Using immunocytochemistry, we investigated whether one of the receptors, mGluR7, whose level of expression is governed by the postsynaptic target, was present in GABAergic terminals and whether such terminals targeted particular cells. A total of 165 interneuron dendritic profiles receiving 466 synapses (82% mGluR7a-positive) were analysed. The presynaptic active zones of most GAD-(77%) or GABA-positive (94%) synaptic boutons on interneurons innervated by mGluR7a-enriched glutamatergic terminals (mGluR7a-decorated) were immunopositive for mGluR7a. GABAergic terminals on pyramidal cells and most other interneurons in str. oriens were mGluR7a-immunonegative. The mGluR7a-decorated cells were mostly somatostatin- and mGluR1alpha-immunopositive neurons in str. oriens and the alveus. Their GABAergic input mainly originated from VIP-positive terminals, 90% of which expressed high levels of mGluR7a in the presynaptic active zone. Parvalbumin-positive synaptic terminals were rare on mGluR7a-decorated cells, but on these neurons 73% of them were mGluR7a-immunopositive. Some type II synapses innervating interneurons were immunopositive for mGluR7b, as were some type I synapses. Because not all target cells of VIP-positive neurons are known it has not been possible to determine whether mGluR7 is expressed in a target-cell-specific manner in the terminals of single GABAergic cells. The activation of mGluR7 may decrease GABA release to mGluR7-decorated cells at times of high pyramidal cell activity, which elevates extracellular glutamate levels. Alternatively, the presynaptic receptor may be activated by as yet unidentified endogenous ligands released by the GABAergic terminals or the postsynaptic dendrites.     

The role of CD36 in peripheral nerve remyelination after crush injury

We previously demonstrated that the deficiency of class A macrophage scavenger receptor type I/II was involved in the delayed phagocytosis of degraded myelin by macrophages in class A macrophage scavenger receptor type I/II knockout mice after crush injury of the sciatic nerve [Naba et al. (2000) Exp. Neurol., 166, 83-89]. In order to elucidate the role of CD36, one of the scavenger receptors, here we inflicted crush injury to the sciatic nerves of CD36 knockout mice and investigated the remyelination after crush injury in comparison with that of class A macrophage scavenger receptor type I/II knockout mice. Although we previously reported a lot of onion-bulbs in class A macrophage scavenger receptor type I/II knockout mice at 3 weeks, the number of onion-bulbs was limited both in CD36 knockout mice and wild-type mice. In the morphometry, the remyelination was seriously delayed, and the infiltrating macrophages into the nerve fascicles were quite frequent in CD36 knockout mice compared with wild-type mice at 3 and 6 weeks postinjury. The immunohistochemistry with the monoclonal antibody reacted with oxidized phosphatidylcholine and oil red O staining were positive in wild-type mice, but were negative in CD36 knockout mice, suggesting that the oxidation of phosphatidylcholine and the generation of neutral lipids in macrophages were disturbed in CD36 knockout mice. We hypothesize that the delayed phagocytosis by macrophages and the defect in reuse of lipids from degraded myelin are related to seriously delayed remyelination and a small number of onion-bulbs in CD36 knockout mice.     

Functional characterization of a neuropeptide F-like receptor from Drosophila melanogaster

A cDNA clone encoding a seven-transmembrane domain, G-protein-coupled receptor (NPFR76F, also called GPCR60), has been isolated from Drosophila melanogaster. Deletion mapping showed that the gene encoding this receptor is located on the left arm of the third chromosome at position 76F. Northern blotting and whole mount in situ hybridization have shown that this receptor is expressed in a limited number of neurons in the central and peripheral nervous systems of embryos and adults. Analysis of the deduced amino acid sequence suggests that this receptor is related to vertebrate neuropeptide Y receptors. This Drosophila receptor shows 62-66% similarity and 32-34% identity to type 2 neuropeptide Y receptors cloned from a variety of vertebrate sources. Coexpression in Xenopus oocytes of NPFR76F with the promiscuous G-protein Galpha16 showed that this receptor is activated by the vertebrate neuropeptide Y family to produce inward currents due to the activation of an endogenous oocyte calcium-dependent chloride current. Maximum receptor activation was achieved with short, putative Drosophila neuropeptide F peptides (Drm-sNPF-1, 2 and 2s). Neuropeptide F-like peptides in Drosophila have been implicated in a signalling system that modulates food response and social behaviour. The identification of this neuropeptide F-like receptor and its endogenous ligand by reverse pharmacology will facilitate genetic and behavioural studies of neuropeptide functions in Drosophila.     

Proton-induced calcitonin gene-related peptide release from rat sciatic nerve axons,in vitro,involving TRPV1

We have shown previously that rat sciatic nerve axons in vitro express sensitivity to capsaicin and heat and responded to these stimuli with a Ca2+-dependent and graded immunoreactive calcitonin gene-related peptide release. Morphological evidence for stimulated vesicular exocytosis and for the vanilloid receptor TRPV1 in the axolemma of the unmyelinated nerve fibres has also been presented. Here we used solutions of low pH, high K+ or 47 degrees C to stimulate isolated desheathed sciatic nerves measuring immunoreactive calcitonin gene-related peptide release. pH 6.1 increased immunoreactive calcitonin gene-related peptide release by 31% over baseline and pH 5.2 and 4.3 caused a log-linear concentration-dependent increase of 137 and 265%, respectively. The effect of pH 3.4 was out of the linear range and not reversible. Stimulation in Ca2+-free solutions and under increased intracellular Ca2+ buffering capacity strongly reduced the proton responses. The TRPV1 antagonists capsazepine and ruthenium red substantially reduced the effects of pH 5.2 but not pH 6.1. Combining a stimulus of 60 mm K+ with the subliminal pH 6.3 reduced the axonal immunoreactive calcitonin gene-related peptide response by 88%. The noxious heat response at pH 6.3, however, was only reduced by 39%, suggesting a hidden sensitization to heat by low pH. This was supported by an effect of capsazepine to reduce the combined response to half, indicative of an involvement of TRPV1 in the sensitization but not in the axonal heat response itself that was found to be resistant to capsazepine. Axonal calcitonin gene-related peptide release is thought to play a physiological role in activity-dependent autoregulation of endoneurial blood flow. Axonal sensitivity to and sensitization by protons may be a pathophysiological mechanism involved in certain peripheral neuropathies.     

Dynamics of Ca(2+) and Na(+) in the dendrites of mouse cerebellar Purkinje cells evoked by parallel fibre stimulation

Ca2+ and Na+ play important roles in neurons, such as in synaptic plasticity. Their concentrations in neurons change dynamically in response to synaptic inputs, but their kinetics have not been compared directly. Here, we show the mechanisms and dynamics of Ca2+ and Na+ transients by simultaneous monitoring in Purkinje cell dendrites in mouse cerebellar slices. High frequency parallel fibre stimulation (50 Hz, 3-50-times) depolarized Purkinje cells, and Ca2+ transients were observed at the anatomically expected sites. The magnitude of the Ca2+ transients increased linearly with increasing numbers of parallel fibre inputs. With 50 stimuli, Ca2+ transients lasted for seconds, and the peak [Ca2+] reached approximately 100 microm, which was much higher than that reported previously, although it was still confined to a part of the dendrite. In contrast, Na+ transients were sustained for tens of seconds and diffused away from the stimulated site. Pharmacological interventions revealed that Na+ influx through alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and Ca2+ influx through P-type Ca channels were essential players, that AMPA receptors did not operate as a Ca2+ influx pathway and that Ca2+ release from intracellular stores through inositol trisphosphate receptors or ryanodine receptors did not contribute greatly to the large Ca2+ transients.