M. Paola Castelli

Selective γ-hydroxybutyric acid receptor ligands increase extracellular glutamate in the hippocampus, but fail to activate G protein and to produce the sedative/hypnotic effect of γ-hydroxybutyric acid

Two γ‐hydroxybutyric acid (GHB) analogues, trans‐γ‐hydroxycrotonic acid (t‐HCA) and γ‐(p‐methoxybenzyl)‐γ‐hydroxybutyric acid (NCS‐435) displaced [3H]GHB from GHB receptors with the same affinity as GHB but, unlike GHB, failed to displace [3H]baclofen from GABAB receptors. The effect of the GHB analogues, GHB and baclofen, on G protein activity and hippocampal extracellular glutamate levels was compared. While GHB and baclofen stimulated 5′‐O‐(3‐[35S]thiotriphospate) [35S]GTPγS binding both in cortex homogenate and cortical slices, t‐HCA and NCS‐435 were ineffective up to 1 mm concentration. GHB and baclofen effect was suppressed by the GABAB antagonist CGP 35348 but not by the GHB receptor antagonist NCS‐382. Perfused into rat hippocampus, 500 nm and 1 mm GHB increased and decreased extracellular glutamate levels, respectively. GHB stimulation was suppressed by NCS‐382, while GHB inhibition by CGP 35348. t‐HCA and NCS‐435 (0.1–1000 µm) locally perfused into hippocampus increased extracellular glutamate; this effect was inhibited by NCS‐382 (10 µm) but not by CGP 35348 (500 µm). The results indicate that GHB‐induced G protein activation and reduction of glutamate levels are GABAB‐mediated effects, while the increase of glutamate levels is a GHB‐mediated effect. Neither t‐HCA nor NCS‐435 reproduced GHB sedative/hypnotic effect in mice, confirming that this effect is GABAB‐mediated. The GHB analogues constitute important tools for understanding the physiological role of endogenous GHB and its receptor.

Central effects of 1,4-butanediol are mediated by GABAB receptors via its conversion into γ-hydroxybutyric acid

Abstract The aliphatic alcohol 1,4-butanediol in converted into γ-hydroxybutyric acid (GHB) via two enzymatic steps: first, it is oxidised by alcohol dehydrogenase in γ-hydroxybutyraldehyde; second, the latter is transformed, likely by aldehyde dehydrogenase, into GHB. Initially, the present study compared the sedative/hypnotic effect of GHB and 1,4-butanediol, measured as loss of righting reflex. 1,4-Butanediol was more potent than GHB, presumably because of a more rapid penetration of the blood brain barrier. Further alcohol dehydrogenase inhibitors, 4-methylpyrazole and ethanol, totally prevented the sedative/hypnotic effect of 1,4-butanediol; the aldehyde dehydrogenase inhibitor disulfiram partially blocked the sedative/hypnotic effect of 1,4-butanediol. Finally, the sedative/hypnotic effect of 1,4-butanediol was antagonised by the GABA B receptor antagonists, SCH 50911 [(2 S )(+)-5,5-dimethyl-2-morpholineacetic acid] and CGP 46381 [(3-aminopropyl)(cyclohexylmethyl)phosphinic acid], but not by the putative GHB receptor antagonist NCS-382 (6,7,8,9-tetrahydro-5-hydroxy-5 H -benzocyclohept-6-ylideneacetic acid), indicating that it is mediated by GABA B but not GHB receptors. Taken together, these results suggest that the sedative/hypnotic effect of 1,4-butanediol is mediated by its conversion in vivo into GHB which, in turn, binds to GABA B receptors. Accordingly 1,4-butanediol, unlike GHB, failed to displace [ 3 H]GHB and [ 3 H]baclofen in brain membranes.

Quantitative autoradiographic distribution of gamma-hydroxybutyric acid binding sites in human and monkey brain

gamma-Hydroxybutyric acid (GHB), a naturally occurring metabolite of GABA, is present in micromolar concentrations in various areas of the mammalian brain. Specific GHB binding sites, uptake system, synthetic and metabolizing enzymes have been identified in CNS. The present study shows the anatomical distribution of GHB binding sites in sections of primate (squirrel monkey) and human brain by radioligand quantitative autoradiography. In both species the highest densities of binding sites were found in the hippocampus, high to moderate densities in cortical areas (frontal, temporal, insular, cingulate and entorhinal) and low densities in the striatum; no binding sites were detected in the cerebellum. High density of GHB binding was found in the monkey amygdala. In addition the binding characteristics of [(3)H]GHB to membrane preparations of human brain cortex were examined. Scatchard analysis and saturation curves revealed both a high (K(d1) 92+/-4.4 nM; B(max1) 1027+/-110 fmol/mg protein) and a low-affinity binding site (K(d2) 916+/-42 nM; B(max2) 8770+/-159 fmol/mg protein). The present study is the first report on the autoradiographic distribution of specific GHB binding sites in the primate and human brain: such distribution is in both species in good agreement with the distribution found in the rat brain.

Regional distribution of 5α-reductase type 2 in the adult rat brain: An immunohistochemical analysis

The enzyme 5α-reductase (5αR) catalyzes the conversion of testosterone and other Δ(4)-3-ketosteroids into their 5α-reduced metabolites. Of the five members of the 5αR family, the type 2 enzyme (5αR2) plays a key role in androgen metabolism, and is abundantly distributed in the urogenital system. Although 5αR2 has been reported to be highly expressed in the brain during early developmental stages, little is currently known on its anatomical and cellular distribution in the adult brain. Thus, the present study was designed to determine the detailed localization of 5αR2 in the adult rat brain, using a highly specific polyclonal antibody against this isoform. Parasagittal and coronal sections revealed 5αR2 immunoreactivity throughout most brain regions, with strong immunolabeling in the layers III and VI of the prefrontal and somatosensory cortex, olfactory bulb, thalamic nuclei, CA3 field of hippocampus, basolateral amygdala and Purkinje cell layer of cerebellum. Lower 5αR2 levels were detected in the hypothalamus and midbrain. Moreover, double labeling fluorescence with confocal laser scanning microscopy (CLSM) revealed that 5αR2 is localized in neurons, but not in glial cells. Specifically, the enzyme was documented in the pyramidal neurons of the cortex by CLSM analysis of simultaneous Golgi-Cox and immunofluorescent staining. Finally, low levels of 5αR2 expression were identified in GABAergic cells across the cortex, hippocampus and striatum. These findings show that, in the adult brain, 5αR2 is distributed in critical regions for behavioral regulation, suggesting that the functional role of this isoform is present throughout the entire lifespan of the individual.

6-Hydroxydopamine lesion in the ventral tegmental area fails to reduce extracellular dopamine in the cerebral cortex

Dopamine and noradrenaline are both involved in modulation of superior cognitive functions that are mainly dependent on frontal cortex activity. Experimental evidence points to parallel variations in extracellular concentrations of catecholamines in the cerebral cortex, which leads us to hypothesize their corelease from noradrenergic neurons. This study aimed to verify this hypothesis, by means of cerebral microdialysis following destruction of dopaminergic innervation in rats. The unilateral injury of dopaminergic neurons, by 6‐hydroxydopamine injection in the ventral tegmental area, dramatically reduced the immunoreactivity for dopamine transporter in the cerebral hemisphere ipsilateral to the lesion. Tissue dopamine content in the ipsilateral nucleus accumbens and medial prefrontal and parietal cortex was also profoundly decreased, whereas noradrenaline was only slightly affected. Despite the lower tissue content in the denervated side, the extracellular dopamine level was not changed in the cortex, although it was markedly decreased in the nucleus accumbens ipsilateral to the lesion. The effect of drugs selective for D2‐dopaminergic (haloperidol) or α2‐noradrenergic (RS 79948) receptors was verified. Haloperidol failed to modify extracellular dopamine in either cortex but increased it in the nucleus accumbens, such an increase being greatly reduced in the denervated side. On the other hand, RS 79948 increased extracellular dopamine and DOPAC in all areas tested, the increases being of the same degree in both intact and lesioned sides. The results strongly support the hypothesis that the majority of extracellular dopamine in the cortex, unlike that in the nucleus accumbens, originates from noradrenergic terminals.

Stereoselectivity of NCS-382 binding to γ-hydroxybutyrate receptor in the rat brain

Abstract γ-Hydroxybutyric acid (GHB), a naturally occurring metabolite of γ-aminobutyric acid (GABA), has been postulated to act both as a specific agonist of GHB receptors and as a weak GABAB receptor agonist. The racemic compound 6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylideneacetic acid (RS-NCS-382), the only available antagonist of GHB receptors, has been resolved in two enantiomers, R- and S-; the potency of the latter to displace 4-hydroxy [2-3-3H] butyric acid ([3H]GHB) and [3H]NCS-382 from GHB receptors, on one hand, and [3H]baclofen from GABAB receptors on the other was compared in rat brain homogenates. R-NCS-382 was found to be twice and 60 times more potent than the RS- and S-forms, respectively, in displacing [3H]GHB and 2 and 14 times, respectively, in displacing [3H]NCS-382 from GHB binding. Neither RS-NCS-382 nor its enantiomers inhibited [3H]baclofen binding up to a concentration of 1 mM. Our results demonstrate that R-NCS-382 is the enantiomer of RS-NCS-382 with higher affinity for GHB receptors.

S amisulpride binds with high affinity to cloned dopamine D3 and D2 receptors

Amisulpride is a substituted benzamide antipsychotic with nanomolar affinity and high selectivity for dopamine D(2) and dopamine D(3) receptors. The interaction of racemic (+/-)RS amisulpride and its two enantiomers (+)R and (-)S with dopamine D(2) and dopamine D(3) receptors subtypes were compared with that of haloperidol. Binding studies were performed using either [3H]spiperone or [3H]nemonapride in baculovirus/Spodoptera frugiperda insect (Sf-9) cell system expressing either the human dopamine recombinant D(2)long (hD(2L)) or the rat dopamine recombinant D(3) (rD(3)) receptors. K(i) values at dopamine rD(3) receptors were similar regardless of the radioligand used, whereas at hD(2L) receptors values were higher using [3H]spiperone than [3H]nemonapride. However, the rank order of compound potency against radiolabeled spiperone or nemonapride both at dopamine hD(2L) and at dopamine rD(3) receptors was similar. (-)S amisulpride displaced [3H]spiperone or [3H]nemonapride binding from both dopamine hD(2L) or dopamine rD(3) receptors, being twofold more potent than the racemic form and 38-19-fold more potent than (+)R enantiomer. Both racemic and the (-)S enantiomer exhibited 2-4 ([3H]spiperone)- and 3-4 ([3H]nemonapride)-fold higher affinity than haloperidol for dopamine rD(3) receptor, respectively. The (+)R enantiomer has weaker affinity with respect to haloperidol for both dopamine hD(2L) and dopamine rD(3) receptors. Our results show that (-)S amisulpride is the active enantiomer of amisulpride, showing high affinity for dopamine D(3) and dopamine D(2) receptors.

Methamphetamine neurotoxicity increases brain expression and alters behavioral functions of CB1 cannabinoid receptors

Cannabis is the most common secondary illicit substance in methamphetamine (METH) users, yet the outcomes of the concurrent consumption of both substances remain elusive. Capitalizing on recent findings on the implication of CB₁ cannabinoid receptors in the behavioral effects of METH, we hypothesized that METH-induced neurotoxicity may alter the brain expression of CB₁, thereby affecting its role in behavioral functions. To test this possibility, we subjected rats to a well-characterized model of METH neurotoxicity (4 mg/kg, subcutaneous × 4 injections, 2 h apart), and analyzed their CB₁ receptor brain expression three weeks later. METH exposure resulted in significant enhancements of CB₁ receptor expression across several brain regions, including prefrontal cortex, caudate-putamen, basolateral amygdala, CA1 hippocampal region and perirhinal cortex. In parallel, a different group of METH-exposed rats was used to explore the responsiveness to the potent cannabinoid agonist WIN 55,212-2 (WIN) (0.5-1 mg/kg, intraperitoneal), within several paradigms for the assessment of emotional and cognitive functions, such as open field, object exploration and recognition, and startle reflex. WIN induced anxiolytic-like effects in METH-exposed rats and anxiogenic-like effects in saline-treated controls. Furthermore, METH-exposed animals exhibited a significantly lower impact of WIN on the attenuation of exploratory behaviors and short-term (90 min) recognition memory. Conversely, METH neurotoxicity did not significantly affect WIN-induced reductions in locomotor activity, exploration time and acoustic startle. These results suggest that METH neurotoxicity may alter the vulnerability to select behavioral effects of cannabis, by inducing distinct regional variations in the expression of CB₁ receptors.

Anti-alcohol and anxiolytic properties of a new chemical entity, GET73

N-[(4-trifluoromethyl)benzyl]4-methoxybutyramide (GET73) is a newly synthesized compound structurally related to the clinically used, alcohol-substituting agent, gamma-hydroxybutyric acid (GHB). The present study was designed to assess whether GET73 may share with GHB the capacity to reduce alcohol intake in rats. Additionally, the effect of treatment with GET73 on anxiety-related behaviors and cognitive tasks in rats was investigated. A series of in vitro binding assays investigated the capacity of GET73 to bind to the GHB binding site and multiple other receptors. GET73 (10−9–10−3 M) failed to inhibit [3H]GHB binding at both high- and low-affinity GHB recognition sites in rat cortical membranes. GET73 displayed minimal, if any, binding at dopamine, serotonin, GABA, and glutamate receptors in membranes from different rat brain areas. Acute treatment with low-to-moderate, non-sedative doses of GET73 (5–50 mg/kg, i.g. or i.p.) (a) reduced alcohol intake and suppressed “alcohol deprivation effect” (a model of alcohol relapse) in selectively bred, Sardinian alcohol-preferring (sP) rats, (b) exerted anxiolytic effects in Sprague-Dawley (SD) and sP rats exposed to the Elevated Plus Maze test, and (c) tended to induce promnestic effects in SD rats exposed to a modified water version of the Hebb–Williams maze test. Although the mechanism of GET73 action is currently unknown, the results of the present study suggest that GET73 has a multifaceted pharmacological profile, including the capacity to reduce alcohol drinking and anxiety-related behaviors in rats.

Δ9-Tetrahydrocannabinol Prevents Methamphetamine-Induced Neurotoxicity

Methamphetamine (METH) is a potent psychostimulant with neurotoxic properties. Heavy use increases the activation of neuronal nitric oxide synthase (nNOS), production of peroxynitrites, microglia stimulation, and induces hyperthermia and anorectic effects. Most METH recreational users also consume cannabis. Preclinical studies have shown that natural (Δ9-tetrahydrocannabinol, Δ9-THC) and synthetic cannabinoid CB1 and CB2 receptor agonists exert neuroprotective effects on different models of cerebral damage. Here, we investigated the neuroprotective effect of Δ9-THC on METH-induced neurotoxicity by examining its ability to reduce astrocyte activation and nNOS overexpression in selected brain areas. Rats exposed to a METH neurotoxic regimen (4×10 mg/kg, 2 hours apart) were pre- or post-treated with Δ9-THC (1 or 3 mg/kg) and sacrificed 3 days after the last METH administration. Semi-quantitative immunohistochemistry was performed using antibodies against nNOS and Glial Fibrillary Acidic Protein (GFAP). Results showed that, as compared to corresponding controls (i) METH-induced nNOS overexpression in the caudate-putamen (CPu) was significantly attenuated by pre- and post-treatment with both doses of Δ9-THC (−19% and −28% for 1 mg/kg pre- and post-treated animals; −25% and −21% for 3 mg/kg pre- and post-treated animals); (ii) METH-induced GFAP-immunoreactivity (IR) was significantly reduced in the CPu by post-treatment with 1 mg/kg Δ9-THC1 (−50%) and by pre-treatment with 3 mg/kg Δ9-THC (−53%); (iii) METH-induced GFAP-IR was significantly decreased in the prefrontal cortex (PFC) by pre- and post-treatment with both doses of Δ9-THC (−34% and −47% for 1 mg/kg pre- and post-treated animals; −37% and −29% for 3 mg/kg pre- and post-treated animals). The cannabinoid CB1 receptor antagonist SR141716A attenuated METH-induced nNOS overexpression in the CPu, but failed to counteract the Δ9-THC-mediated reduction of METH-induced GFAP-IR both in the PFC and CPu. Our results indicate that Δ9-THC reduces METH-induced brain damage via inhibition of nNOS expression and astrocyte activation through CB1-dependent and independent mechanisms, respectively.