Norberto Garcia-Cairasco

New insights from the use of pilocarpine and kainate models

Local or systemic administration of pilocarpine and kainate in rodents leads to a pattern of repetitive limbic seizures and status epilepticus, which can last for several hours. A latent period follows status epilepticus and precedes a chronic phase, which is characterized by the occurrence of spontaneous limbic seizures. These distinct features, in a single animal preparation, of an acute damage induced by status epilepticus, a silent interval between injury and the onset of spontaneous seizures, and a chronic epileptic state have allowed antiepileptic drug (AED) studies with different purposes, (a) in the acute phase, identification of compounds with efficacy against refractory status epilepticus and/or neuroprotection against damage induced by sustained seizures; (b) in the latent period, identification of agents with a potential for preventing epileptogenesis and/or against seizure-induced long-term behavioral deficits and (c) in the chronic phase, testing drugs effective against partial and secondarily generalized seizures. Studies on pilocarpine and kainate models have pointed out that some AEDs or other compounds exert an antiepileptogenic effect. The analogy of the latent phase of pilocarpine and kainate models with the acquisition of amygdala kindling should encourage testing of drugs that have proved to suppress the evolution of amygdala kindling. Drug testing in the chronic phase should not address only the suppression of secondarily generalized motor seizures. Most of current tools used to quantify spontaneous seizure events need to be coupled to electrophysiology and more sophisticated systems for recording and analyzing behavior.

Neuroethological and morphological (Neo-Timm staining) correlates of limbic recruitment during the development of audiogenic kindling in seizure susceptible Wistar rats

Acute audiogenic seizures are a model of generalized tonic-clonic seizures, induced by high intensity acoustic stimulation in genetically susceptible rodents. The neural substrate are sensory motor brainstem nuclei. Recruitment of forebrain structures takes places upon repetition of acoustically evoked seizures. The term audiogenic kindling means forebrain kindling evoked by repeated brainstem seizures and has been described in several strains of genetically epilepsy-prone rats. Thus, the present work was conducted in order to test the hypothesis that audiogenic kindling recruits the forebrain, which may be behaviorally evaluated and associated with morphological changes as well. The behavioral sequences observed during the development of audiogenic kindling were assessed by neuroethological methods (cluster analysis), with the ETHOMATIC program. Seizure severity indexes (brainstem and limbic seizures) and latencies of wild running and tonic-clonic seizures were measured to quantify seizure evolution. Densitometric analysis of Neo-Timm staining was used for assessing morphological changes associated with audiogenic kindling. In group I, II resistant (R) and 16 susceptible (S) animals were stimulated (120 dB) 21 times, and allowed a 10 day recovery period prior to retesting. In group II, 22 R and 20 S were stimulated 60 times, and allowed a 2 month recovery period prior to retesting. Repetition of the acoustic stimulation in group I and group II susceptible animals led to a progressive and statistically significant attenuation of the behaviors associated with brainstem seizures and a concomitant increased expression of the behaviors associated with limbic seizures. After either a 10 day (group I) or 2 month (group II) recovery period, acoustic stimulation preferentially evoked brainstem-associated behaviors and seizures rather than limbic ones in the audiogenic susceptible animals, although in some animals overlapped brainstem and limbic seizures were detected. Latencies for the wild running and tonic seizures after acoustic stimulation significantly increased during audiogenic kindling for both group I and group II susceptible animals. The quantitative ethological evaluation in both group I and group II, illustrated by flowcharts, showed the evolution of the kindling installation by the presence of limbic seizure clusters, competing in time with the original tonic-clonic clusters. Expression of limbic seizures by group I animals, after acoustic stimulation, was not associated with changes in the mossy fiber Neo-Timm staining pattern of these animals. In group II however, Neo-Timm staining revealed mossy fiber sprouting in the ventral hippocampus (but not in the dorsal), and a significant change in the optical density of amygdaloid nuclei and perirhinal cortex in susceptible animals as compared to resistant ones. In conclusion, audiogenic kindling effectively recruits forebrain structures, responsible for the appearance of limbic seizures. It is possible that the paradigm used in group I was subthreshold for the development of clear-cut synaptic reorganization in the hippocampal mossy fiber system, since the behavioral patterns reverted ten days after the last seizure induction. In group II, however, an increased number of evoked seizures and a more prolonged time after the last chronic seizure showed structural re-arrangements in amygdala, perirhinal cortex and hippocampus, associated with permanence in terms of behavioral data (lack of regression of limbic seizures to control values).

A critical review on the participation of inferior colliculus in acoustic-motor and acoustic-limbic networks involved in the expression of acute and kindled audiogenic seizures

The main goal of this article is to review the key role that the inferior colliculus plays in the expression of acoustic-motor and acoustic-limbic integration involved, respectively, in acute and chronic audiogenic seizures. In order to put this in context, we will review the behavioral characterization of acute and chronic audiogenic seizures, neuroanatomical substrates, neurochemistry, neuropharmacology, electrophysiology, as well as the cellular and molecular mechanisms involved in their expression. Secondly, we will also correlate our results, collected from audiogenic seizures susceptible rats, before and after the genetic selection of our own audiogenic susceptible strain, and from those sensitized by lesions or drug microinjections, with those pertinent from the international literature. In brief, genetic or sensitized animals express acute audiogenic seizures as a wild running behavior preceding the onset of tonic-clonic seizures. The latter can have several presentations including opistotonus and fore- and hindlimb tonic hyperextensions, followed by clonic convulsions of fore- and hindlimbs. Chronic (kindled) audiogenic seizures change this behavioral expression, with similar patterns such as those present in temporal lobe epileptic seizures, intermingled with the original audiogenic seizure pattern, which is known to be dependent on brainstem networks.

Animal models of epilepsy: use and limitations.

Epilepsy is a chronic neurological condition characterized by recurrent seizures that affects millions of people worldwide. Comprehension of the complex mechanisms underlying epileptogenesis and seizure generation in temporal lobe epilepsy and other forms of epilepsy cannot be fully acquired in clinical studies with humans. As a result, the use of appropriate animal models is essential. Some of these models replicate the natural history of symptomatic focal epilepsy with an initial epileptogenic insult, which is followed by an apparent latent period and by a subsequent period of chronic spontaneous seizures. Seizures are a combination of electrical and behavioral events that are able to induce chemical, molecular, and anatomic alterations. In this review, we summarize the most frequently used models of chronic epilepsy and models of acute seizures induced by chemoconvulsants, traumatic brain injury, and electrical or sound stimuli. Genetic models of absence seizures and models of seizures and status epilepticus in the immature brain were also examined. Major uses and limitations were highlighted, and neuropathological, behavioral, and neurophysiological similarities and differences between the model and the human equivalent were considered. The quest for seizure mechanisms can provide insights into overall brain functions and consciousness, and animal models of epilepsy will continue to promote the progress of both epilepsy and neurophysiology research.

Behavioral, Morphologic, and Electroencephalographic Evaluation of Seizures Induced by Intrahippocampal Microinjection of Pilocarpine

Summary:  Purpose: We studied, by means of video‐EEG and neo‐Timm histochemistry, the behavioral, electrophysiologic, and structural characteristics of seizures induced by intrahippocampal microinjection of pilocarpine (HIP‐PILO), a selective model of temporal lobe epilepsy (TLE).

Midbrain substrates of audiogenic seizures in rats

Audiogenic seizures (AS) are a rodent model of generalized tonic-clonic seizures, induced in susceptible (S) animals by high intensity (110 dB) acoustic stimulation. Resistant (R) animals do not respond to the sound with any seizure-related behavior, but they display facial automatisms and grooming clusters. Genetic selection and neuroethology are the basic tools used in our laboratory to perform behavioral analysis of AS S and R animals. Based upon selective lesion and microinjection (GABA, clobazam, NMDA) studies of substantia nigra (SN), inferior colliculus (IC), superior colliculus (SC), and on specific knife cuts at midcollicular levels, we have suggested differential roles for these substrates in the origin and spreading of AS. The IC central nucleus is suggested to be the most critical area involved in the afferent pathway whose activation is necessary for AS origin. IC cortical nuclei seem to be the most important structures involved in the transduction of sensory to motor activity. SC, SN and other reticular subnuclei are suggested to be modulators or components of the efferent pathway. Although the midbrain is considered to be the only network necessary for acute AS origin, both emotion-linked acoustic memories and plastic changes linked to audiogenic kindling involve midbrain-forebrain connections. This paper reviews the behavioral manifestations of acute and chronic AS, our contribution to the knowledge of some AS neurobiological midbrain substrates and the suggested implications of midbrain-forebrain interactions typical of AS kindling.

Hippocampal cell proliferation and epileptogenesis after audiogenic kindling are not accompanied by mossy fiber sprouting or Fluoro-Jade staining.

Repetitive sound-induced seizures, known as audiogenic kindling (AK), gradually induce the transference of epileptic activity from brainstem to forebrain structures along with behavioral changes. The aim of our work was to correlate the behavioral changes observed during the AK with possible alterations in neuronal proliferation, cell death, hippocampal mossy fiber sprouting and in the EEG pattern of Wistar audiogenic rats, a genetically susceptible strain from our laboratory. Susceptible and non-susceptible animals were submitted to repeated sound stimulations for 14-16 days and hippocampal mitotic activity was studied through the incorporation of bromodeoxyuridine (BrdU). Cell death and mossy fiber sprouting were assessed, respectively, by using Fluoro-Jade and Timm staining, 2 and 32 days after the last kindling stimulation. In addition, we used immunofluorescent double labeling for a glial and a mitotic marker to evaluate newly born cell identity. Some animals had hippocampus and amygdala electrodes for EEG recordings. Our results show that kindled animals with 6-11 generalized limbic seizures (class IV-V) had increased cell proliferation in the dentate gyrus when compared with animals with zero or one to three seizures. BrdU-positive cells labeled on day 2 and on day 32 were both GFAP negative. In the later group, rounded and well-defined BrdU-positive/GFAP-negative nuclei were seen in different portions of the granule cell layer. We did not observe any Fluoro-Jade or differential Timm staining in kindled animals at both killing times. However, EEG recordings showed intense epileptic activity in the hippocampus and amygdala of all animals with limbic seizures.Therefore, our data indicate that AK-induced limbic epileptogenicity is able to increase the hippocampal mitotic rate, even though it does not seem to promote neuronal death or mossy fiber sprouting in the supragranular layer of the dentate gyrus.

Intrinsic neural circuits between dorsal midbrain neurons that control fear-induced responses and seizure activity and nuclei of the pain inhibitory system elaborating postictal antinociceptive processes: a functional neuroanatomical and neuropharmacological study.

The blockade of GABA-mediated Cl(-) influx with pentylenetetrazol (PTZ) was used in the present work to induce seizures in Rattus norvegicus. The aim of this work was to study the involvement of monoamines in the antinociception induced by convulsions elicited by peripheral administration of PTZ (64 mg/kg). The analgesia was measured by the tail-flick test in seven or eight Wistar rats per group. Convulsions were followed by statistically significant increase in the tail-flick latencies (TFL), at least for 120 min of the postictal period. Peripheral administration of methysergide (0.5, 1, 2, and 3 mg/kg) caused a significant decrease in the TFL in seizing animals, as compared to controls, in all postictal periods studied. These findings were corroborated by the pretreatment with ketanserin, a 5-HT(2A/2C)-serotonergic/alpha(1)-noradrenergic receptors antagonist, at the same doses. Peripheral administration of yohimbine (0.5, 1, 2, and 3 mg/kg), alpha(2)-noradrenergic antagonist, also decreased the postictal analgesia either at initial or more terminal periods of the postictal analgesia. These data were corroborated with peripheral administrations of propranolol, a beta-noradrenergic receptor blocker that caused a decrease in the postictal analgesia consistently in later stages (after the first 20-min post-tonic-clonic convulsive reactions) of the post-seizure analgesia, except for the highest dose. These results indicate that monoamines may be involved in the postictal analgesia. The blockade of 5-HT(2A/2C)-serotoninergic, alpha(1)-noradrenergic, or alpha(2)-noradrenergic receptors before tonic clonic seizure-induced analgesia antagonized the increase in the nociceptive threshold caused by seizures in initial steps of the temporal antinociceptive curve, as compared to the blockade of beta-noradrenergic ones. These findings suggest that the recruitment of alpha-noradrenergic receptor and serotonergic receptors was made immediately after convulsions and in other initial periods of the postictal analgesia, as compared to the involvement of beta-noradrenergic receptor. Neurochemical lesions of the locus coeruleus (LC) and neuronal damage of the dorsal raphe nucleus induced a significant decrease of the postictal analgesia, suggesting the involvement of these nuclei in this antinociceptive process. The functional neuroanatomical study of the neural link between the mesencephalic tectum and nuclei of the central pain inhibitory system showed evidence for the interconnection between superior colliculus, both dorsal and ventral periaqueductal gray matter (PAG), and inferior colliculus. Defensive substrates of the inferior colliculus, also involved with wild running and epilepsy, send inputs toward dorsal raphe nucleus and locus coeruleus. Since these nuclei are rich in monoamines and send neural connections toward other monoaminergic nuclei of the brainstem involved with the control of the nociceptive inputs in the dorsal horn of the spinal cord, the present results offer a neuroanatomical and psychopharmacological basis for the antinociceptive processes following tonic-clonic seizures.

Limbic epileptogenicity, cell loss and axonal reorganization induced by audiogenic and amygdala kindling in wistar audiogenic rats (WAR strain).

Audiogenic seizures are a model of generalized tonic-clonic brainstem-generated seizures. Repeated induction of audiogenic seizures, in audiogenic kindling (AuK) protocols, generates limbic epileptogenic activity. The present work evaluated associations between permanence of AuK-induced limbic epileptogenicity and changes in cell number/gluzinergic terminal reorganization in limbic structures in Wistar audiogenic rats (WARs). Additionally, we evaluated histological changes after only amygdala kindling (AmK) and only AuK, and longevity of permanence of AuK-induced limbic epileptogenicity, up to 160 days. WARs and Wistar non-susceptible rats were submitted to AuK (80 stimuli) followed by both 50 days without acoustic stimulation and AmK (16 stimuli), only AmK and only AuK. Cell counting and gluzinergic terminal reorganization were assessed, respectively, by using Nissl and neo-Timm histochemistries, 24 h after the last AmK stimulus. Evaluation of behavioral response to a single acoustic stimulus after AuK and up to 160 days without acoustic stimulation was done in another group. AuK-induced limbic epileptogenicity developed in parallel with a decrease in brainstem-type seizure severity during AuK. AmK was facilitated after AuK. Permanence of AuK-induced limbic epileptogenicity was associated with cell loss only in the rostral lateral nucleus of amygdala. Roughly 20 generalized limbic seizures induced by AuK were neither associated with hippocampal cell loss nor mossy fiber sprouting (MFS). AmK developed with cell loss in hippocampal and amygdala nuclei but not MFS. Main changes of gluzinergic terminals after kindling protocols were observed in amygdala, perirhinal and piriform cortices. AuK and AuK-AmK induced a similar number and type of seizures, higher than in AmK. AmK and AuK-AmK were associated with broader cell loss than AuK. Data indicate that permanent AuK-induced limbic epileptogenicity is mainly associated to gluzinergic terminal reorganization in amygdala but not in the hippocampus and with no hippocampal cell loss. Few AmK-induced seizures are associated to broader and higher cell loss than a higher number of AuK-induced seizures.

Reduced exploratory activity of audiogenic seizures suceptible Wistar rats

Patients with epilepsy and animals with experimental usually show behavioral changes such as increased anxiety. Audiogenic seizures (AS) are a model of generalized tonic-clonic limbic seizures induced by sound stimulation in genetically susceptible animals. The objective of this paper was to evaluate the exploratory activity of an inbred strain derived from Wistar progenitors that has been selected in our laboratory for AS susceptibility. The exploratory activity of audiogenic seizures susceptible (S) and resistant (R) Wistar rats was measured in two situations: an open arena and the elevated plus maze, an animal model of anxiety. S animals displayed a reduced exploration in both the open arena (reduced total distance moved) and the elevated plus maze (reduced number of enclosed-arm entries). In the latter there was also a decrease in open-arm exploration, particularly of the distal part of these arms. This effect persists even when the effect of a decreased number of enclosed-arm entries is removed by analysis of covariance. Therefore, the results indicate that audiogenic seizure genetically susceptible Wistar rats display a reduced exploration of novel environments. Moreover, the results with the elevated plus maze suggest that these animals are more anxious than AS resistant rats.