Carla Cannizzaro

Hampered long-term depression and thin spine loss in the nucleus accumbens of ethanol-dependent rats

Significance This paper examines the intimate neuroarchitecture of the nucleus accumbens shell region and how it affects synaptic plasticity in alcohol-dependent rats. To do so, a simultaneous morphometrical/immunofluorescence method was applied to visualize various types of dendritic spines and patch-clamp techniques to detect changes in synaptic currents. Using these tools, we show a selective loss of “long thin” spines accompanied by an impaired long-term depression (LTD) in alcohol-dependent rats. Dopaminergic and glutamatergic signaling are similarly altered. The results highlight the role of long thin dendritic spines in the genesis of LTD in alcohol dependence. Alcoholism involves long-term cognitive deficits, including memory impairment, resulting in substantial cost to society. Neuronal refinement and stabilization are hypothesized to confer resilience to poor decision making and addictive-like behaviors, such as excessive ethanol drinking and dependence. Accordingly, structural abnormalities are likely to contribute to synaptic dysfunctions that occur from suddenly ceasing the use of alcohol after chronic ingestion. Here we show that ethanol-dependent rats display a loss of dendritic spines in medium spiny neurons of the nucleus accumbens (Nacc) shell, accompanied by a reduction of tyrosine hydroxylase immunostaining and postsynaptic density 95-positive elements. Further analysis indicates that “long thin” but not “mushroom” spines are selectively affected. In addition, patch-clamp experiments from Nacc slices reveal that long-term depression (LTD) formation is hampered, with parallel changes in field potential recordings and reductions in NMDA-mediated synaptic currents. These changes are restricted to the withdrawal phase of ethanol dependence, suggesting their relevance in the genesis of signs and/or symptoms affecting ethanol withdrawal and thus the whole addictive cycle. Overall, these results highlight the key role of dynamic alterations in dendritic spines and their presynaptic afferents in the evolution of alcohol dependence. Furthermore, they suggest that the selective loss of long thin spines together with a reduced NMDA receptor function may affect learning. Disruption of this LTD could contribute to the rigid emotional and motivational state observed in alcohol dependence.

[3H]-DA release evoked by low pH medium and internal H+ accumulation in rat hypothalamic synaptosomes: involvement of calcium ions.

The pH fluctuations have been often interpreted as an insufficient regulation or as a consequence of the onset of pathological events, such as ischemia, in which a significant decrease in pH levels occurs. Neurotransmitter release appears to be affected by pH drop significantly. In this study, we investigated the effect of an extracellular and an intracellular acidification on tritiated dopamine release ([3H]-DA release), from superfused rat hypothalamic synaptosomes. When compared to basal release, extracellular acidification, due to a reduction in the external pH of the nominally carbonic-free superfusion media, provoked a significant increase in [3H]-DA release that showed a sensitiveness to calcium omission. Intraterminal acidification, obtained blocking the Na(+)/H(+) exchanger by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) and 5-(N,N-dimethyl)-amiloride (DMA), induced a significant increase in [3H]-DA outflow which occurred in a calcium-dependent manner (80% inhibition in absence of calcium from superfusion media). To further promote an intraterminal acidification through a H(+) inner accumulation, the proton ionophore nigericin was used. At every dose employed (10 microM), this compound induced a significant increase in [3H]-DA outflow, compared to basal release. Nigericin-evoked [3H]-DA release showed a 50% decrease when calcium was omitted from superfusion media. When BAPTA-AM, a chelator of intracellular calcium, was added, nigericin-evoked [3H]-DA was completely abolished. These data indicate that [3H]-DA release can be induced by extracellular acidification due to a lowering of external pH and by an intraterminal acidification due to an internal proton accumulation. The mechanism that can trigger this exocytotic process appears to depend on calcium presence, and in particular, on an increased intraterminal calcium availability.

Evidences of cannabinoids-induced modulation of paroxysmal events in an experimental model of partial epilepsy in the rat

The anticonvulsant effect of cannabinoids (CB) has been shown to be mediated by the activation of the CB(1) receptor. This study evaluates the anticonvulsant activity of (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo[1,2,3-de]-1,4-benzoxazin-6-Yl]-1-naphthalenylmethanone (WIN55,212-2, CB agonist) alone or preceded by the administration of N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251, selective CB(1) antagonist) in an experimental in vivo model of complex partial seizures (maximal dentate gyrus activation - MDA) in the rat. WIN55,212-2 (21mgkg(-1)) exerted an anticonvulsant effect, significantly reduced by the pre-treatment with AM251 (1mgkg(-1), 30 min interval). Surprisingly, AM251, administered alone at the same dose, failed to induce any modification in MDA responses. Our data suggest the involvement of the CB system in the inhibitory control of hyperexcitability phenomena in a model of acute partial epilepsy. Although the MDA model per se does not induce a basal activation of CB(1) receptors, as suggested by the lack of efficacy of AM251 when administered alone, the partial suppression of WIN55,212-2-induced effects in rats pre-treated with AM251 allows to hypothesise that the WIN55,212-2-induced antiepileptic effect is strictly linked to an increased CB(1) receptor activation or to the involvement of further receptor subtypes.

Long-lasting handling affects behavioural reactivity in adult rats of both sexes prenatally exposed to diazepam

Environmental stressors can substantially affect the adaptive response of rats to novelty in a sexually dimorphic manner. Gender-related differences are also observed in neurochemical and behavioural patterns of adult rats following prenatal exposure to diazepam (DZ). In the present study the behavioural reactivity to novelty is investigated in open field (OF) and in acoustic startle reflex (ASR) tests, in non handled (NH), short-lasting handled (SLH) and long-lasting handled (LLH) adult male and female rats prenatally exposed to DZ. A single daily s.c. injection of DZ (1.5 mg/kg) over gestation days 14-20 decreases GABA/BDZ receptor function in both sexes, as shown by the decreased electrographic hippocampal response to DZ and the increased response to picrotoxin, after intra-locus coeruleus injection of the two compounds. In OF NH DZ-exposed males display a lower total distance travelled (TDT), a higher rearing frequency (RF) and a greater number of transitions in the centre of the arena (CNT) compared to NH rats prenatally exposed to vehicle. Conversely, NH DZ-exposed females show slight changes in TDT and RF and a greater reduction in CNT and in the amount of time spent in the centre of the arena (CAT). These effects are associated with an increase in the peak amplitude of the ASR in both sexes. Short-lasting handling slightly influences DZ-evoked effects in animals of both sexes. In DZ-exposed males long-lasting handling attenuates the reduction in TDT and the enhancement in RF, prevents the increase in CNT and reduces the peak amplitude of ASR. In DZ-exposed females, long-lasting handling increases TDT and RF, induces a lower avoidance of the centre of the arena, and does not modify the peak amplitude of ASR, when compared to controls. These findings indicate that prenatal exposure to DZ differently affects behavioural reactivity in adult male and female rats, and suggest that a long-lasting handling is able to attenuate some behavioural deficits induced by prenatal DZ exposure.

Prevalence of headache in patients with Behçet's disease without overt neurological involvement

The aims of the present study were to evaluate the prevalence of headache and the frequency of different headache syndromes in patients with Behçets Disease (BD) without neurological involvement and to investigate the relationship with other clinical, and behavioural variables. Twenty-seven BD patients and 27 control subjects underwent a validated semistructured questionnaire based on the International Headache Society criteria. Levels of anxiety and depression, disease activity, and current medication were collected. Headache occurred in 88.9% of BD patients. There was no difference in the prevalence of the different headache syndromes between BD patients and controls. Only migraine without aura (MwA) was significantly more frequent in BD patients than controls (44.4% vs. 11.1%, respectively, P = 0.013). No relationship was found between MwA and clinical, and behavioural variables. Among headache syndromes, MwA showed the highest frequency in BD. A vascular or neuronal subclinical dysfunction could justify this association. A careful interview for migraine might be included in the diagnostic work-up of BD.

Presynaptic effects of anandamide and WIN55,212-2 on glutamatergic nerve endings isolated from rat hippocampus.

We examined the effects of the endocannabinoide-anandamide (AEA), the synthetic cannabinoid, WIN55,212-2, and the active phorbol ester, 4-beta-phorbol 12-myristate 13-acetate (4-beta-PMA), on the release of [(3)H]d-Aspartate ([(3)H]d-ASP) from rat hippocampal synaptosomes. Release was evoked with three different stimuli: (1) KCl-induced membrane depolarization, which activates voltage-dependent Ca(2+) channels and causes limited neurotransmitter exocytosis, presumably from ready-releasable vesicles docked in the active zone; (2) exposure to the Ca(2+) ionophore-A23187, which causes more extensive transmitter release, presumably from intracellular reserve vesicles; and (3) K(+) channel blockade by 4-aminopyridine (4-AP), which generates repetitive depolarization that stimulates release from both ready-releasable and reserve vesicles. AEA produced concentration-dependent inhibition of [(3)H]d-ASP release stimulated with 15 mM KCl (E(max)=47.4+/-2.8; EC(50)=0.8 microM) but potentiated the release induced by 4-AP (1mM) (+22.0+/-1.3% at 1 microM) and by A23187 (1 microM) (+98.0+/-5.9% at 1 microM). AEAs enhancement of the [(3)H]d-ASP release induced by the Ca(2+) ionophore was mimicked by 4-beta-PMA, which is known to activate protein kinase C (PKC), and the increases produced by both compounds were completely reversed by synaptosome treatment with staurosporine (1 microM), a potent PKC blocker. In contrast, WIN55,212-2 inhibited the release of [(3)H]d-ASP evoked by KCl (E(max)=47.1+/-2.8; EC(50)=0.9 microM) and that produced by 4-AP (-26.0+/-1.5% at 1 microM) and had no significant effect of the release induced by Ca(2+) ionophore treatment. AEA thus appears to exert a dual effect on hippocampal glutamatergic nerve terminals. It inhibits release from ready-releasable vesicles and potentiates the release observed during high-frequency stimulation, which also involves the reserve vesicles. The latter effect is mediated by PKC. These findings reveal novel effects of AEA on glutamatergic nerve terminals and demonstrate that the effects of endogenous and synthetic cannabinoids are not always identical.

Inhibition by anandamide and synthetic cannabimimetics of the release of [3H]D-aspartate and [3H]GABA from synaptosomes isolated from the rat hippocampus.

Cannabinoids (CB) can act as retrograde synaptic mediators of depolarization-induced suppression of inhibition or excitation in hippocampus. This mechanism may underlie the impairment of some cognitive processes produced by these compounds, including short-term memory formation in the hippocampus. In this study, we investigated several compounds known to interact with CB receptors, evaluating their effects on K+-evoked release of [3H]d-aspartate ([3H]d-ASP) and [3H]GABA from superfused synaptosomes isolated from the rat hippocampus. [3H]d-ASP and [3H]GABA release were inhibited to different degrees by the synthetic cannabinoids WIN 55,212-2; CP 55,940, and arachidonyl-2′-chloroethylamide/N-(2-chloroethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (ACEA), as well as by the endocannabinoids, anandamide (AEA), and 2-arachidonoylglycerol (2-AG). Both types of release were also inhibited by capsaicin. The inhibition produced by each of the cannabinoid compounds and capsaicin was unaffected by capsazepine or by the CB1-receptor antagonists AM-251 and SR141716A. The mechanism underlying AEA- and synthetic CB-induced inhibition of the release of [3H]GABA and [3H]d-ASP from rat hippocampal synaptosomes might not involve activation of presynaptic CB1 receptors.

Acetaldehyde Oral Self‐Administration: Evidence from the Operant‐Conflict Paradigm

BACKGROUND Acetaldehyde (ACD), ethanols first metabolite, has been reported to interact with the dopaminergic reward system, and with the neural circuits involved in stress response. Rats self-administer ACD directly into cerebral ventricles, and multiple intracerebroventricular infusions of ACD produce conditioned place preference. Self-administration has been largely employed to assess the reinforcing and addictive properties of most drugs of abuse. In particular, operant conditioning is a valid model to investigate drug-seeking and drug-taking behavior in rats. METHODS This study was aimed at the evaluation of (i) the motivational properties of oral ACD in the induction and maintenance of an operant-drinking behavior; (ii) ACD effect in a conflict situation employing the punishment-based Geller-Seifter procedure; and (iii) the onset of a relapse drinking behavior, following ACD deprivation. The lever-pressing procedure in a sound-attenuated operant-conditioning chamber was scheduled into 3 different periods: (i) training-rewarded responses with a fixed ratio 1; (ii) conflict-rewarded responses periodically associated with a 0.2 mA foot-shock; and (iii) relapse-rewarded lever presses following 1-week ACD abstinence. RESULTS Our results show that oral self-administrated ACD induced: a higher rate of punished responses in Geller-Seifter procedures; and the establishment of a relapse behavior following ACD deprivation. CONCLUSIONS In conclusion, our results indicate that ACD is able to induce an operant-drinking behavior, which is also maintained besides the conflict procedure and enhanced by the deprivation effect, supporting the hypothesis that ACD itself possesses motivational properties, such as alcohol and other substances of abuse.

Ethanol Modulates Corticotropin Releasing Hormone Release From the Rat Hypothalamus: Does Acetaldehyde Play a Role?

BACKGROUND AND METHODS Ethanol (EtOH) activates hypothalamic-pituitary-adrenal (HPA) axis, resulting in adrenocorticotropin hormone, glucocorticoid release, and in modifications of the response of the axis to other stressors. The initial site of EtOH action within the HPA system seems to be the hypothalamus. Thus, to determine the mechanisms responsible for these effects, we investigated: (i) whether EtOH was able to release corticotrophic releasing hormone (CRH) from incubated hypothalamic explants; (ii) whether acetaldehyde (ACD), its first metabolite formed in the brain by catalase activity, might play a role in EtOH activity. To this aim, rat hypothalamic explants were incubated with: (i) medium containing EtOH at 32.6 x 10(3) microM; (ii) different concentration of ACD (1, 3, 10, and 30 microM); (iii) EtOH plus 3amino-1,2,4-triazole (3AT, 32 x 10(3) microM) an inhibitor of cerebral catalase; (iv) ACD plus D-penicillamine (DP, 50.3 x 10(3) microM) an ACD-trapping agent. CRH levels were evaluated by a radioimmunoassay. RESULTS Incubation with EtOH induced a 7-fold increase in CRH secretion, with respect to basal levels; ACD was able to stimulate CRH release in a dose-dependent manner; the inhibition of cerebral catalase by 3AT blocked EtOH-induced CRH outflow; the inactivation of ACD by DP reverted the ACD-stimulating effect on CRH secretion. CONCLUSIONS These data show that both EtOH and acetaldehyde are able to increase hypothalamic CRH release from the rat hypothalamus and that acetaldehyde itself appears to be the mediator of EtOH activity.

Prenatal exposure to diazepam and alprazolam, but not to zolpidem, affects behavioural stress reactivity in handling-naïve and handling-habituated adult male rat progeny.

A gentle long-lasting handling produces persistent neurochemical and behavioural changes and attenuates the impairment in the behavioural reactivity to novelty induced by the prenatal exposure to diazepam (DZ) in adult male rat progeny. This study investigated the consequences of a late prenatal treatment with three GABA/BDZ R agonists (DZ) alprazolam (ALP) and zolpidem (ZOLP)), on different stress-related behavioural patterns, in non-handled (NH), short-lasting handled (SLH) and long-lasting handled (LLH) adult male rats exposed to forced swim test (FST), acoustic startle reflex (ASR) and Vogel test (VT). The effects on motor activity were evaluated in the open field and in the Skinner box. The seizure sensitivity to picrotoxin (PTX) was investigated as an index of the functional state of GABA/BDZ Rs. A single daily s.c. injection of DZ (1.25-2.50 mg/kg) and ALP (0.125-0.250 mg/kg) over gestational days 14-20 induced a decrease in immobility time in the FST in NH rats, no change in SLH rats and an increase in LLH rats; DZ induced an increase in the peak amplitude of the ASR in NH rats, no change in SLH rats and a reduction in LLH rats; ALP was ineffective in all groups. DZ and ALP reduced the number of punished licks in the VT in NH, SLH and LLH rats while the unpunished licks were not modified. DZ decreased locomotion and the lever pressing responses while ALP increased them. DZ and ALP increased the seizure sensitivity to PTX (2.5-4.0 mg/kg i.p.). These findings indicate a convergence on anxiety-related behaviours in the effects of prenatal exposure to DZ and ALP and a differentiation on motor activity. Long-lasting handling was able to overcompensate the increased behavioural stress reactivity induced by the prenatal exposure to DZ and ALP.