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Specific neurophysiological seizure patterns in patients with focal epilepsy depend on cerebral location and the underlying neuropathology. Location-specific patterns have been also reported in experimental models. Two focal seizure patterns, named p-type and l-type, typical of neocortical and mesial temporal regions were identified in both patients explored with intracerebral EEG and in animal models. These two patterns were recorded in the olfactory regions and in the entorhinal cortex after either 4AP or BMI administration. Here we mapped epileptiform activities in other cortices to verify the existence of specific epileptiform patterns. Field potentials were simultaneously recorded at multiple locations in olfactory, limbic and neocortical regions of the isolated guinea pig brain after arterial administration of either 4AP or BMI. Most neocortical areas did not generate new distinctive focal seizure-like event (SLE), beside the p-type and l-type patterns. Spiking activity was typically recorded after BMI in all new analyzed regions, whereas SLEs were commonly observed during 4AP perfusion. We confirmed the presence of reproducible region-specific epileptiform patterns in all explored cortical areas and demonstrated that strongly inter-connected areas generate similar SLEs. Our study suggests that p- and l-type SLE represent the most common focal seizure patterns during acute manipulations with pro-epileptic compounds.

The Na

The Na+/K+/Cl- cotransporter-1 (NKCC1) and the K+/Cl- cotransporter-2 (KCC2) set the transmembrane Cl- gradient in the brain, and are implicated in epileptogenesis. We studied the postnatal distribution of NKCC1 and KCC2 in wild-type (WT) mice, and in a mouse model of sleep-related epilepsy, carrying the mutant β2-V287L subunit of the nicotinic acetylcholine receptor (nAChR). In WT neocortex, immunohistochemistry showed a wide distribution of NKCC1 in neurons and astrocytes. At birth, KCC2 was localized in neuronal somata, whereas at subsequent stages it was mainly found in the somatodendritic compartment. The cotransporters' expression was quantified by densitometry in the transgenic strain. KCC2 expression increased during the first postnatal weeks, while the NKCC1 amount remained stable, after birth. In mice expressing β2-V287L, the KCC2 amount in layer V of prefrontal cortex (PFC) was lower than in the control littermates at postnatal day 8 (P8), with no concomitant change in NKCC1. Consistently, the GABAergic excitatory to inhibitory switch was delayed in PFC layer V of mice carrying β2-V287L. At P60, the amount of KCC2 was instead higher in mice bearing the transgene. Irrespective of genotype, NKCC1 and KCC2 were abundantly expressed in the neuropil of most thalamic nuclei since birth. However, KCC2 expression decreased by P60 in the reticular nucleus, and more so in mice expressing β2-V287L. Therefore, a complex regulatory interplay occurs between heteromeric nAChRs and KCC2 in postnatal forebrain. The pathogenetic effect of β2-V287L may depend on altered KCC2 amounts in PFC during synaptogenesis, as well as in mature thalamocortical circuits.

We studied the effect of hypocretin 1 (orexin A) in the frontal area 2 (Fr2) of the murine neocortex, implicated in the motivation-dependent goal-directed tasks. In layer V, hypocretin stimulated the spontaneous excitatory postsynaptic currents (EPSCs) on fast-spiking (FS) interneurons. The effect was accompanied by increased frequency of miniature EPSCs, indicating that hypocretin can target the glutamatergic terminals. Moreover, hypocretin stimulated the spontaneous inhibitory postsynaptic currents (IPSCs) on pyramidal neurons, with no effect on miniature IPSCs. This action was prevented by blocking 1) the ionotropic glutamatergic receptors; 2) the hypocretin receptor type 1 (HCRTR-1), with SB-334867. Finally, hypocretin increased the firing frequency in FS cells, and the effect was blocked when the ionotropic glutamate transmission was inhibited. Immunolocalization confirmed that HCRTR-1 is highly expressed in Fr2, particularly in layer V-VI. Conspicuous labeling was observed in pyramidal neuron somata and in VGLUT1+ glutamatergic terminals, but not in VGLUT2+ fibers (mainly thalamocortical afferents). The expression of HCRTR-1 in GABAergic structures was scarce. We conclude that 1) hypocretin regulates glutamate release in Fr2; 2) the effect presents a presynaptic component; 3) the peptide control of FS cells is indirect, and probably mediated by the regulation of glutamatergic input onto these cells.

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is a focal epilepsy with attacks typically arising in the frontal lobe during non-rapid eye movement (NREM) sleep. It is characterized by clusters of complex and stereotyped hypermotor seizures, frequently accompanied by sudden arousals. Cognitive and psychiatric symptoms may be also observed. Approximately 12% of the ADNFLE families carry mutations on genes coding for subunits of the heteromeric neuronal nicotinic receptors (nAChRs). This is consistent with the widespread expression of these receptors, particularly the α4β2(*) subtype, in the neocortex and thalamus. However, understanding how mutant nAChRs lead to partial frontal epilepsy is far from being straightforward because of the complexity of the cholinergic regulation in both developing and mature brains. The relation with the sleep-waking cycle must be also explained. We discuss some possible pathogenetic mechanisms in the light of recent advances about the nAChR role in prefrontal regions as well as the studies carried out in murine models of ADNFLE. Functional evidence points to alterations in prefrontal GABA release, and the synaptic unbalance probably arises during the cortical circuit maturation. Although most of the available functional evidence concerns mutations on nAChR subunit genes, other genes have been recently implicated in the disease, such as KCNT1 (coding for a Na(+)-dependent K(+) channel), DEPD5 (Disheveled, Egl-10 and Pleckstrin Domain-containing protein 5), and CRH (Corticotropin-Releasing Hormone). Overall, the uncertainties about both the etiology and the pathogenesis of ADNFLE point to the current gaps in our knowledge the regulation of neuronal networks in the cerebral cortex.

By regulating the neocortical excitability, nicotinic acetylcholine receptors (nAChRs) control vigilance and cognition and are implicated in epileptogenesis. Modulation of gamma-aminobutyric acid (GABA) release often accompanies these processes. We studied how nAChRs regulate GABAergic transmission in the murine neocortex with immunocytochemical and patch-clamp methods. The cholinergic fibers densely innervated the somatosensory, visual, motor, and prefrontal cortices (PFC). Laminar distribution was broadly homogeneous, especially in the PFC. The cholinergic terminals were often adjacent to the soma and dendrites of GABAergic interneurons, but well-differentiated synapses were rare. Tonically applied nicotine (1-100 microM) increased the frequency of spontaneous GABAergic inhibitory postsynaptic currents (IPSCs) on pyramidal neurons in PFC layer V. The contribution of nAChR types was assessed by using 1 microM dihydro-beta-erythroidine (DHbetaE), to block heteromeric nAChRs, and 10 nM methyllycaconitine (MLA), to block homomeric nAChRs. Both inhibitors antagonized the effect of nicotine on IPSCs, suggesting that mixed nAChR types control pyramidal neuron inhibition in layer V. To determine whether nAChRs are expressed on basket cells' terminals, we studied miniature IPSCs (mIPSCs). These were revealed using 0.5 microM tetrodotoxin and 50 microM Cd(2+) to isolate the GABAergic terminals from the action potential drive. The nicotinic stimulation of mIPSCs was antagonized by DHbetaE, but not MLA, indicating that heteromeric nAChRs prevail in GABAergic terminals. Immunocytochemistry confirmed the expression of nAChRs on basket cells' somata and terminals. Finally, when the ionotropic glutamatergic transmission was blocked, nicotine partially inhibited the IPSCs, an effect counteracted by both DHbetaE and MLA. Therefore, a fraction of nAChRs are capable of activating GABAergic interneurons that in turn inhibit other GABAergic interneurons, thereby reducing the IPSCs. We conclude that heteromeric nAChRs control GABA release presynaptically, whereas mixed nAChRs regulate both excitation and inhibition of interneurons, the balance depending on the overall glutamatergic drive.

Highlights • α4β2∗ nAChRs stimulate IPSCs in FS interneurons, in layer V of the mouse PFC (Fr2). • In P16–P63 mice, nicotine increased both IPSC and mIPSC frequencies. • GABAergic terminals adjacent to PV+ cells expressed α4 nAChR. • The percentage of FS cells with somatic α4β2∗ currents decreased with age. • Hence, nAChRs may be able to induce local circuit disinhibition in Fr2 PFC., Nicotinic acetylcholine receptors (nAChRs) produce widespread and complex effects on neocortex excitability. We studied how heteromeric nAChRs regulate inhibitory post-synaptic currents (IPSCs), in fast-spiking (FS) layer V neurons of the mouse frontal area 2 (Fr2). In the presence of blockers of ionotropic glutamate receptors, tonic application of 10 μM nicotine augmented the spontaneous IPSC frequency, with minor alterations of amplitudes and kinetics. These effects were studied since the 3rd postnatal week, and persisted throughout the first two months of postnatal life. The action of nicotine was blocked by 1 μM dihydro-β-erythroidine (DHβE; specific for α4∗ nAChRs), but not 10 nM methyllycaconitine (MLA; specific for α7∗ nAChRs). It was mimicked by 10 nM 5-iodo-3-[2(S)-azetidinylmethoxy]pyridine (5-IA; which activates β2∗ nAChRs). Similar results were obtained on miniature IPSCs (mIPSCs). Moreover, during the first five postnatal weeks, approximately 50% of FS cells displayed DHβE-sensitive whole-cell nicotinic currents. This percentage decreased to ∼5% in mice older than P45. By confocal microscopy, the α4 nAChR subunit was immunocytochemically identified on interneurons expressing either parvalbumin (PV), which mainly labels FS cells, or somatostatin (SOM), which labels the other major interneuron population in layer V. GABAergic terminals expressing α4 were observed to be juxtaposed to PV-positive (PV+) cells. A fraction of these terminals displayed PV immunoreactivity. We conclude that α4β2∗ nAChRs can produce sustained regulation of FS cells in Fr2 layer V. The effect presents a presynaptic component, whereas the somatic regulation decreases with age. These mechanisms may contribute to the nAChR-dependent stimulation of excitability during cognitive tasks as well as to the hyperexcitability caused by hyperfunctional heteromeric nAChRs in sleep-related epilepsy.