Rossella Dall'Olio

Structural, neurochemical and behavioural consequences of neonatal blockade of NMDA receptor through chronic treatment with CGP 39551 or MK-801

Recent evidence suggests that NMDA receptors may be involved in survival of neurons and establishment of correct connectivity during development. We have treated rat pups from postnatal day 1 to 22 with daily s.c. injections of a competitive (CGP 39551) and a non-competitive (MK-801) antagonist of the NMDA receptor. Body weight of treated rats was decreased by 50-65% at postnatal day 24 and by 25-32% at 70 days of age. Brain weight was decreased by 16-24% at both ages. Among the different brain regions, the cerebellum and striatum appeared more decreased in size than the cortex and hippocampus. Only few minor, and in some cases transient, differences were measured in the cerebellum, the hippocampus and the cortex for a battery of neurochemical markers related to cholinergic, GABAergic and glutamatergic transmission as well as to astrocyte and oligodendrocyte activity. When tested in actometric cages from postnatal days 28 to 60, treated rats exhibited a dramatic increase of spontaneous locomotor activity which was maximal in 28-day-old animals (380% and 250% of control values in CGP 39551 and MK-801 groups, respectively) and was still significant at 60 days of age. Therefore, long-lasting alteration of motor behaviour is obtained by the schedule of chronic treatment adopted for the present experiments. Our results suggest that blockade of NMDA receptors during the critical period of brain maturation may result in permanent alteration of neural circuits.

Effect of chronic treatment with dizocilpine (MK-801) on the behavioral response to dopamine receptor agonists in the rat

The D2 or D1 dopamine receptor blockers (−)-sulpiride or SCH 23390 antagonized, in a dose dependent manner, the hypermotility induced by the N-methyl-d-aspartate (NMDA) receptor antagonist, MK-801 (0.25 mg/kg IP). MK-801 induced hyperactivity was not detected when rats were observed on days 7, 14 or 21 of 21 daily injections of MK-801. This lack of hyperactivity was also noted 5 days after the last administration of the repeated treatment with MK-801. The hypermotility induced by the D2 dopamine receptor agonist LY 171555 (0.3 mg/kg IP) was reduced 5 days following repeated treatment (21 days) with MK-801, while no change in the behavioral responses to the selective D1 agonist, SKF 38393, or the mixed D1/D2 agent apomorphine was detected. The results, although suggesting the involvement of dopaminergic pathways in the behavioral effect of MK-801, are conflicting with regard to the underlying mechanisms and to the adaptive changes of dopaminergic system following repeated NMDA receptor blockade.

Time course of rat motility response to apomorphine: A simple model for studying preferential blockade of brain dopamine receptors mediating sedation

The present work proposes a simple behavioral method for studying the ability of certain neuroleptics to block preferentially dopamine receptors subserving sedation. The model is based on the temporally biphasic motor response induced in rats by a single critical dose of apomorphine. It was chosen from a preliminary apomorphine dose-response study which showed that the same doses between 6.25 and 625 μg/kg affected rat motility differently according to whether the animals were “naive” or “familiarized” to the apparatus for 90 min before administering the drug. When the motility response of naive rats to 300 μg/kg of apomorphine was recorded immediately after SC injection, an initial (1–5 min) inhibition and a subsequent (20–45 min) stimulation of motility were obtained. (-)-Sulpiride (1.25–50 mg/kg) was found to be approximately 6-fold more effective in counteracting the apomorphine inhibition than stimulation of locomotion. Haloperidol (0.005–0.1 mg/kg) incompletely antagonized apomorphine inhibition and markedly blocked stimulation, which suggests that it has no preferential activity on dopamine receptors subserving sedation. The results were in accordance with those obtained by other authors with different paradigms, and indicated that the time course of the rat motility response to a single dose of apomorphine may constitute a useful model for detecting selective influences on different dopamine receptors.

Changes in behavioural responses to the combined administration of D1 and D2 dopamine agonists in normosensitive and D1 supersensitive rats

The selective D1 receptor stimulant SKF 38393 dose-dependently increased grooming time in rats without affecting locomotor activity or eliciting stereotyped behaviour. The selective D2 receptor agonist LY 171555 induced a dose-dependent increase in rat motility, a marked decrease in grooming time and a low occurrence of stereotyped behaviour. Concurrent administration of the two selective agonists induced high-degree stereotyped responses and reductions in locomotor and grooming behaviours. Rats withdrawn from repeated treatment with the selective D1 receptor blocker SCH 23390 (0.05 mg/kg twice daily for 21 days; 7 days of washout) did not exhibit any change of locomotor and grooming responses to threshold doses of LY 171555 and SKF 38393 given alone or in combination. On the contrary, a significantly greater occurrence of high-degree stereotyped responses to the combination of the two selective agonists was observed. The data support the view that D1 and D2 receptors have a cooperative role in the generation of stereotypies and suggest that D1 receptor supersensitivity needs D2 stimulation to be revealed.

Neurotoxicity of polyamines and pharmacological neuroprotection in cultures of rat cerebellar granule cells.

We have studied in a well-characterized in vitro neuronal system, cultures of cerebellar granule cells, the toxicity of polyamines endogenously present in the brain: spermine, spermidine, and putrescine. Twenty-four-hour exposure of mature (8 days in vitro) cultures to 1-500 microM spermine resulted in a dose-dependent death of granule cells, with the half-maximal effect being reached below 50 microM concentration. Putrescine was moderately toxic but only at 500 microM concentration. Spermidine was tested at 50 and 100 microM concentration and its toxicity was evaluated to be about 50% that of spermine. Neuronal death caused by spermine occurred, at least in part, by apoptosis. Spermine toxicity was completely prevented by competitive (CGP 39551) and noncompetitive (MK-801) antagonists of the NMDA receptor, but was unaffected by a non-NMDA antagonist (NBQX) or by antagonists of the polyamine site present on the NMDA receptor complex, such as ifenprodil. A partial protection from spermine toxicity was obtained through the simultaneous presence of free radical scavengers or through inhibition of the free radical-generating enzyme nitric oxide synthase, known to be partially effective against direct glutamate toxicity. The link between spermine toxicity and glutamate was further strengthened by the fact that, under culture conditions in which glutamate toxicity was ineffective or much reduced, spermine toxicity was absent or very much decreased. Exposure to spermine was accompanied by a progressive accumulation of glutamate in the medium of granule cell cultures. This was attributed to glutamate leaking out from dying or dead cells and was substantially prevented by the simultaneous presence of MK-801 or CGP 39551. The present results demonstrate that polyamines are toxic to granule cells in culture and that this toxicity is mediated through the NMDA receptor by interaction of exogenously added polyamines with endogenous glutamate released by neurons in the medium. The involvement of brain polyamines, in particular spermine and spermidine, in excitotoxic neuronal death is strongly supported by our present results.

Responses to selective D-1 and D-2 agonists after repeated treatment with selective D-1 and D-2 antagonists

This study was aimed at achieving a better understanding of the functional role of D-1 and D-2 receptors in some dopamine-mediated behaviors. Hypermotility, grooming behavior and stereotyped behavior were induced, respectively, by LY 171555 (D-2 agonist), SKF 38393 (D-1 agonist) and apomorphine (mixed agonist). Acute pretreatment either with the D-1 selective antagonist SCH 23390 (0.02 mg/kg) or with the D-2 receptor blocker YM 09151-2 (0.02 mg/kg, IP) blocked all these behaviors, suggesting the existence of functional interactions between D-1 and D-2 receptors. Striatal membranes prepared from rats receiving repeated administrations with SCH 23390 (0.05 mg/kg, twice daily for 21 days) showed an increase in the number of D-1 but not of D-2 receptors. On the contrary the repeated treatments with YM 09151-2 increased only the Bmax values of D-2 receptors. While the D-1 supersensitive rats showed only enhancement of apomorphine-induced stereotyped behavior, the D-2 supersensitive rats exhibited an increase of both apomorphine-elicited stereotypy and LY 171555-elicited hypermotility. SKF 38393-induced grooming was unaffected by any pretreatments. Moreover when D-2 supersensitive rats were acutely pretreated with SCH 23390, the enhancement of apomorphine-induced stereotyped behavior was abolished. It is concluded that the behavioral expression of D-1 receptor supersensitivity requires the simultaneous activation of D-1 and D-2 receptors.

Long-lasting tolerance to alcohol following a history of dependence.

Tolerance to alcohol effects is one of the defining features of clinical alcohol dependence. Here, we hypothesized that the post‐dependent state may include tolerance to sedative‐hypnotic alcohol actions. To address this question, we used a recently developed animal model in which repeated cycles of alcohol intoxication and withdrawal trigger long‐lasting behavioral plasticity. This animal model shares important features with the clinical condition. Animals were exposed to 7 weeks of intermittent alcohol vapor, allowed to recover for 3 weeks, and tested in protracted abstinence to exclude contributions from acute withdrawal. Post‐dependent and control rats were injected with a hypnotic dose of alcohol (3 g/kg), and the loss of righting reflex (LORR) was recorded, blood alcohol levels were monitored, and the elimination rate was calculated. Post‐dependent animals showed a decrease in LORR. Alcohol metabolism and elimination kinetics did not differ between groups. In conclusion, a history of alcohol dependence induces long‐lasting hypnotic tolerance. This process may play an important role in maintaining the dependent state.

The non-competitive NMDA receptor blocker dizocilpine potentiates serotonergic function.

Forepaw treading induced in rats by the 5HT1A agonist 8-OH-DPAT, and head shakes caused by the administration of the 5HT2A receptor against DOI, and by the 5HT precursor (-)5HTP, were significantly increased by pretreatment with the non-competitive N-methyl-D-aspartate (NMDA) antagonist dizocilpine. Dizocilpine administration also significantly increased the locomotor activity induced by the serotonin agonists. The competitive NMDA receptor antagonist CGP 43487 increased only the head shakes induced by DOI, but did not alter the behavior elicited by 8-OH-DPAT, or (-)5HTP, and did not modify locomotor responses to any of the agonists used. The dizocilpine-induced potentiation of head shakes elicited by DOI and (-)5HTP was inhibited by the 5HT2 agonist ketanserin, but was not modified by the selective dopamine D1 and D2 receptor blockers SCH 23390 and (-)sulpiride. The dopamine receptor antagonists did, however, counteract the dizocilpine facilitation of both forepaw treading induced by 8-OH-DPAT, and the locomotor response to all the serotonergic agonists. The results indicate that, unlike competitive NMDA receptor antagonists, the non-competitive antagonists enhanced the expression of serotonergic stimulation, and suggest that a glutamate deficiency could contribute to the pathogenesis of schizophrenia, not only through dopaminergic, but also through serotonergic, hyperactivity.

The NMDA positive modulator D-cycloserine potentiates the neuroleptic activity of D1 and D2 dopamine receptor blockers in the rat

According to the view that N-methyl-d-aspartate (NMDA) agonists could be seen as putative therapeutic agents in schizophrenia, the present study was aimed at investigating whether the NMDA positive modulatord-cycloserine (DCS) could show neuroleptic activity. When given alone, DCS (1.5, 3, 6, 12 mg/kg) failed to affect the stereotyped behavior induced by 0.5 mg/kg SC apomorphine, a test routinely used to detect neuroleptic activity. Nevertheless, the administration of different doses of DCS (1.5, 3, 6 mg/kg) in combination with the D1 dopamine receptor blocker SCH 23390 or the D2 antagonist YM 09151-2, both given in doses which by themselves were ineffective in blocking apomorphine elicited behavior, induced a dose- dependent neuroleptic effect. Furthermore, the positive NMDA modulator allowed (−)-sulpiride, which given alone never antagonized the apomorphine-induced stereotypy, to exhibit a full neuroleptic activity. The lower dose of DCS effective in potentiating antipsychotic effect of dopaminergic blockers also counteracted the behavioral response (hypermotility) induced by the NMDA negative modulator MK-801 (0.25 mg/kg), thus indicating the specificity of DCS effect. The results strengthen the view that drugs which increase NMDA receptor function could be a useful supplement in the therapy of psychotic disorders.

Chronic treatment with MK-801 decreases D2 dopamine receptor function in rat striatum

Present results show that a single treatment with dizocilpine (MK-801, 0.25 mg/kg IP) failed to modify the specific binding to D1 or D2 DA receptors. In contrast, repeated administrations for 3 weeks resulted in a statistically significant decrease of [3H]Spiroperidol binding to cortical or striatal membranes but did not change the number or the apparent affinity of [3H]MK-801 binding in well-washed cortical membranes. Consistent reduction in specific D2 receptor mediated behavior was obtained. The data suggest that the changes in DAergic function following repeated administrations with MK-801 could be suggestive of potential therapeutic uses of negative allosteric drugs in some DA related dysfunctions.