Pierluigi Saba

Differential distribution of functional cannabinoid CB1 receptors in the mouse gastroenteric tract.

Recently, the gastrointestinal pharmacology of cannabinoid CB(1) receptors has been extensively explored. We employed western blotting and immunohistochemistry techniques to study the distribution of the cannabinoid CB(1) receptor protein in the mouse gastroenteric tract. The cannabinoid CB(1) receptor peptide was detected by western blotting only in its glycosylated form (63 kDa) with a significant differential distribution. The highest levels of expression were detected in the stomach and in the colon, while the pyloric valve was devoid of any cannabinoid CB(1) receptor protein. The immunohistochemical study showed intense cannabinoid CB(1) receptor immunoreactivity in ganglia subadjacent to the gastric epithelium and in the smooth muscle layers of both the small and large intestine. Only the small intestine showed (-)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)-phenyl]-4-(3-hydroxylpropyl) cyclohexan-1-ol) ([3H]CP 55,940) specific binding (27%). These receptors mediated pharmacologically significant effects since the cannabinoid CB(1) receptor agonist R(-)-7-hydroxy-delta-6-tetra-hydrocannabinol-dimethylheptyl (HU 210) dose dependently inhibited gastrointestinal transit up to 70%, while the cannabinoid CB(1) receptor antagonist N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide (SR 141716A) increased gastrointestinal transit. Moreover, the dose of 0.3 microg/kg of HU 210, devoid per se of any activity on mouse intestinal propulsion, blocked the increased gastroenteric transit induced by the cannabinoid CB(1) antagonist SR 141716A.

Stimulation of the locus coeruleus elicits noradrenaline and dopamine release in the medial prefrontal and parietal cortex

Our previous studies have suggested that dopamine and noradrenaline may be coreleased from noradrenergic nerve terminals in the cerebral cortex. To further clarify this issue, the effect of electrical stimulation of the locus coeruleus on extracellular noradrenaline, dopamine and DOPAC in the medial prefrontal cortex, parietal cortex and caudate nucleus was analysed by microdialysis in freely moving rats. Stimulation of the locus coeruleus for 20 min with evenly spaced pulses at 1 Hz failed to modify cortical catecholamines and DOPAC levels. Stimulation with bursts of pulses at 12 and 24 Hz increased, in a frequency‐related manner, not only noradrenaline but also dopamine and DOPAC in the two cortices. In both cortices noradrenaline returned to baseline within 20 min of stimulation, irrespective of the stimulation frequency, whereas dopamine returned to normal within 20 and 60 min in the medial prefrontal cortex and within 60 and 80 min in the parietal cortex after 12 and 24 Hz stimulation, respectively. DOPAC remained elevated throughout the experimental period. Phasic stimulation of the locus coeruleus at 12 Hz increased noradrenaline in the caudate nucleus as in the cerebral cortices but was totally ineffective on dopamine and DOPAC. Tetrodotoxin perfusion into the medial prefrontal cortex dramatically reduced noradrenaline and dopamine levels and suppressed the effect of electrical stimulation. These results indicate that electrical stimulation‐induced increase of dopamine is a nerve impulse exocytotic process and suggest that cortical dopamine and noradrenaline may be coreleased from noradrenergic terminals.

Central dopaminergic transmission is selectively increased in the limbic system of rats chronically exposed to antidepressants

Repeated electroconvulsive shock (ECS) exposure produced a decrease of [3H]SCH 23390 binding sites and a reduced response of adenylate cyclase activity to dopamine D-1 receptor stimulation in the rat limbic area analogous to that previously observed in rats chronically treated with imipramine. These effects were completely prevented by the repeated administration of a small dose of alpha-methyl-p-tyrosine (alpha-MPT), associated with the tricyclic compound. Increased dopaminergic transmission seems to be involved in the mechanism of antidepressant action. Rats chronically treated with imipramine showed a decrease of dihydroxyphenylacetic acid (DOPAC) concentration restricted to the limbic area. Finally, both imipramine and desipramine blocked the uptake of [3H]dopamine in the limbic system with a 100-fold greater potency than that observed in the basal ganglia.

Co-release of noradrenaline and dopamine in the cerebral cortex elicited by single train and repeated train stimulation of the locus coeruleus

BackgroundPrevious studies by our group suggest that extracellular dopamine (DA) and noradrenaline (NA) may be co-released from noradrenergic nerve terminals in the cerebral cortex. We recently demonstrated that the concomitant release of DA and NA could be elicited in the cerebral cortex by electrical stimulation of the locus coeruleus (LC). This study analyses the effect of both single train and repeated electrical stimulation of LC on NA and DA release in the medial prefrontal cortex (mPFC), occipital cortex (Occ), and caudate nucleus. To rule out possible stressful effects of electrical stimulation, experiments were performed on chloral hydrate anaesthetised rats.ResultsTwenty min electrical stimulation of the LC, with burst type pattern of pulses, increased NA and DA both in the mPFC and in the Occ. NA in both cortices and DA in the mPFC returned to baseline within 20 min after the end of the stimulation period, while DA in the Occ reached a maximum increase during 20 min post-stimulation and remained higher than baseline values at 220 min post-stimulation. Local perfusion with tetrodotoxin (TTX, 10 μM) markedly reduced baseline NA and DA in the mPFC and Occ and totally suppressed the effect of electrical stimulation in both areas.A sequence of five 20 min stimulations at 20 min intervals were delivered to the LC. Each stimulus increased NA to the same extent and duration as the first stimulus, whereas DA remained elevated at the time next stimulus was delivered, so that baseline DA progressively increased in the mPFC and Occ to reach about 130 and 200% the initial level, respectively.In the presence of the NA transport (NAT) blocker desipramine (DMI, 100 μM), multiple LC stimulation still increased extracellular NA and DA levels.Electrical stimulation of the LC increased NA levels in the homolateral caudate nucleus, but failed to modify DA level.ConclusionThe results confirm and extend that LC stimulation induces a concomitant release of DA and NA in the mPFC and Occ.The different time-course of LC-induced elevation of DA and NA suggests that their co-release may be differentially controlled.

Sub-chronic treatment with classical but not atypical antipsychotics produces morphological changes in rat nigro-striatal dopaminergic neurons directly related to "early onset" vacuous chewing.

In the present work, we investigated if an impairment of dopaminergic neurons after subchronic haloperidol treatment might be a possible physiopathologic substrate of the ‘early onset’ vacuous chewing movements (VCMs) in rats. For this purpose, different antipsychotics were used to analyse a possible relationship between VCMs development and morphological alterations of tyrosine‐hydroxylase‐immunostained (TH‐IM) neurons.

Disulfiram stimulates dopamine release from noradrenergic terminals and potentiates cocaine-induced dopamine release in the prefrontal cortex

RationaleDisulfiram efficacy in treatment of cocaine addiction is attributed to the inhibition of dopamine-β-hydroxylase and reduction in brain noradrenaline (NA)/dopamine (DA) ratio.ObjectivesUsing microdialysis, we investigated if disulfiram causes DA release from noradrenergic terminals and modifies cocaine-induced DA release.ResultsDisulfiram reduced extracellular NA in the medial prefrontal (mPF) cortex, occipital cortex, accumbens and caudate nuclei, while it markedly increased DA not only in mPF but also in the occipital cortex, despite its scanty dopaminergic afferences, and modestly increased DA in the accumbens and caudate nuclei, despite their dense dopaminergic innervation. Disulfiram-induced DA accumulation was reversed in both cortices by tetrodotoxin infusion and by systemic administration of the α2-adrenoceptor agonist clonidine, but was not modified by the α2-adrenoceptor antagonist RS 79948 or the D2-like agonist quinpirole. Disulfiram prevented cocaine-induced NA release in the mPF cortex and nucleus accumbens, potentiated cocaine-induced DA release in the mPF cortex but failed to modify cocaine effect in the nucleus accumbens. DA release induced by disulfiram-cocaine combination in the mPF cortex was prevented by clonidine but not by quinpirole.ConclusionsWe suggested that disulfiram, by removing NA-mediated inhibitory control on noradrenergic terminals, causes an unrestrained cocaine-induced DA release from those terminals in the mPF cortex. In the accumbens and caudate nuclei, “allogenic” DA concentration might be clouded by DA originated from dopaminergic terminals. The possible role of “allogenic” DA in disulfiram ability to prevent stress-induced reinstatement of cocaine seeking is discussed.

Inhibition of 5α-reductase in the nucleus accumbens counters sensorimotor gating deficits induced by dopaminergic activation

Cogent evidence highlights a key role of neurosteroids and androgens in schizophrenia. We recently reported that inhibition of steroid 5α-reductase (5αR), the rate-limiting enzyme in neurosteroid synthesis and androgen metabolism, elicits antipsychotic-like effects in humans and animal models, without inducing extrapyramidal side effects. To elucidate the anatomical substrates mediating these effects, we investigated the contribution of peripheral and neural structures to the behavioral effects of the 5αR inhibitor finasteride (FIN) on the prepulse inhibition (PPI) of the acoustic startle reflex (ASR), a rat paradigm that dependably simulates the sensorimotor gating impairments observed in schizophrenia and other neuropsychiatric disorders. The potential effect of drug-induced ASR modifications on PPI was excluded by measuring this index both as percent (%PPI) and absolute values (ΔPPI). In both orchidectomized and sham-operated rats, FIN prevented the %PPI deficits induced by the dopamine (DA) receptor agonists apomorphine (APO, 0.25mg/kg, SC) and d-amphetamine (AMPH, 2.5mg/kg, SC), although the latter effect was not corroborated by ΔPPI analysis. Conversely, APO-induced PPI deficits were countered by FIN infusions in the brain ventricles (10μg/1μl) and in the nucleus accumbens (NAc) shell and core (0.5μg/0.5μl/side). No significant PPI-ameliorating effect was observed following FIN injections in other brain regions, including dorsal caudate, basolateral amygdala, ventral hippocampus and medial prefrontal cortex, although a statistical trend was observed for the latter region. The efflux of DA in NAc was increased by systemic, but not intracerebral FIN administration. Taken together, these findings suggest that the role of 5αR in gating regulation is based on post-synaptic mechanisms in the NAc, and is not directly related to alterations in DA efflux in this region.

Chronic imipramine reduces [3H]SCH 23390 binding and DA sensitive adenylate cyclase in the limbic system

[3H]SCH 23390 binding and dopamine (DA)-stimulated adenylate cyclase activity were measured in brain membrane preparations from rats chronically treated with imipramine (10 mg/kg twice daily for 14 days). [3H]SCH 23390 binding sites were decreased by 27% in the limbic system but only 14% in the striatum. The responsiveness of adenylate cyclase to DA was reduced by 38% in the limbic system but was not modified in the striatum. Concomitant treatment with alpha-methyltyrosine (alpha-MPT) (50 mg/kg daily for 14 days) prevented the imipramine-induced reduction in both [3H]SCH 23390 binding sites and the responsiveness of adenylate cyclase to DA.

Haloperidol, but not clozapine, produces dramatic catalepsy in Δ9‐THC‐treated rats: possible clinical implications

The effect on rat catalepsy induced by Δ9‐tetrahydrocannabinol (Δ9‐THC) in association with haloperidol (HP) or clozapine (CLOZ) administration was investigated. Δ9‐THC dose‐dependently increased HP (0.05–1 mg kg−1, s.c.)‐induced rat catalepsy, while no catalepsy was observed after CLOZ (1–20 mg kg−1, s.c.) or Δ9‐THC+CLOZ administration. The CB1 antagonist SR141716A (0.5–5 mg kg−1, i.p.) reversed the increase mediated by Δ9‐THC on HP‐induced catalepsy. The D2 agonist quinpirole completely reversed the catalepsy induced by both HP and HP+Δ9‐THC; however, higher doses of quinpirole were needed in the presence of Δ9‐THC. The M1 antagonist scopolamine and α2 antagonist yohimbine were able to reduce the catalepsy induced by HP and HP+Δ9‐THC in a similar manner. CLOZ and the 5‐HT2A/2C antagonists ritanserin, RS102221 and SB242084 were more effective in antagonizing HP than HP+Δ9‐THC‐induced catalepsy. HP and CLOZ failed to inhibit in vitro [3H]CP‐55,940 binding, while Δ9‐THC and SR141716A did not show an appreciable affinity for the D2 receptor. It was suggested that the different effects on rat catalepsy induced by Δ9‐THC following HP or CLOZ administration may depend on the receptor‐binding profiles of the two antipsychotics. The preferential use of CLOZ rather than HP in the treatment of psychotic symptoms in cannabis abusers was discussed.

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.