In recent years, several mutations have been identified in the CHRNA4 and CHRNB2 gene, coding for the α4 and β2 subunits of nAChRs, respectively. These mutations are resposible for the onset of around 12% of the cases of an idiopathic form of epilepsy, the nocturnal frontal lobe epilepsy (NFLE). Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is a partial epilepsy characterized by short nocturnal seizure episodes, occurring during stage II of sleep. The attacks originate in the frontal lobe and may lead to cognitive and psycological impairment. The mutations identified to date show a common gain of receptor function often caused by hypersensitivity to ligand binding and retarded desensitization. Recently, Manfredi and colleagues generated, in a FVB background, a mouse model carrying an identified human β2 nAChR subunit mutation (β2-V287L) in a tetracycline-controlled expression system (Tet-Off system). This model is interesting not only for its similarities to human ADNFLE symptoms, but also for its contribution to study the developmental effect of the mutation. During our investigations, we performed whole-cell patch-clamp recordings on layer V neurons in the Fr2 region of PFC brain slices obtained from mature FVB mice. In the first part of our work, we focused on the regulation of fast-spiking (FS) interneurons by heteromeric nAChRs in wild-type animals. The Fr2 region constitutes the main output channel to subcortical structures and is crucial for spread of synchronized activity. Pyramidal neurons in layer V are tightly controlled by a dense network of GABAergic cells and, in particular, FS cells are the main responsible of feed-forward inhibition in the neocortex. Taken toghether, our results show that α4β2* nAChRs regulate GABA release onto FS cells in layer V, which reveals a potentially potent mechanism to stimulate physiological excitability as well as cause pathological hyperexcitability in prefrontal areas. In the second part of our study, we investigated the excitatory/inhibitory balance in mature PFC circuits of mice expressing β2-V287L nAChR subunits. In particular, we studied the nAChR-dependent glutamate and GABA release on PFC layer V pyramidal neurons, by measuring spontaneous excitatory and inhibitory post-synaptic currents (EPSCs, IPSCs) in the presence or in the absence of nicotine. In the absence of nicotine, neither excitatory nor inhibitory transmission showed major differences between β2-V287L and control mice, indicating that the expression of mutant nicotinic receptors doesn’t affect the basal excitatory/inhibitory ratio. In mature (older than P28) mice expressing β2-V287L subunits, we observed a potentiation of both EPSCs and IPSCs stimulation in response to 10 μM nicotine, compared to the control littermates. Then, we focused on the reciprocal inhibition sensed by the two major types of GABAergic neurons, the Fast Spiking interneurons and the Regular Spiking Non Pyramidal neurons. In transgenic animals, we observed different levels of basal inhibitory tone and opposite reactions after nicotine administration. Taken toghether, these data suggest the presence of altered connection ratios among interneurons that can lead to hyperexcitability during periods of intense PFC stimulation, as in stage II of sleep. For these reasons, ADNFLE has to be treated as a developmental disease and not as a channelopathy.

(2016). Cholinergic transmission in the cerebral cortex of a conditional murine model of ADNFLE. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2016).

Cholinergic transmission in the cerebral cortex of a conditional murine model of ADNFLE

MENEGHINI, SIMONE
2016-06-30

Abstract

In recent years, several mutations have been identified in the CHRNA4 and CHRNB2 gene, coding for the α4 and β2 subunits of nAChRs, respectively. These mutations are resposible for the onset of around 12% of the cases of an idiopathic form of epilepsy, the nocturnal frontal lobe epilepsy (NFLE). Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is a partial epilepsy characterized by short nocturnal seizure episodes, occurring during stage II of sleep. The attacks originate in the frontal lobe and may lead to cognitive and psycological impairment. The mutations identified to date show a common gain of receptor function often caused by hypersensitivity to ligand binding and retarded desensitization. Recently, Manfredi and colleagues generated, in a FVB background, a mouse model carrying an identified human β2 nAChR subunit mutation (β2-V287L) in a tetracycline-controlled expression system (Tet-Off system). This model is interesting not only for its similarities to human ADNFLE symptoms, but also for its contribution to study the developmental effect of the mutation. During our investigations, we performed whole-cell patch-clamp recordings on layer V neurons in the Fr2 region of PFC brain slices obtained from mature FVB mice. In the first part of our work, we focused on the regulation of fast-spiking (FS) interneurons by heteromeric nAChRs in wild-type animals. The Fr2 region constitutes the main output channel to subcortical structures and is crucial for spread of synchronized activity. Pyramidal neurons in layer V are tightly controlled by a dense network of GABAergic cells and, in particular, FS cells are the main responsible of feed-forward inhibition in the neocortex. Taken toghether, our results show that α4β2* nAChRs regulate GABA release onto FS cells in layer V, which reveals a potentially potent mechanism to stimulate physiological excitability as well as cause pathological hyperexcitability in prefrontal areas. In the second part of our study, we investigated the excitatory/inhibitory balance in mature PFC circuits of mice expressing β2-V287L nAChR subunits. In particular, we studied the nAChR-dependent glutamate and GABA release on PFC layer V pyramidal neurons, by measuring spontaneous excitatory and inhibitory post-synaptic currents (EPSCs, IPSCs) in the presence or in the absence of nicotine. In the absence of nicotine, neither excitatory nor inhibitory transmission showed major differences between β2-V287L and control mice, indicating that the expression of mutant nicotinic receptors doesn’t affect the basal excitatory/inhibitory ratio. In mature (older than P28) mice expressing β2-V287L subunits, we observed a potentiation of both EPSCs and IPSCs stimulation in response to 10 μM nicotine, compared to the control littermates. Then, we focused on the reciprocal inhibition sensed by the two major types of GABAergic neurons, the Fast Spiking interneurons and the Regular Spiking Non Pyramidal neurons. In transgenic animals, we observed different levels of basal inhibitory tone and opposite reactions after nicotine administration. Taken toghether, these data suggest the presence of altered connection ratios among interneurons that can lead to hyperexcitability during periods of intense PFC stimulation, as in stage II of sleep. For these reasons, ADNFLE has to be treated as a developmental disease and not as a channelopathy.
BECCHETTI, ANDREA
nAChRs, epilepsy, ADNFLE
BIO/09 - FISIOLOGIA
English
Scuola di Dottorato in Medicina Traslazionale e Molecolare
SCUOLA DI DOTTORATO IN MEDICINA TRASLAZIONALE E MOLECOLARE (DIMET) - 72R
28
2014/2015
(2016). Cholinergic transmission in the cerebral cortex of a conditional murine model of ADNFLE. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2016).
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/10281/116649
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