G. Nisticò

Evidence that locus coeruleus is the site where clonidine and drugs acting at α1- and α2-adrenoceptors affect sleep and arousal mechanisms

1 The behavioural and electrocortical (ECoG) effects of clonidine were studied after microinjection into the third cerebral ventricle, or microinfusion into some specific areas of the rat brain rich in noradrenaline‐containing cell bodies (locus coeruleus) or into areas receiving noradrenergic terminals (dorsal hippocampus, amygdaloid complex, thalamus, frontal and sensimotor cortex). 2 The ECoG effects were continuously analysed and quantified by means of a Berg‐Fourier analyser as total power and as power in preselected bands of frequency. 3 Clonidine (9.4 to 75 nmol) given into the third cerebral ventricle produced behavioural sedation and sleep and a dose‐dependent increase in ECoG total voltage power as well as in the lower frequency bands. Much lower doses were required to produce similar behavioural and ECoG spectrum power effects after either unilateral or bilateral microinfusion of clonidine into the locus coeruleus. 4 Doses of clonidine equimolar to those given into the third cerebral ventricle, were almost ineffective in inducing behavioural and ECoG sleep after their microinfusion into the dorsal hippocampus. In addition, a dose (0.56 nmol) of clonidine which, given into the locus coeruleus, produced marked behavioural sleep and ECoG synchronization, lacked effects when given into the ventral or anterior thalamus, into the amygdaloid complex or onto the frontal and sensimotor cortex. 5 The behavioural and ECoG spectrum power effects of clonidine given into the third cerebral ventricle or into the locus coeruleus were prevented by antagonists of α2‐adrenoceptors but not by α1‐adrenoceptor antagonists. 6 Intraventricular microinjection, or microinfusion into the locus coeruleus, of yohimbine, a selective α2‐adrenoceptor antagonist, produced behavioural arousal, increase in locomotor and exploratory activity, tachypnoea and ECoG desynchronization with a significant reduction in total voltage power. Similar stimulatory effects were also observed after microinjection of phentolamine into the same sites. 7 No significant effects on behaviour and ECoG activity were evoked after intraventricular injection or microinfusion into the locus coeruleus of prazosin or methoxamine.

Anticonvulsant effects of some calcium entry blockers in DBA/2 mice

1 The behavioural and anticonvulsant effects of several drugs acting by various mechanisms on calcium‐channels or affecting intracellular Ca2+ concentrations were studied after both systemic and intracerebroventricular administration in DBA/2 mice, a strain genetically susceptible to sound‐induced seizures. 2 The anticonvulsant effects were evaluated on seizures evoked by means of auditory stimulation (109 dB) in animals placed singly under a perspex dome. 3 Flunarizine and dihydropyridine derivatives, belonging to class I of calcium entry blockers, administered intraperitoneally, were the most potent compounds. 4 Diltiazem, a benzothiazepine derivative belonging to class III, and HA 1004, a calcium antagonist, acting by inhibiting Ca2+ mobilization from intracellular stores, injected intraperitoneally, were 3–7.6 fold and 5.8–10.7 fold less potent than flunarizine respectively. 5 Verapamil and methoxyverapamil, two phenylalkylamine derivatives, given intraperitoneally, were completely ineffective in preventing sound‐induced seizures in DBA/2 mice. In addition, high doses of verapamil and its methoxyderivative occasionally produced spontaneous tonic‐clonic seizures. 6 After intracerebroventricular administration of the hydrosoluble calcium entry blockers, belonging to different classes, the anticonvulsant effects were similar to those observed after systemic administration. 7 The systemic administration of Bay K 8644, a dihydropyridine analogue, having the ability to stimulate calcium entry into cells produced a dose‐dependent increase in clonic and tonic convulsions and other neurological side effects. 8 The present results strongly support the idea that some Ca2+ antagonists may be useful in human epilepsy.

Behavioural and ECoG spectrum changes induced by intracerebral infusion of interferons and interleukin 2 in rats are antagonized by naloxone

Rat interferon, alpha-interferon, interleukin 2 and recombinant interleukin-2 injected into the third cerebral ventricle produced typical behavioural sedation and/or sleep and ECoG synchronization in rats while beta-interferon produced no behavioural sleep or ECoG synchronization. A slight sedation was observed after the largest dose of beta-interferon only. During sleep induced by lymphokines, a dose-dependent increase in total voltage power as well as in the 0.5-3, 4-7 and 12-16 Hz frequent bands was observed. Much smaller doses were required to produce similar behavioural and ECoG spectrum effects after infusion of interferons and interleukin-2 into the locus coeruleus. No significant behavioural and ECoG changes were obtained after infusion of the same doses of interferons and interleukin-2 into other areas of the brain (caudate nucleus, dorsal hippocampus, substantia nigra pars compacta, ventromedial hypothalamus). The behavioural and ECoG effects of alpha-interferon, rat interferon and interleukin-2 were blocked in animals pretreated with naloxone. These results are consistent with the hypothesis that the behavioural and ECoG effects of these lymphokines are mediated at locus coeruleus level by stimulation of opiate receptors.

GABA of CNS origin in the rat anterior pituitary inhibits prolactin secretion.

Hypothalamic hormones synthesised in nuclei within the medial basal hypothalamus (MBH) and extrahypothalamic regions and released from nerve endings at the level of the median eminence (ME) into the hypophysial portal capillary system control anterior pituitary (AP) function1. The median eminence is a key area for neuroendocrine regulation as it receives projections of neurotransmitter pathways which modulate functions such as synthesis and release of hypothalamic hormones2. In addition, transmitters released from the ME into the stalk portal blood may act directly at the AP level to control hormone secretion—this has been demonstrated for dopamine (DA) with respect to prolactin (PRL) secretion3 and it has been suggested that DA itself may represent the so far elusive PRL-inhibiting factor (PIF)2,4,5. Recently, evidence has been presented which suggests that γ-aminobutyric acid (GABA) may also have an important inhibitory role in regulating PRL secretion in the rat. Both GABA and muscimol, a powerful agonist at GABA receptors6, inhibited PRL secretion when injected into urethane anaesthetised female7 or freely moving male rats8. In view of the poor penetrability of the blood-brain barrier (BBB) by both amino acids9 and the recently described specific GABA receptor sites in rat AP10, the above data support the notion that GABA may act directly at the level of the AP to inhibit PRL secretion. This proposition is reinforced by the demonstration that both GABA11 and muscimol12 inhibit PRL secretion from rat isolated APs, an effect which is blocked by the antagonist picrotoxin. We report here that GABA, derived from the central nervous sytem (CNS), is present in the rat anterior pituitary, and we provide evidence that GABA has a functional role in the control of PRL secretion.

Anticonvulsant properties of flunarizine on reflex and generalized models of epilepsy.

The anticonvulsant activity of 1-bis(4-fluorophenyl)methyl-4-(3-phenyl-2-propenyl)-piperazine, flunarizine, was studied after intraperitoneal administration in DBA/2 mice (seizures induced by sound), intravenous administration in Papio papio (myoclonus induced by photic stimulation) and oral administration in Wistar rats (seizures induced by cefazolin). Protection against sound-induced seizures was observed after intraperitoneal administration of flunarizine (5-40 mg/kg). The ED50 for suppression of tonic, clonic and wild running phases of sound-induced seizures was 3.3, 9.8 and 17.5 mg/kg, respectively. This protective action was significantly reduced by pretreatment with aminophylline (50 mg/kg, i.p.). In photosensitive baboons flunarizine (0.5-1.0 mg/kg, i.v.) provided partial protection against myoclonic responses to stroboscopic stimulation. After flunarizine (2 mg/kg, i.v.) this protection lasted for more than 5 hr (and was complete at 2-3 hr). Cefazolin-induced seizures in rats were prevented by administration of flunarizine (20-40 mg/kg, orally). The ED50 for the suppression of tonic and clonic seizures evoked by subsequent intravenous administration of cefazolin was 25 mg/kg. The protective effects of flunarizine (40 mg/kg, orally) were maximal after 3-6 hr and were maintained for 16-24 hr. Behavioural effects of flunarizine included signs of sedation in both mice and rats. Tolerance to the antiepileptic effects of flunarizine was not seen after chronic treatment in rats. The role of purinergic receptors and of calcium entry blockade in the anticonvulsant action of flunarizine requires further study.

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.

Epileptogenic activity of some β-lactam derivatives: Structure-activity relationship

The epileptogenic activity of several derivatives of beta-lactam was compared following their intracerebroventricular administration in rats. At a dose of 0.033 mumol/kg cefazolin was the most powerful epileptogenic compound among the drugs tested; dramatic seizure signs (nodding, clonic convulsions and sometimes escape responses) were observed repeatedly. It was approximately three times more potent than benzylpenicillin and other similar compounds, such as ceftriaxone, cefoperazone and cefamandole. No epileptogenic signs were observed with equimolar doses of cefotaxime, cefonicid and ceftizoxime. All these derivatives differ from the substitution at position 3 and at position 7 of 7-aminocephalosporanic acid. The more convulsant compounds (i.e. cefazolin and ceftezole) are both derivatives of tetrazol and show a marked similarity with pentylentetrazol. In addition, aztreonam, a compound having only the beta-lactam ring substituted with a heterocyclic ring at the 4 position, possessed convulsant properties like those of cefoperazone and cefamandole. This suggests that the beta-lactam ring is able to produce epileptogenic activity and it seems likely that substitutions at the 7-aminocephalosporanic or 6-aminopenicillanic acid may increase or reduce the epileptogenic properties of beta-lactam derivatives.

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.

Behavioural and neuropathological effects produced by tetanus toxin injected into the hippocampus of rats.

The behavioural effects of tetanus toxin, injected into the rostral hippocampus, have been studied in rats. A single dose (1000 mouse minimum lethal doses; n = 10) of the toxin produced tail rigidity, hunched back and sound- and touch-evoked stimuli, 48 hr after the injection in all rats treated and these culminated in generalized convulsions 5-7 days later. Seizures were also observed 4 days after the injection of 2000 MLDs (n = 10), whereas a dose of 500 MLDs (n = 10) was ineffective. Similarly, dose- and time-dependent lethal effects were observed. In comparison to the contralateral (untreated) hippocampus, tetanus toxin (1000 MLDs; n = 3) produced a statistically significantly reduction in the number of cells in the CA1 pyramidal cell layer of the injected side, 7 and 10 days after the injection. No changes were observed in other sectors (CA2 and CA3 areas) of the hippocampus. In conclusion, the present experiments have shown that the focal injection of tetanus toxin into the hippocampus produced dose- and time-dependent behavioural stimulation and lethal effects in rats.

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.