Autosomal Dominant Nocturnal Frontal Lobe Epilepsy (ADNFLE) is a focal epilepsy characterized by hyperkinetic seizures frequently arising in the frontal lobe during sleep. The ADNFLE families often bear mutations on genes coding for subunits of the nicotinic cerebral acetylcholine receptors (nAChRs), that regulate excitability and neurotransmitter release. By regulating arousal, the cholinergic system modulates the sleep-waking cycle and is implicated in cognitive processes, besides exerting a crucial role during synaptogenesis. A widespread cerebral nAChR subtype is α4β2, and the first ADNFLE-linked mutation found on the β2 subunit was β2-V287L. In this study, we applied immunohistochemical and electrophysiological methods to a murine model of ADNFLE conditionally expressing β2-V287L, which develops spontaneous seizures during slow-wave sleep, only when the transgene is expressed during brain development (Manfredi et al., 2009). First, we studied whether the expression of β2-V287L could alter the maturation of the GABAergic system, which is known to regulate synaptogenesis during early postnatal stages. We focused on the most important Cl- cotransporter, the K+/Cl- cotransporter-2 (KCC2), that sets the transmembrane Cl- gradient in the brain. The balance of abundance and activity of KCC2 is implicated in epileptogenesis as well as in the compensatory responses observed in hyperexcitable networks. Hence, we studied the postnatal distribution of this protein in wild-type (WT) and β2-V287L mice by means of immunohistochemical staining and densitometric analysis. In β2-V287L mice, the KCC2 amount in layer V of prefrontal cortex (PFC) was lower than in the control littermates at postnatal day 8 (P8). Consistently, electrophysiological recordings on pyramidal neurons showed that the GABAergic excitatory to inhibitory switch was delayed in PFC layer V of mice carrying the transgene. At later stages, however (P60), the amount of KCC2 in PFC layer V was instead higher in transgenic mice, accompanied by a decreased KCC2 expression in the reticular thalamic nucleus (RT). These data suggest that β2-V287L could produce stable alterations of the PFC synaptic network, by delaying the GABAergic switch, whereas the late reversal of KCC2 expression in β2-V287L could be a compensatory response to hyperexcitability or a direct contribution to seizure facilitation in the adult thalamocortical network (Chapter 2). In PFC, layer V is the most prone to develop seizures. In normal conditions, layer V pyramidal cell activity is tightly controlled by parvalbumin-positive (PV+) fast-spiking (FS) cells and somatostatin-positive (SOM+) regular-spiking non-pyramidal (RSNP) cells, the two most abundant GABAergic interneuron populations in this region. We thus studied the spontaneous excitatory (EPSC) and inhibitory (IPSC) postsynaptic currents by applying patch-clamp methods to pyramidal, FS and RSNP neurons in murine brain slices. In mice expressing β2-V287L, the ratio between the basal frequencies of EPSCs and IPSCs increased in pyramidal neurons, whereas an opposite effect was observed in FS (but not in RSNP) cells. This suggests that i) a higher basal excitatory input is present in mice carrying the transgene, and ii) this effect could be due to an impairment of the normal inhibitory feedback produced by FS interneurons on pyramidal cells. To better determine the cellular basis of these observations, we estimated the number of both PV+ and SOM+ neurons and of the GABAergic and glutamatergic synaptic terminals contacting these neurons. To this purpose, we used immunohistochemical staining and 3D reconstruction of neurons and terminals. We found no significant changes in the GABAergic cell populations, nor in the GABAergic terminals. However, PV+ cells displayed a significant increase of glutamatergic terminals, suggesting that the functional decrease of glutamatergic input onto FS cells was not caused by a decreased synaptic density, but by a decreased efficacy of glutamatergic transmission onto FS cells (Chapter 3). A parallel analysis was focused on the main cerebral cholinergic nuclei and thalamic RT showing no significant differences in mice bearing the transgene (Chapter 4). Our chronic model of hyperexcitability opens the way to future studies on the role of β2-V287L on synapse formation and how it can be pharmacologically modulated to attempt preventive therapeutic approaches in epilepsy. In the secondary project of my work, we carried out a preliminary study of the protein α-synuclein in both WT and β2-V287L mice. The physiological role of α-synuclein and the reason of its accumulation in neurodegenerative pathologies, such as Parkinson’s disease (PD) and dementia with Lewy bodies (DLB), is unknown. Both PD and DLB also show non-motor manifestations, such as sleep dysfunction and EEG alterations, which in DLB became frequently epileptic seizures. We thus hypothesize a functional link between α-synuclein and the cholinergic system. We quantified α-synuclein in CTRL and β2-V287L mice, using immunofluorescence methods on the corpus striatum (CS) and the somatosensory cortex. Moreover, a colocalization analysis was done for GABA and glutamate vesicular transporters with α-synuclein, to check if the colocalization index was altered in epileptic mice. We found a significant decrease of α-synuclein expression in the dorsolateral CS of the epileptic mice, and an increase of the colocalization ratio in GABAergic synapses of the dorsomedial CS. These preliminary results suggest the existence of a functional relationship between α-synuclein and nAChRs (Chapter 5).

NEURONAL NICOTINIC RECEPTORS AND EPILEPSY: A MORPHO-FUNCTIONAL STUDY ON A CONDITIONAL MURINE MODEL / D. Modena ; tutor: A. Amadeo ; coordinatore: C. Sforza. - : . Università degli Studi di Milano, 2020 Jan 20. ((32. ciclo, Anno Accademico 2019. [10.13130/modena-debora_phd2020-01-20].

NEURONAL NICOTINIC RECEPTORS AND EPILEPSY: A MORPHO-FUNCTIONAL STUDY ON A CONDITIONAL MURINE MODEL

D. Modena
2020

Abstract

Autosomal Dominant Nocturnal Frontal Lobe Epilepsy (ADNFLE) is a focal epilepsy characterized by hyperkinetic seizures frequently arising in the frontal lobe during sleep. The ADNFLE families often bear mutations on genes coding for subunits of the nicotinic cerebral acetylcholine receptors (nAChRs), that regulate excitability and neurotransmitter release. By regulating arousal, the cholinergic system modulates the sleep-waking cycle and is implicated in cognitive processes, besides exerting a crucial role during synaptogenesis. A widespread cerebral nAChR subtype is α4β2, and the first ADNFLE-linked mutation found on the β2 subunit was β2-V287L. In this study, we applied immunohistochemical and electrophysiological methods to a murine model of ADNFLE conditionally expressing β2-V287L, which develops spontaneous seizures during slow-wave sleep, only when the transgene is expressed during brain development (Manfredi et al., 2009). First, we studied whether the expression of β2-V287L could alter the maturation of the GABAergic system, which is known to regulate synaptogenesis during early postnatal stages. We focused on the most important Cl- cotransporter, the K+/Cl- cotransporter-2 (KCC2), that sets the transmembrane Cl- gradient in the brain. The balance of abundance and activity of KCC2 is implicated in epileptogenesis as well as in the compensatory responses observed in hyperexcitable networks. Hence, we studied the postnatal distribution of this protein in wild-type (WT) and β2-V287L mice by means of immunohistochemical staining and densitometric analysis. In β2-V287L mice, the KCC2 amount in layer V of prefrontal cortex (PFC) was lower than in the control littermates at postnatal day 8 (P8). Consistently, electrophysiological recordings on pyramidal neurons showed that the GABAergic excitatory to inhibitory switch was delayed in PFC layer V of mice carrying the transgene. At later stages, however (P60), the amount of KCC2 in PFC layer V was instead higher in transgenic mice, accompanied by a decreased KCC2 expression in the reticular thalamic nucleus (RT). These data suggest that β2-V287L could produce stable alterations of the PFC synaptic network, by delaying the GABAergic switch, whereas the late reversal of KCC2 expression in β2-V287L could be a compensatory response to hyperexcitability or a direct contribution to seizure facilitation in the adult thalamocortical network (Chapter 2). In PFC, layer V is the most prone to develop seizures. In normal conditions, layer V pyramidal cell activity is tightly controlled by parvalbumin-positive (PV+) fast-spiking (FS) cells and somatostatin-positive (SOM+) regular-spiking non-pyramidal (RSNP) cells, the two most abundant GABAergic interneuron populations in this region. We thus studied the spontaneous excitatory (EPSC) and inhibitory (IPSC) postsynaptic currents by applying patch-clamp methods to pyramidal, FS and RSNP neurons in murine brain slices. In mice expressing β2-V287L, the ratio between the basal frequencies of EPSCs and IPSCs increased in pyramidal neurons, whereas an opposite effect was observed in FS (but not in RSNP) cells. This suggests that i) a higher basal excitatory input is present in mice carrying the transgene, and ii) this effect could be due to an impairment of the normal inhibitory feedback produced by FS interneurons on pyramidal cells. To better determine the cellular basis of these observations, we estimated the number of both PV+ and SOM+ neurons and of the GABAergic and glutamatergic synaptic terminals contacting these neurons. To this purpose, we used immunohistochemical staining and 3D reconstruction of neurons and terminals. We found no significant changes in the GABAergic cell populations, nor in the GABAergic terminals. However, PV+ cells displayed a significant increase of glutamatergic terminals, suggesting that the functional decrease of glutamatergic input onto FS cells was not caused by a decreased synaptic density, but by a decreased efficacy of glutamatergic transmission onto FS cells (Chapter 3). A parallel analysis was focused on the main cerebral cholinergic nuclei and thalamic RT showing no significant differences in mice bearing the transgene (Chapter 4). Our chronic model of hyperexcitability opens the way to future studies on the role of β2-V287L on synapse formation and how it can be pharmacologically modulated to attempt preventive therapeutic approaches in epilepsy. In the secondary project of my work, we carried out a preliminary study of the protein α-synuclein in both WT and β2-V287L mice. The physiological role of α-synuclein and the reason of its accumulation in neurodegenerative pathologies, such as Parkinson’s disease (PD) and dementia with Lewy bodies (DLB), is unknown. Both PD and DLB also show non-motor manifestations, such as sleep dysfunction and EEG alterations, which in DLB became frequently epileptic seizures. We thus hypothesize a functional link between α-synuclein and the cholinergic system. We quantified α-synuclein in CTRL and β2-V287L mice, using immunofluorescence methods on the corpus striatum (CS) and the somatosensory cortex. Moreover, a colocalization analysis was done for GABA and glutamate vesicular transporters with α-synuclein, to check if the colocalization index was altered in epileptic mice. We found a significant decrease of α-synuclein expression in the dorsolateral CS of the epileptic mice, and an increase of the colocalization ratio in GABAergic synapses of the dorsomedial CS. These preliminary results suggest the existence of a functional relationship between α-synuclein and nAChRs (Chapter 5).
AMADEO, ALIDA
SFORZA, CHIARELLA
ADNFLE; epilepsy; nicotinic acetylcholine receptors; mouse model
Settore BIO/13 - Biologia Applicata
Settore BIO/17 - Istologia
NEURONAL NICOTINIC RECEPTORS AND EPILEPSY: A MORPHO-FUNCTIONAL STUDY ON A CONDITIONAL MURINE MODEL / D. Modena ; tutor: A. Amadeo ; coordinatore: C. Sforza. - : . Università degli Studi di Milano, 2020 Jan 20. ((32. ciclo, Anno Accademico 2019. [10.13130/modena-debora_phd2020-01-20].
Doctoral Thesis
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