Gang Zhu

Defective function of GABA-containing synaptic vesicles in mice lacking the AP-3B clathrin adaptor

AP-3 is a member of the adaptor protein (AP) complex family that regulates the vesicular transport of cargo proteins in the secretory and endocytic pathways. There are two isoforms of AP-3: the ubiquitously expressed AP-3A and the neuron-specific AP-3B. Although the physiological role of AP-3A has recently been elucidated, that of AP-3B remains unsolved. To address this question, we generated mice lacking μ3B, a subunit of AP-3B. μ3B−/− mice suffered from spontaneous epileptic seizures. Morphological abnormalities were observed at synapses in these mice. Biochemical studies demonstrated the impairment of γ-aminobutyric acid (GABA) release because of, at least in part, the reduction of vesicular GABA transporter in μ3B−/− mice. This facilitated the induction of long-term potentiation in the hippocampus and the abnormal propagation of neuronal excitability via the temporoammonic pathway. Thus, AP-3B plays a critical role in the normal formation and function of a subset of synaptic vesicles. This work adds a new aspect to the pathogenesis of epilepsy.

Staphylococcal enterotoxin induces emesis through increasing serotonin release in intestine and it is downregulated by cannabinoid receptor 1

Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus are the most recognizable bacterial superantigenic toxins causing food poisoning in humans throughout the world. However, it remains unclear how SEs induce emesis and its emetic signal pathway. We investigated a mechanism of SEA‐induced emesis using a small emetic animal model, house musk shrew. SEA‐induced emesis in the animals was inhibited by a 5‐hydroxytryptamine (5‐HT) synthesis inhibitor and a 5‐HT3 receptor antagonist. SEA could increase 5‐HT release in the small intestine. Pre‐treatment with 5,7‐dihydroxytryptamine (5,7‐DHT) markedly inhibited SEA‐induced emesis. SEA‐induced emesis was also abolished by surgical vagotomy. Furthermore, cannabinoid (CB) receptor agonists inhibited SEA‐induced emesis, and the action was reversed by a CB1 antagonist. Both 5‐HT release and CB1 receptor expression were found in the mucosal and myenteric plexus of the intestine. Moreover, a CB1 receptor agonist significantly decreased the 5‐HT release in the intestine. These results demonstrate that SEA induces 5‐HT release in intestine, rather than in brain, and that the 5‐HT3 receptors on vagal afferent neurons are essential for SEA‐stimulated emesis. In addition, SEA‐induced emesis is downregulated by the CB system through decreasing 5‐HT release in intestine.

Age-dependent modulation of hippocampal excitability by KCNQ-channels.

Recently, mutations of KCNQ2 or KCNQ3, members of the KCNQ-related K(+)-channel (KCNQ-channel) family, were identified as cause of benign familial neonatal convulsions (BFNC). However, the exact pathogenic mechanisms of age-dependent development and spontaneous remission of BFNC remain to be elucidated. To clarify the age-dependent etiology of BFNC, we determined age-dependent functional switching of KCNQ-channels, GABAergic- and glutamatergic-transmission in rat hippocampus. The effects of inhibitors of KCNQ-channel, GABA- and glutamate-receptors on propagation of neuronal-excitability and neurotransmitter release were determined by 64-channel multielectrode-dish (MED64), whole-cell recording, in vitro release technique and in vivo microdialysis biosensor, using rat hippocampus from day of birth (P0) to postnatal-day 56 (P56). Inhibition of KCNQ-channels enhanced depolarization-induced glutamate and GABA releases during P0-P7, but not during P14-P28. Inhibition of KCNQ-channels magnified neuronal-excitability propagation from P0 to P14: maximal at P3, but this effect disappeared by P28. GABA(A)-receptor inhibition surprisingly reduced neuronal-excitability propagation during P0-P3, but not at P7. AMPA/glutamate-receptors inhibition reduced propagation of neuronal-excitability throughout the study period. KCNQ-channels inhibition shortened spike-frequency adaptation, but this stimulation was more predominant during P<7 than P>14. During the first week of life, KCNQ-channels performed as a predominant inhibitory system, whereas after this period GABAergic-transmission switched from excitatory to inhibitory function. Contrary, glutamatergic-transmission has acquired as excitatory function from P0. These findings suggest that the pathogenic mechanisms of age-dependent development and spontaneous remission of BFNC are, at least partially, associated with the interaction between age-dependent reduction of inhibitory KCNQ-channel activity and age-dependent functional switching of the GABAergic-system from excitatory to inhibitory action in neonatal CNS.

Rats Harboring S284L Chrna4 Mutation Show Attenuation of Synaptic and Extrasynaptic GABAergic Transmission and Exhibit the Nocturnal Frontal Lobe Epilepsy Phenotype

Mutations of genes encoding α4, β2, or α2 subunits (CHRNA4, CHRNB2, or CHRNA2, respectively) of nAChR [neuronal nicotinic ACh (acetylcholine) receptor] cause nocturnal frontal lobe epilepsy (NFLE) in human. NFLE-related seizures are seen exclusively during sleep and are characterized by three distinct seizure phenotypes: “paroxysmal arousals,” “paroxysmal dystonia,” and “episodic wandering.” We generated transgenic rat strains that harbor a missense mutation S284L, which had been identified in CHRNA4 in NFLE. The transgenic rats were free of biological abnormalities, such as dysmorphology in the CNS, and behavioral abnormalities. The mRNA level of the transgene (mutant Chrna4) was similar to the wild type, and no distorted expression was detected in the brain. However, the transgenic rats showed epileptic seizure phenotypes during slow-wave sleep (SWS) similar to those in NFLE exhibiting three characteristic seizure phenotypes and thus fulfilled the diagnostic criteria of human NFLE. The therapeutic response of these rats to conventional antiepileptic drugs also resembled that of NFLE patients with the S284L mutation. The rats exhibited two major abnormalities in neurotransmission: (1) attenuation of synaptic and extrasynaptic GABAergic transmission and (2) abnormal glutamate release during SWS. The currently available genetically engineered animal models of epilepsy are limited to mice; thus, our transgenic rats offer another dimension to the epilepsy research field.

Effects of zonisamide on neurotransmitter exocytosis associated with ryanodine receptors.

To clarify the antiepileptic and neuroprotective actions of zonisamide (ZNS), we determined acute effects of ZNS on exocytosis of GABA and glutamate associated with ryanodine-receptor (Ryr) in rat hippocampus using microdialysis. ZNS increased basal GABA release concentration-dependently without affecting basal glutamate release; however, K(+)-evoked glutamate and GABA releases were reduced by ZNS concentration-dependently. Inhibition of Ryr reduced K(+)-evoked GABA and glutamate releases without affecting their basal releases. Ryanodine affected GABA and glutamate releases biphasic concentration-dependently: lower concentration of ryanodine increased both basal and K(+)-evoked releases of GABA and glutamate, whereas higher concentration reduced them. The therapeutically relevant concentration of ZNS inhibited ryanodine-induced GABA and glutamate releases, and abolished the inflection point in concentration-response curve for ryanodine on neurotransmitter exocytosis. These data suggest that ZNS elevates seizure threshold via enhancement of GABAergic transmission during resting stage. ZNS inhibits propagation of epileptic hyperexcitability and Ryr-associated neuronal damage during neuronal hyperexcitable stage. These demonstrations indicate that the indirect inhibition of Ryr activities by ZNS during neuronal hyperexcitability appear to be involved in the mechanisms of action of antiepileptic and neuroprotective actions of ZNS.

Pharmacological discrimination between effects of carbamazepine on hippocampal basal, Ca2+‐ and K+‐evoked serotonin release

To elucidate mechanisms of hippocampal serotonin release and possible mechanisms of clinical action of carbamazepine (CBZ), we determined interaction between antagonists of N‐type (ω‐conotoxin GVIA:GVIA), P‐type (ω‐agatoxin IVA:IVA) Ca2+ channels, Na+ channel (tetrodotoxin: TTX) and CBZ on hippocampal basal, Ca2+‐ and K+‐evoked serotonin releases, using microdialysis in freely moving rats. Basal release was reduced by TTX, GVIA and IVA (GVIA>IVA). Ca2+‐evoked release was reduced by GVIA but unaffected by TTX and IVA. K+‐evoked release was reduced by TTX, GVIA and IVA (GVIA

Effects of interleukin-1β on hippocampal glutamate and GABA releases associated with Ca2+-induced Ca2+ releasing systems

Recent clinical and basic studies have demonstrated that hyperactivation of interleukin-1beta (IL-1beta) plays important roles in generation of febrile and epileptic seizures. To clarify this mechanism, the present study determined the effects of IL-1beta on Ca2+-associated releases of glutamate and GABA in mouse hippocampus. Both basal and K+-evoked GABA releases were regulated by Ca2+ influx and Ca2+-induced Ca2+ releasing system (CICR). The K+-evoked glutamate release was also regulated by Ca2+ influx and CICR, whereas basal glutamate release was not affected by them. IL-1beta increased basal releases of glutamate and GABA depending on the activation of Ca2+ influx and ryanodine receptor (RyR)-sensitive CICR, but reduced K+-evoked releases depending on Ca2+ influx, RyR-sensitive and inositol 1,4,5-trisphosphate receptor (IP3R)-sensitive CICRs. During neuronal hyperexcitability, the effect of IL-1beta on GABA release was more predominantly modulated by Ca2+ influx and RyR-sensitive CICR than that on glutamate. These results indicate that hyperactivation of IL-1beta leads to imbalance between glutamatergic and GABAergic transmission via toxic overload response of Ca2+ influx and CICR.

Biphasic actions of topiramate on monoamine exocytosis associated with both soluble N-ethylmaleimide-sensitive factor attachment protein receptors and Ca2+-induced Ca2+-releasing systems

To explore the pharmacological mechanisms of topiramate (TPM), we determined the effects of TPM on monoamine (dopamine and serotonin) exocytosis associated with N-ethylmaleimide-sensitive factor attachment protein receptors and Ca(2+)-induced Ca(2+)-releasing systems, including inositol-triphosphate receptor and ryanodine receptor in freely moving rat pre-frontal cortex using in vivo microdialysis. During resting stage, Ca(2+) output from endoplasmic reticulum Ca(2+) store via inositol-triphosphate receptor regulates syntaxin-associated monoamine exocytosis mechanism, whereas during neuronal hyperexcitable stage, Ca(2+) output via ryanodine receptor regulates synaptobrevin-associated monoamine exocytosis mechanism. Basal monoamine releases were increased and decreased by therapeutically relevant and supratherapeutic concentration of TPM, respectively. The therapeutic-relevant concentration of TPM increased Ca(2+)-evoked release concentration-dependently; however, its stimulatory effect was attenuated in the supratherapeutic range. The K(+)-evoked releases were reduced by TPM concentration-dependently (from therapeutic to supratherapeutic ranges). The therapeutic-relevant concentration of TPM-induced elevation of basal release was reduced by cleavage with syntaxin and inhibition of inositol-triphosphate receptor predominantly, by cleavage with SNAP-25 and synaptobrevin weakly, but not by ryanodine receptor inhibitor. The therapeutic-relevant concentration of TPM-induced elevation of Ca(2+)-evoked release was reduced by cleavage with syntaxin and inositol-triphosphate receptor inhibitor selectively. The therapeutic-relevant concentration of TPM-induced reduction of K(+)-evoked monoamine release was abolished by cleavage with synaptobrevin, but was not affected by cleavage with SNAP-25 or synaptobrevin. The stimulatory effect of ryanodine receptor agonist on K(+)-evoked monoamine release was reduced by TPM, whereas that of inositol-triphosphate receptor agonist was not affected by TPM. Therefore, these results indicate that the combination of the effects of TPM on exocytosis mechanisms associated with SNARE and Ca(2+)-induced Ca(2+)-releasing systems, enhancement of inositol-triphosphate receptor/syntaxin and inhibition of ryanodine receptor/synaptobrevin in pre-frontal cortex, may be involved in clinical actions of TPM.

Protein kinase associated with gating and closing transmission mechanisms in temporoammonic pathway

The entorhinal cortex (EC) is a major source of afferent input to the hippocampus via the perforant and temporoammonic pathways; however, the detailed transmission mechanism in the temporoammonic pathway remains to be clarified. Thus, we determined interaction among GABA(A), AMPA/glutamate receptors and protein kinases (PKA and PKC) in the exocytosis of GABA and glutamate using multiprobe microdialysis, as well as propagation of neuronal excitability using optical recording in the EC-Hippocampal formation. Multiprobe microdialysis demonstrated that EC-evoked GABA release in ventral CA1 was predominantly regulated by the PKC-related rather than PKA-related exocytosis mechanism and was augmented by the activation of glutamatergic transmission. Contrary to GABA release, EC-evoked glutamate release was predominantly regulated by PKA-related rather than PKC-related mechanisms and was suppressed by activation of GABAergic transmission. Optical recording demonstrated that there are two sub-pathways in the temporoammonic pathway; direct projects from EC layers (II-IV) to dendrites on pyramidal cells and GABAergic interneurons in ventral hippocampal CA1. PKC activation enhanced trisynaptic transmission, whether the GABA(A) receptor was functional or blocked, whereas PKC activation enhanced and inhibited temporoammonic transmission when the GABA(A) receptor was functional and blocked, respectively. Thus, GABAergic inhibition, which is regulated by PKC activity, in the temporoammonic pathway is more significant than that in the trisynaptic pathway.

Interaction between carbamazepine, zonisamide and voltage-sensitive Ca2+ channel on acetylcholine release in rat frontal cortex

To clarify the mechanisms of action of antiepileptic drugs (AEDs), carbamazepine (CBZ) and zonisamide (ZNS), on exocytosis mechanisms, the present study determined the concentration-dependent action of CBZ and ZNS, as well as the interaction between these AEDs and voltage-sensitive Ca(2+) channel (VSCC) activity on basal, Ca(2+)- and K(+)-evoked acetylcholine (ACh) release in frontal cortex of freely moving rat using in vivo microdialysis. Perfusion with therapeutic-relevant concentrations of CBZ and ZNS increased basal ACh release, which was regulated by N-type VSCC predominantly and P-type VSCC weakly, whereas supratherapeutic-relevant concentrations of these AEDs reduced this release. The 3.4 mM Ca(2+)-evoked release, which was regulated by N-type VSCC selectively, but not by P-type VSCC, was increased by therapeutic-relevant concentrations of CBZ and ZNS, whereas this release was reduced by supratherapeutic-relevant concentrations of them. The 50 mM K(+)-evoked release, which was regulated by P-type VSCC predominantly and N-type VSCC weakly, was decreased by CBZ and ZNS, in a concentration-dependent manner. These findings indicate that the interplay between enhancement of basal ACh release and reduction of depolarization-related ACh release in the frontal cortex are at least partially involved in a common mechanism of antiepileptic action between CBZ and ZNS.