Chromaffin cells: the peripheral brain.

Chromaffin cells probably are the most intensively studied of the neural crest derivates. They are closely related to the nervous system, share with neurons some fundamental mechanisms and thus were the ideal model to study the basic mechanisms of neurobiology for many years. The lessons we have learned from chromaffin cell biology as a peripheral model for the brain and brain diseases pertain more than ever to the cutting edge research in neurobiology. Here, we highlight how studying this cell model can help unravel the basic mechanisms of cell renewal and regeneration both in the central nervous system (CNS) and neuroendocrine tissue and also can help in designing new strategies for regenerative therapies of the CNS.

Effects of TNF-α and IL-1 ß on the activation of genes related to inflammatory, immune responses and cell death in immortalized human HaCat keratinocytes.

The present experiments were designed to characterize by microarray analysis the transcriptional responses of human keratinocytes (HaCat) to TNF-α and IL-1 ß, given alone or in combination, in order to better understand the mechanisms underlying inflammatory, immune responses and cell death in which both cytokines play a pathophysiological role. Significant differences in the percentage and quality of genes dysregulated by TNF-α and IL-1 ß were shown. Both cytokines activated a series of genes involved in inflammatory, immune response as well as in cell death. In our experimental conditions, TNF-α, in contrast to IL-1 ß, did not induce a significant level of apoptosis in keratinocytes. However, given together both cytokines produced a significant decrease in apoptotic cells and synergistic transcriptional response which was due to the activation of several specific genes occurring after application of each cytokine. TNF-α and IL-1 ß evoked apoptotic effect and transcriptional responses were linked to the stimulation of their specific receptors since a pre-treatment with monoclonal antibodies vs TNF-α and/or IL-1 ß receptors was able to significantly reduce them.

Neuroprotective effect of hydrogen peroxide on an in vitro model of brain ischaemia.

BACKGROUND AND PURPOSE: Reactive oxygen species (ROS) have been postulated to play a crucial role in the pathogenesis of ischaemia-reperfusion injury. Among these, hydrogen peroxide (H(2)O(2)) is known to be a toxic compound responsible for free-radical-dependent neuronal damage. In recent years, however, the 'bad reputation' of H(2)O(2) and other ROS molecules has changed. The aim of this study was to assess the protective role of H(2)O(2) and modification in its endogenous production on the electrophysiological and morphological changes induced by oxygen/glucose deprivation (OGD) on CA1 hippocampal neurons. EXPERIMENTAL APPROACH: Neuroprotective effects of exogenous and endogenous H(2)O(2) were determined using extracellular electrophysiological recordings of field excitatory post synaptic potentials (fEPSPs) and morphological studies in a hippocampal slice preparation. In vitro OGD was delivered by switching to an artificial cerebrospinal fluid solution with no glucose and with oxygen replaced by nitrogen. KEY RESULTS: Neuroprotection against in vitro OGD was observed in slices treated with H(2)O(2) (3 mM). The rescuing action of H(2)O(2) was mediated by catalase as pre-treatment with the catalase inhibitor 3-amino-1,2,4-triazole blocked this effect. More interestingly, we showed that an increase of the endogenous levels of H(2)O(2), due to a combination of an inhibitor of the glutathione peroxidase enzyme and addition of Cu,Zn-superoxide dismutase in the tissue bath, prevented the OGD-induced irreversible depression of fEPSPs. CONCLUSIONS AND IMPLICATIONS: Taken together, our results suggest new possible strategies to lessen the damage produced by a transient brain ischaemia by increasing the endogenous tissue level of H(2)O(2).

Is interleukin 2 a neuromodulator in the brain?

A bidirectional flow of information exists between the CNS and the neuroendocrine and immune systems, representing an important homeostatic mechanism in the body. Lymphokines and other products of immunocompetent cells seem to play a crucial role in this communication and seem to exert powerful effects on neurones in the brain. In this article, Giuseppe Nisticò and Giovambattista De Sarro describe the central effects following interleukin 2 (IL-2) microinfusion into several areas of the rat brain. The locus coeruleus seems to be the main site in the brain through which IL-2 exerts soporific effects. In addition, the possible transducing mechanisms coupling IL-2 receptor stimulation and the electroencephalogram (EEG) spectrum power responses elicited from the locus coeruleus seem to involve stimulation of specific receptors coupled to adenylate cyclase through a Gi protein.

Tetanus toxin as a neurobiological tool to study mechanisms of neuronal cell death in the mammalian brain.

Tetanus toxin is a potent clostridial neurotoxin responsible for causing spastic paralysis in humans, often accompanied by seizures and death. The tetanic syndrome is believed to originate from a disinhibitory action of the toxin in the CNS. To produce its effects, tetanus toxin undergoes retrograde, intra-axonal transport to the CNS, where it blocks preferentially the release of gamma-aminobutyric acid and glycine, two inhibitory neurotransmitters. These effects stem from the cleavage of synaptobrevin, a constitutive small-vesicle protein, by tetanus toxin, whose zinc-dependent metalloprotease characteristics recently have been recognized. Blockade of inhibitory transmission produces a predominance of excitatory amino acid neurotransmission, which is responsible for the neurodegenerative effect caused by tetanus toxin after intrahippocampal injection in rats. In fact, hippocampal damage can effectively be prevented by reduction of glutamate-mediated excitatory transmission, thus suggesting that unopposed excitation may be the underlying mechanism for neuronal cell death.

NGF restores decrease in catalase activity and increases superoxide dismutase and glutathione peroxidase activity in the brain of aged rats.

The effects of ageing on the activity of copper-zinc superoxide dismutase (SOD), selenium-dependent and independent glutathione peroxidase (GSH-Px) and catalase in several areas of the brain in 3-, 12-, and 24-month-old rats were studied. In addition, the effects of a subacute intracerebroventricular treatment of NGF (1 microgram daily for 28 consecutive days) on SOD, GSH-Px, and catalase activity in the same areas of the brain were assessed. The effects of ageing on the activities of antioxidant enzymes varied considerably in the different brain areas studied. Copper-zinc SOD was alone in being unaffected by ageing. Intraventricular infusion of NGF significantly increased SOD activity in the prefrontal cortex, hypothalamus, caudate nucleus, and mesencephalon of 24-month-old rats. Selenium-dependent GSH-Px activity did not significantly change in 12-month-old rats but it increased in the lower brain stem of 24-month-old animals. In comparison to vehicle-treated rats, NGF significantly increased selenium-dependent GSH-Px activity in all brain areas studied in 12- and 24-month-old rats. Catalase activity decreased significantly in the majority of the brain areas studied in 12- and 24-month-old rats. NGF completely restored the fall in catalase activity in 12- and 24-month-old animals to levels similar to those occurring in young rats. In conclusion, the present experiments show, for the first time, that long-term intraventricular administration of NGF significantly increases in old animals the activity of key enzymes involved in the metabolic degradation of superoxide radicals and hydrogen peroxide.

Age-related changes in Cu,Zn superoxide dismutase, Se-dependent and -independent glutathione peroxidase and catalase activities in specific areas of rat brain.

Oxidative injury of tissues involves both accumulation of damage due to persistent oxidative stress and loss of the proper balance of antioxidative enzymes. These events may produce a faster rate of tissue senescence. In this regard, we have assayed the antioxidative enzyme activities (Cu,Zn superoxide dismutase, glutathione peroxidase and catalase), in various areas of rat brain (prefrontal cortex, parietal cortex, hippocampus, hypothalamus, caudate nucleus, mesencephalon and lower brain stem) for the age groups of 3, 6, 12, 24 months. The results obtained show that the levels of antioxidant enzyme activities differed considerably in the various brain parts studied. Furthermore, changes in the specific activities of superoxide dismutase, catalase, and glutathione peroxidase did not follow the same pattern as a function of aging. In particular, in prefrontal cortex and caudate nucleus, superoxide dismutase and glutathione peroxidase activities did not change, while catalase activity decreased. In parietal cortex and mesencephalon, superoxide dismutase and glutathione peroxidase activities increased, but the catalase activity decreased in parietal cortex and did not change in mesencephalon. In lower brain stem, the activities of glutathione peroxidase and catalase decreased in 3-12-month-old rats. The activity of glutathione peroxidase was increased in the hippocampus and was decreased in hypothalamus during aging. In this area the catalase activity was also significantly diminished.

Effects of tetanus toxin after intracerebral microinjection are antagonized by drugs enhancing GABAergic transmission in adult fowls.

In adult fowls (Gallus domesticus), the behavioural effects of small doses of tetanus toxin, after unilateral microinjection into the nucleus mesencephalicus profundus (NMP), homologous to the mammalian substantia nigra, were studied. A rich pattern of stereotyped movements, vocalization, ipsilateral head-neck rotation, wing abduction, occasional ipsilateral circling and escape responses from the box were observed; the intensity of such symptomatology was dose-related and fully reversible within approx. 4 hr. Pretreatment with sodium valproate (100 and 200 mg/kg, i.m.) or with ethanolamine-sulphate (EOS, 1.6 mumol into the NMP) completely prevented the effects of tetanus toxin. The present findings show that fowls can represent an ideal species for studying acute central effects of tetanus toxin and more interestingly that drugs enhancing GABAergic mechanisms are able to prevent the effects of tetanus toxin.

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.

Age-related alterations in cardiovascular responsiveness to clonidine infused into the nucleus tractus solitarii in rats.

The cardiovascular response to the unilateral injection of clonidine into the nucleus tractus solitarii in old compared to young rats was evaluated. In 3-month old rats clonidine (0.25, 0.5 and 1 microgram) injected into the nucleus tractus solitarii in anaesthetized rats produced a significant fall in blood pressure (BP) and a significant decrease in heart rate (HR). In contrast, in 12 month old rats the maximum fall in blood pressure and heart rate was significantly less than in young animals. In addition, in older rats (24 month old) clonidine, at the same or larger doses given into the nucleus tractus solitarii did not produce any significant change in the cardiovascular parameters studied. In conclusion, the present experiments provide evidence that during ageing there is a progressive decrease in the cardiovascular response to alpha 2-adrenoceptor stimulation in the nucleus tractus solitarii. In addition, it is conceivable that such a decrease and subsequently the lack in response may be related to a progressive decrease in the number and/or affinity of the specific alpha 2-adrenoceptor binding sites at this level.

Behavioural and electrocortical changes induced by muscimol in rats withdrawn from chronic treatment with diazepam.

In rats withdrawn from a chronic treatment with diazepam, the effects of muscimol, given into the III cerebral ventricle, on behaviour and spectrum power of activity in the electrocorticogram (ECoG) were studied. In comparison to control rats which received only muscimol, in rats pretreated with diazepam (1 mg/kg/day for 30 consecutive days) the behavioural and ECoG effects of muscimol were significantly reduced or abolished. In fact, in rats pretreated with diazepam a small dose (50 ng) of muscimol did not affect behaviour or ECoG activity, in contrast to control animals in which the same dose produced, after a period of locomotor stimulation and ECoG desynchronization, typical and long-lasting behavioural sedation or sleep accompanied by a significant increase in total voltage power and in the lower frequency bands in the ECoG. In addition, larger doses (100 and 200 ng) of muscimol, which in control rats produced a typical biphasic pattern of ECoG and behavioural changes, i.e. an initial period of ECoG desynchronization and behavioural stimulation, followed by a second period of behavioural and ECoG sleep, in animals pretreated with diazepam, produced only an increase in total voltage power and in the lower frequency bands in the ECoG resembling the effects of the smaller (50 ng) dose. The present experiments suggest that, after chronic stimulation of benzodiazepine receptors a decrease in sensitivity of receptors for gamma-aminobutyric acid (GABA) occurs, since the effects of muscimol on behaviour and spectrum power were significantly reduced or abolished.

Intra-nigral infusion of Cu-free superoxide dismutase prevents paraquat-induced behavioural stimulation and ECoG epileptogenic discharges in rats.

In adult rats, with cannulae chronically implanted by a stereotactic instrument into the substantia nigra (pars compacta), the electrocortical (ECoG) and behavioural effects elicited by intranigral infusion of paraquat and the prevention of these effects by prior administration into the same site of different types of superoxide dismutase, were studied. Paraquat (50 micrograms) produced an intense pattern of behavioural stimulation, contralateral circling and repetitive discharges of high voltage ECoG spikes. The effects of paraquat were abolished in all of the animals pretreated into the same site with copper-free superoxide dismutase. Pretreatment with native Cu, Zn-superoxide dismutase prolonged significantly the latency of onset but did not prevent the behavioural stimulation and ECoG spikes evoked by paraquat. On the contrary, pretreatment with albumin or saline did not confer any protection against the neurotoxicological changes induced by paraquat. In conclusion, the present experiments showed that motor, ECoG and lethal effects of paraquat were completely prevented by Cu-free superoxide dismutase, suggesting that the central effects of this herbicide are in some way related to the release in the brain of copper and/or other transition metal ions.

High vulnerability of dentate granule cells to the neuropathological effects induced by intrahippocampal injection of tetanus toxin.

The behavioural and neuropathological effects of tetanus toxin, injected into the dentate gyrus, were studied in rats. The monolateral injection of a single dose (1000 mouse minimum lethal doses, MLDs; n = 14 rats) of tetanus toxin produced time-dependent behavioural stimulation. Wet-dog shakes and facial stereotypy were observed 3-4 days after the injection, culminating 4-5 days after treatment, in "limbic motor seizures". Ten days after injection, histological examination revealed death of dentate granule cells in the tetanus toxin-treated side but not in the contralateral, control side (treated with neutralized toxin). This effect was observed in all rats (n = 5) receiving tetanus toxin in the dentate gyrus and no damage was reported in other sectors (e.g. CA1 and CA3 pyramidal cell layers) of the hippocampus. Quantification of the neuronal damage yielded an approximately 70% reduction (P less than 0.01) in the number of granule cells in the toxin-injected dentate gyrus, compared with the control side. This was greater than that previously reported (30% reduction) in the CA1 pyramidal cell layer of rats receiving the same dose of toxin, into the regio superior of the hippocampus. In conclusion, the present experiments have shown that the focal injection of tetanus toxin into the dentate gyrus produced behavioural excitation and selective death of dentate granule cells.

Epileptogenic activity of some beta-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.

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.

Antagonists of N-methyl-D-aspartate receptors block seizures induced by putrescine in the deep prepiriform cortex.

The role of excitatory amino acid receptors in the genesis of motor and electrocortical seizures, elicited by administration of the polyamine putrescine into the deep prepiriform cortex, has been evaluated in rats. Motor and electrocortical seizures occurred in rats receiving unilateral local injections into the deep prepiriform cortex, of putrescine (10 or 20 nmol). The selective N-methyl-D-aspartate receptor antagonist, 2-amino-7-phosphonoheptanoate (AP7), injected previously (15 min) into the deep prepiriform cortex, prevented the development of seizures induced by putrescine, injected at the same site. In addition, dizocilpine (MK-801), a non-selective NMDA antagonist or ifenprodil, a specific inhibitor of the polyamine site at the NMDA receptor, when injected into the deep prepiriform cortex, 15 min prior to putrescine, significantly protected against seizures elicited by this polyamine. A subconvulsant dose of putrescine (5 nmol) potentiated the convulsant effects of NMDA, when injected into the deep prepiriform cortex. These data indicate a potential role of polyamines in the genesis of seizures, elicited from the deep prepiriform cortex. They further suggest that activation of the polyamine site, located at excitatory amino acid NMDA receptors, within the deep prepiriform cortex, may contribute to the genesis of seizure activity in this area.

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.

Inhibition by pertussis toxin of the soporific effects induced by stimulation of dopamine D2 autoreceptors in the ventral tegmental area in rats.

The behavioural and ECoG spectrum power effects of agonists at dopamine D2 autoreceptors, both after systemic or intracerebral administration, were studied in rats. It was shown that the bilateral injection of apomorphine or (+) 3PPP (0.1, 0.5 and 1.0 nmol for each compound) into the ventral tegmental area produced behavioural and ECoG sleep, accompanied by a statistically-significant increase in ECoG total spectrum power. These effects were completely antagonized by a pretreatment (24, 48 or 72 hr before) with pertussis toxin (0.34 and 3.4 micrograms), given into the same site. Similarly, behavioural sleep and an increase in ECoG total voltage power, produced by systemic administration of apomorphine (263 nmol/kg i.p.), were abolished by pertussis toxin (3.4 micrograms) injected bilaterally into the ventral tegmental area 24, 48 or 72 hr before. In conclusion, the present results suggest that behavioural and ECoG spectrum power effects, triggered by stimulation of dopamine D2 autoreceptors in the ventral tegmental area of rats, seem to be linked to the inhibition of adenylate cyclase activity through a Gi protein and or to other biochemical events linked to Gi proteins.

Age-dependent ECoG spectrum power alterations are reduced by phosphatidylserine in rats.

The present experiments were carried out in order to characterize the changes in ECoG spectrum power occurring in old compared with young rats. In addition, it was planned to ascertain whether chronic treatment with phosphatidylserine affected possible ECoG changes occurring in aged animals. In comparison to 3 months-old rats, the ECoG activity of 13-15 months-old rats showed spontaneous single or bursts of monophasic and biphasic spikes. In addition, spectrum analysis revealed a significant increase in the power of the lowest frequency band (0.25-3 Hz), accompanied by a decrease in 6-9, 9-12 and 12-16 Hz power. Chronic oral or parenteral treatment with phosphatidylserine gradually reversed the changes in ECoG spectrum power occurring in old rats and produced, at the ECoG level, a distribution pattern of single frequency, bands similar to that observed in young animals.

Behavioural and electrocortical effects after injection of two ornithine decarboxylase inhibitors into several areas of the brain in the rat.

The behavioural and electrocortical effects of alpha-methylornithine and alpha-difluoromethylornithine, two ornithine decarboxylase inhibitors, were evaluated after their infusion into several areas of the brain in the rat. Both compounds induced, in dose-dependent manner, similar epileptogenic effects, stereotyped behaviour and postural asymmetry, depending upon the site of injection. Unilateral injection of DFMO into the entopeduncular nucleus or the substantia nigra pars reticulata, caused an increase in locomotor activity. Unilateral injection of DFMO into the caudate nucleus or the substantia nigra pars compacta had no effect on locomotor activity. Among the areas of the brain studied, the most sensitive site from which electrocortical epileptogenic disorders were evoked, was the hippocampus. The GABA agonist muscimol, infused prior to DFMO, resulted in an anticonvulsant action, while DFMO, infused prior to putrescine, increased the convulsant properties of putrescine. In conclusion, these results indicate that DFMO and alpha-methylornithine produced epileptogenic effects after their intracerebral infusion and suggest that may act as analogues of putrescine.