Mapping region-specific seizure-like patterns in the in vitro isolated guinea pig brain.

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.

Enhanced thalamo-hippocampal synchronization during focal limbic seizures.

OBJECTIVE: The key factors that promote the termination of focal seizures have not been fully clarified. The buildup of neuronal synchronization during seizures has been proposed as one of the possible activity-dependent, self-limiting mechanisms. We investigate if increased thalamo-cortical coupling contributes to enhance synchronization during the late phase of focal seizure-like events (SLEs) generated in limbic regions. METHODS: Recordings were simultaneously performed in the nucleus reuniens of the thalamus, in the hippocampus and in the entorhinal cortex of the isolated guinea pig brain during focal bicuculline-induced SLEs with low voltage fast activity at onset. RESULTS: Spectral coherence and cross-correlation analysis demonstrated a progressive thalamo-cortical entrainment and synchronization in the generation of bursting activity that characterizes the final part of SLEs. The hippocampus is the first activated structure at the beginning of SLE bursting phase and thalamo-hippocampal synchronization is progressively enhanced as SLE develops. The thalamus takes the lead in generating the bursting discharge as SLE end approaches. SIGNIFICANCE: As suggested by clinical studies performed during pre-surgical intracranial monitoring, our data confirm a role of the midline thalamus in leading the synchronous bursting activity at the end of focal seizures in the mesial temporal regions.

Postnatal Changes in K+/Cl- Cotransporter-2 Expression in the Forebrain of Mice Bearing a Mutant Nicotinic Subunit Linked to Sleep-Related Epilepsy.

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 ß2-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 ß2-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 ß2-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 ß2-V287L. Therefore, a complex regulatory interplay occurs between heteromeric nAChRs and KCC2 in postnatal forebrain. The pathogenetic effect of ß2-V287L may depend on altered KCC2 amounts in PFC during synaptogenesis, as well as in mature thalamocortical circuits.

α4ß2∗ nicotinic receptors stimulate GABA release onto fast-spiking cells in layer V of mouse prefrontal (Fr2) cortex.

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 10µM 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 1µM dihydro-ß-erythroidine (DHßE; specific for α4∗ nAChRs), but not 10nM methyllycaconitine (MLA; specific for α7∗ nAChRs). It was mimicked by 10nM 5-iodo-3-[2(S)-azetidinylmethoxy]pyridine (5-IA; which activates ß2∗ nAChRs). Similar results were obtained on miniature IPSCs (mIPSCs). Moreover, during the first five postnatal weeks, approximately 50% of FS cells displayed DHßE-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 α4ß2∗ 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.

Nocturnal frontal lobe epilepsy with paroxysmal arousals due to CHRNA2 loss of function.

OBJECTIVE: We assessed the mutation frequency in nicotinic acetylcholine receptor (nAChR) subunits CHRNA4, CHRNB2, and CHRNA2 in a cohort including autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) and sporadic nocturnal frontal lobe epilepsy (NFLE). Upon finding a novel mutation in CHRNA2 in a large family, we tested in vitro its functional effects. METHODS: We sequenced all the coding exons and their flanking intronic regions in 150 probands (73 NFLE, 77 ADNFLE), in most of whom diagnosis had been validated by EEG recording of seizures. Upon finding a missense mutation in CHRNA2, we measured whole-cell currents in human embryonic kidney cells in both wild-type and mutant α2ß4 and α2ß2 nAChR subtypes stimulated with nicotine. RESULTS: We found a c.889A>T (p.Ile297Phe) mutation in the proband (≈0.6% of the whole cohort) of a large ADNFLE family (1.2% of familial cases) and confirmed its segregation in all 6 living affected individuals. Video-EEG studies demonstrated sleep-related paroxysmal epileptic arousals in all mutation carriers. Oxcarbazepine treatment was effective in all. Whole-cell current density was reduced to about 40% in heterozygosity and to 0% in homozygosity, with minor effects on channel permeability and sensitivity to nicotine. CONCLUSION: ADNFLE had previously been associated with CHRNA2 dysfunction in one family, in which a gain of function mutation was demonstrated. We confirm the causative role of CHRNA2 mutations in ADNFLE and demonstrate that also loss of function of α2 nAChRs may have pathogenic effects. CHRNA2 mutations are a rare cause of ADNFLE but this gene should be included in mutation screening.

The role of nicotinic acetylcholine receptors in autosomal dominant nocturnal frontal lobe epilepsy.

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 α4ß2(*) 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.

Hypocretin (orexin) regulates glutamate input to fast-spiking interneurons in layer V of the Fr2 region of the murine prefrontal cortex.

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.

Regulation of glutamate release by heteromeric nicotinic receptors in layer V of the secondary motor region (Fr2) in the dorsomedial shoulder of prefrontal cortex in mouse.

We studied how nicotinic acetylcholine receptors (nAChRs) regulate glutamate release in the secondary motor area (Fr2) of the dorsomedial murine prefrontal cortex, in the presence of steady agonist levels. Fr2 mediates response to behavioral situations that require immediate attention and is a candidate for generating seizures in the frontal epilepsies caused by mutant nAChRs. Morphological analysis showed a peculiar chemoarchitecture and laminar distribution of pyramidal cells and interneurons. Tonic application of 5 µM nicotine on Layer V pyramidal neurons strongly increased the frequency of spontaneous glutamatergic excitatory postsynaptic currents. The effect was inhibited by 1 µM dihydro-ß-erythroidine (which blocks α4-containing nAChRs) but not by 10 nM methyllicaconitine (which blocks α7-containing receptors). Excitatory postsynaptic currents s were also stimulated by 5-iodo-3-[2(S)-azetidinylmethoxy]pyridine, selective for ß2-containing receptors, in a dihydro-ß-erythroidine -sensitive way. We next studied the association of α4 with different populations of glutamatergic terminals, by using as markers the vesicular glutamate transporter type (VGLUT) 1 for corticocortical synapses and VGLUT2 for thalamocortical projecting fibers. Immunoblots showed higher expression of α4 in Fr2, as compared with the somatosensory cortex. Immunofluorescence showed intense VGLUT1 staining throughout the cortical layers, whereas VGLUT2 immunoreactivity displayed a more distinct laminar distribution. In Layer V, colocalization of α4 nAChR subunit with both VGLUT1 and VGLUT2 was considerably stronger in Fr2 than in somatosensory cortex. Thus, in Fr2, α4ß2 nAChRs are expressed in both intrinsic and extrinsic glutamatergic terminals and give a major contribution to control glutamate release in Layer V, in the presence of tonic agonist levels.

Tonic modulation of GABA release by nicotinic acetylcholine receptors in layer V of the murine prefrontal 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.

Na+-activated K+ current contributes to postexcitatory hyperpolarization in neocortical intrinsically bursting neurons.

The ionic mechanisms underlying the termination of action-potential (AP) bursts and postburst afterhyperpolarization (AHP) in intrinsically bursting (IB) neocortical neurons were investigated by performing intracellular recordings in thin slices of rat sensorimotor cortex. The blockade of Ca(2+)-activated K(+) currents enhanced postburst depolarizing afterpotentials, but had inconsistent and minor effects on the amplitude and duration of AHPs. On the contrary, experimental conditions resulting in reduction of voltage-dependent Na(+) entry into the cells caused a significant decrease of AHP amplitude. Slice perfusion with a modified artificial cerebrospinal fluid in which LiCl (40 mM) partially replaced NaCl had negligible effects on the properties of individual APs, whereas it consistently increased burst length and led to an approximately 30% reduction in the amplitude of AHPs following individual bursts or short trains of stimulus-induced APs. Experiments performed by partially replacing Na(+) ions with choline revealed a comparable reduction in AHP amplitude associated with an inhibition of bursting activity. Moreover, in voltage-clamp experiments carried out in both in situ and acutely isolated neurons, partial substitution of extracellular NaCl with LiCl significantly and reversibly reduced the amplitude of K(+) currents evoked by depolarizing stimuli above-threshold for Na(+)-current activation. The above effect of Na(+)-to-Li(+) substitution was not seen when voltage-gated Na(+) currents were blocked with TTX, indicating the presence of a specific K(+)-current component activated by voltage-dependent Na(+) (but not Li(+)) influx. The above findings suggest that a Na(+)-activated K(+) current recruited by the Na(+) entry secondary to burst discharge significantly contributes to AHP generation and the maintenance of rhythmic burst recurrence during sustained depolarizations in neocortical IB neurons.