G. Bagetta

Tacrine-induced seizures and brain damage in LiCl-treated rats can be prevented by Nω-nitro-L-arginine methyl ester

The effects of tacrine (5 mg/kg i.p.), a potent acetylcholinesterase inhibitor, were studied in rats pretreated (24 h beforehand) with a single dose (12 mEq/kg i.p.) of LiCl. Tacrine and LiCl were ineffective when given individually. Tacrine elicited seizures and brain damage in 90% of the rats treated. The intracerebroventricular microinfusion of N omega-nitro-L-arginine methyl ester (300 micrograms given 24 h after LiCl administration) significantly reduced the seizures and brain damage produced by tacrine (given 15 min later). These experiments suggest that the seizures and brain damage elicited by tacrine may be due, in part, to increased nitric oxide production in the brain.

Evidence that the HIV-1 coat protein gp120 causes neuronal apoptosis in the neocortex of rat via a mechanism involving CXCR4 chemokine receptor.

The HIV-1 coat protein, gp120 (100 ng given intracerebroventricularly (i.c.v.) daily for seven consecutive days) causes DNA fragmentation in the brain neocortex of rat. In neocortical cells bearing ultrastructural features typical of apoptosis, electron microscopy revealed specific immunopositivity for neurofilament cytoskeletal proteins, suggesting the neuronal nature of dying cells. Neuronal apoptosis by gp120 implicates CXCR4 chemokine receptors; in fact, in rats receiving a single daily, non-neurotoxic, dose of SDF-1alpha (0.25 pmoles given i.c.v. for 7 days before gp120), the natural ligand of CXCR4 receptor, apoptosis was significantly hindered. The mechanism of SDF-1alpha protection involves inhibition of gp120-enhanced expression of IL-1beta, a cytokine implicated in the mechanisms of apoptosis induced by the viral protein in the neocortex of rat.

Neurotoxic Effects Induced by Intracerebral and Systemic Injection of Paraquat in Rats

1 The neurotoxic effects elicited by paraquat after systemic and intracerebral injection were studied in rats. 2 Intrahippocampal microinfusion of paraquat (0.1 μmol) produced behavioural stimulation and electrocortical (ECoG) excitation followed, at 24 h, by multifocal brain damage. Similarly, microinfusion of paraquat (0.2-0.4 μmol) into the locus coeruleus, substantia nigra or into the raphe nuclei, where noradrenergic, dopaminergic and serotonergic neurons are present, respectively, elicited potent excitotoxic effects (n=6 rats per dose and area). A lower dose (0.01 μmol) of the herbicide or injection of the vehicle (1.0 μl) did not produce any behavioural, ECoG or neurodegenerative effect. 3 After systemic administration, paraquat (20 mg kg-1 s.c.) evoked limbic motor seizures and ECoG epileptogenic discharges; in 10 out of 15 treated rats neuronal cell death was observed in the pyriform cortex, but not in other brain regions. A dose of 5 mg kg-1 was ineffective. 4 Among the regions of the brain studied, high concentrations of paraquat were detected in the pyriform cortex 24 h after systemic administration (5.0 and 20 mg kg -1 s.c.) lower levels being observed in the caudate nucleus. 5 In conclusion, paraquat, given systemically or intracerebrally in rats produces neurodegenerative effects.

Behavioural and electrocortical spectrum power effects of growth hormone releasing factor in rats

The effects on behaviour and electrocortical spectrum power of intracerebroventricular, intrahippocampal and intracaudate injections of human pancreatic growth hormone releasing Factor-40 (hpGRF) (10-100 ng) were studied in rats. The hpGRF, given into the third cerebral ventricle or into the dorsal hippocampus (50-100 ng), in freely-moving rats, produced behavioural sedation accompanied by electrocortical synchronization and an increase in the total voltage power with a predominant increase in the lower frequency bands. On the contrary, unilateral injection of hpGRF (75 ng) into the head of the caudate nucleus produced an increase in locomotor activity, marked postural changes, episodes of contralateral circling and an intense pattern of stereotyped movements. In all, these results indicate that, besides its specific endocrinological effects, hpGRF possesses, in small doses, marked behavioural and electrocortical actions, the mechanism(s) of which still remain to be elucidated.

Exploitation of the HIV-1 coat glycoprotein, gp120, in neurodegenerative studies in vivo

Neuronal loss has often been described at post‐mortem in the brain neocortex of patients suffering from AIDS. Neuroinvasive strains of HIV infect macrophages, microglial cells and multinucleated giant cells, but not neurones. Processing of the virus by cells of the myelomonocytic lineage yields viral products that, in conjunction with potentially neurotoxic molecules generated by the host, might initiate a complex network of events which lead neurones to death. In particular, the HIV‐1 coat glycoprotein, gp120, has been proposed as a likely aetiologic agent of the described neuronal loss because it causes death of neurones in culture. More recently, it has been shown that brain neocortical cell death is caused in rat by intracerebroventricular injection of a recombinant gp120 coat protein, and that this occurs via apoptosis. The latter observation broadens our knowledge in the pathophysiology of the reported neuronal cell loss and opens a new lane of experimental research for the development of novel therapeutic strategies to limit damage to the brain of patients suffering from HIV‐associated dementia.

Microinfusion of clonidine and yohimbine into locus coeruleus alters EEG power spectrum: effects of aging and reversal by phosphatidylserine

1 The behavioural and electrocortical (ECoG) power spectrum effects of clonidine, and yohimbine, an agonist and an antagonist at α2‐adrenoceptors, after their unilateral microinfusion into the rat locus coeruleus (LC) in young (50–70 days old) and old (13–15 months old) rats were studied. 2 Clonidine (0.09, 0.19, 0.28 and 0.56 nmol) microinfused into the LC of young rats induced dose‐dependent behavioural and ECoG slow wave sleep (SWS) with a significant increase in total voltage power and power in the lower frequency bands. In contrast, yohimbine (1.3 and 2.6 nmol) infused into the LC of young rats produced ECoG desynchronization and a significant decrease in total voltage power. 3 In contrast to young rats, clonidine (0.19 and 0.28 nmol) given into the LC did not affect behaviour and the ECoG power spectrum in old rats. However, after higher doses of clonidine (0.56 and 1.2 nmol) a small and short‐lasting period of behavioural and ECoG SWS was still evident. Similarly, in old rats yohimbine, at a dose (1.3 nmol) which was stimulative in young animals, did not significantly affect behaviour and ECoG power spectrum. Higher doses of yohimbine (2.6 and 5.2 nmol) were required to induce behavioural and ECoG changes similar to those observed with lower doses of yohimbine in young rats. 4 Chronic treatment with phosphatidylserine (30mgkg−1, orally, daily for 21 and 30 days), was able gradually to restore in old rats, in comparison with a vehicle‐treated group, the responsiveness of α2‐adrenoceptors to clonidine and yohimbine given into the LC.

Evidence that increases of mitochondrial immunoreactive IL‐1β by HIV‐1 gp120 implicate in situ cleavage of pro‐IL‐1β in the neocortex of rat

Immunoelectron microscopy analysis of brain tissue sections and rat‐specific sandwich ELISA allowed the localization of interleukin‐1β (IL‐1β) immunoreactivity in the mitochondria and cytosol of neocortical tissue preparations from the brain of naive, untreated, rats and rats receiving a single daily injection into one lateral cerebral ventricle (i.c.v.) of bovine serum albumin (BSA; 100u2003ng/day) for seven consecutive days. Interestingly, seven days i.c.v. treatment with the HIV‐1 coat protein gp120 (100u2003ng/day) enhances IL‐1β immunoreactivity in the cellular fractions studied. Elevation of mitochondrial immunoreactive IL‐1β levels seems to originate from the conversion operated by the interleukin converting enzyme (ICE) of mitochondrial pro‐IL‐1β; in fact, IL‐1β increases reported in the ELISA experiments were paralleled by a decrease of the mitochondrial pro‐IL‐1β 31‐kDa band in conjunction with enhanced expression of the p20 component of activated ICE. In conclusion, the present results demonstrate that gp120‐enhanced neocortical expression of IL‐1β originates, at least in part, from in situ cleavage of mitochondrial pro‐IL‐1β and suggest that this, together with the central role of the mitochondrion in the expression of programmed cell death, may be important for apoptosis induced by the viral coat protein in the brain of rats.

Systemic administration of lithium chloride and tacrine increases nitric oxide synthase activity in the hippocampus of rats

We planned to ascertain whether the administration of the anticholinesterase, tacrine (5 mg/kg i.p.), to rats pretreated 24 h before with lithium chloride (LiCl; 12 mEq/kg i.p.) produced any change in nitric oxide (NO) synthase activity in the hippocampus. A significant increase in hippocampal Ca(2+)-calmodulin-dependent NO synthase activity occurred 15 min after tacrine injection and was blocked by atropine (5 mg/kg i.p. given 15 min before tacrine) and by N omega-nitro-L-arginine methyl ester (300 micrograms given into one lateral cerebral ventricle 10 min before tacrine), a NO synthase inhibitor. A consistent cyclic guanosine 3,5-monophosphate (cGMP) accumulation was also seen. In conclusion, the present results show that tacrine given to LiCl-pretreated rats produces a significant increase in NO synthase activity in the hippocampus and this may be responsible, at least in part, for seizures and related brain damage elicited by these drugs.

Different profile of electrocortical power spectrum changes after micro-infusion into the locus coeruleus of selective agonists at various opioid receptor subtypes in rats

1 The effects of various opioid receptor agonists given directly by means of a chronically implanted cannula into the locus coeruleus (LC) on behaviour and ECoG activity, continuously analysed, and quantified as total power spectrum (0–16 Hz) and in preselected frequency bands (0–3; 3–6; 6–9; 9–12 and 12–16 Hz), were studied in rats. 2 Dermorphin (0.05, 0.5, 1, 2 and 5 pmol) and Tyr‐d‐Ala‐Gly‐N‐Me‐Phe‐Gly‐ol (DAMGO; 1, 10, 30, 100 pmol and 1 nmol), two typical μ‐receptor agonists, applied unilaterally or bilaterally directly into the LC, produced a typical dose‐dependent ECoG synchronization with a significant increase in total power spectrum as well as in the lower frequency bands. Dermorphin was found to be approximately 30 times more powerful than DAMGO in producing similar quantitative ECoG changes. 3 d‐Ala‐d‐Leu‐Thr‐Gly‐Gly‐Phe‐Leu (DADLE; 1, 10, 50 and 100 pmol), a selective δ‐receptor agonist, micro‐infused into the LC produced dose‐dependent behavioural soporific effects and ECoG increase in total power spectrum as well as in 3–6, 6–9, 9–12 Hz frequency bands. In comparison to dermorphin, the ECoG power spectrum effects of DADLE were 10 fold less potent, whereas in comparison to DAMGO it was approximately 3 times more potent. A lower dose (0.1 pmol) was ineffective in changing behaviour and ECoG power spectrum. 4 The microinfusion into the LC of U 50, 488H, a selective κ‐opioid receptor agonist, (0.25, 1, 2.5, 5 and 10 pmol) produced a typical pattern characterized by a first short‐lasting (3–25 min) phase of behavioural arousal and ECoG desynchronization, followed by a longer lasting (20–130 min according to the dose) phase of behavioural sleep and ECoG synchronization. A lower dose (0.1 pmol) was ineffective in changing behaviour and ECoG power spectrum. 5 Dextromethorphan and ketamine, two selective agonists at σ‐receptors given into the LC (1, 5 and 10 pmol) induce behavioural arousal, increase in locomotor activity and an intense pattern of stereotyped movements. However, by increasing the dose of ketamine (50 and 100 pmol), marked sedation, postural changes and an increase in low frequency ECoG bands, sometimes associated with high amplitude fast frequency potentials, were observed. 6 Naloxone applied directly into the LC (1 and 2 pmol 15 min before) was able to prevent the behavioural and ECoG effects induced by dermorphin, DAMGO and DADLE. Higher doses of naloxone (10 pmol into the LC) were however, required to antagonize the behavioural and ECoG soporific effects induced by the κ‐receptor agonist U 50,488H. In contrast, naloxone (10 pmol into the LC) was unable to prevent or reduce the behavioural and ECoG effects induced by subsequent administration into the same site of dextromethorphan and ketamine. 7 In conclusion, the present experiments confirm that behavioural and ECoG effects elicited following stimulation of μ‐, δ‐, κ‐ and σ‐opioid receptors located in the LC are quite different. Activation of μ‐, δ‐and κ‐receptors induced sedative effects whereas dextromethorphan and ketamine, two σ‐receptor agonists, induced behavioural arousal and ECoG desynchronization. In addition, the present results strongly support the crucial role played by opioid mechanisms, in the locus coeruleus, in the mediation of the soporific effects of drugs acting as agonists at opioid receptors.

The effect of hydroxylation of linoleoyl amides on their cannabinomimetic properties

As yet, the physiological significance of hydroxylation of anandamide and linoleoyl amides is unknown. Therefore, we investigated whether hydroxylation of ODNHEtOH and ODNH2 influences their binding abilities to the CB‐1 receptor and whether it alters their reactivity towards a fatty acid amide hydrolase (FAAH) from rat brain. Neither the fatty acid amides nor their hydroxylated derivatives were able to displace the potent cannabinoid [3H]CP 55.940 from the CB‐1 receptor (K i>1 μM). Hydroxylation of ODNHEtOH resulted in a strong reduction of the maximum rate of hydrolysis by a FAAH, but the affinity of FAAH for the substrate remained of the same order of magnitude. Hydroxylation of ODNH2 led to a decrease in the affinity of FAAH for the substrate, but its maximum rate of conversion was unaffected. Furthermore, hydroxylation of ODNHEtOH enhanced its capacity to inhibit competitively the hydrolysis of anandamide. The resulting prolonged lifetime of anandamide and other fatty acid amide derivatives may have a considerable impact on cellular signal transduction.