The role of locus coeruleus in the antiepileptic activity induced by vagus nerve stimulation.

Stimulation of the vagus nerve produces antiepileptic effects. This is used clinically to treat drug-refractory epilepsies. The mechanisms responsible for these effects depend on the activation of vagal afferents reaching the nucleus of the solitary tract. This review focuses on the neuroanatomy of the nucleus of the solitary tract and its relation with the nucleus locus coeruleus as a preferential anatomical substrate in producing antiepileptic effects. In fact, following the transient or permanent inactivation of locus coeruleus neurons, some antiepileptic effects of vagus nerve stimulation are lost. The activation of locus coeruleus per se is known to limit the spread of a seizure and the duration of a variety of seizure types. This is due to the fine chemical neuroanatomy of norepinephrine pathways that arise from the locus coeruleus, which produce widespread changes in cortical areas. These changes may be sustained by norepinephrine alone, or in combination with its co-transmitters. In addition, vagus nerve stimulation may prevent seizures by activating the serotonin-containing dorsal raphe neurons.

Lithium delays progression of amyotrophic lateral sclerosis.

ALS is a devastating neurodegenerative disorder with no effective treatment. In the present study, we found that daily doses of lithium, leading to plasma levels ranging from 0.4 to 0.8 mEq/liter, delay disease progression in human patients affected by ALS. None of the patients treated with lithium died during the 15 months of the follow-up, and disease progression was markedly attenuated when compared with age-, disease duration-, and sex-matched control patients treated with riluzole for the same amount of time. In a parallel study on a genetic ALS animal model, the G93A mouse, we found a marked neuroprotection by lithium, which delayed disease onset and duration and augmented the life span. These effects were concomitant with activation of autophagy and an increase in the number of the mitochondria in motor neurons and suppressed reactive astrogliosis. Again, lithium reduced the slow necrosis characterized by mitochondrial vacuolization and increased the number of neurons counted in lamina VII that were severely affected in saline-treated G93A mice. After lithium administration in G93A mice, the number of these neurons was higher even when compared with saline-treated WT. All these mechanisms may contribute to the effects of lithium, and these results offer a promising perspective for the treatment of human patients affected by ALS.

Protein clearing pathways in ALS.

In the present review a large amount of experimental and clinical studies on ALS are discussed in an effort to dissect common pathogenic mechanisms which may provide novel information and potential therapeutic strategies for motor neuron degeneration.Protein clearing systems play a critical role in motor neuron survival during excitotoxic stress, aging and neurodegenerative disorders. Among various mechanisms which clear proteins from the cell recent studies indicate autophagy as the most prominent pathway to promote survival of motor neurons.Autophagy regulates the clearance of damaged mitochondria, endoplasmic reticulum and misfolded proteins in eukaryotic cells. Upon recruitment of the autophagy pathway, an autophagosome is produced and directed towards lysosomal degradation.Here we provide evidence that in both genetic and sporadic amyotrophic lateral sclerosis (ALS, the most common motor neuron disorder) a defect in the autophagy machinery is common. In fact, swollen, disrupted mitochondria and intracellular protein aggregates accumulate within affected motor neurons. These structures localize within double membrane vacuoles, autophagosomes, which typically cluster in perinuclear position. In keeping with this, when using autophagy inhibitors or suppressing autophagy promoting genes, motor symptoms and motor neuron death are accelerated. Conversely stimulation of autophagy alleviates motor neuron degeneration.Therefore, autophagy represents an important target when developing novel treatments in ALS.

Autophagy activation in glutamate-induced motor neuron loss.

Recent literature demonstrated that exposure to excitatory amino acid in specific experimental conditions might produce a defect in the autophagy pathway. Such an effect was observed in motor neurons exposed chronically to glutamate agonists. On the other hand, it is well known that glutamate induces motor neuron death and this is supposed to play a key role in the physiopathology of motor neuron loss in amyotrophic lateral sclerosis (ALS). Similarly, a defective recruitment of autophagy was recently documented in ALS. In the present study we found that exposure of motor neurons to kainic acid produces intracellular changes associated with defective autophagy. In this experimental conditions, pharmacological activation of autophagy rescues the loss of motor neurons.

A systematic study of brainstem motor nuclei in a mouse model of ALS, the effects of lithium.

Transgenic mice expressing the human superoxide dismutase 1 (SOD-1) mutant at position 93 (G93A) develop a phenotype resembling amyotrophic lateral sclerosis (ALS). In fact, G93A mice develop progressive motor deficits which finally lead to motor palsy and death. This is due to the progressive degeneration of motor neurons in the ventral horn of the spinal cord. Although a similar loss is reported for specific cranial motor nuclei, only a few studies so far investigated degeneration in a few brainstem nuclei. We recently reported that chronic lithium administration delays onset and duration of the disease, while reducing degeneration of spinal motor neuron. In the present study, we extended this investigation to all somatic motor nuclei of the brain stem in the G93A mice and we evaluated whether analogous protective effects induced by lithium in the spinal cord were present at the brain stem level. We found that all motor but the oculomotor nuclei were markedly degenerated in G93A mice, and chronic treatment with lithium significantly attenuated neurodegeneration in the trigeminal, facial, ambiguus, and hypoglossal nuclei. Moreover, in the hypoglossal nucleus, we found that recurrent collaterals were markedly lost in G93A mice while they were rescued by chronic lithium administration.

Intracellular pathways underlying the effects of lithium.

This is a short overview focusing on the biochemical interactions underlying the protective effects of lithium at the neuronal level. These include lithium modulation of autophagy, growth factors, excitotoxicity, and a variety of mechanisms underlying cell death, neurogenesis, and neuronal differentiation. All these effects represent the result of a multifaceted pharmacology, which is becoming more and more complex. Nonetheless, when trying to dissect the various mechanisms of action of lithium, two primary targets emerge: glycogen synthase kinase 3beta and phosphatidylinositol phosphatase. The numerous lithium effects on biochemical systems are placed downstream of these two main mechanisms. At several steps, these mechanisms interconnect to each other, thus making it difficult to keep distinct the biochemical cascades promoted by lithium. In this way, it is not surprising that, despite being described as different phenomena at the behavioral level, molecular mechanisms underlying the effects of lithium on mood, motor activity, and sensitization overlap with those responsible for neuroprotection and neurorestoration. It is likely that the ancestral role of this ion as a modulator of cell survival, cell growth, movement, and mood is the consequence of a few molecular mechanisms operating in different neuronal networks, where a variety of cascade events take place. This review is an attempt to elucidate the primary effects of lithium to interconnect the simpler targets to the most complex pharmacological effects.

Genetic or pharmacological blockade of noradrenaline synthesis enhances the neurochemical, behavioral, and neurotoxic effects of methamphetamine.

N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) lesions of the locus coeruleus, the major brain noradrenergic nucleus, exacerbate the damage to nigrostriatal dopamine (DA) terminals caused by the psychostimulant methamphetamine (METH). However, because noradrenergic terminals contain other neuromodulators and the noradrenaline (NA) transporter, which may act as a neuroprotective buffer, it was unclear whether this enhancement of METH neurotoxicity was caused by the loss of noradrenergic innervation or the loss of NA itself. We addressed the specific role of NA by comparing the effects of METH in mice with noradrenergic lesions (DSP-4) and those with intact noradrenergic terminals but specifically lacking NA (genetic or acute pharmacological blockade of the NA biosynthetic enzyme dopamine beta-hydroxylase; DBH). We found that genetic deletion of DBH (DBH-/- mice) and acute treatment of wild-type mice with a DBH inhibitor (fusaric acid) recapitulated the effects of DSP-4 lesions on METH responses. All three methods of NA depletion enhanced striatal DA release, extracellular oxidative stress (as measured by in vivo microdialysis of DA and 2,3-dihydroxybenzoic acid), and behavioral stereotypies following repeated METH administration. These effects accompanied a worsening of the striatal DA neuron terminal damage and ultrastructural changes to medium spiny neurons. We conclude that NA itself is neuroprotective and plays a fundamental role in the sensitivity of striatal DA terminals to the neurochemical, behavioral, and neurotoxic effects of METH.

Activation of brain metabolism and fos during limbic seizures: the role of locus coeruleus.

The noradrenergic nucleus Locus Coeruleus (LC) densely innervates limbic structures. In rats, the damage to LC by the neurotoxin DSP-4, converts episodic limbic seizures induced by bicuculline infusion in the anterior piriform cortex (APC) into self-sustaining status epilepticus (SE). SE induced by this approach is similar to SE induced by co-infusing cyclothiazide and bicuculline into APC in rats bearing an intact LC. As opposed to other commonly used rat SE models (e.g. systemic kainate or pilocarpine), this approach allows one to analyze the effects of SE on brain regions which are solely due to spreading of seizure activity, rather than to direct effect of systemic chemoconvulsant. We evaluated the expression of Fos protein (an immediate early gene product), and the local cerebral metabolic rates for [14C] 2-deoxyglucose (lCMRglc), in rats following SE induced either by cyclothiazide+bicuculline or by DSP-4+bicuculline. We demonstrated that regional Fos expression after SE does not parallel the increase in lCMRglc, in LC-lesioned rats. In DSP-4+bicuculline rats there is an overall lower expression of the protein as compared with the cyclothiazide+bicuculline or bicuculline alone groups; even more, such a difference co-exists with an higher lCMRglc in the DSP-4+bicuculline-treated rats in some regions, as compared with the other groups. These data show that LC neurons play an important role in determining immediate early genes expression even in conditions of strong pathological activation, such as limbic SE. This might have relevant effects in the plastic mechanisms related with epileptogenesis.

Alpha-synuclein and autophagy as common steps in neurodegeneration.

The major component of Lewy bodies in Parkinson's disease (PD) is alpha-synuclein, which is considered as a substrate of the ubiquitin-proteasome (UP) system, although autophagy seems to be equally involved. Here we discuss the co-existence of alpha-synuclein and proteins belonging to the UP system within autophagic granules, further developing as neuronal inclusions. We hypothesize that, following slight insults, both UP and autophagy are induced; if toxic stimuli are prolonged, these pathways are overwhelmed and cell death occurs. We then indicate a protective role of autophagy in PD and suggest it as a therapeutic target to slow down the progression of the disease.

The "Parkinsonian heart": from novel vistas to advanced therapeutic approaches in Parkinson's disease.

The present manuscript reviews novel data on the progressive involvement of different regions of the central nervous system as well as peripheral nerves in Parkinson's disease. Most of these regions are involved in the regulation of the autonomic nervous system, and their damage is concomitant with the specific loss of sympathetic cardiac axon terminals. This causes a cardiovascular dysfunction, which occurs solely in Parkinsonian patients. In order to specify the peculiarity of this cardiovascular alteration we coined the term "Parkinsonian Heart". This is characterized by a severe loss of the physiological noradrenergic innervation and a slight impairment of central autonomic control and it is often characterized by drug-induced morpho-functional alterations. In fact, the current dopamine substitution therapy could make worse such an already abnormal heart. For instance, structure-activity studies on dopamine substitutive drugs report that dopamine agonists belonging to the class of ergot derivatives may produce, with a high frequency, valvular fibrosis in Parkinsonian patients. These effects recently became a major issue and led to consider all ergot dopamine agonists as dangerous for the treatment of Parkinson's disease. In the present review we re-describe the effects of dopamine agonist within the specific context of the Parkinsonian heart. In line with this, additional factors need to be considered: 1--The lack of noradrenergic innervation which might play a significant role in the fibrogenic mechanism. 2--The ergot structure per se, which is not sufficient, but it is rather the ability to act as agonist at 5HT(2B) or alpha-noradrenergic receptors to determine the fibrotic reaction. Therefore, we suggest that binding to these receptor subtypes, joined with the lack of endogenous noradrenergic innervation, might synergize to produce the cardiac fibrosis.

Noradrenaline in Parkinson's disease: from disease progression to current therapeutics.

The loss of the neurotransmitter noradrenaline occurs constantly in Parkinson's disease. This is supposed to worsen disease progression, either by increasing the vulnerability of dopamine-containing neurons or by reducing the recovery once they are damaged. Novel data also show that the loss of noradrenergic innervation facilitates the onset of dyskinesia occurring in Parkinsonian patients during dopamine replacement therapy. In the first part of the manuscript we review the evidence showing the loss of the noradrenergic system as an early event in the natural history of Parkinsonism. This evidence is discussed in light of novel reports showing the deleterious effects produced by the noradrenergic deficit on the survival of nigral dopamine neurons. In particular, we analyze the biochemical and morphological changes produced in the nigrostriatal system by the loss of endogenous noradrenaline. In a dedicated paragraph we specifically evaluate the cross affinity between dopamine and noradrenaline systems. In fact, this is critical during dopamine/noradrenaline replacement therapy in Parkinson's disease. In the last part, we overview novel therapeutic approaches aimed at restoring the activation of noradrenaline receptors to reduce the dyskinesia occurring in the treatment of Parkinson's disease.

Fine ultrastructure and biochemistry of PC12 cells: a comparative approach to understand neurotoxicity.

The PC12 cell line is commonly used as a tool to understand the biochemical mechanisms underlying the physiology and degeneration of central dopamine neurons. Despite the broad use of this cell line, there are a number of points differing between PC12 cells and dopamine neurons in vivo which are missed out when translating in vitro data into in vivo systems. This led us to compare the PC12 cells with central dopamine neurons, aiming at those features which are predictors of in vivo physiology and degeneration of central dopamine neurons. We carried out this comparison, either in baseline conditions, following releasing or neurotoxic stimuli (i.e. acute or chronic methamphetamine), to end up with therapeutic agents which are suspected to produce neurotoxicity (l-DOPA). Although the neurotransmitter pattern of PC12 cells is close to dopamine neurons, ultrastructural morphometry demonstrates that, in baseline conditions, PC12 cells possess very low vesicles density, which parallels low catecholamine levels. Again, compartmentalization of secretory elements in PC12 cells is already pronounced in baseline conditions, while it is only slightly affected following catecholamine-releasing stimuli. This low flexibility is caused by the low ability of PC12 cells to compensate for sustained catecholamine release, due both to non-sufficient dopamine synthesis and poor dopamine storage mechanisms. This contrasts markedly with dopamine-containing neurons in vivo lending substance to opposite findings between these compartments concerning the sensitivity to a number of neurotoxins.

The comet assay as a method of assessment of neurotoxicity: usefulness for drugs of abuse.

Comet assay is a quick and versatile method for assessing DNA damage in individual cells. It allows the detection of single and double DNA strand breaks, as well as the presence of alkali labile sites. DNA breaks may represent the direct effect of some damaging agent, or they may be intermediates in cellular repair. DNA strand breaks may also come from the action of free radicals generated by oxidative stress processes. The present article summarizes some data from our and other groups underlining the suitability of the Comet assay in assessing neurotoxicity and its potential in evaluating drugs of abuse-related genotoxicity.

Overexpression of alpha-synuclein following methamphetamine: is it good or bad?

alpha-Synuclein is a presynaptic protein involved in various degenerative disorders now defined as synucleinopathies. These include neurological diseases that share a few pathological features consisting of aggregates of both normal and altered alpha-synuclein within specific neuronal populations and/or glial cells. The prototype of synucleinopathies is represented by Parkinson's disease (PD) in which alpha-synuclein is identified as a constant component of neuronal pale eosinophilic inclusions: "the Lewy Bodies." In the present article, we discuss the potential significance of amphetamine-induced overexpression of alpha-synuclein in light of clinical findings showing neurodegeneration following overexpression of alpha-synuclein and recent experimental studies that measured increased expression of alpha-synuclein following amphetamine derivatives.

Inclusion dynamics in PC12 is comparable between amphetamines and MPTP.

In previous studies it was demonstrated that amphetamine derivatives and 1-methyl article-4-phenylpyridinium produce neuronal cell bodies. In the present work, we compared the fine ultrastructure of the intracellular inclusions induced by these different neurotoxic treatments. In particular, we compared the dynamical changes occurring when a mild toxic stimulus acts for different time intervals. For this purpose, we exposed catecholamine-synthesizing PC12 cells to different amphetamine derivatives (methamphetamine and 3,4-methylenedioxymethamphetamine), or 1-methyl-4-phenylpyridinium ion, which represents the active metabolite of the neurotoxin 1-methyl-4-phenyl-1,3,4,6-tetrahydropyridine. Despite inclusions that are elicited by different mechanisms depending on the specific neurotoxin, their ultrastructural features are similar and there is a high parallelism in their temporal evolution. This suggests that formation of inclusions is a multi-step process that might be elicited by different stimuli and, once triggered, leads to the same final effect.

MDMA and seizures: a dangerous liaison?

In the past decades, there was a massive increase in the abuse of methylenedioxymethamphetamine (MDMA) in the Western countries. Seizure onset after MDMA is considered to be related mainly to its acute systemic effects (e.g., hyponatremia and hyperthermia). However, additional mechanisms might concur to it as well. Experiments aimed at disclosing the basis for such an acute effect have the advantage of profiting of controlled conditions and the "pure" compounds, as opposed to the limits of clinical data which are biased by several confounding factors. Amphetamines exert profound effect on different monoaminergic systems, which might participate to lowering of seizure threshold. Chronic effects of MDMA abuse on seizure threshold have not been explored in detail so far. Recent data showed that in mice receiving small, repeated doses of MDMA, a persisting pro-convulsant effect toward limbic seizures and metabolic hyperexcitability can be observed. In the present article, we reviewed these studies and we report our preliminary experimental data documenting the lack of mossy fiber sprouting at short time intervals following MDMA, when seizure susceptibility is already present.

MDMA induces caspase-3 activation in the limbic system but not in striatum.

Several studies, carried out in chronic (+/-) 3,4-methylenedioxymethamphetamine (MDMA) abusers, have shown memory loss and cognitive impairment, as well as persistent electroencephalographic changes. This suggests that, at least in humans, forebrain areas, including the limbic system, might be altered by MDMA. Consistently, recent experimental evidences suggest that, in rodents, MDMA, besides effects on the basal ganglia, produces alterations in the hippocampus. Therefore, the aim of the present article was to investigate whether treatment with MDMA produces activation of the caspase-3 enzyme, which is part of an enzymatic pathway involved in cell death, within limbic areas (i.e., hippocampus, amygdala, and piriform cortex) and striatum. A marked induction of caspase-3 activity was demonstrated in the amygdala and hippocampus, although MDMA did not affect caspase-3 activity neither in the striatum nor in the frontal cortex. These data indicate that limbic structures possess a high sensitivity to MDMA with respect to the activation of at least one step in the apoptotic pathway. Potential implications and pitfalls of such an experimental observation are reported.

Comparison between heroin and heroin-cocaine polyabusers: a psychopathological study.

The concomitant use of cocaine by heroin-dependent subjects, or by patients on methadone maintenance treatment, is a relevant phenomenon that determines the negative consequences on health, social adjustment, and outcome of opioid addiction treatment. Little is known about the patterns of co-use of these two substances and the pathophysiological alterations following this condition. Only a few studies have evaluated the neurochemical effects in subjects carrying this specific pattern of abuse. Similarly, the impact of cocaine abuse on psychiatric and social function in subjects already affected by opioid addiction is still poorly understood and further studies are necessary to investigate this specific area that could profoundly affect methadone maintenance treatment. The aim of this article is to investigate the psychopathological symptoms of heroin-cocaine abuse in a group of heroin addicts applying for treatment. Results show a direct relationship between cocaine abuse and a higher rate of psychiatric disorders, but a negative correlation with the severity of self-rated psychopathology.

Morphological evidence that xenon neuroprotects against N-methyl-DL-aspartic acid-induced damage in the rat arcuate nucleus: a time-dependent study.

The hyperactivation of glutamate receptors, especially those of the N-methyl-d-aspartate subtype (NMDA), can induce excess calcium entry into cells, leading to neuronal death. Since the anesthetic gas xenon behaves as an NMDA antagonist, the present article investigated, by distinct morphological approaches and after different times, the possible neuroprotectant effects of this gas in a model of neuronal damage induced by N-methyl-dl-aspartic acid (NMA) on rat arcuate nucleus. Rats were assigned to the following groups: controls; xenon exposure; NMA treatment; or xenon exposure + NMA treatment. Animals were placed in an experimental cage and after 10 min a mixture of xenon (or nitrogen) 70% and oxygen 30% was delivered. After 3 h, 1, 2, 5, or 7 days from gas exposure, rats were euthanized and the whole brain was removed and processed for either transmission electron microscopy or light microscopy. In the arcuate nucleus from NMA-treated animals only 40-60% of cell population survived in all times with several degenerating neurons giving the typical appearance of a "bull's eye." At ultrastructural level, chromatin margination, nuclear shrinkage, mitochondria with matrix dilution, dilated endoplasmic cisternae, and electrondense cytoplasm were detected. Xenon alone did not induce changes, but reduced of about 50% NMA-induced cell loss as well as degenerating neurons, with the maximal neuroprotection at 7 days. These results confirm that in the rat arcuate nucleus NMA can induce a severe neuronal damage that is already marked after 3 h. Xenon significantly reduced the neuronal damage at all times and can be then regarded as a promising neuroprotectant agent.

Convergent roles of alpha-synuclein, DA metabolism, and the ubiquitin-proteasome system in nigrostriatal toxicity.

Recent studies disclosed the relevance of specific molecules for the onset of Parkinson's disease (PD) and for the composition of neuronal inclusions. The scenario which is now emerging leads to identify a potential common pathway named the ubiquitin-proteasome (UP) system. In line with this, striatal or systemic inhibiton of the UP system causes experimental Parkinsonism characterized by the formation of neuronal inclusions. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which is also a complex I inhibitor, has been used for decades to produce experimental Parkinsonism with no evidence for neuronal inclusions in rodents. This leaves open the question whether neuronal inclusions need an alternative mechanism or the inhibition of complex I needs to be carried out continuously to build up inclusions. In the present article, we administered continuously MPTP. In these experimental conditions we compared the neurological consequence of intermittent versus continuous MPTP. In both cases we observed a severe dopamine (DA) denervation and cell loss. However, when MPTP was delivered continuously, spared DA nigral neurons develop ubiquitin, parkin, and alpha-synuclein positive inclusions, which are not detectable after intermittent dosing. The onset of Parkinsonism is associated with inhibition of the UP system. We compared these results with those obtained with amphetamine derivative in vivo and in vitro in which occurrence of neuronal inclusions was associated with inhibition of the UP system and we evaluated the role of DA metabolism in inducing these effects.