Ketamine alters cortical integration of GABAergic interneurons and induces long-term sex-dependent impairments in transgenic Gad67-GFP mice.

Ketamine, a non-competitive N-methyl-D-aspartate (NMDA) antagonist, widely used as an anesthetic in neonatal pediatrics, is also an illicit drug named Super K or KitKat consumed by teens and young adults. In the immature brain, despite several studies indicating that NMDA antagonists are neuroprotective against excitotoxic injuries, there is more and more evidence indicating that these molecules exert a deleterious effect by suppressing a trophic function of glutamate. In the present study, we show using Gad67-GFP mice that prenatal exposure to ketamine during a time-window in which GABAergic precursors are migrating results in (i) strong apoptotic death in the ganglionic eminences and along the migratory routes of GABAergic interneurons; (ii) long-term deficits in interneuron density, dendrite numbers and spine morphology; (iii) a sex-dependent deregulation of γ-aminobutyric acid (GABA) levels and GABA transporter expression; (iv) sex-dependent changes in the response to glutamate-induced calcium mobilization; and (v) the long-term sex-dependent behavioral impairment of locomotor activity. In conclusion, using a preclinical approach, the present study shows that ketamine exposure during cortical maturation durably affects the integration of GABAergic interneurons by reducing their survival and differentiation. The resulting molecular, morphological and functional modifications are associated with sex-specific behavioral deficits in adults. In light of the present data, it appears that in humans, ketamine could be deleterious for the development of the brain of preterm neonates and fetuses of addicted pregnant women.

Influence of glial cells in the dopamine releasing effect resulting from the stimulation of striatal delta-opioid receptors.

Recent data indicate that striatal dopamine release induced by stimulation of delta-opioid receptors is a consequence of glutamate release. However, glial cells, which mainly support glutamate uptake and are involved in glutamate signaling and potentially express delta-opioid receptors, could participate to this effect. The present study investigates the contribution of glial cells in the releasing effects of [d-Pen2, d-Pen5]-enkephalin (DPDPE) by using the gliotoxin l-alpha-aminoadipate (l-alpha AA). Initially, we evaluated the early influence of l-alpha AA local infusion (10 microg/microL) on dialysate levels of glutamate and dopamine under basal or DPDPE treatment conditions. l-alpha AA produced a significant increase of glutamate and dopamine in dialysates (+76% and +50% respectively) and the concomitant infusion of DPDPE (10 microM) significantly enhanced this effect in an additive manner (+110% and +44% respectively). Secondly, we assessed the DPDPE effects on striatal glutamate and dopamine dialysate levels, 2 days after an intra-striatal injection of l-alpha AA which produced destruction of glial cells. This lesion, decreasing the basal glutamate dialysate level as well as its tissue content (by 55% and 36% respectively), prevented the increase in glutamate and dopamine extracellular levels induced by DPDPE. This result confirmed that the DPDPE-induced dopamine release requires an initial glutamate release. However, this effect could reflect a major disruption of glutamatergic transmission caused by the toxin, as suggested by the local infusion of glutamine (2.5 mM) which, in lesioned rats, prevented the decrease in the basal extracellular content of glutamate and restored the DPDPE-induced increase in glutamate and dopamine dialysate levels. Therefore, these results indicate that, although glial cells are essential to maintain functional glutamatergic neurotransmission, they are not directly involved in the process by which stimulation of striatal delta-opioid receptors induces extracellular glutamate release and, consecutively, dopamine release.

[Assessment of the in vitro and in vivo toxicity of 3,4-dihydroxyphenylacetaldehyde (DOPAL)]. / Recherche d'une toxicité du 3,4-dihydroxyphénylacétaldéhyde (DOPAL) in vitro et in vivo.

This work was carried out in order to evaluate the in vitro and in vivo toxicity of 3,4-dihydroxyphenylacetaldehyde (DOPAL). This aldehyde is formed from dopamine (DA) by monoamine oxidases (MAO) and is mainly oxidised to 3,4-dihydroxyphenylacetic acid by brain aldehyde dehydrogenases (ALDH), or eventually reduced to 3,4-dihydroxyphenylethanol by aldose/aldehyde reductases. In vitro, catecholaminergic SH-SY5Y cells were incubated with DA and disulfiram (DSF), an irreversible inhibitor of ALDH. As evidenced by MTT assays, a 24-h treatment with 10(-4) M DA and/or 10(-6) M DSF followed by a 24-h incubation in a drug-free medium evidenced that the toxicity of each of these drugs was potentiated by the second drug. HPLC measurements demonstrated that this drug association induced an early DOPAL production that could result in a delayed cell toxicity. For in vivo studies, male Sprague-Dawley rats were treated with L-DOPA-benserazide, which increases the production of DOPAL by MAO, and DSF. An acute injection of DSF (100mg/kg i.p.) and L-DOPA/benserazide (100mg/kg+25mg/kg, 24h later) significantly increased the DOPAL striatal level. However, a 30-day treatment with DSF (100mg/kg i.p., once every two days) and L-DOPA/benserazide (100mg/kg+25mg/kg, twice a day) did not affect both indexes used to assess the integrity of the nigro-striatal dopaminergic terminals (i.e. the striatal content in DA and the binding to the vesicular monoamine transporter on striatal membranes). These results do not support the hypothesis of a DOPAL toxicity and argue against the toxicity of L-DOPA therapy.

Contextual fear conditioning is associated with an increase of acetylcholine release in the hippocampus of rat.

The effects of contextual fear conditioning on the release of acetylcholine (ACh) in the hippocampus of freely moving rats was assessed using microdialysis. Measures were carried out during both acquisition and retention testing (re-exposure to the conditioning chamber) and compared between animals that either received foot-shocks as unconditioned stimulus (conditioned group) or no foot-shocks (control group) during acquisition. Results showed that during acquisition, hippocampal ACh extracellular level was increased with respect to baseline but that this increase was of similar magnitude in both groups. By contrast, re-exposure to the conditioning chamber the day after (retention testing) produced a significantly greater increase in ACh extracellular level in the conditioned (that, otherwise, displayed conditioned freezing behavior to contextual cues), than in the control group (which displayed virtually no freezing). This enhanced hippocampal ACh release seems to result from the greater hippocampal processing of contextual stimuli in conditioned animals with respect to controls.

Acute interactions between L-DOPA and the neurotoxic effects of 1-methyl-4-phenylpyridinium or 6-hydroxydopamine in mice.

We have compared the effects of an i.p. pretreatment with L-DOPA (200 mg/kg) associated with benserazide (25 mg/kg) on neurotoxic effects of either 6-hydroxydopamine (6-OHDA) (50 microg, 10 microl per mouse) or 1-methyl-4-phenylpyridinium (MPP+) (17.5 microg, 10 microl per mouse). The striatal dopamine (DA) content, the vesicular monoamine transporter (VMAT2) density, as well as the hypothalamic norepinephrine (NE) content were measured 8 days after treatments. The L-DOPA-benserazide pretreatment worsened by 65% the 6-OHDA-induced depletion in striatal DA. On the contrary, it reduced by 42% the MPP+-induced depletion in striatal DA and by 54% the MPP+-induced decrease in VMAT2 density. It was noticed that the L-DOPA-benserazide pretreatment did not modify the marked decrease in hypothalamic NE content induced by 6-OHDA.

The specific dopamine uptake inhibitor GBR 12783 improves learning of inhibitory avoidance and increases hippocampal acetylcholine release.

The specific dopamine uptake inhibitor, GBR 12783 was tested on the retention performance of a one-trial passive avoidance test. For a moderate electric shock intensity, GBR 12783 (10 mg/kg), injected before acquisition session, improved retention performance. Scopolamine (0.125-0.5 mg/kg) completely blocked the promnesic effect of GBR 12783. Moreover, GBR 12783 increased hippocampal acetylcholine release in vivo. These data suggest that the promnesic effect of GBR 12783 is mediated by an increase in the septo-hippocampal cholinergic transmission.

Plasma 3-methoxy-4-hydroxyphenylglycol and homovanillic acid measurements in deficit and nondeficit forms of schizophrenia.

BACKGROUND: Discrepancies in the biochemical research on negative symptoms in schizophrenia may be ascribed to the lack of differentiation into primary and secondary negative symptoms. We have used Carpenter's criteria to define the deficit syndrome of schizophrenia as the presence of enduring and primary negative symptoms and measured catecholaminergic parameters in deficit as compared with nondeficit schizophrenics. METHODS: We have investigated plasma homovanillic acid (pHVA) and 3-methoxy-4-hydroxyphenylglycol (pMHPG) concentrations in 34 DSM-III-R neuroleptic-treated schizophrenic patients who were classified into deficit (n = 14) and nondeficit (n = 20) forms of schizophrenia. All these patients were in a stable clinical and therapeutic status for the preceding 12 months. RESULTS: The 14 deficit schizophrenic patients had lower plasma levels of pHVA and higher plasma concentrations of pMHPG from 9 AM to 12 AM as compared with the 20 nondeficit schizophrenic patients. The two groups did not differ on any demographic, therapeutic, or clinical variable considered. CONCLUSIONS: Our data are consistent with the postulated distinct pathophysiological basis for the deficit syndrome of schizophrenia and suggest that opposite alterations in the pHVA or pMHPG levels may reflect specific changes in noradrenergic and dopaminergic functions in these deficit patients.

Mechanism of the hypothermic effect of MPP+ administered centrally in mice.

The neurotoxin methyl phenyl pyridinium (MPP+) was administered intracerebroventricularly (i.c.v.) to mice. From the 1.25 microg dose per mouse, MPP+ elicited a dose-dependent hypothermic effect from doses as low as 1.25 microg per mouse. The minimal lethal dose was determined to be between 17.5 and 20 microg per mouse. The hypothermia induced by 2.5 microg MPP+ was unaffected by pretreatment with propranolol (8 mg/kg, i.p.), scopolamine (5 mg/kg, s.c.) and haloperidol (250 microg/kg, i.p.). It was decreased by yohimbine (4 mg/kg, s.c.), idazoxan (5 mg/kg, s.c.) and desipramine (20 mg/kg, i.p.). In mice injected i.c.v. with 6 hydroxydopamine (50 microg, 8 days before testing with MPP+ 2.5 microg), a significant reduction in the hypothermic effect of MPP+ was observed. A similar 6 OHDA injection has been shown previously to reduce by about 40% the DA striatal content of DA and by about 70% the hypothalamic content of NE. On the contrary, in mice injected with MPP+ (17.5 microg, 8 days before testing with 50 microg 6 OHDA) there was no modification in the hypothermic effect of 6 OHDA (50 microg). This injection of MPP+ reduced by about 40% the striatal content of DA but did not affect the hypothalamic content of NE. It is concluded that MPP+ decreases body temperature, at least in part, by acting as an indirect NE agonist, which stimulates alpha2 adrenoreceptors. In contrast, this agent in the present experimental conditions, does not destroy NE neurons in opposition to its action on DA neurons.

Involvement of cholinergic neurons in the release of dopamine elicited by stimulation of mu-opioid receptors in striatum.

The involvement of striatal cholinergic neurons in the release of dopamine (DA) elicited by the mu-opioid receptor agonist DAGO ([D-Ala2, NMePhe4-Gly5(ol)]enkephalin) was explored. The striatal release of DA was measured by microdialysis in rats anesthetized with chloral hydrate. When infused in the striatum, through the microdialysis probe, DAGO increased the extracellular levels of DA. The previous injection in striatum of AF 64-A, a toxin for cholinergic neurons, or the concomitant infusion of the M2-muscarinic antagonist methoctramine abolished the effect of DAGO on the DA release. It is concluded that stimulation of mu-opioid receptors, by inhibiting the acetylcholine release which stimulates tonically M2-muscarinic receptors likely associated with dopaminergic nerve endings, indirectly increases the striatal DA release.

Association of DNA polymorphism in the first intron of the tyrosine hydroxylase gene with disturbances of the catecholaminergic system in schizophrenia.

We examined whether there are clinical or biological differences in chronic schizophrenic patients sharing a rare variant allele (a perfect ten tetranucleotide repeats allele of the human TH01 microsatellite) in the tyrosine hydroxylase (TH) gene. For that purpose, clinical parameters (PANSS subscores) and plasma measurements (homovanillic acid and 3-methoxy-4-hydroxy-phenylglycol (MHPG)) were analyzed in five schizophrenic patients sharing the rare allele and 19 schizophrenic patients who did not possess this allele. The mean concentration of plasma HVA and plasma MHPG were significantly lower in the group of schizophrenic patients sharing the rare allele. No other group differences were observed between both groups. These results suggest that this TH gene polymorphism may be associated with disturbances of the catecholaminergic pathway.

Rapid and long lasting reduction of the hypothermic effect of a D2 dopamine agonist after an intracerebroventricular injection of 6-hydroxydopamine.

In mice pretreated intracerebroventricularly (i.c.v.) with 6-hydroxydopamine (6OHDA) (50 micrograms per mouse), as soon as the hypothermia elicited by the neurotoxin had vanished (3 hr), the hypothermic effect induced by the direct D2 dopamine receptor agonist RU 24926 (1 mg/kg, s.c.), was almost completely suppressed. This reduction in hypothermic effect was observed more than 1 month after the 6OHDA injection. On the 3rd day after 6OHDA injection, this reduction was observed for all tested doses of RU 24926 (0.25-2 mg/kg). It was prevented when an i.p. administration of the norepinephrine uptake inhibitor desipramine (20 mg/kg) was performed 30 min before the 6OHDA i.c.v. injection. It was not modified when an i.p. administration of the dopamine uptake inhibitor GBR 12783 (20 mg/kg) was performed 30 min before the 6OHDA i.c.v. injection. The 6OHDA i.c.v. injection modified significantly neither the dopamine nor the serotonin hypothalamic contents. On the contrary it resulted in a marked decrease (-73%) of the norepinephrine hypothalamic content, which was unchanged by the administration of GBR 12783 (20 mg/kg, i.p.) 30 min before 6OHDA, but completely prevented by desipramine (20 mg/kg, i.p.) 30 min before 6OHDA i.c.v. injection. It is concluded that the hypothermic effect resulting from the stimulation of D2 dopamine receptors involves a central norepinephrine transmission, which is very rapidly altered after 6OHDA administration.

Involvement of glutamate receptors in the striatal enkephalin-induced dopamine release.

In anesthetized rats, the intrastriatal infusion of the delta-opioid receptor agonist, [D-Pen2,D-Pen5]enkephalin, increased the extracellular concentration of dopamine. This effect was abolished by the NMDA receptor antagonist, 3-[(+/-)-2-carboxypiperazine-4-yl]propyl-1-phosphonate, but was unchanged by the AMPA (D,L-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate) and kainate receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione. This suggests that the dopamine release induced by the delta-opioid agonist depends critically on the involvement of glutamatergic transmission via NMDA receptors.

Kainic acid lesion of the striatum increases dopamine release but reduces 3-methoxytyramine level.

Kainic acid lesion of the striatum leads to the disruption of the striatonigral regulatory loop. Although microdialysis showed an increase in dopamine release, two days after kainic acid injection, 3-methoxytyramine levels are dramatically decreased. This suggests that the postsynaptic membrane-bound catechol-O-methyltransferase (COMT), but not the glial COMT, is responsible for the formation of measured 3-methoxytyramine.

Differential effect of intrastriatal kainic acid on the modulation of dopamine release by mu- and delta-opioid peptides: a microdialysis study.

The present study investigated the effects of a striatal lesion induced by kainic acid on the striatal modulation of dopamine (DA) release by mu- and delta-opioid peptides. The effects of [D-Pen2,D-Pen5]-enkephalin (DPDPE) and [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAGO), two highly selective delta- and mu-opioid agonists, respectively, were studied by microdialysis in anesthetized rats. In control animals both opioid peptides, administered locally, significantly increased extracellular DA levels. The effects of DPDPE were also observed in animals whose striatum had been previously lesioned with kainic acid. In contrast to the effects of the delta agonist, the significant increase induced by DAGO was no longer observed in lesioned animals. These results suggest that delta-opioid receptors modulating the striatal DA release, in contrast to mu receptors, are not located on neurons that may be lesioned by kainic acid.

Local enkephalins tonically modulate dopamine release in the striatum: a microdialysis study.

Kelatorphan, an inhibitor of the enkephalin-degrading enzymes, infused by microdialysis (10(-6) M) in the striatum of anaesthetized rats, significantly increased dopamine (DA) output but left dihydroxyphenylacetic acid and homovanillic acid extracellular levels unchanged. The local application of naltrexone (10(-6) M) prevented the effect of kelatorphan on DA release. These data indicate that local enkephalins tonically modulate DA release in rat striatum.

Role of delta opioid receptors in the effects of inhibitors of enkephalin-degrading peptidases on the horizontal and vertical components of locomotion in mice.

In the present study we report the effects of inhibitors of enkephalin-degrading peptidases on spontaneous locomotion in mice and the involvement of delta opioid receptors in these effects. Animals received intracerebroventricularly (i.c.v.) or intravenously (i.v.) enkephalinase inhibitors (thiorphan and acetorphan), aminopeptidase inhibitors (bestatin and carbaphethiol) or mixed peptidase inhibitors (kelatorphan). The i.c.v. co-administration of bestatin and thiorphan (50 micrograms + 50 micrograms) induced an increase in both the horizontal and vertical components of locomotion. A similar pattern was observed after the i.c.v. administration of kelatorphan (8.5-50 micrograms) or the i.v. co-administration of acetorphan and carbaphethiol (5 mg/kg + 10 mg/kg). The opiate antagonist naltrexone (1 mg/kg, s.c.) failed to reverse the excitolocomotor effects of kelatorphan or of bestatin and thiorphan and antagonized only partially the effects of acetorphan and carbaphethiol. Naloxone (2 mg/kg-10 mg/kg, s.c.) partially reversed the increase in locomotion elicited by bestatin and thiorphan. The pretreatment with the delta opioid antagonists ICI 154,129 (20 micrograms, i.c.v.) or ICI 174,864 (2-4 micrograms, i.c.v.) strongly decreased the effects of all the peptidase inhibitors we tested. These results suggest that endogenous enkephalins may control via delta opioid receptors the horizontal and vertical components of locomotor activity in mice.

MU and delta opioid receptors control differently the horizontal and vertical components of locomotor activity in mice.

The present study investigated the role of mu and delta opioid receptors in the control of the horizontal and vertical components of locomotion. Mice received intracerebroventricularly (i.c.v.) enkephalin analogs specific for either the mu or delta opioid receptors. The administration of the specific mu agonist [D-Ala2-NMePhe4-Gly5(ol)] enkephalin (DAGO) induced a dose-dependent increase in horizontal activity and a decrease in vertical activity. The specific delta agonist [D-Pen2,D-Pen5] enkephalin (DPDPE) increased both components of motor activity. The opiate antagonist naltrexone reversed the effects of DAGO, but did not influence the effects of DPDPE on motor activity. The pretreatment with the delta opiate antagonist ICI 154, 129 completely reversed the effects of DPDPE on locomotion but antagonized only partially the effects of DAGO on locomotion. These results indicate that the two components of locomotor activity--horizontal and vertical activity--are modulated differently by the stimulation of mu or delta opioid receptors.

Role of endogenous enkephalins in locomotion and nociception studied with peptidase inhibitors in two inbred strains of mice (C57BL/6J and DBA/2J).

Acetorphan, a parenterally active enkephalinase inhibitor, induced dose-dependently a naloxone-reversible analgesia on the hot-plate jump test in DBA/2J (DBA2) mice but was devoid of effects in C57BL/6J (C57) mice. By contrast, acetorphan increased locomotion in both strains; however, the DBA2 strain was much more sensitive than C57 mice to the locomotor stimulant effect. The increased locomotion was antagonized by naloxone in both strains. These data suggest that endogenous enkephalins modulate nociception and locomotion in the two inbred strains differently.