Laura Uva

An Excitatory Loop with Astrocytes Contributes to Drive Neurons to Seizure Threshold

Studies in rodent brain slices suggest that seizures in focal epilepsies are sustained and propagated by the reciprocal interaction between neurons and astroglial cells

In Vivo and In Vitro Effects of Pilocarpine: Relevance to Ictogenesis

Objectives: A common experimental model of status epilepticus (SE) utilizes intraperitoneal administration of the cholinergic agonist pilocarpine preceded by methyl‐scopolamine treatment. Currently, activation of cholinergic neurons is recognized as the only factor triggering pilocarpine SE. However, cholinergic receptors are also widely distributed systemically and pretreatment with methyl‐scopolamine may not be sufficient to counteract the effects of systemically injected pilocarpine. The extent of such peripheral events and the contribution to SE are unknown and the possibility that pilocarpine also induces SE by peripheral actions is yet untested.

Acute induction of epileptiform discharges by pilocarpine in the in vitro isolated guinea-pig brain requires enhancement of blood–brain barrier permeability

Systemic application of the muscarinic agonist, pilocarpine, is commonly utilized to induce an acute status epilepticus that evolves into a chronic epileptic condition characterized by spontaneous seizures. Recent findings suggest that the status epilepticus induced by pilocarpine may be triggered by changes in the blood-brain barrier (BBB) permeability. We tested the role of the BBB in an acute pilocarpine model by using the in vitro model brain preparation and compared our finding with in vivo data. Arterial perfusion of the in vitro isolated guinea-pig brain with <1 mM pilocarpine did not cause epileptiform activity, but rather reduced synaptic transmission and induced steady fast (20-25 Hz) oscillatory activity in limbic cortices. These effects were reversibly blocked by co-perfusion of the muscarinic antagonist atropine sulfate (5 microM). Brain pilocarpine measurements in vivo and in vitro suggested modest BBB penetration. Pilocarpine induced epileptiform discharges only when perfused with compounds that enhance BBB permeability, such as bradykinin (n=2) or histamine (n=10). This pro-epileptic effect was abolished when the BBB-impermeable muscarinic antagonist atropine methyl bromide (5 microM) was co-perfused with histamine and pilocarpine. In the absence of BBB permeability enhancing drugs, pilocarpine induced epileptiform activity only after arterial perfusion at concentrations >10 mM. Ictal discharges correlated with a high intracerebral pilocarpine concentration measured by high pressure liquid chromatography. We propose that acute epileptiform discharges induced by pilocarpine treatment in the in vitro isolated brain preparation are mediated by a dose-dependent, atropine-sensitive muscarinic effect promoted by an increase in BBB permeability. Pilocarpine accumulation secondary to BBB permeability changes may contribute to in vivo ictogenesis in the pilocarpine epilepsy model.

Propagation Dynamics of Epileptiform Activity Acutely Induced by Bicuculline in the Hippocampal–Parahippocampal Region of the Isolated Guinea Pig Brain

Summary:  Purpose: Aim of the study is to investigate the involvement of parahippocampal subregions in the generation and in the propagation of focal epileptiform discharges in an acute model of seizure generation in the temporal lobe induced by arterial application of bicuculline in the in vitro isolated guinea pig brain preparation.

Polysynaptic olfactory pathway to the ipsi- and contralateral entorhinal cortex mediated via the hippocampus

Interactions between olfactory cortices and the hippocampus support sensory discrimination and spatial learning functions. The olfactory input accesses the hippocampal formation via a polysynaptic pathway mediated by the lateral and rostral entorhinal cortex (EC). We recently demonstrated that following repetitive stimulation of the lateral olfactory tract (LOT) at 2-8 Hz, a delayed response (onset at circa 60 ms) was evoked in the caudal portion of the EC, identified as medial EC, that does not receive a direct olfactory input. By performing simultaneous laminar profile analysis in the EC and in different hippocampal subfields, we conclusively demonstrate that the delayed EC response evoked by repetitive ipsilateral LOT stimulation is headed by the sequential activation of the dentate gyrus and the CA3/CA1 subfields in the septal and temporal hippocampus. Repetitive stimulation of the contralateral LOT also induced an EC response that peaked at 76.28+/-2.42 ms (n=15). Current source density analysis and time-delay analysis of simultaneous field potential laminar profiles performed from the EC and from DG, CA3 and CA1 hippocampal subfields suggested that the contralateral EC response is mainly carried by an intrahippocampal CA3-CA3 commissural pathway. Contralateral LOT stimulation also induced a later EC component (delay >100 ms) generated in the superficial layers, mediated either by local associative interactions or by extrahippocampal circuits. The opportunity to activate the ipsi- and contralateral olfactory pathways in the same experiment and to record field potentials profiles simultaneously in different structures of both hemispheres in the isolated guinea-pig brain confirms that this preparation is unique and is particularly suitable for investigating the system physiology of the limbic region. The present study demonstrates that patterned stimulation of the olfactory input that mimics sniffing patterns during odor discrimination induces a diffuse activation of both ipsi- and contralateral hippocampi and ECs. The findings contribute to the understanding the physiological mechanisms that underlie associative interactions between olfactory and non-olfactory cortical inputs converging into the mesial temporal region.

Synchronous GABAA-receptor-dependent potentials in limbic areas of the in-vitro isolated adult guinea pig brain

Epileptiform discharges are known to reflect the hypersynchronous glutamatergic activation of cortical neurons. However, experimental evidence has revealed that epileptiform synchronization is also contributed to by population events mediated by GABAA receptors. Here, we analysed the spatial distribution of GABAA‐receptor‐dependent interictal events in the hippocampal/parahippocampal region of the adult guinea pig brain isolated in vitro. We found that arterial perfusion of this preparation with 4‐aminopyridine caused the appearance of glutamatergic‐independent interictal potentials that were reversibly abolished by GABAA receptor antagonism. Laminar profiles and current source density analysis performed in different limbic areas demonstrated that these GABAA‐receptor‐mediated events were independently generated in different areas of the hippocampal/parahippocampal formation (most often in the medial entorhinal cortex) and propagated between interconnected limbic structures of both hemispheres. Finally, intracellular recordings from principal neurons of the medial entorhinal cortex demonstrated that the GABAergic field potential correlated to inhibitory postsynaptic potentials (membrane potential reversal, −68.12 ± 8.01 mV, n = 5) that were interrupted by ectopic spiking. Our findings demonstrate that, in an acute seizure model developed in the adult guinea pig brain, hypersynchronous GABAA‐receptor‐mediated interictal events are generated from independent sources and propagate within limbic cortices in the absence of excitatory synaptic transmission. As spared or enhanced inhibition was reported in models of epilepsy, our data may support a role of GABA‐mediated signaling in ictogenesis and epileptogenesis.

Synchronous Inhibitory Potentials Precede Seizure-Like Events in Acute Models of Focal Limbic Seizures

Interictal spikes in models of focal seizures and epilepsies are sustained by the synchronous activation of glutamatergic and GABAergic networks. The nature of population spikes associated with seizure initiation (pre-ictal spikes; PSs) is still undetermined. We analyzed the networks involved in the generation of both interictal and PSs in acute models of limbic cortex ictogenesis induced by pharmacological manipulations. Simultaneous extracellular and intracellular recordings from both principal cells and interneurons were performed in the medial entorhinal cortex of the in vitro isolated guinea pig brain during focal interictal and ictal discharges induced in the limbic network by intracortical and brief arterial infusions of either bicuculline methiodide (BMI) or 4-aminopyridine (4AP). Local application of BMI in the entorhinal cortex did not induce seizure-like events (SLEs), but did generate periodic interictal spikes sensitive to the glutamatergic non-NMDA receptor antagonist DNQX. Unlike local applications, arterial perfusion of either BMI or 4AP induced focal limbic SLEs. PSs just ahead of SLE were associated with hyperpolarizing potentials coupled with a complete blockade of firing in principal cells and burst discharges in putative interneurons. Interictal population spikes recorded from principal neurons between two SLEs correlated with a depolarizing potential. We demonstrate in two models of acute limbic SLE that PS events are different from interictal spikes and are sustained by synchronous activation of inhibitory networks. Our findings support a prominent role of synchronous network inhibition in the initiation of a focal seizure.

Topographic distribution of direct and hippocampus‐ mediated entorhinal cortex activity evoked by olfactory tract stimulation

Olfactory information is central for memory‐related functions, such as recognition and spatial orientation. To understand the role of olfaction in learning and memory, the distribution and propagation of olfactory tract‐driven activity in the parahippocampal region needs to be characterized. We recently demonstrated that repetitive stimulation of the olfactory tract in the isolated guinea pig brain preparation induces an early direct activation of the rostrolateral entorhinal region followed by a delayed response in the medial entorhinal cortex (EC), preceded by the interposed activation of the hippocampus. In the present study we performed a detailed topographic analysis of both the early and the delayed entorhinal responses induced by patterned stimulation of the lateral olfactory tract in the isolated guinea pig brain. Bi‐dimensional maps of EC activity recorded at 128 recording sites with 4 × 4 matrix electrodes (410 µm interlead separation) sequentially placed in eight different positions, showed (i) an early (onset at 16.09 ± 1.2 ms) low amplitude potential mediated by the monosynaptic LOT input, followed by (ii) an associative potential in the rostral EC which originates from the piriform cortex (onset at 33.2 ± 2.3 ms), and (iii) a delayed potential dependent on the previous activation of the hippocampus. The sharp component of the delayed response had an onset latency between 52 and 63 ms and was followed by a slow wave. Laminar profile analysis demonstrated that in the caudomedial EC the delayed response was associated with two distinct current sinks located in deep and in superficial layers, whereas in the rostrolateral EC a small‐amplitude sink could be detected in the superficial layers exclusively. The present report demonstrates that the output generated by the hippocampal activation is unevenly distributed across different EC subregions and indicates that exclusively the medial and caudal divisions receive a deep‐layer input from the hippocampus. In the rostrolateral EC, specific network interactions may be generated by the convergence of the direct olfactory input and the olfaction‐driven hippocampal output.

Network Dynamics During the Progression of Seizure-Like Events in the Hippocampal–Parahippocampal Regions

Seizure patterns in temporal lobe epilepsies have been described both in humans and in animal models. The involvement of specific hippocampal-parahippocampal subregions in the initiation and progression of temporal lobe seizures is not defined yet. We analyzed limbic network dynamics during seizures induced by 3-min arterial perfusion of 50 µM bicuculline in the in vitro isolated guinea pig brain preparation. As for human and animal temporal lobe epilepsies, 2 seizure types characterized at onset by either fast activity (FA) or hypersynchronous activity (HSA) were observed in our acute model. Simultaneous extracellular recordings were performed from ventral hippocampal-parahippocampal subregions with multichannel electrodes, and laminar analysis and propagation directions were computed to define reciprocal interactions during seizures. FA seizures started with fast oscillations generated in CA1-subiculum and entorhinal cortex, followed by irregular spikes and progressively regular bursts well defined in all subfields, with the exception of pre- and parasubiculum that do not participate in seizure activity. Dentate gyrus was not involved at FA seizure onset and became prominent during the transition to bursting in both FA and HSA patterns. HSA seizures were similar to FA events, but lacked initial FA. During seizures, reliable and steady propagation within the intra-hippocampal re-entrant loop was observed.

Distribution of the Olfactory Fiber Input Into the Olfactory Tubercle of the In Vitro Isolated Guinea Pig Brain

The olfactory tubercle (OT) is a cortical component of the olfactory system involved in reward mechanisms of drug abuse. This region covers an extensive part of the rostral ventral cerebrum and is relatively poorly studied. The intrinsic network interactions evoked by olfactory input are analyzed in the OT of the in vitro isolated guinea pig brain by means of field potential analysis and optical imaging of voltage-sensitive signals. Stimulation of the lateral olfactory tract induces a monosynaptic response that progressively decreases in amplitude from lateral to medial. The monosynaptic input induces a disynaptic response that is proportionally larger in the medial portion of the OT. Direct stimulation of the piriform cortex and subsequent lesion of this pathway showed the existence of an associative disynaptic projection from the anterior part of the piriform cortex to the lateral part of the OT that integrates with the component mediated by the local intra-OT collaterals. Optical and electrophysiological recordings of the signals evoked by stimulation of the olfactory tract during arterial perfusion with the voltage-sensitive dye di-2-ANEPEQ confirmed the pattern of distribution of the mono and disynaptic responses in the OT. Finally, current source density analysis of laminar profiles recorded with 16-channel silicon probes confirmed that the monosynaptic and disynaptic potentials localize in the most superficial and the deep portions of the plexiform layer I, as suggested by previous reports. This study sets the standard for further analysis of the modulation of network properties in this largely unexplored brain region.