Cholesterol 24-hydroxylase is a novel pharmacological target for anti-ictogenic and disease modification effects in epilepsy.

Therapies for epilepsy mainly provide symptomatic control of seizures since most of the available drugs do not target disease mechanisms. Moreover, about one-third of patients fail to achieve seizure control. To address the clinical need for disease-modifying therapies, research should focus on targets which permit interventions finely balanced between optimal efficacy and safety. One potential candidate is the brain-specific enzyme cholesterol 24-hydroxylase. This enzyme converts cholesterol to 24S-hydroxycholesterol, a metabolite which among its biological roles modulates neuronal functions relevant for hyperexcitability underlying seizures. To study the role of cholesterol 24-hydroxylase in epileptogenesis, we administered soticlestat (TAK-935/OV935), a potent and selective brain-penetrant inhibitor of the enzyme, during the early disease phase in a mouse model of acquired epilepsy using a clinically relevant dose. During soticlestat treatment, the onset of epilepsy was delayed and the number of ensuing seizures was decreased by about 3-fold compared to vehicle-treated mice, as assessed by EEG monitoring. Notably, the therapeutic effect was maintained 6.5 weeks after drug wash-out when seizure number was reduced by about 4-fold and their duration by 2-fold. Soticlestat-treated mice showed neuroprotection of hippocampal CA1 neurons and hilar mossy cells as assessed by post-mortem brain histology. High throughput RNA-sequencing of hippocampal neurons and glia in mice treated with soticlestat during epileptogenesis showed that inhibition of cholesterol 24-hydroxylase did not directly affect the epileptogenic transcriptional network, but rather modulated a non-overlapping set of genes that might oppose the pathogenic mechanisms of the disease. In human temporal lobe epileptic foci, we determined that cholesterol 24-hydroxylase expression trends higher in neurons, similarly to epileptic mice, while the enzyme is ectopically induced in astrocytes compared to control specimens. Soticlestat reduced significantly the number of spontaneous seizures in chronic epileptic mice when was administered during established epilepsy. Data show that cholesterol 24-hydroxylase contributes to spontaneous seizures and is involved in disease progression, thus it represents a novel target for chronic seizures inhibition and disease-modification therapy in epilepsy.

Scn1a gene reactivation after symptom onset rescues pathological phenotypes in a mouse model of Dravet syndrome.

Dravet syndrome is a severe epileptic encephalopathy caused primarily by haploinsufficiency of the SCN1A gene. Repetitive seizures can lead to endurable and untreatable neurological deficits. Whether this severe pathology is reversible after symptom onset remains unknown. To address this question, we generated a Scn1a conditional knock-in mouse model (Scn1a Stop/+) in which Scn1a expression can be re-activated on-demand during the mouse lifetime. Scn1a gene disruption leads to the development of seizures, often associated with sudden unexpected death in epilepsy (SUDEP) and behavioral alterations including hyperactivity, social interaction deficits and cognitive impairment starting from the second/third week of age. However, we showed that Scn1a gene re-activation when symptoms were already manifested (P30) led to a complete rescue of both spontaneous and thermic inducible seizures, marked amelioration of behavioral abnormalities and normalization of hippocampal fast-spiking interneuron firing. We also identified dramatic gene expression alterations, including those associated with astrogliosis in Dravet syndrome mice, that, accordingly, were rescued by Scn1a gene expression normalization at P30. Interestingly, regaining of Nav1.1 physiological level rescued seizures also in adult Dravet syndrome mice (P90) after months of repetitive attacks. Overall, these findings represent a solid proof-of-concept highlighting that disease phenotype reversibility can be achieved when Scn1a gene activity is efficiently reconstituted in brain cells.

Targeting oxidative stress improves disease outcomes in a rat model of acquired epilepsy.

Epilepsy therapy is based on antiseizure drugs that treat the symptom, seizures, rather than the disease and are ineffective in up to 30% of patients. There are no treatments for modifying the disease-preventing seizure onset, reducing severity or improving prognosis. Among the potential molecular targets for attaining these unmet therapeutic needs, we focused on oxidative stress since it is a pathophysiological process commonly occurring in experimental epileptogenesis and observed in human epilepsy. Using a rat model of acquired epilepsy induced by electrical status epilepticus, we show that oxidative stress occurs in both neurons and astrocytes during epileptogenesis, as assessed by measuring biochemical and histological markers. This evidence was validated in the hippocampus of humans who died following status epilepticus. Oxidative stress was reduced in animals undergoing epileptogenesis by a transient treatment with N-acetylcysteine and sulforaphane, which act to increase glutathione levels through complementary mechanisms. These antioxidant drugs are already used in humans for other therapeutic indications. This drug combination transiently administered for 2 weeks during epileptogenesis inhibited oxidative stress more efficiently than either drug alone. The drug combination significantly delayed the onset of epilepsy, blocked disease progression between 2 and 5 months post-status epilepticus and drastically reduced the frequency of spontaneous seizures measured at 5 months without modifying the average seizure duration or the incidence of epilepsy in animals. Treatment also decreased hippocampal neuron loss and rescued cognitive deficits. Oxidative stress during epileptogenesis was associated with de novo brain and blood generation of high mobility group box 1 (HMGB1), a neuroinflammatory molecule implicated in seizure mechanisms. Drug-induced reduction of oxidative stress prevented HMGB1 generation, thus highlighting a potential novel mechanism contributing to therapeutic effects. Our data show that targeting oxidative stress with clinically used drugs for a limited time window starting early after injury significantly improves long-term disease outcomes. This intervention may be considered for patients exposed to potential epileptogenic insults.

Changes of dimension of EEG/ECoG nonlinear dynamics predict epileptogenesis and therapy outcomes.

The lack of early biomarkers of epileptogenesis precludes a sound prediction of epilepsy development after acute brain injuries and of the natural course of the disease thus impairing the development of antiepileptogenic treatments. We investigated whether the dimensional changes of nonlinear dynamics in EEG/ECoG signals, that were recorded in the early aftermath of different epileptogenic injuries, provide a measure to be exploited as a sensitive prognostic and predictive biomarker for epilepsy. Using three different models of epilepsy in two rodent species, we report a common and significant decrease of nonlinear dynamics dimension in EEG/ECoG tracings during early epileptogenesis. In particular, the magnitude of this dimensional decrease predicts the severity of ensuing epilepsy, and this measure is modulated by disease-modifying or antiepileptogenic treatments. The broad application of EEG/ECoG monitoring in epilepsy underlines the translational value of these findings for enriching the population of patients at risk for developing epilepsy in clinical investigations.

Inhibition of monoacylglycerol lipase terminates diazepam-resistant status epilepticus in mice and its effects are potentiated by a ketogenic diet.

OBJECTIVE: Status epilepticus (SE) is a life-threatening and commonly drug-refractory condition. Novel therapies are needed to rapidly terminate seizures to prevent mortality and morbidity. Monoacylglycerol lipase (MAGL) is the key enzyme responsible for the hydrolysis of the endocannabinoid 2-arachidonoylglycerol (2-AG) and a major contributor to the brain pool of arachidonic acid (AA). Inhibiting of monoacylglycerol lipase modulates synaptic activity and neuroinflammation, 2 mediators of excessive neuronal activation underlying seizures. We studied the effect of a potent and selective irreversible MAGL inhibitor, CPD-4645, on SE that was refractory to diazepam, its neuropathologic sequelae, and the mechanism underlying the drug's effects. METHODS: Diazepam-resistant SE was induced in adult mice fed with standard or ketogenic diet or in cannabinoid receptor type 1 (CB1) receptor knock-out mice. CPD-4645 (10 mg/kg, subcutaneously) or vehicle was dosed 1 and 7 h after status epilepticus onset in video-electroencephalography (EEG) recorded mice. At the end of SE, mice were examined in the novel object recognition test followed by neuronal cellloss analysis. RESULTS: CPD-4645 maximal plasma and brain concentrations were attained 0.5 h postinjection (half-life = 3.7 h) and elevated brain 2-AG levels by approximately 4-fold. CPD-4645 administered to standard diet-fed mice progressively reduced spike frequency during 3 h postinjection, thereby shortening SE duration by 47%. The drug immediately abrogated SE in ketogenic diet-fed mice. CPD-4645 rescued neuronal cell loss and cognitive deficit and reduced interleukin (IL)-1ß and cyclooxygenase 2 (COX-2) brain expression resulting from SE. The CPD-4645 effect on SE was similar in mice lacking CB1 receptors. SIGNIFICANCE: MAGL represents a novel therapeutic target for treating status epilepticus and improving its sequelae. CPD-4645 therapeutic effects appear to be predominantly mediated by modulation of neuroinflammation.

High Mobility Group Box 1 is a novel pathogenic factor and a mechanistic biomarker for epilepsy.

Approximately 30% of epilepsy patients experience seizures that are not controlled by the available drugs. Moreover, these drugs provide mainly a symptomatic treatment since they do not interfere with the disease's mechanisms. A mechanistic approach to the discovery of key pathogenic brain modifications causing seizure onset, recurrence and progression is instrumental for designing novel and rationale therapeutic interventions that could modify the disease course or prevent its development. In this regard, increasing evidence shows that neuroinflammation is a pathogenic factor in drug-resistant epilepsies. The High Mobility Group Box 1 (HMGB1)/Toll-like receptor 4 axis is a key initiator of neuroinflammation following brain injuries leading to epilepsy, and its activation contributes to seizure mechanisms in animal models. Recent findings have shown dynamic changes in HMGB1 and its isoforms in the brain and blood of animals exposed to acute brain injuries and undergoing epileptogenesis, and in surgically resected epileptic foci in humans. HMGB1 isoforms reflect different pathophysiological processes, and the disulfide isoform, which is generated in the brain during oxidative stress, is implicated in seizures, cell loss and cognitive dysfunctions. Interfering with disulfide HMGB1-activated cell signaling mediates significant therapeutic effects in epilepsy models. Moreover, both clinical and experimental data suggest that HMGB1 isoforms may serve as mechanistic biomarkers for epileptogenesis and drug-resistant epilepsy. These novel findings suggest that the HMGB1 system could be targeted to prevent seizure generation and may provide clinically useful prognostic biomarkers which may also predict the patient's response to therapy.

Inflammation and Epilepsy: Preclinical Findings and Potential Clinical Translation.

BACKGROUND: The lack of treatments which can prevent epilepsy development or improve disease prognosis represents an unmet and urgent clinical need. The development of such drugs requires a deep understanding of the mechanisms underlying disease pathogenesis. In the last decade, preclinical studies in models of acute seizures and of chronic epilepsy highlighted that neuroinflammation arising in brain areas of seizure onset and generalization is a key contributor to neuronal hyper-excitability underlying seizure generation. Microglia and astrocytes are pivotal cells involved in both the induction and perpetuation of the inflammatory response to epileptogenic injuries or seizures; other cell contributors are neurons, cell components of the blood brain barrier and leukocytes. METHODS: From the clinical standpoint, neuroinflammation is now considered an hallmark of epileptogenic foci in various forms of focal onset pharmacoresistant epilepsies. Moreover, pharmacological studies in animal model with drugs targeting specific inflammatory molecules, and changes in intrinsic seizure susceptibility of transgenic mice with perturbed neuroinflammatory mechanisms, have demonstrated that neuroinflammation is not a bystander phenomenon but has a pathogenic role in seizures, cell loss and neurological co-morbidities. Understanding the role of neuroinflammation in seizure pathogenesis is instrumental for a mechanism-based discovery of selective therapies targeting the epilepsy causes rather than its symptoms, thereby allowing the development of novel disease-modifying treatments. Notably, clinical translation of laboratory findings may take advantage of anti-inflammatory drugs already in medical use for peripheral autoinflammatory or autoimmune disorders. CONCLUSION: This review reports key preclinical and clinical findings supporting a role for brain inflammation in the pathogenesis of seizures. It also highlights the emerging proof-of-concept studies showing signs of clinical efficacy of target-specific anti-inflammatory interventions in epilepsies of differing etiologies. We will discuss the need for biomarkers and novel clinical trial designs for anti-inflammatory therapies that have a mechanism of action very different than standard antiepileptic drugs.

Targeting oxidative stress improves disease outcomes in a rat model of acquired epilepsy.

Epilepsy therapy is based on antiseizure drugs that treat the symptom, seizures, rather than the disease and are ineffective in up to 30% of patients. There are no treatments for modifying the disease-preventing seizure onset, reducing severity or improving prognosis. Among the potential molecular targets for attaining these unmet therapeutic needs, we focused on oxidative stress since it is a pathophysiological process commonly occurring in experimental epileptogenesis and observed in human epilepsy. Using a rat model of acquired epilepsy induced by electrical status epilepticus, we show that oxidative stress occurs in both neurons and astrocytes during epileptogenesis, as assessed by measuring biochemical and histological markers. This evidence was validated in the hippocampus of humans who died following status epilepticus. Oxidative stress was reduced in animals undergoing epileptogenesis by a transient treatment with N-acetylcysteine and sulforaphane, which act to increase glutathione levels through complementary mechanisms. These antioxidant drugs are already used in humans for other therapeutic indications. This drug combination transiently administered for 2 weeks during epileptogenesis inhibited oxidative stress more efficiently than either drug alone. The drug combination significantly delayed the onset of epilepsy, blocked disease progression between 2 and 5 months post-status epilepticus and drastically reduced the frequency of spontaneous seizures measured at 5 months without modifying the average seizure duration or the incidence of epilepsy in animals. Treatment also decreased hippocampal neuron loss and rescued cognitive deficits. Oxidative stress during epileptogenesis was associated with de novo brain and blood generation of disulfide high mobility group box 1 (HMGB1), a neuroinflammatory molecule implicated in seizure mechanisms. Drug-induced reduction of oxidative stress prevented disulfide HMGB1 generation, thus highlighting a potential novel mechanism contributing to therapeutic effects. Our data show that targeting oxidative stress with clinically used drugs for a limited time window starting early after injury significantly improves long-term disease outcomes. This intervention may be considered for patients exposed to potential epileptogenic insults.

Inhibitory effects of beta-amyloid on the nicotinic receptors which stimulate glutamate release in rat hippocampus: the glial contribution.

We investigated on the neuronal nicotinic acetylcholine receptor subtypes involved in the cholinergic control of in vivo hippocampal glutamate (GLU), aspartate (ASP) and inhibitory γ-aminobutyric acid (GABA) overflow. We also investigated on the possible contribution of nicotinic acetylcholine receptors subtypes present on astrocytes in the regulation of the three neurotransmitter amino acids overflow using hippocampal gliosomes and on the effects of beta-amyloid (Aß) 1-40 on the nicotinic control of amino acid neurotransmitter release. Nicotine was able to enhance the in vivo overflow of the three amino acids being more potent in stimulating GLU overflow. The α7 selective agonist PHA543613 induced an overflow very similar to that of nicotine. The α4ß2 selective agonist 5IA85380 was significantly less potent in inducing GLU overflow while the overflow of ASP and GABA were almost inconsistent. Aß1-40 inhibited the neurotransmitter overflow stimulated by PHA543613 but not the one evoked by 5IA85380. In hippocampal gliosomes nicotine elicited selectively GLU overflow which was also evoked by 5IA85380 and by the α7 selective agonist choline. Nicotine- and choline-induced glutamate overflow in gliosomes was inhibited by Aα1-40. In conclusion nicotine administration in vivo elicits hippocampal GLU release mostly through α7 nicotinic acetylcholine receptors likely present both on neurons and astrocytes. Aß inhibitory effect on the nicotinic-control of GLU release seems to depend primarily to the inhibition of α7 nicotinic acetylcholine receptors functional responses.

Dangerous liaisons between beta-amyloid and cholinergic neurotransmission.

The review examines the multifaceted interactions between cholinergic transmission and beta-amyloid suggesting a continuum in the action of the peptide that at low concentrations (picomolar-low nanomolar) may directly stimulate nicotinic cholinergic receptor while desensitizing them at increasing concentrations (high nanomolar-low micromolar). In addition high beta-amyloid concentrations may reduce the synaptic release of several neurotransmitters, including glutamate, aspartate, GABA, glycine and dopamine, when the release is elicited through cholinergic stimulation but not following depolarization. The effect of beta-amyloid has been observed both in vitro and in vivo in at least three different brain areas (nucleus accumbens, striatum, hippocampus) suggesting that the peptide may exert some general effects even if not all the brain areas have been evaluated. In turn the activation of cholinergic receptors may affect the amyloid precursor protein processing diverting the metabolism toward non-amyloidogenic products. These actions, dissociated from those described in the case of high beta-amyloid concentrations leading to neurotoxic oligomers, may participate to cause dysfunctions in the neurotransmitter activity, in turn leading, at least from a theoretical point of view, to early neuropsychiatric disturbances in the disease. Complexively these observations underscore novel relationships between two main players in Alzheimer's disease pathogenesis that are beta-amyloid and cholinergic transmission. Also emerges the inherent difficulty of targeting beta-amyloid in a context in which the peptide exerts several actions beyond neurotoxicity.

Prolonged nicotine exposure down-regulates presynaptic NMDA receptors in dopaminergic terminals of the rat nucleus accumbens.

The presynaptic control of dopamine release in the nucleus accumbens (NAc) by glutamate and acetylcholine has a profound impact on reward signaling. Here we provide immunocytochemical and neurochemical evidence supporting the co-localization and functional interaction between nicotinic acetylcholine receptors (nAChRs) and N-methyl-D-aspartic acid (NMDA) receptors in dopaminergic terminals of the NAc. Most NAc dopaminergic terminals possessed the nAChR α4 subunit and the pre-exposure of synaptosomes to nicotine (30 µM) or to the α4ß2-containing nAChR agonist 5IA85380 (10 nM) selectively inhibited the NMDA (100 µM)-evoked, but not the 4-aminopyridine (10 µM)-evoked, [(3)H] dopamine outflow; this inhibition was blunted by mecamylamine (10 µM). Nicotine and 5IA85380 pretreatment also inhibited the NMDA (100 µM)-evoked increase of calcium levels in single nerve terminals, an effect prevented by dihydro-ß-erythroidine (1 µM). This supports a functional interaction between α4ß2-containing nAChR and NMDA receptors within the same terminal, as supported by the immunocytochemical co-localization of α4 and GluN1 subunits in individual NAc dopaminergic terminals. The NMDA-evoked [(3)H]dopamine outflow was blocked by MK801 (1 µM) and inhibited by the selective GluN2B-selective antagonists ifenprodil (1 µM) and RO 25-6981 (1 µM), but not by the GluN2A-preferring antagonists CPP-19755 (1 µM) and ZnCl2 (1 nM). Notably, nicotine pretreatment significantly decreased the density of biotin-tagged GluN2B proteins in NAc synaptosomes. These results show that nAChRs dynamically and negatively regulate NMDA receptors in NAc dopaminergic terminals through the internalization of GluN2B receptors.

Beta Amyloid Differently Modulate Nicotinic and Muscarinic Receptor Subtypes which Stimulate in vitro and in vivo the Release of Glycine in the Rat Hippocampus.

Using both in vitro (hippocampal synaptosomes in superfusion) and in vivo (microdialysis) approaches we investigated whether and to what extent ß amyloid peptide 1-40 (Aß 1-40) interferes with the cholinergic modulation of the release of glycine (GLY) in the rat hippocampus. The nicotine-evoked overflow of endogenous GLY in hippocampal synaptosomes in superfusion was significantly inhibited by Aß 1-40 (10 nM) while increasing the concentration to 100 nM the inhibitory effect did not further increase. Both the Choline (Ch; α7 agonist; 1 mM) and the 5-Iodo-A-85380 dihydrochloride (5IA85380, α4ß2 agonist; 10 nM)-evoked GLY overflow were inhibited by Aß 1-40 at 100 nM but not at 10 nM concentrations. The KCl evoked [(3)H]GLY and [(3)H]Acetylcholine (ACh) overflow were strongly inhibited in presence of oxotremorine; however this inhibitory muscarinic effect was not affected by Aß 1-40. The effects of Aß 1-40 on the administration of nicotine, veratridine, 5IA85380, and PHA543613 hydrochloride (PHA543613; a selective agonist of α7 subtypes) on hippocampal endogenous GLY release in vivo were also studied. Aß 1-40 significantly reduced (at 10 µM but not at 1 µM) the nicotine-evoked in vivo release of GLY. Aß 1-40 (at 10 µM but not at 1 µM) significantly inhibited the PHA543613 (1 mM)-elicited GLY overflow while was ineffective on the GLY overflow evoked by 5IA85380 (1 mM). Aß 40-1 (10 µM) did not produce any inhibitory effect on nicotine-evoked GLY overflow both in the in vitro and in vivo experiments. Our results indicate that (a) the cholinergic modulation of the release of GLY occurs by the activation of both α7 and α4ß2 nicotinic ACh receptors (nAChRs) as well as by the activation of inhibitory muscarinic ACh receptors (mAChRs) and (b) Aß 1-40 can modulate cholinergic evoked GLY release exclusively through the interaction with α7 and the α4ß2 nAChR nicotinic receptors but not through mAChR subtypes.

In vitro exposure to nicotine induces endocytosis of presynaptic AMPA receptors modulating dopamine release in rat nucleus accumbens nerve terminals.

Here we provide functional and immunocytochemical evidence supporting the presence on Nucleus Accumbens (NAc) dopaminergic terminals of cyclothiazide-sensitive, alfa-amino-3-hydroxy-5-methyl-4-isoxazolone propionate (AMPA) receptors, which activation causes Ca²âº-dependent [³H]dopamine ([³H]DA) exocytosis. These AMPA receptors cross-talk with co-localized nicotinic receptors (nAChRs), as suggested by the finding that in vitro short-term pre-exposure of synaptosomes to 30 µM nicotine caused a significant reduction of both the 30 µM nicotine and the 100 µM AMPA-evoked [³H]DA overflow. Entrapping pep2-SVKI, a peptide known to compete for the binding of GluA2 subunit to scaffolding proteins involved in AMPA receptor endocytosis, in NAC synaptosomes prevented the nicotine-induced reduction of AMPA-mediated [³H]DA exocytosis, while pep2-SVKE, used as negative control, was inefficacious. Immunocytochemical studies showed that a significant percentage of NAc terminals were dopaminergic and that most of these terminals also posses GluA2 receptor subunits. Western blot analysis of GluA2 immunoreactivity showed that presynaptic GluA2 proteins in NAc terminals were reduced in nicotine-pretreated synaptosomes when compared to the control. The nACh-AMPA receptor-receptor interaction was not limited to dopaminergic terminals since nicotine pre-exposure also affected the presynaptic AMPA receptors controlling hippocampal noradrenaline release, but not the presynaptic AMPA receptors controlling GABA and acetylcholine release. These observations could be relevant to the comprehension of the molecular mechanisms at the basis of nicotine rewarding.

Dual effect of beta-amyloid on α7 and α4ß2 nicotinic receptors controlling the release of glutamate, aspartate and GABA in rat hippocampus.

BACKGROUND: We previously showed that beta-amyloid (Aß), a peptide considered as relevant to Alzheimer's Disease, is able to act as a neuromodulator affecting neurotransmitter release in absence of evident sign of neurotoxicity in two different rat brain areas. In this paper we focused on the hippocampus, a brain area which is sensitive to Alzheimer's Disease pathology, evaluating the effect of Aß (at different concentrations) on the neurotransmitter release stimulated by the activation of pre-synaptic cholinergic nicotinic receptors (nAChRs, α4ß2 and α7 subtypes). Particularly, we focused on some neurotransmitters that are usually involved in learning and memory: glutamate, aspartate and GABA. METHODOLOGY/FINDINGS: WE USED A DUAL APPROACH: in vivo experiments (microdialysis technique on freely moving rats) in parallel to in vitro experiments (isolated nerve endings derived from rat hippocampus). Both in vivo and in vitro the administration of nicotine stimulated an overflow of aspartate, glutamate and GABA. This effect was greatly inhibited by the highest concentrations of Aß considered (10 µM in vivo and 100 nM in vitro). In vivo administration of 100 nM Aß (the lowest concentration considered) potentiated the GABA overflow evoked by nicotine. All these effects were specific for Aß and for nicotinic secretory stimuli. The in vitro administration of either choline or 5-Iodo-A-85380 dihydrochloride (α7 and α4ß2 nAChRs selective agonists, respectively) elicited the hippocampal release of aspartate, glutamate, and GABA. High Aß concentrations (100 nM) inhibited the overflow of all three neurotransmitters evoked by both choline and 5-Iodo-A-85380 dihydrochloride. On the contrary, low Aß concentrations (1 nM and 100 pM) selectively acted on α7 subtypes potentiating the choline-induced release of both aspartate and glutamate, but not the one of GABA. CONCLUSIONS/SIGNIFICANCE: The results reinforce the concept that Aß has relevant neuromodulatory effects, which may span from facilitation to inhibition of stimulated release depending upon the concentration used.

Different presynaptic nicotinic receptor subtypes modulate in vivo and in vitro the release of glycine in the rat hippocampus.

In the present study, using an in vivo approach (a microdialysis technique associated to HPLC with fluorimetric detection) and in vitro purified hippocampal synaptosomes in superfusion, we investigated the glycinergic transmission in the hippocampus, focusing on the nicotinic control of glycine (GLY) release. The acute administration of nicotine in vivo was able to evoke endogenous GLY release in the rat hippocampus. The specific nicotinic agonists PHA-543613 hydrochloride (PHA543613) selective for the α7 nicotinic receptor subtype administered in vivo also elicited GLY release in a similar extent, while the α4ß2 agonist 5-IA85380 dihydrochloride (5IA85380) was less effective. Nicotine elicited GLY overflow also from hippocampal synaptosomes in vitro. This overflow was Ca(2+)-dependent and inhibited by methyllycaconitine (MLA), but was not modified by dihydro-beta-erythroidine (DHßE, 1 µM). Choline(Ch)-evoked GLY overflow was Ca(2+) dependent, unaltered in presence of DHßE and blocked by methyllycaconitine (MLA). Additionally, 5IA85380 elicited a GLY overflow, which in turn was Ca(2+) dependent, was significantly inhibited by DHßE but was unaffected by MLA. The GLY overflow produced by these nicotinic agonists quantitatively resembles that evoked by 9 mM KCl. The effects of a high concentration of 5IA85380 (1mM), in the presence of 2 µM DHßE, on the release of GLY was also studied comparatively to that on glutamate and aspartate release. The nicotinic agonist 5IA85380 tested at high concentration (1mM) was able to produce a stimulatory effect of endogenous release of the three amino acids, even in the presence of 2 µM DHßE, indicating the existence of a DHßE resistant, α4ß2 nAChR subtype with a functional role in the modulation of GLY, ASP, and GLU release. Our results show that in the rat hippocampus the release of GLY is, at least in part, of neuronal origin and is modulated by the activation of both α7 and α4ß2 (low and high affinity) nAChR subtypes.