No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Neurons in the amygdala play an important role in the neuronal network mediating a clonic form of audiogenic seizures both before and after audiogenic kindling
Abstract
Previous studies showed that neuronal network nuclei for behaviorally different forms of audiogenic seizure (AGS) exhibit similarities and important differences. The amygdala is involved differentially in tonic AGS as compared to clonic AGS networks. The role of the lateral amygdala (LAMG) undergoes major changes after AGS repetition (AGS kindling) in tonic forms of AGS. The present study examined the role of LAMG in a clonic form of AGS [genetically epilepsy-prone rats (GEPR-3s)] before and after AGS kindling using bilateral microinjection and chronic neuronal recordings. AGS kindling in GEPR-3s results in facial and forelimb (F&F) clonus, and this behavior could be blocked following bilateral microinjection of a NMDA antagonist (2-amino-7-phosphonoheptanoate) without affecting generalized clonus. Higher AP7 doses blocked both generalized clonus and F&F clonus. LAMG neurons in GEPR-3s exhibited only onset type neuronal responses both before and after AGS kindling, unlike LAMG neurons in normal rats and a tonic form of AGS. A significantly greater LAMG neuronal firing rate occurred after AGS kindling at high acoustic intensities. The latency of LAMG neuronal firing increased significantly after AGS kindling. Burst firing occurred during wild running and generalized clonic behaviors before and after AGS kindling. Burst firing also occurred during F&F clonus after AGS kindling. These findings indicate that LAMG neurons play a critical role in the neuronal network for generalized clonus as well as F&F clonus in GEPR-3s, both before and after AGS kindling, which contrasts markedly with the role of LAMG in tonic AGS.
... Additionally, fully kindled GEPR-9s have their post-tonic clonic seizure abolished after administration of a selective adenylyl cyclase inhibitor directly into the lateral amygdala nucleus [84]. Similar results were also observed in kindled GEPR-3s who had limbic seizures prevented by administering the NMDA receptor antagonist AP7 into the same structure [85]. Thus, the increased TRPV1 expression endogenously detected in the hippocampus and BLA of WARs is in line with previous neuroplastic alterations and add new information regarding epilepsy susceptibility and calcium mobilization in these limbic brain sites. ...
Epilepsies are neurological disorders characterized by chronic seizures and their related
neuropsychiatric comorbidities, such as anxiety. The Transient Receptor Potential Vanilloid type-1 (TRPV1) channel has been implicated in the modulation of seizures and anxiety-like behaviors in preclinical models. Here, we investigated the impact of chronic epileptic seizures in anxiety-like behavior and TRPV1 channels expression in a genetic model of epilepsy, theWistar Audiogenic Rat (WAR) strain. WARs were submitted to audiogenic kindling (AK), a preclinical model of temporal lobe epilepsy (TLE) and behavioral tests were performed in the open-field (OF), and light-dark box (LDB) tests 24 h after AK.WARs displayed increased anxiety-like behavior and TRPV1R expression in the hippocampal CA1 area and basolateral amygdala nucleus (BLA) when compared to control Wistar rats. Chronic seizures increased anxiety-like behaviors and TRPV1 and FosB expression in limbic and brainstem structures involved with epilepsy and anxiety comorbidity, such as the hippocampus, superior colliculus, and periaqueductal gray matter. Therefore, these results highlight previously unrecognized alterations in TRPV1 expression in brain structures involved with TLE and anxiogenic-like behaviors in a genetic model of epilepsy, the WAR strain, supporting an important role of TRPV1 in the modulation of neurological disorders and associated neuropsychiatric comorbidities.
... Glutamatergic neurotransmission in the amygdala has bi-directional impact on AGS in GEPRs. Blockade reduces the clonic component of AGS in non-kindled GEPR-3, suppresses facial and forelimb clonus in audiogenic kindled GEPR-3; by contrast, NMDA agonists produce facial and forelimb clonus following AGS in GEPR-3 and post-tonic clonus in GEPR-9 (Raisinghani, 2003;Raisinghani and Faingold, 2005). WAR display mild mossy fiber sprouting in the hippocampus and neuroplastic changes in amygdala and perirhinal cortex following audiogenic kindling (Garcia-Cairasco et al., 1996). ...
Acoustically evoked seizures (e.g., audiogenic seizures or AGS) are common in models of inherited epilepsy and occur in a variety of species including rat, mouse, and hamster. Two models that have been particularly well studied are the genetically epilepsy prone rat (GEPR-3) and the Wistar Audiogenic Rat (WAR) strains. Acute and repeated AGS, as well as comorbid conditions, displays a close phenotypic overlap in these models. Whether these similarities arise from convergent or divergent structural changes in the brain remains unknown. Here, we examined the brain structure of Sprague Dawley (SD) and Wistar (WIS) rats, and quantified changes in the GEPR-3 and WAR, respectively. Brains from adult, male rats of each strain (n=8-10 per group) were collected, fixed, and embedded in agar and imaged using a 7-T Bruker MRI. Post-acquisition analysis included voxel-based morphometry (VBM), diffusion tensor imaging (DTI), and manual volumetric tracing. In the VBM analysis, GEPR-3 displayed volumetric changes in brainstem structures known to be engaged by AGS (e.g., superior and inferior colliculus, periaqueductal grey) and in forebrain structures (e.g., striatum, septum, nucleus accumbens). WAR displayed volumetric changes in superior colliculus, and a broader set of limbic regions (e.g., hippocampus, amygdala/piriform cortex). The only area of significant overlap in the two strains was the midline cerebellum: both GEPR-3 and WAR showed decreased volume compared to their control strains. In the DTI analysis, GEPR-3 displayed decreased fractional anisotropy (FA) in the corpus callosum, posterior commissure and commissure of the inferior colliculus (IC). WAR displayed increased FA only in the commissure of IC. These data provide a biological basis for further comparative and mechanistic studies in the GEPR-3 and WAR models, as well as provide additional insight into commonalities in the pathways underlying AGS susceptibility and behavioral comorbidity.
... In the present study, increases in activity at SML structures, including the PAG, AMG, PRF, MRF, and SNr, were found, which is consistent with previous findings on other AGSz networks (Deransart et al., 2001;Faingold, 2012;Feng and Faingold, 2002;Millan et al., 1988;N'Gouemo and Faingold, 1998). The increased activity at the AMG in the present study also suggests a potential role in AGSz and S-IRA, which is consistent with its role as a requisite structure in the neuronal network that mediates other forms of AGSz (Garcia-Cairasco, 2002; Raisinghani and Faingold, 2005). The AMG is also known to modulate respiration (Bondarenko et al., 2014;Masaoka et al., 2014;Sugita et al., 2015;Zhang et al., 2009). ...
Sudden unexpected death in epilepsy (SUDEP) is a major concern for patients with epilepsy. In most witnessed cases of SUDEP generalized seizures and respiratory failure preceded death, and pre-mortem neuroimaging studies in SUDEP patients observed changes in specific subcortical structures. Our study examined the role of subcortical structures in the DBA/1 mouse model of SUDEP using manganese-enhanced magnetic resonance imaging (MEMRI). These mice exhibit acoustically-evoked generalized seizures leading to seizure-induced respiratory arrest (S-IRA) that results in sudden death unless resuscitation is rapidly instituted. MEMRI data in the DBA/1 mouse brain immediately after acoustically-induced S-IRA were compared to data in C57 (control) mice that were exposed to the same acoustic stimulus that did not trigger seizures. The animals were anesthetized and decapitated immediately after seizure in DBA/1 mice and after an equivalent time in control mice. Comparative T1 weighted MEMRI images were evaluated using a 14T MRI scanner and quantified. We observed significant increases in activity in DBA/1 mice as compared to controls at previously-implicated auditory (superior olivary complex) and sensorimotor-limbic [periaqueductal gray (PAG) and amygdala] networks and also in structures in the respiratory network. The activity at certain raphe nuclei was also increased, suggesting activation of serotonergic mechanisms. These data are consistent with previous findings that enhancing the action of serotonin prevents S-IRA in this SUDEP model. Increased activity in the PAG and the respiratory and raphe nuclei suggest that compensatory mechanisms for apnea may have been activated by S-IRA, but they were not sufficient to prevent death. The present findings indicate that changes induced by S-IRA in specific subcortical structures in DBA/1 mice are consistent with human SUDEP findings. Understanding the changes in brain activity during seizure-induced death in animals may lead to improved approaches directed at prevention of human SUDEP.
... The main reason for the discrepancy between the efficacy on epilepsy and peripheral neuropathic pain could be the possibility that LCS was found to exhibit a marked use-dependent effect on Na v channels in neurons, as described in our paper. During an epileptic seizure, in contrast to the chronic neuropathic pain, active neurons involved usually have a much highfrequency burst firing pattern under which its inhibitory action on Na v channels can be greatly enhanced (Yamane et al., 2007;Raisinghani and Faingold, 2005). ...
The effect of lacosamide (LCS), a functionalized molecule with anti-convulsant properties, on ion channels was investigated, with the aid of patch clamp technology and simulation modeling. In NSC-34 neuronal cells, LCS was found to block voltage-gated Na(+) current (INa) in a frequency- and concentration-dependent manner. With two-step voltage protocol, minimal change in the steady-state inactivation of INa was found in the presence of LCS. However, with repetitive stimulation, the pulse-to-pulse reduction in peak current was shown to be exponential, with a rate linearly related to both the inter-stimulus interval and the LCS concentration. In addition, the frequency-dependent blocking properties were modeled by considering the drug interaction with a voltage-dependent mixture of NaV channels harboring either an accessible or an inaccessible binding site. LCS also increased the dimension of inactivation space of NaV-channel states, thereby producing the adaptive response of neurons to previous firing. LCS (30 μM) had no effects on the non-inactivating component of INa, while it slightly decreased the amplitude of delayed-rectifier K(+) current. Moreover, LCS suppressed the peak amplitude of INa in embryonic cortical neurons. In HEK293T cells which expressed SCN5A, LCS attenuated the peak amplitude of INa, in a concentration-dependent fashion. The unique effects of LCS on NaV currents presented here may contribute to its in vivo modulation of cellular excitability.
Copyright © 2014. Published by Elsevier Ltd.
... Normal levels of serum constituents clearly excluded the possibility that these abnormal excitabilities are resulted from a systemic metabolic disorder (Suzuki et al. 2007). In contrast to the widely distributed patterns of vacuole formation (spongy degeneration) observed in the central nervous systems of tremor (Kitada et al. 2000) and spontaneously epileptic rats (SER) (Inui et al. 1990), vacuolization localized to a specific region, such as the hippocampus or amygdala, has not been reported in other genetic epilepsy models, whereas close relationships between epileptogenesis and pathophysiological changes in hippocampus and amygdala have been reported (Buckmaster 2004; Galvis-Alonso et al. 2004; Raisinghani and Faingold 2005). Although the molecular basis by which the Wwox mutation causes vacuolization in lde/lde rats is unknown, WWOX has been reported to prevent Tau phosphorylation in hippocampus, which is essential for neurofibrillary tangle formation in the neurons of patients with Alzheimer's disease (Sze et al. 2004). ...
The lde/lde rat is characterized by dwarfism, postnatal lethality, male hypogonadism, a high incidence of epilepsy and many vacuoles in the hippocampus and amygdala. We used a candidate approach to identify the gene responsible for the lde phenotype and assessed the susceptibility of lde/lde rats for audiogenic seizures. Following backcross breeding of lethal dwarfism with epilepsy (LDE) to Brown Norway rats, the lde/lde rats with an altered genetic background showed all pleiotropic phenotypes. The lde locus was mapped to a 1.5-Mbp region on rat chromosome 19 that included the latter half of the Wwox gene. Sequencing of the full-length Wwox transcript identified a 13-bp deletion in exon 9 in lde/lde rats. This mutation causes a frame shift, resulting in aberrant amino acid sequences at the C-terminal. Western blotting showed that both the full-length products of the Wwox gene and its isoform were present in normal testes and hippocampi, whereas both products were undetectable in the testes and hippocampi of lde/lde rats. Sound stimulation induced epileptic seizures in 95% of lde/lde rats, with starting as wild running (WR), sometimes progressing to tonic-clonic convulsions. Electroencephalogram (EEG) analysis showed interictal spikes, fast waves during WR and burst of spikes during clonic phases. The Wwox protein is expressed in the central nervous system (CNS), indicating that abnormal neuronal excitability in lde/lde rats may be because of a lack of Wwox function. The lde/lde rat is not only useful for understanding the multiple functions of Wwox but is also a unique model for studying the physiological function of Wwox in CNS.
Source: PubMed
Self Archiving Restrictions
... A potential mechanism for these structural changes includes the anatomic and functional connections to the hippocampus in patients with medial TLE (2)(3)(4)(5)(6)(7)(8). The studies by Bonilha et al. and Gonçalves Pereira et al. support the hypothesis that direct connections between neuronal structures and the mesial temporal lobe account for the temporal lobe and thalamic structural alterations. ...
Voxel-based Morphometry of the Thalamus in Patients with Refractory Medial Temporal Lobe Epilepsy
Bonilha L, Rorden C, Castellano G, Cendes F, Li LM
Neuroimage 2005;25:1016–1021
Previous research has suggested that patients with refractory medial temporal lobe epilepsy (MTLE) show gray matter atrophy both within the temporal lobes and in the thalamus. However, these studies have not distinguished between different nuclei within the thalamus. We examined whether thalamic atrophy correlates with the nuclei's connections to other regions in the limbic system. T 1 -weighted MRI scans were obtained from 49 neurologically healthy control subjects and 43 patients diagnosed with chronic refractory MTLE that was unilateral in origin (as measured by ictal EEG and hippocampal atrophy observed on MRI). Measurements of gray matter concentration (GMC) were made by using automated segmentation algorithms. GMC was analyzed both voxel by voxel (preserving spatial precision) as well as using predefined regions of interest. Voxel-based morphometry revealed intense GMC reduction in the anterior portion relative to posterior thalami. Furthermore, thalamic atrophy was greater ipsilateral to the MTLE origin than on the contralateral side. Here we demonstrate that the thalamic atrophy is most intense in the thalamic nuclei that have strong connections with the limbic hippocampus. This finding suggests that thalamic atrophy reflects this region's anatomic and functional association with the limbic system rather than a general vulnerability to damage.
Ipsilateral and Contralateral MRI Volumetric Abnormalities in Chronic Unilateral Temporal Lobe Epilepsy and Their Clinical Correlates
Seidenberg M, Kelly KG, Parrish J, Geary E, Dow C, Rutecki P, Hermann B
Epilepsia 2005;46:420–430
Purpose
To assess the presence, extent, and clinical correlates of quantitative MR volumetric abnormalities in ipsilateral and contralateral hippocampus, and temporal and extratemporal lobe regions in unilateral temporal lobe epilepsy (TLE).
Methods
In total, 34 subjects with unilateral left ( n = 15) or right ( n = 19) TLE were compared with 65 healthy controls. Regions of interest included the ipsilateral and contralateral hippocampus as well as temporal, frontal, parietal, and occipital lobe gray and white matter. Clinical markers of neurodevelopmental insult (initial precipitating insult, early age of recurrent seizures) and chronicity of epilepsy (epilepsy duration, estimated number of lifetime generalized seizures) were related to MR volume abnormalities.
Results
Quantitative MR abnormalities extend beyond the ipsilateral hippocampus and temporal lobe with extratemporal (frontal and parietal lobe) reductions in cerebral white matter, especially ipsilateral but also contralateral to the side of seizure onset. Volumetric abnormalities in ipsilateral hippocampus and bilateral cerebral white matter are associated with factors related to both the onset and the chronicity of the patients’ epilepsy.
Conclusions
These cross-sectional findings support the view that volumetric abnormalities in chronic TLE are associated with a combination of neurodevelopmental and progressive effects, characterized by a prominent disruption in ipsilateral hippocampus and neural connectivity (i.e., white matter volume loss) that extends beyond the temporal lobe, affecting both ipsilateral and contralateral hemispheres. MR Volumetric Analysis of the Piriform Cortex and Cortical Amygdala in Drug-refractory Temporal Lobe Epilepsy Gonçalves Pereira PM, Insaustid R, Artacho-Pérulad E, Salmenperäe T, Kälviäinene R, Pitkänen A AJNR Am J Neuroradiol 2005;26:319–332
Purpose
The assessment of patients with temporal lobe epilepsy (TLE) traditionally focuses on the hippocampal formation. These patients, however, may have structural abnormalities in other brain areas. Our purpose was to develop a method to measure the combined volume of the human piriform cortex and cortical amygdala (PCA) by using MRI and to investigate PCA atrophy.
Methods
The definition of anatomic landmarks on MRIs was based on histologic analysis of 23 autopsy control subjects. Thirty-nine adults with chronic TLE and 23 age-matched control subjects were studied. All underwent high-spatial-resolution MRI at 1.5 T, including a tilted T 1 -weighted 3D dataset. The PCA volumes were compared with the control values and further correlated with hippocampal, amygdale, and entorhinal cortex volumes.
Results
The normal volume was 530 ± 59 mm ³ (422-644) (mean ± 1 SD [range]) on the right and 512 ± 60 mm ³ (406-610) on the left PCA (no asymmetry, and no age or sex effect). The intraobserver and interobserver variability were 6% and 8%, respectively. In right TLE patients, the mean right PCA volume was 18% smaller than that in control subjects ( p < 0.001) and 15% smaller than in left TLE ( p < 0.001). In left TLE, the mean left PCA volume was 16% smaller than in control subjects ( p < 0.001) and 19% smaller than in right TLE ( p < 0.001). Overall, 18 (46%) of the 39 patients had a greater than 20% volume reduction in the ipsilateral PCA. Bilateral atrophy was found in 7 (18%) of 39. Patients with hippocampal volumes of at least 2 SDs below the control mean had an 18% reduction in the mean PCA volume compared with patients without hippocampal atrophy ( p < 0.001). Ipsilaterally, hippocampal ( r = 0.756, p < 0.01), amygdaloid ( r = 0.548, p < 0.01), and entorhinal ( r = 0.500, p < 0.01) volumes correlated with the PCA volumes.
Conclusions
The quantification of PCA volume with MRI showed that the PCA is extensively damaged in chronic TLE patients, particularly in those with hippocampal atrophy.
Neuronal networks involved in the epilepsies have been widely studied. The epilepsies are central nervous system disorders characterized by abnormal firing patterns of neurons within neuronal networks that are specific to the type of epilepsy. Critical brain sites or "hubs" within these networks and a variety of network control mechanisms have been identified in the various experimental forms of epilepsy. These epilepsy networks include models of developmental epilepsy, absence epilepsy, kindling epilepsy, status epilepticus, and generalized human epilepsy. One of the problems with much of the network-related information in these epilepsy models and human data is that the onset of the seizure is unpredictable, so the actual network nucleus (hub) for seizure initiation and the propagation pathway of the seizure through the network nuclei are uncertain. However, there are several animal models and certain forms of human epilepsy that are triggered by external (sensory) stimuli, which allows seizure onset to be under experimental control ("reflex" epilepsy). In most models of reflex epilepsy, the seizures are audiogenic seizures (AGS) that are induced by acoustic stimuli. Genetic AGS models have been studied in great detail, which allow comparisons of network hubs and mechanisms. These models include genetically epilepsy-prone rats (GEPR-9s and GEPR-3s) derived from the Sprague-Dawley strain and the Wistar-derived Strasbourg and Brazilian audiogenic rats and Russian (Krushinski-Molodkina) rats. Susceptibility to AGS can also be induced, and these models include thyroid deficiency-induced, ischemia-induced, and ethanol withdrawal-induced AGS. The induced models exhibit similar behavioral patterns of AGS, and these induced models have shed further light on the common hubs and mechanisms of AGS. AGS network elements involve the lower brainstem auditory network up through the critical hub in the inferior colliculus (IC), which projects onto other brainstem sites, including the deep layers of the superior colliculus, the pontine reticular formation, the periaqueductal gray, and the substantia nigra reticulata. Projections from these latter sites to the spinal cord generate the convulsive behaviors via overactivation of normal midbrain locomotor networks. Each of the sites plays a sequential dominant role during the behavior changes that occur during the convulsion. Glutamatergic and GABAergic mechanisms in certain of these network hubs, particularly in the IC, have been shown to be critically involved in initiating network activation. In addition, certain sites in the forebrain, including the amygdala, are differentially implicated in the different genetic AGS models. These different forms of AGS exhibit similar, but not identical, patterns of convulsive behavior, which are mediated by analogous but not identical network sites. The similarities and differences in network sites and mechanisms discovered with this comparative approach may provide a template for improved understanding of the "variations on a theme" seen in many human brain disorders that exhibit overlapping spectra of symptoms but do not display identical behavioral patterns.
Psychiatric illness is very commonly associated with epilepsy, both in adults as well as in children and adolescents; however, the etiology of psychiatric conditions in persons with epilepsy is still controversial. Although the understanding of psychiatric comorbidity has vastly improved over the past century, in many cases, it is difficult to resolve whether psychiatric illness is coincidental or associated with the underlying seizure disorder. Despite numerous reports confirming an overrepresentation of psychiatric illness associated with epilepsy, many patients do not receive mental health treatment. Unfortunately, in some cases, the psychiatric comorbidity may be more impairing to quality of life than the seizure themselves.The consistently high level of psychiatric comorbidity suggests that epilepsy is a complicated illness that may have neuropsychiatric symptoms well beyond discrete seizures.Epileptologists and advocacy groups have raised awareness of the need for an interdisciplinary approach to management of epilepsy. The existing literature tends to focus upon one of three potential explanations for psychiatric comorbidity: symptoms related to psychosocial stress of chronic disease, symptoms related to medication side effects, and symptoms directly related to epilepsy pathophysiology. Although the evidence base is limited regarding treatment for the most common comorbidities of depression, anxiety, attention and cognitive disorders, recent studies have been encouraging in terms of outlining practical treatment approaches in the context of specific epilepsy factors.This chapter addresses historical and theoretical characteristics of psychiatric illness associated with epilepsy as well as strategies for managing the most common psychiatric comorbidities.
Young, but not adult, Fmr1 knockout (KO) mice display audiogenic seizures (AGS) that can be prevented by inhibiting extracellular signal-regulated kinases 1/2 (ERK1/2) phosphorylation. In order to identify the cerebral regions involved in these phenomena, we characterized the response to AGS in Fmr1 KO mice and wild type (WT) controls at postnatal day (P) 45 and P90. To characterize the diverse response to AGS in various cerebral regions, we evaluated the activity markers FosB/ΔFosB and phosphorylated ERK1/2 (p-ERK1/2). Wild running (100% of tested mice) followed by clonic/tonic seizures (30%) were observed in P45 Fmr1 KO mice, but not in WT mice. In P90 Fmr1 KO mice, wild running was only present in 25% of tested animals. Basal FosB/ΔFosB immunoreactivity was higher (P<0.01 vs WT) in the CA1 and subiculum of P45 Fmr1 KO mice. Following the AGS test, FosB/ΔFosB expression consistently increased in most of the analyzed regions in both groups at P45, but not at P90. Interestingly, FosB/ΔFosB immunoreactivity was significantly higher in P45 Fmr1 KO mice in the medial geniculate body (P<0.05 vs WT) and CA3 (P<0.01). Neurons presenting with immunopositivity to p-ERK1/2 were more abundant in the subiculum of Fmr1 KO mice in control condition (P<0.05 vs WT, in both age groups). In this region, p-ERK1/2-immunopositive cells significantly decreased (-75%, P<0.01) in P90 Fmr1 KO mice exposed to the AGS test, but no changes were found in P45 mice or in other brain regions. In both age groups of WT mice, p-ERK1/2-immunopositive cells increased in the subiculum after exposure to the acoustic test. Our findings illustrate that FosB/ΔFosB markers are overexpressed in the medial geniculate body and CA3 in Fmr1 KO mice experiencing AGS, and that p-ERK1/2 is markedly decreased in the subiculum of Fmr1 KO mice resistant to AGS induction. These findings suggest that resilience to AGS is associated with dephosphorylation of p-ERK1/2 in the subiculum of mature Fmr1 KO mice.
Seizures and Neuronal Synchronization: Increased or Decreased Relative to Interictal Values?The ‘Focus’ (‘Ictio-centric’) vs the Network Theory in IctiogenesisReferences
An understanding of the in vivo spatial emergence of abnormal brain activity during spontaneous seizure onset is critical to future early seizure detection and closed-loop seizure prevention therapies. In this study, we use Granger causality (GC) to determine the strength and direction of relationships between local field potentials (LFPs) recorded from bilateral microelectrode arrays in an intermittent spontaneous seizure model of chronic temporal lobe epilepsy before, during, and after Racine grade partial onset generalized seizures. Our results indicate distinct patterns of directional GC relationships within the hippocampus, specifically from the CA1 subfield to the dentate gyrus, prior to and during seizure onset. Our results suggest sequential and hierarchical temporal relationships between the CA1 and dentate gyrus within and across hippocampal hemispheres during seizure. Additionally, our analysis suggests a reversal in the direction of GC relationships during seizure, from an abnormal pattern to more anatomically expected pattern. This reversal correlates well with the observed behavioral transition from tonic to clonic seizure in time-locked video. These findings highlight the utility of GC to reveal dynamic directional temporal relationships between multichannel LFP recordings from multiple brain regions during unprovoked spontaneous seizures.
Voltage-gated calcium (Ca(2+)) channels are thought to play an important role in epileptogenesis and seizure generation. Here, using the whole cell configuration of patch-clamp techniques, we report on the modifications of biophysical and pharmacological properties of high threshold voltage-activated Ca(2+) channel currents in inferior colliculus (IC) neurons of the genetically epilepsy-prone rats (GEPR-3s). Ca(2+) channel currents were measured by depolarizing pulses from a holding potential of - 80 mV using barium (Ba(2+)) as the charge carrier. We found that the current density of high threshold voltage-activated Ca(2+) channels was significantly larger in IC neurons of seizure-naive GEPR-3s compared to control Sprague-Dawley rats, and that seizure episodes further enhanced the current density in the GEPR-3s. The increased current density was reflected by both a - 20 mV shifts in channel activation and a 25% increase in the non-inactivating fraction of channels in seizure-naive GEPR-3s. Such changes were reduced by seizure episodes in the GEPR-3s. Pharmacological analysis of the current density suggests that upregulation of L-, N- and R-type of Ca(2+) channels may contribute to IC neuronal hyperexcitability that leads to seizure susceptibility in the GEPR-3s.
We investigated the effect of Rosa damascena Mill, essential oil on the development of induced amygdala kindling seizures. Male Wistar rats were implanted with one tripolar and two monopolar electrodes in right basolateral amygdala and dura surface, respectively. The control group was injected solvent of essential oil and two experimental groups were injected 750 and 1000 mg kg(-1) of essential oil (ip), 30 min before a daily kindling stimulation. The number of stimulations required for the first appearance of seizure stages was significantly larger in two experimental groups than in control group. Mean after discharge duration was significantly different and essential oil reduced the increase of after discharge duration. Mean after discharge amplitude was also shorter in the groups treated with essential oil than in control group. Duration time for 5th stage of seizure at fully-kindled rats was significantly shorter in two experimental groups than control group. These results suggest that Rosa damascena essential oil significantly retarded the development of seizure stages and possesses the ability to counteract kindling acquisition. The flavonoids of Rosa damascena may act via GABAA receptors as previous studies have proposed for flavonoids of other medicinal plants. More detailed studies are recommended to define the effective component(s) of Rosa on different types of epilepsy.
CNS neuronal networks are known to control normal physiological functions, including locomotion and respiration. Neuronal networks also mediate the pathophysiology of many CNS disorders. Stimulation therapies, including localized brain and vagus nerve stimulation, electroshock, and acupuncture, are proposed to activate "therapeutic" neuronal networks. These therapeutic networks are dormant prior to stimulatory treatments, but when the dormant networks are activated they compete with pathophysiological neuronal networks, disrupting their function. This competition diminishes the disease symptoms, providing effective therapy for otherwise intractable CNS disorders, including epilepsy, Parkinson's disease, chronic pain, and depression. Competition between stimulation-activated therapeutic networks and pathophysiological networks is a major mechanism mediating the therapeutic effects of stimulation. This network interaction is hypothesized to involve competition for "control" of brain regions that contain high proportions of conditional multireceptive (CMR) neurons. CMR regions, including brainstem reticular formation, amygdala, and cerebral cortex, have extensive connections to numerous brain areas, allowing these regions to participate potentially in many networks. The participation of CMR regions in any network is often variable, depending on the conditions affecting the organism, including vigilance states, drug treatment, and learning. This response variability of CMR neurons is due to the high incidence of excitatory postsynaptic potentials that are below threshold for triggering action potentials. These subthreshold responses can be brought to threshold by blocking inhibition or enhancing excitation via the paradigms used in stimulation therapies. Participation of CMR regions in a network is also strongly affected by pharmacological treatments (convulsant or anesthetic drugs) and stimulus parameters (strength and repetition rate). Many studies indicate that treatment of unanesthetized animals with antagonists (bicuculline or strychnine) of inhibitory neurotransmitter (GABA or glycine) receptors can cause CMR neurons to become consistently responsive to external inputs (e.g., peripheral nerve, sensory, or electrical stimuli in the brain) to which these neurons did not previously respond. Conversely, agents that enhance GABA-mediated inhibition (e.g., barbiturates and benzodiazepines) or antagonize glutamate-mediated excitation (e.g., ketamine) can cause CMR neurons to become unresponsive to inputs to which they responded previously. The responses of CMR neurons exhibit extensive short-term and long-term plasticity, which permits them to participate to a variable degree in many networks. Short-term plasticity subserves termination of disease symptoms, while long-term plasticity in CMR regions subserves symptom prevention. This network interaction hypothesis has value for future research in CNS disease mechanisms and also for identifying therapeutic targets in specific brain networks for more selective stimulation and pharmacological therapies.
Perirhinal cortex (PRh) is strongly implicated in neuronal networks subserving forebrain-driven partial onset seizures, but whether PRh plays a role in generalized onset seizures is unclear. The moderate seizure severity substrain of genetically epilepsy-prone rats (GEPR-3s) exhibits generalized onset clonic audiogenic seizures (AGS), but following repetitive AGS (AGS kindling), an additional behavior, facial and forelimb (F&F) clonus emerges immediately following generalized clonus. F&F clonus is thought to be driven from forebrain structures. The present in vivo study used PRh focal blockade or extracellular PRh neuronal recording with simultaneous behavioral observations to examine the role played by PRh in AGS neuronal networks before and after AGS kindling in GEPR-3s. Bilateral microinjection of an NMDA receptor antagonist [2-amino-7-phosphonoheptanoic acid, AP7 (0.2-7.5 nmol/side)] into PRh did not affect generalized clonus before or after AGS kindling. However, complete and reversible blockade of only the F&F clonic seizure behavior was induced by AP7 (1 and 7.5 nmol) in AGS-kindled GEPR-3s. Significant increases in PRh neuronal responses to acoustic stimuli occurred after AGS kindling. Tonic PRh neuronal firing patterns appeared during generalized clonus before and after AGS kindling. During F&F clonus, burst firing, an indicator of increased excitability, appeared in PRh neurons. These neurophysiological and microinjection findings support a critical role of PRh in generation of this AGS kindling-induced convulsive behavior. These data are the first indication that PRh participates importantly in the neuronal network for AGS as a result of AGS kindling and demonstrate a previously unknown involvement of PRh in generalized onset seizures.
The caudal pontine reticular formation nucleus (cPRF) is implicated in seizure propagation to the spinal cord in several forms of generalized convulsive seizures, including audiogenic seizures (AGS). Focal microinjection studies implicate cPRF as a requisite neuronal network site subserving generalized AGS in the moderate severity substrain of genetically epilepsy-prone rats (GEPR-3s). AGS in GEPR-3s culminate in generalized clonus, but daily repetition of AGS (AGS kindling) results in an additional seizure behavior, facial and forelimb (F and F) clonus, not seen prior to kindling. This study examined cPRF neuronal firing changes and seizure behaviors during AGS in GEPR-3s. We examined extracellular cPRF neuronal responses to acoustic stimuli (12 kHz) and observed neuronal firing during AGS. cPRF neurons exhibited onset responses to acoustic stimuli before and after AGS kindling. After AGS kindling, increased neuronal firing occurred, and response latencies were prolonged. Tonic neuronal firing occurred during generalized clonus, which changed to burst firing after AGS kindling. Burst firing also occurred during F and F clonus. Increased neuronal firing and the change from tonic to burst firing suggest that AGS kindling involves increased cPRF excitability. These data support an important role for cPRF neurons in generation of generalized clonus in unkindled GEPR-3s, which is increased by AGS kindling. The increased cPRF response latency might reflect a greater role of rostral components of the AGS neuronal network in transmission of acoustic responses to cPRF. This study also suggests that cPRF neurons may be involved in F and F clonus, which was unexpected since F and F clonus is thought to originate primarily in forebrain structures.
A broad spectrum of learning/memory, social interaction, and affective behavioral measures serve as functional correlates for neurobiological changes in seizure-prone animals as well as in epileptic clinical populations. The utility of such measures is demonstrated by their ability to distinguish anomalous characteristics in developing organisms predisposed to seizure onset, as well as to discriminate prior seizure history in organisms with established pathology. For instance, typical findings that generalize across species suggest that seizure-experienced organisms exhibit a variety of deficits in cognitive function as well as inappropriate social neglect and aggression. Behavioral testing batteries have also proven useful in assessing neural mechanisms for seizure induction, subcortical neural circuits, and neuropeptide modulators, for example, as well as in identifying neural pathology resulting from prior seizure activity. However, the wanton application of behavioral tests can also produce false positives in the identification of seizure-related disorders unless alternative performance and motivational hypotheses are discounted effectively. Accordingly, the present review attempts to provide the reader interested in behavioral phenotyping and characterization of seizure-prone rats and mice with a roadmap for rational selection, implementation, and interpretation of data from behavior assays while highlighting potential successes and pitfalls inherent in employing functional correlates of brain activity using animal models of epilepsy.
1. Intracellular current-clamp recordings were obtained from neurons of the basolateral amygdala (BLA) in an in vitro slice preparation from control and kindled animals. Postsynaptic potentials, elicited by stimulation of the stria terminalis (ST) or lateral amygdaloid nucleus (LA), were used to investigate the role of excitatory and inhibitory amino acid transmission in kindling-induced epileptiform activity. The contributions of glutamatergic and GABAergic receptor subtypes were analyzed by use of the non-N-methyl-D-aspartate (non-NMDA) antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), the NMDA antagonist DL-2-amino-5-phosphonovaleric acid (APV), and the GABAA antagonist bicuculline methiodide (BMI). 2. The synaptic waveform evoked in control neurons consisted of an excitatory postsynaptic potential (EPSP), a fast inhibitory postsynaptic potential (f-IPSP), and a slow inhibitory postsynaptic potential (s-IPSP). Stimulation of the ST or LA pathways evoked a burst-firing response in BLA neurons contralateral from the site of stimulation of kindled animals. 3. APV (50 microM) reduced, but CNQX (10 microM) completely blocked, the burst-firing response in BLA neurons from kindled animals and bicuculline-induced bursting in control neurons. 4. Kindling significantly increased the amplitude of both the slow NMDA- and the fast non-NMDA-receptor-mediated components of synaptic transmission (s- and f-EPSPs, respectively). Furthermore, the stimulus intensities required to evoke EPSPs just subthreshold for action potential generation were significantly lower in slices from kindled animals. 5. In kindled neurons no significant change was observed in the membrane input resistance and resting membrane potential or in the number of action potentials elicited in response to depolarizating current injection. 6. Kindling resulted in a pathway-specific loss of ST- and LA-evoked feedforward GABAergic synaptic transmission and of spontaneous IPSPs. In the same BLA neurons, direct GABAergic inhibition via stimulation of the LA was not affected by kindling. 7. The enhanced glutamatergic transmission was not due to disinhibition, because, in the presence of BMI (and CNQX to prevent BMI-induced bursting), the s-EPSP amplitude was still greater in kindled than in control neurons. 8. These results provide evidence that the epileptiform activity observed in BLA neurons after kindling results from an increase in excitatory NMDA- and non-NMDA-receptor-mediated glutamatergic transmission and a decrease in inhibitory gamma-aminobutyric acid (GABA)-receptor-mediated transmission; the enhanced excitatory transmission cannot be accounted for by reduced inhibition.(ABSTRACT TRUNCATED AT 400 WORDS)
The lateral amygdaloid nucleus (AL) is anatomically connected with sensory processing structures in the thalamus and cortex and is believed to be critically involved in emotional processing by virtue of these connections. In order to understand further how auditory projections to AL contribute to emotional processing, acoustic response properties of single AL neurons were characterized in rats. Recordings were also made in the posterior striatum dorsal to AL. Many cells in AL and the striatum could be driven by broad-band auditory stimulation with white noise or clicks. Initial onset latencies were typically between 12 and 25 msec. Most cells also had later responses (60-150 msec), and a few only had late responses. In frequency receptive field tests, different classes of cells were identified. One group had relatively clear frequency preferences. Thresholds for these relatively tuned cells tended to be somewhat higher in AL than in the striatum. Frequency preferences for AL cells were always above 10 kHz. Although most striatal cells had preferences for frequencies above 10 kHz, some cells were found with frequencies below 10 kHz as well. A second group of acoustically responsive neurons, much more common in AL than in the striatum, showed no frequency specificity (untuned cells). These responded to a wide range of frequencies, even at intensities near threshold. A third group, found mainly in AL (approximately 60% of the total population of cells examined in AL), exhibited rapid habituation to auditory stimuli. These tended to have high thresholds (80-100 dB). Because these cells habituated so quickly, frequency specificity could not be determined. Responses in AL and the striatum were compared with responses in the "specific" auditory relay nucleus of the thalamus, the ventral division of the medial geniculate body, where cells had shorter onset latencies, narrower tuning functions, and lower-intensity thresholds than cells in AL and striatal areas. These findings show that cells in AL exhibit a wide range of auditory tuning properties and suggest that information processing in the amygdala might be fruitfully studied as a direct extension of processing in sensory afferent structures.
Unit activity was recorded from cells and cell clusters in the amygdala and striatum in response to electrical stimulation of the medial geniculate body (MGB) in rats anesthetized with chloral hydrate. Responses were mostly excitatory and were evoked against a relatively silent background (i.e., the units seldom fired between stimuli). The shortest latency responses were recorded in the caudate putamen (CPU), lateral amygdaloid nucleus (AL), and amygdalostriatal transition area (AST). Longer latency responses were obtained from neurons in the basolateral (ABL), basomedial (ABM), and central (ACE) nuclei of the amygdala. Moreover, while responses were evoked in AL, AST, and CPU with 300-500 microA stimuli delivered once every 10 sec, more intense and higher-frequency stimuli were required to obtain responses in ABL, ABM, and ACE. These findings are consistent with anatomical tracing studies showing that AL, AST, and CPU receive direct projections from the MGB and related acoustic processing areas of the thalamus but that ACE, ABL, and ABM do not.
Previous studies indicate that daily repetition of audiogenic seizures (AGS) leads to audiogenic 'kindling' with increased seizure duration and additional seizural behaviors. The present study examined the neuronal correlates of this phenomenon. Extracellular single neuron firing and concomitant convulsive behaviors associated with 14 repetitive AGS were evaluated in the genetically epilepsy-prone rat severe seizure strain (GEPR-9). An increase in the number of acoustically-evoked action potentials in neurons of the central nucleus of inferior colliculus (ICc) was observed by the second day of AGS repetition, and peaked at day four. The ICc responses remained at similar enhanced level through day 14. ICc neuronal responses were completely absent for approximately two min post-ictally after a single AGS in all animals, but 80% of the animals undergoing repetitive AGS consistently exhibited neuronal firing in this post-ictal period. Post-tonic clonus and an increased duration of post-ictal behavioral depression were also observed with repetitive AGS. The increased ICc neuronal firing was observed prior to the appearance of the post-tonic clonus component of repetitive AGS. This suggests that the ICc neuronal firing increase may subserve, at least, the initial increase in AGS severity. However, changes in neuronal firing in nuclei of the neuronal network for AGS efferent to the ICc may be responsible for the increased AGS severity that occurs after the fourth day of AGS repetition.
Audiogenic seizures, a model of brainstem epilepsy, are characterized by a tonic phase (sustained muscular contraction fixing the limbs in a flexed or extended position) associated with a short cortical electroencephalogram flattening. When sound-susceptible rats are exposed to repeated acoustic stimulations, kindled audiogenic seizures, characterized by a clonic phase (facial and forelimb repetitive jerks) associated with cortical spike-waves, progressively appear, suggesting that repetition of brainstem seizures causes a propagation of the epileptic discharge toward the forebrain. In order to determine the structures through which this propagation occurs, four kinds of experiments were performed in non-epileptic rats and in sound-susceptible rats exposed to single or repeated sound stimulations. The following results were obtained:
There is extensive evidence that ethanol interacts with a variety of neurotransmitters. Considerable research indicates that the major actions of ethanol involve enhancement of the effects of gamma-aminobutyric acid (GABA) at GABAA receptors and blockade of the NMDA subtype of excitatory amino acid (EAA) receptor. Ethanol increases GABAA receptor-mediated inhibition, but this does not occur in all brain regions, all cell types in the same region, nor at all GABAA receptor sites on the same neuron, nor across species in the same brain region. The molecular basis for the selectivity of the action of ethanol on GaBAA receptors has been proposed to involve a combination of benzodiazepine subtype, beta 2 subunit, and a splice variant of the gamma 2 subunit, but substantial controversy on this issue currently remains. Chronic ethanol administration results in tolerance, dependence, and an ethanol withdrawal (ETX) syndrome, which are mediated, in part, by desensitization and/or down-regulation of GABAA receptors. This decrease in ethanol action may involve changes in subunit expression in selected brain areas, but these data are complex and somewhat contradictory at present. The sensitivity of NMDA receptors to ethanol block is proposed to involve the NMDAR2B subunit in certain brain regions, but this subunit does not appear to be the sole determinant of this interaction. Tolerance to ethanol results in enhanced EAA neurotransmission and NMDA receptor upregulation, which appears to involve selective increases in NMDAR2B subunit levels and other molecular changes in specific brain loci. During ETX a variety of symptoms are seen, including susceptibility to seizures. In rodents these seizures are readily triggered by sound (audiogenic seizures). The neuronal network required for these seizures is contained primarily in certain brain stem structures. Specific nuclei appear to play a hierarchical role in generating each stereotypical behavioral phases of the convulsion. Thus, the inferior colliculus acts to initiate these seizures, and a decrease in effectiveness of GABA-mediated inhibition in these neurons is a major initiation mechanism. The deep layers of superior colliculus are implicated in generation of the wild running behavior. The pontine reticular formation, substantia nigra and periaqueductal gray are implicated in generation of the tonic-clonic seizure behavior. The mechanisms involved in the recruitment of neurons within each network nucleus into the seizure circuit have been proposed to require activation of a critical mass of neurons. Achievement of critical mass may involve excess EAA-mediated synaptic neurotransmission due, in part, to upregulation as well as other phenomena, including volume (non-synaptic diffusion) neurotransmission. Effects of ETX on receptors observed in vitro may undergo amplification in vivo to allow the excess EAA action to be magnified sufficiently to produce synchronization of neuronal firing, allowing participation of the nucleus in seizure generation. GABA-mediated inhibition, which normally acts to limit excitation, is diminished in effectiveness during ETX, and further intensifies this excitation.
The lateral nucleus of the amygdala (LA), a key component of the fear conditioning circuitry, receives a rapid but relatively impoverished auditory input from the auditory thalamus and a slower but richer input from the auditory cortex. We examined in urethane anesthetized rats whether individual cells in the LA receive convergent inputs from these two areas, and whether different postsynaptic receptors contribute to the temporally separated excitations over the two pathways. With both extracellular and intracellular recordings, individual cells could be activated by stimulation of each pathway. In extracellular recordings iontophoretic application of the N-methyl-D-aspartate (NMDA) receptor antagonist APV and the L-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor antagonist CNQX demonstrated that synaptic transmission in both pathways depends on AMPA receptors, whereas transmission in the thalamic pathway also depends on the involvement of NMDA receptors. The involvement of NMDA receptors in synaptic activation of the LA from the thalamus but not the cortex was confirmed in intracellular recordings using systemic injections of the NMDA antagonist MK-801. The slow time course of NMDA currents could provide LA cells with a mechanism to integrate the inputs arriving rapidly from the thalamus and somewhat later from the cortex, thus allowing the LA to integrate signals in the two pathways during the acquisition and expression of conditioned fear reactions.
Despite the veritable spate of books on the epilepsies and individual aspects thereof over the past few years, the appearance of Wyllie's tome sets a new standard for comprehensiveness on the subject of treatment of epilepsy. The authorship includes 128 individuals, most of whom have made significant contributions in their individual areas of expertise. Yet, this book is much more than a Name-Dropper's Guide to the Galaxy of Epileptology. It brings together the collective experience of those actively engaged in both research and the clinical areas covered, providing a remarkably up-to-date account whose currency has been enhanced by the fast-track treatment of editor and publisher.The book has six main sections. Section 1 is devoted to basic mechanisms including neuroanatomy, neurophysiology, and principles of genetics and epidemiology. I found the chapters on the basic mechanisms of epileptogenesis and natural history of seizures of particular value in clarifying areas that frequently
Repetition of seizures appears to increase severity in a number of seizure models, but the nature of these severity increases has not been elucidated in naturally occurring genetic epilepsy models. The genetically epilepsy-prone rat (GEPR) is highly susceptible to many seizure provoking stimuli, and high intensity acoustic stimuli induce audiogenic seizures (AGS). The role of forebrain structures in AGS in the GEPR has not been clear, and the presence of cortical epileptiform EEG activity in the GEPR is controversial. The present study examined the effects of 21 daily AGS repetitions on behavior and EEG activity recorded from the cortex of two GEPR substrains that exhibit moderate (GEPR-3) or severe AGS (GEPR-9). The results indicated that AGS repetition induced seizure severity increases in both GEPR substrains and resulted in prominent cortical epileptiform EEG activity. The AGS behavioral patterns remained distinctly different in the two substrains throughout seizure repetition. In each substrain a different additional behavioral phase was expressed; the GEPR-9 exhibited post-tonic clonus, and the GEPR-3 exhibited facial and forelimb clonus. These findings indicate that seizure repetition results in expansion of the neuronal network subserving AGS to involve forebrain structures. The medial geniculate body and amygdala appear to be part of this expanded network, and long-term potentiation, which was reported in the pathway between the latter brain structures, may be involved. These data suggest that recruitment of forebrain structures into the AGS neuronal network appears to be essential for production of the additional ictal behaviors evoked by AGS repetition.
The purpose of this study was to advance our understanding of the anatomical organization of sensory projections to the amygdala, and specifically to identify potential interactions within the amygdala between thalamic and cortical sensory projections of a single sensory modality. Thus, interconnections between the amygdala and acoustic processing areas of the thalamus and cortex were examined in the rat using WGA-HRP as an anterograde and a retrograde axonal tracer. Injections placed in medial aspects of the medial geniculate body (MGB) produced anterograde transport to the lateral nucleus of the amygdala and to adjacent areas of the striatum. Injections of primary auditory cortex (TE1) produced no transport to amygdala. In contrast, injections ventral to TE1 involving TE3 and perirhinal periallocortex (PRh) produced anterograde transport in the subcortical forebrain that was indistinguishable from that produced by the MGB injections. The TE3 and PRh injections also resulted in retrograde transport to primary auditory cortex and to MGB, thus confirming the involvement of these ventral cortical areas in auditory functions. Injections of the lateral nucleus of the amygdala resulted in retrograde transport back to the medial areas of MGB and to temporal cortical areas PRh, TE3, and the ventral most part of TE1. Thus, auditory processing regions of the thalamus and cortex give rise to overlapping (possibly convergent) projections to the lateral nucleus of the amygdala. These projections may allow diverse auditory signals to act on common ensembles of amygdaloid neurons and may therefore play a role in the integration of sensory messages leading to emotional reactions.
Numerous studies have implicated the substantia nigra pars reticulata (SNR) in the initiation and behavioral expression of kindled seizures. In immobilized, amygdala-kindled animals, SNR neurons have been shown to enter an intense burst-firing pattern during afterdischarge (AD). Taken together these findings raised the possibility that the SNR facilitates the expression of kindled seizures by directly propagating seizure activity into target structures. In this study we examined the relationship between activation of SNR neurons and the electrical (EEG) and behavioral (clonic motor) expression of kindled seizures using both immobilized and unrestrained animals. The principal findings were that: (1) in both immobilized and unrestrained animals the SNR neurons of kindled, but not control, animals were recruited into a burst-firing pattern during AD; (2) the onset of burst-firing was delayed until after the onset of AD; and (3) the onset of burst-firing was not correlated with the onset of rhythmic motor seizure activity. These findings support the idea that the development of kindling is associated with recruitment of SNR neurons into a seizure propagating network. However, these data suggest that activation of SNR neurons is not necessary for the expression of clonic motor activity and does not lower seizure threshold.
Full limbic seizures were kindled in rats by repeated, bi-daily microinjections of glutamate (1.5 mumol) into the basolateral amygdala. Co-administration of the N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-7-phosphonoheptanoic acid with the glutamate, prevented the development of both electroencephalographic and motor signs of the kindling response. These results indicate an important functional role for NMDA receptors in the development of excitatory amino acid-induced kindling.
The genetically epilepsy-prone (GEP) rat is susceptible to seizure induction by acoustic stimuli. The inferior colliculus (IC) has been implicated as being critically important in audiogenic seizure susceptibility based on lesion, electrical stimulation, and focal implantation experiments. The current study determined that GEP rats were most susceptible to seizure induction by pure tone bursts at 100 dB at a frequency of 12 kHz. IC neurons in the GEP rat exhibited a significantly elevated incidence of a particular response pattern at 12 kHz and at characteristic frequency. This pattern consisted of a peak at the beginning and end of the stimulus (onset-offset response). This response pattern only occurred with high intensity stimuli approximating those which induce seizures and may represent an afterdischarge phenomenon. The response threshold was significantly elevated and tuning characteristics were also significantly altered in IC neurons of GEP rats as compared to normal IC neurons. The latter two findings may be related to the deficit of hearing which is reported in the GEP rat. The increased incidence of onset-offset responses may be due to a decreased efficacy of inhibition in the GEP rat neurons as compared to normal rat neurons.
Kindling is an animal model of epilepsy and neuronal plasticity produced by periodic electrical stimulation of the brain. Electrophysiologic studies indicate that this phenomenon is associated with increased participation of N-methyl-D-aspartate (NMDA) receptors in excitatory synaptic transmission. Biochemical studies suggest that a change intrinsic to the NMDA receptor-channel complex may contribute to the increase in NMDA receptor-mediated synaptic transmission. We tested this idea by measuring the binding of 3-[(+)-2-(carboxypiperazin-4-yl)][1,2-3H]propyl-1-phosphonic acid ([3H]CPP), [3H]glycine, and tritiated N-[(1-thienyl)cyclohexyl]piperidine [( 3H]TCP) to rat hippocampal membranes. In this preparation these ligands are selective for the NMDA receptor, the strychnine-insensitive glycine receptor, and the NMDA receptor-gated ion channel, respectively. Kindling increased the density of CPP, glycine, and TCP binding sites in hippocampal membranes by 47%, 42%, and 25%, respectively. No significant changes were detected in the affinity of these binding sites. Surprisingly, alterations in the glycine binding site were detected in animals sacrificed 1 month but not 1 day after the final kindling stimulation. Thus, delayed upregulation of the NMDA receptor-channel complex may be one molecular mechanism that maintains the long-lasting hyperexcitability of hippocampal neurons in kindled animals.
Fully developed limbic seizures were kindled by repeated (every second day) microinjections of an L-glutamate plus L-aspartate (Glu/Asp) mixture (1:3 ratio; 1.5 mumol total dose). Glu alone (1.5 mumol) or Asp alone (1.5 mumol), into the rat amygdala. This excitatory amino acid (EAA)-induced kindling was durable, persisting for at least several months, and showed strong positive transfer to electrical kindling. Fully EAA kindled seizures were inhibited by focally applied NMDA-receptor antagonists. EAA kindling and electrical kindling are shown to have many similar properties. This strongly suggests that they may also have neurochemical mechanisms in common. These results further highlight the important role of EAAs in basic mechanisms involved in the generation and expression of epilepsy.
This study was designed to analyze in vitro the changes in synaptic potentials that occur in neurons of the basolateral amygdala 4-6 weeks after kindling in vivo. The following 3 phenomena were observed in basolateral neurons which were contralateral to the kindling site: (1) spontaneous epileptiform bursting; (2) evoked epileptiform bursting or 'extra' evoked synaptic potentials; and (3) the absence of GABAergic inhibitory postsynaptic potentials either spontaneous or evoked. Epileptiform bursting, spontaneous and evoked, and 'extra' evoked synaptic potentials were depressed by NMDA receptor antagonists and were recorded in normal physiological solution. These data suggest that the amygdala is an area of the brain particularly sensitive to epileptogenesis.
Previous studies indicate that the inferior colliculus is the brain stem auditory nucleus most sensitive to the chemical blockade of audiogenic seizures in the genetically epilepsy-prone rat. Other auditory structures do not appear to be as important. This study attempted to define the efferent pathways involved in propagation of the seizure from the colliculus to the spinal cord where the motor components of the convulsion are generated. This study also determined whether certain nuclei which have been implicated in the propagation of seizures in other epilepsy models are involved in audiogenic seizures. The excitant amino acid antagonist, 2-amino-7-phosphonoheptanoate, was infused bilaterally into several of those sites. The drug was effective in significantly reducing seizure severity with infusion of 5 nmol bilaterally into the midbrain and the pontine reticular formation or the substantia nigra. However, similar drug doses were not effective when infused into the entopeduncular nucleus even though prominent behavioral effects were observed with this infusion. Infusion of 2-amino-7-phosphonoheptanoate into the prepiriform cortex resulted in a small but significant reduction in seizure severity. These results suggest that inhibition of excitatory transmission within the substantia nigra and the reticular formation effectively blocks the output pathway for the audiogenic seizures, whereas the role of the prepiriform cortex in this process is relatively minor.
The afferent pathway involved in initiation of audiogenic seizures in the genetically epilepsy-prone rat was investigated by bilateral microinfusion of the excitant amino acid antagonist 2-amino-7-phosphonoheptanoate into the major brain stem and subcortical nuclei of the auditory system. This antagonist has been shown to possess anticonvulsant properties in other seizure models, and an excitant amino acid has been implicated as a putative neurotransmitter in several of these nuclei. Seizure severity was significantly reduced following infusion of this agent into the cochlear nucleus, superior olivary complex, inferior colliculus, and medial geniculate body. Many of these animals exhibited a complete blockade of seizures. The smallest effective dose in the cochlear nucleus and the medial geniculate body was 5 nmol per side. The smallest effective dose in the olive was 1 nmol, and in the inferior colliculus 0.1 nmol per side was protective. The onset of anticonvulsant effectiveness was earliest in the inferior colliculus. These findings showed that the inferior colliculus was the most sensitive auditory center to the anticonvulsant action of 2-amino-7-phosphonoheptanoate and that imbalance between inhibitory and excitatory transmission within this brain structure may be crucial in the initiation of audiogenic seizures in the genetically epilepsy-prone rat.
A strain of Wistar rats was inbred for susceptibility to audiogenic seizures characterized by one or two wild running fits followed by tonic dorsiflexion with open mouth and then a catatonic state. During the tonic phase, the cortical EEG was flat for 1 to 2 sec, then changed to a slow, regular low-amplitude discharge, 9 to 12 c/s, for 25 to 60 sec. In these rats exposed to 40 daily 90-sec auditory stimuli, behavior and EEG changed. The wild running became disorganized by myoclonic jerks of the limbs and body. In some animals, the tonic extension disappeared and a myoclonic seizure developed progressively, with facial and forelimb clonus, and rearing and falling. In others, the tonic phase was followed by a generalized clonic phase. The EEG during the myoclonic and tonic-clonic seizures showed high-amplitude rhythmic spikes, polyspikes and spike-waves, 1 to 10 c/s, for 40 to 120 sec, often outlasting the sound stimulus. The effects of ethosuximide, carbamazepine and phenytoin were the same on primary and modified audiogenic seizures. The progressive behavioral and EEG modifications of audiogenic seizures following repeated auditory stimuli suggest that kindling had developed, the seizures being propagated from the brain stem to forebrain structures.
The EEG of 20 Wistar rats inbred for audiogenic seizures was recorded during 40 daily auditory stimuli 90 s long. The first stimuli provoked wild running, with no cortical EEG abnormality, and then a tonic phase with a characteristic EEG of a brief flat trace 2 to 3 s long followed by low-amplitude regular activity, 10 to 12 c/s, lasting 40 to 60 s. The lack of paroxysmal EEG patterns suggests that the cortex plays only a minor role in audiogenic seizure development. After 5 to 15 daily stimuli, the EEG during the running period exhibited brief spike and spike-wave discharges preceding the EEG pattern of the tonic phase. After a few more daily stimuli these paroxysmal discharges progressively increased in amplitude and duration, overlapping with the regular activity of the tonic phase. After 20 to 30 stimuli, only high-amplitude spikes and spike-waves, 1 to 10 c/s, were seen for 40 to 120 s. The modified EEG persisted 2 to 4 months after daily stimulation was discontinued. Thus, with stimulus repetition, a paroxysmal discharge progressively involved cortical structures. These data suggest that repetition of audiogenic seizures induced a phenomenon related to kindling in Wistar rats susceptible to sound-induced epilepsy.
A strain of Wistar rats was inbred in our laboratory for its susceptibility to sound. The seizures are characterized by one or two wild running fits which terminate in a tonic dorsiflexion with open mouth, followed by a catatonic state. During the tonic phase of the seizure, the cortical EEG is flattened for 2 to 3 s. Then, a slow and regular low-voltage (9-12 c/s) activity is observed during 40 to 60 s. When these animals are submitted to daily sound-stimulations, the behavioral as well as the EEG manifestations of the audiogenic seizures change progressively. After 5 to 30 exposures, the wild running becomes disorganized by occurrence of myoclonic jerks of the limbs and the body. In some animals, the tonic extension disappears and a myoclonic seizure develops progressively with facial and forelimb clonus, rearing and falling. In other animals, the tonic phase still occurs and is followed by a generalized clonic phase. During both the myoclonic and the tonicoclonic seizures, rhythmic spikes, polyspikes and spike and waves of high amplitude (1-10 c/s) during 40 to 120 s are observed on EEG recordings. These EEG modifications often outlast the sound stimulation. The pharmacological reactivity in rats exposed to single or repeated audiogenic seizures is similar: phenytoin and carbamazepine suppress both kinds of seizures at low doses whereas ethosuximide is efficacious only at high doses. In order to know whether the repeated exposure to sound or the repetition of seizures are responsible of the observed changes in audiogenic seizures, animals susceptible to sound were exposed daily to the seizure-inducing sound after previous injection of Diazepam, which prevented them from convulsing. On the other hand, sound susceptible animals were injected daily with a dose of PTZ inducing one or several convulsions without exposure to sound. None of these treatments ever facilitated the development of kindled audiogenic seizures. The progressive modification of behavioral and EEG modifications occurring when audiogenic seizures are repeated suggests that kindling has developed, the seizure extending from the brainstem to forebrain structures.
The Genetically Epilepsy-Prone Rat (GEPR) is rapidly gaining support as a model of epilepsy. In addition to a marked sensitivity to both sound-induced and hyperthermic seizures, GEPRs exhibit unusual sensitivity to a number of seizure-provoking modalities, including various forms of electrical and chemical stimulation. The existence of a moderate seizure colony (GEPR-3) and a severe seizure colony (GEPR-9) allows pathophysiological studies of seizure susceptibility and severity. The consistency of seizures within each colony allows for comparisons in seizure naive GEPRs and seizure experienced GEPRs. The consistent seizure responses of the GEPR are also ideal for the testing of anticonvulsant drugs. Further, the relative potencies of anticonvulsant drugs between the two colonies of GEPRs predict the clinical efficacies of traditional antiepileptic drugs and may be able to predict novel anticonvulsants.
Intraperitoneal injection of pilocarpine (380 mg/kg) produces motor limbic seizures in rats. Focal injection into the prepiriform cortex (PC) of an N-methyl-D-aspartate receptor antagonist, 2-amino-7-phosphonoheptanoic acid (APH), 1-10 pmol, potently protects against these seizures and their pathological consequences. Sites similarly sensitive to the protective action of APH are found along a substantial part of the anterior-posterior extension of the piriform cortex. More caudal injection sites, located at the level of lateral septum are less sensitive. The anticonvulsant action of APH along the extent of the PC is localised in the vicinity of the injection site, as shown by autoradiography following focal injection of tritiated APH.
A bilateral mechanical lesion of the midbrain and pontine tegrnentum was found to abolish completely the tonic components of sound‐induced seizures in genetically epilepsy‐prone rats (GEPR) that display tonicclonic seizures. Correlations between varied lesion placements and effects on maximal audiogenic seizures provided evidence that damage to the nucleus reticularis pontis oralis (RPO) of the midbrain and pontine reticular formation (RF) was responsible for the seizure‐attenuating effects. Moreover, electrolytic lesions of the pontine RF involving the RPO nucleus were found to abolish the tonic components of the maximal audiogenic seizure. Additionally, bilateral mechanical lesions involving the RPO nucleus were found to attenuate the clonic components of sound‐induced seizures in GEPR that display only running seizures and clonus. These findings are consistent with previous studies showing that pontine tegmental lesions attenuate the tonic components of maximal electroshock‐ and pentylenetetrazol‐induced seizures, and lend further support to the hypothesis that all generalized tonic seizures share a common neural substrate. The role of the brainstem RF in tonic versus clonic convulsions is discussed in light of the present findings.
ll a éeé démontré au'une lésion mécanique bilatérale de la calotte mesencephalique et pontine abolit complètement les composantes toniques des crises induites par le bruit chez les rats génétiquement prédisposés (GEPR) qui ont des crises tonico‐cloniques. L‘étude des correlations entre différentes localisations de la lésion et ses effets sur les crises audiogènes maximum a montré que l'atteinte du nucleus reticularis pontis ovalis (RPO) et de la formation réticulaire pontine (RF) était responsable de cet effet d'atténtuation des crises. De plus on a montré que des lésions électrolytiques de la RF pontine intéressant le noyau RPO abolissaient aussi les composantes toniques des crises audiogènes maximum. Enfin, des léesions mécaniques bilatérales intéressant le RPO atténuent les composantes cloniques des crises induites par le bruit chez les rats GEP qui ont seulement des “crises de course” et des clonies. Ces données sont en accord avec les études précédentes montrant que les lésions de la calotte pontine atténuent les composantes toniques des crises maximum induites par l’électrochoc maximal et par pentylenetetrazol, et conduisent à formuler l'hypothèse que toutes les crises généralisées toniques partagent un substratum neural commun. le rôle de la RF du tronc cérébral dans les convulsions cloniques par rapport aux convulsions toniques est discutéà la lumière de ces données.
RESUMEN
Una lesión mecänica bilateral del tegmento mesencefälico y pontino produjo una abolicion completa de los componentes tónicos de crisis inducidas por sonidos (audiogénicos) en ratas genéticamente predispuestas a la epilepsía (RGPE) que presentaban crisis tónicoclónicas. Correlaciones entre diferentes localizaciones y efectos de la lesion en las crisis audiogénicas proporcionaron evidencia de que el dano del núecleo reticular anterior del puente (RPO) y formación reticular pontina fué responsable de los efectos atenuantes de las crisis. Además lesiones electrolíticas de la formación reticular pontina afectando el núcleo RPO mostraron abolición de los componentes tónicos de crisis audiogénica mäxima. Adicionalniente, lesiones mecánicas bilaterales afectando la RPO atenuaron los components clónicos de las crisis comiciales inducidas por sonidos en ratas GPE que mostraban sólamente crisis cursivas y clonus. Estos hallazgos son comparables con estudios previos que evidenciaban que las lesiones del tegmento pontino atenuaban los componentes tonicos de las crisis tónicas inducidas por electroshock y pentylentetrazoly llevan además a sustentar la hipótesis de que todas las crisis tónicas generalizadas tienen un sustrato neural común. Se discute et papel de la formación reticular del tronco cerebral en las convulsions tónicas versus clónicas a la luz de 10s presentes hallazgos.
Chronic recordings of amygdaloid neurons were performed on freely moving rats following kindling. Satisfactory recordings were obtained from 22 amygdaloid neurons of the contralateral amygdala before, during, and after unilateral kindling. Kindling stimulations were given once per hour. Seven cells disappeared during kindling. Seven cells were recorded during the full course of kindling. These units showed (a) an increase in spontaneous firing, (b) a development of high‐frequency bursts (the peak interval of the interval histogram decreased from 18 to 2 ms), and (c) high‐frequency firings during spontaneous activity that were similar to the firings recording during afterdischarge.
RÉSUMÉ
Un enregistrement chronique des neurones du noyau amygdalien a été effectué chez des rats libres de leurs mouvements soumis à l'effet d'embrasement. Un enregistrement satisfaisant a été obtenu pour 22 neurones amygdaliens de l'amygdale controlatérale, avant, pendant et après un embrasement amygdalien unilatéral. Les stimulations étaient pratiquées une fois par heure. Sept cellules ont pu étre enregistrées pendant toute la durée de l'expérience. Au niveau de ces cellules, l'on observe: (1) une augmentation des décharges spontanées, (2) l'apparition de décharges à haute fréquence, l'intervalle entre les pics diminuant de 18 msec à 2 msec, et (3) des décharges à haute fréquence pendant l'activité spontanée, décharges similaires à celles obtenues pendant une post‐décharge.
ZUSAMMENFASSUNG
Chronische Aufzeichnungen von Neuronen des Amygdalons wurden bei frei sich bewegenden Ratten in Anschluß an das Kindeln durch‐geführt. Befriedigende neuronale Ableitungen wurden von 22 Neuronen des kontralateralen Amygdalons vor, während und nach einseitigem Kindeln gemacht. Die Kindlingreize wurden einmal pro Stunde gegeben. Sieben Zellen verschwanden während des Kindelns. Sieben Zellen wurden während des gesamten Kindelns abgeleitet. Die Einheiten zeigen 1. eine Zunahme der spontanen Entladungen, 2. eine Entwicklung hochfrequenter bursts; ihr peak Intervall nahm im Histogramm von 18 msec auf 2 msec ab. 3. Hochfrequente Entladungen während Spontan‐aktivität ähnelten den Aufzeichnungen während der Nachentladungen.
Corticocortical and corticoamygdaloid connections of temporal cortext and perirhinal cortex (PRh) were examined in the rat with the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L). Iontophoretic injections of PHA-L into area TE1 resulted in columnar axonal terminations in surrounding and contralateral regions of temporal neocortex and in the striatum, but not in the amygdala. Within temporal neocortex, labeled fibers were present locally in adjacent regions of TE1, as well as in TE2d, TE1v, TE3v, and TE2c. Injection of cortical areas TE1v, TE3v, and TE2c, which received projections from TE1, or injections of perirhinal periallocortex, which received projections from TE1v, TE2v, and TE3v, resulted in projections to the amygdala. The pattern of corticocortical and corticoamygdaloid projections differed among the divisions of auditory cortex. TE1 exhibited extensive ipsilateral and contralateral projections to temporal cortical regions and no projections to the amygdala. In contrast, areas of temporal neocortex ventral and posterior to TE1, including TE1v, TE3v, TE2c, and PRh, had more limited ipsi- and contralateral corticocortical projections but had an increased connectivity with the subcortical forebrain, especially the lateral nucleus of the amygdala (AL). There was a topographic organization to the AL afferents. The dorsal subdivision of AL received projections from TE1v, TE3v, TE2c, and PRh, while the ventrolateral division received projections from TE3v, TE2c, and PRh. The ventromedial division received projections only from PRh, which, unlike other temporal cortical areas, also projected to the basolateral and basomedial nuclei of the amygdala. These findings define the complete sequence of connections linking primary auditory cortex with the amygdala in the rat. In addition, the findings indicate that the ventral portion of TE1, designated TE1v, has connections that distinguish it from dorsal TE1, namely, dense projections to AL and a diminished number of corticocortical projections ipsilaterally and contralaterally. Finally, the results suggest a topographic organization to the cortical terminations within the amygdala.
In the present study we analyzed the organization of the thalamocortical projections of the specific auditory relay nucleus of the thalamus, the ventral division of the medial geniculate body (MGv), using the anterograde axonal tracer Phaseolus vulgaris leucoagglutinin. All injections of MGv produced dense labeling of axonal fibers in temporal cortex. In all cases, labeled axons were predominantly concentrated in cortical layers III and IV and, to a lesser extent, at the junction of layers V and VI. Injections confined to the medial regions of MGv, and specifically to the ovoid nucleus of MGv (OV, pars ovoidea), resulted in anterograde labeling of TE1, with minor labeling of the ventral quarter of TE1, designated subarea TE1v. Injections placed in lateral regions of MGv and occupying the lateral ventral subnucleus (LV), or injections in the mediolateral center of MGv and occupying parts of LV and OV, also resulted in labeling of area TE1 and minor labeling of TE1v. However, these injections also produced labeling in areas TE2 and TE3. Thus, area TE1 (excluding subarea TE1v) receives heavy projections from all aspects of MGv and appears to be the core target of MGv. While regions of MGv also project to surrounding cortical belt areas, these projections tend to be lighter and to vary depending on the region of MGv examined. These results, together with other connectional findings, and cytoarchitectonic and physiological studies, suggest that TE1 (possibly excluding subarea TE1v) is the primary auditory cortex in the rat.
Changes in gene expression after kindled seizures were examined using microdissection of discrete brain areas and Northern and slot blot analyses. Experimental animals were kindled with either of two protocols: (1) a paradigm in which 50 Hz/10 s stimulus trains were delivered every 30 min through hippocampal electrodes (12 stimulations every other day for 4 days) and (2) a traditional approach in which 50 Hz/10 s stimulus trains were given to the hippocampus three times daily for 16 days. Rats were sacrificed 24 h or 30 days after the last kindled seizure. We first examined the possibility that kindling may affect transcription of mRNA for neurotransmitter receptors. We found significant decreases (22-58%) in AMPA/kainate activated glutamate receptor mRNAs (GluR1, -2, -3 mRNAs) in hippocampus, amygdala/entorhinal cortex and in frontoparietal cortex 24 h but not 30 days after rapidly kindled seizures. However, changes in GABA receptor alpha 1, alpha 2, alpha 4 or beta 1 mRNAs were not observed in any brain region 30 days after traditional kindling or 24 h after rapidly kindled seizures. In addition, we tested whether changes in the expression of proenkephalin could be detected after kindling. We found significant increases (1.7-10 fold) in proenkephalin mRNA in the frontoparietal cortex, hippocampus and in the amygdala/entorhinal cortex 24 h but not 30 days after rapidly kindled seizures. Our findings suggest that changes in glutamate receptor and proenkephalin gene expression are robust, acute sequelae to kindled seizures and may be involved in kindling.
We examined whether the NMDA class of excitatory amino acid receptors contribute to synaptic transmission in the pathway connecting the medial geniculate body (MGB) with the lateral nucleus of the amygdala (LA) using extracellular single unit recordings and microiontophoresis. Cells were identified in LA on the basis of responsivity to electrical stimulation of the MGB. For each cell, a level of current was found for the iontophoretic ejection of the NMDA antagonist AP5 that blocked responses elicited by iontophoresis of NMDA, but had no effect on responses elicited by AMPA. Iontophoresis of AP5 with this level of current blocked the excitatory response elicited by MGB stimulation in most cells tested. Microinfusion of AP5 (25, 50, or 100 microM) also blocked the responses. Additional studies tested individual cells with both AP5 and the AMPA antagonist CNQX and showed that blockade of either NMDA or AMPA receptors interferes with synaptic transmission. Finally, iontophoretic ejection of either AP5 or CNQX blocked short-latency (< 25 ms) responses elicited in LA by peripheral auditory stimulation. Together, these results suggest that the synaptic evocation of action potentials in the thalamo-amygdala pathway depends on both NMDA and non-NMDA receptors. We hypothesize that non-NMDA receptors are most likely required to depolarize the cell sufficiently to remove the blockade of NMDA channels by magnesium and NMDA receptors are required to further depolarize the membrane to the level required for action potential generation.
The tissue content and the interstitial fluid levels of glutamate, aspartate, GABA, glutamine, glycine, and serine were studied in amygdaloid-kindled rat brain. Interstitial levels were studied in vivo before and during stage 5 full limbic seizures using microdialysis. Slices of amygdala from kindled and sham-operated animals were used to study baseline and KCl-evoked release in vitro. The contents of these amino acids were measured in slices of amygdala, hippocampus, and cerebral cortex from kindled and sham-operated animals. Kindled brains showed two- to threefold higher levels of glutamate, aspartate, and GABA and 12-fold higher levels of glutamine than sham-operated controls. Correlating with this, interstitial fluid levels of glutamate were two- to threefold higher from kindled amygdala than from control both in vivo (microdialysis) and in vitro (superfusion). GABA levels in interstitial fluid from kindled amygdala were reduced by 67% compared with control amygdala.
The purpose of this study was to further our understanding of the contribution of auditory thalamoamygdala projections to conditioned emotional memories formed when auditory and noxious somatosensory stimuli are associated. Single unit activity was recorded in the acoustic thalamus of chloral hydrate-anesthetized rats in response to auditory (white noise, clicks, tones) and somatosensory (foot-shock) stimulation. The thalamic areas focused on were the medial division of the medial geniculate body (MGm), the suprageniculate nucleus (SG), and the posterior intralaminar nucleus (PIN), thalamic areas that receive inputs from both the inferior colliculus and the spinal cord and that project to the lateral nucleus of the amygdala (AL). For comparison, recordings were also made from the specific thalamocortical relay nucleus, the ventral division of the medial geniculate body (MGv), which receives projections from the inferior colliculus but not from the spinal cord. Auditory but not somatosensory responses were recorded from MGv, while both auditory and somatosensory responses were frequently found in MGm, PIN, and SG. In these areas, convergent auditory and somatosensory responses were more frequently found rostrally than caudally. Within a thalamic subregion, the acoustic response properties of the convergence cells were not different from the response properties of unimodal auditory cells. Some cells that responded to somatosensory but not auditory stimuli showed a potentiated response when tested with simultaneous presentation of auditory and somatosensory stimuli. In some studies, thalamic cells that project to the amygdala were antidromically activated by stimulation of the AL. Consistent with anatomical tracing results, antidromically activated cells were found in MGm, PIN, and SG, but not in MGv. Antidromically activated cells were more likely to respond to auditory stimuli than to somatosensory stimuli, but unimodal somatosensory and convergence cells were also found. These findings, which provide the first characterization of acoustic response properties of multimodal cells in the auditory thalamus and of cells in the auditory thalamus that project to amygdala, suggest insights into the emotional functions of the thalamoamygdala pathway.
The behavioral and EEG concomitants of kindling produced by daily electrical stimulation of the inferior colliculus have been recorded in three series of Wistar rats: (1) non epileptic controls (NE), (2) rats susceptible to audiogenic seizures (AS), (3) acoustically susceptible rats with prior kindling of audiogenic seizures by repeated sound exposure (KAS). Repeated collicular stimulation produced behavioral and EEG changes which were similar in the AS and the NE rats. The tonic seizure without cortical discharges elicited by the first stimulation progressively changed into tonic-clonic seizures with sustained cortical EEG discharges after more than 20 stimulations. In the KAS group, the electrical collicular kindling was clearly accelerated: kindled tonic-clonic seizures and their EEG discharges already occurred after one to five electrical stimulations. Similarly, after completion of electrical collicular kindling in AS, sound stimulations immediately induced characteristic kindled audiogenic seizures. The immediate reciprocal positive transfer observed between kindling of audiogenic seizures and kindling of seizures induced by electrical stimulation of the inferior colliculus suggests that kindling of these two brain-stem seizures involves similar structures and mechanisms.
Limbic seizures were kindled by repeated, daily intra-amygdaloid microinjections of N-methyl-D-aspartate (NMDA; 2 nmol). The seizures, and accompanying afterdischarges, closely resembled those seen following electrical kindling of the amygdala. As with electrical kindling, co-administration of the competitive NMDA receptor antagonist DL-2-amino-7-phosphonoheptanoic acid (AP7; 70 nmol) prevented the development of seizure activity. NMDA-induced kindling was durable, lasting at least 1 month, and showed positive transfer to electrical kindling. Fully kindled seizures were inhibited by co-administration of the potent NMDA receptor antagonist DL-[E]-2-amino-4-methyl-5-phosphono-3-pentenoic acid (CGP 37849) with the agonist. These results strongly support a role for NMDA receptors in kindling epileptogenesis.
The genetically epilepsy-prone rat (GEPR-9) exhibits elevated seizure sensitivity and audiogenic seizures (AGS). The pontine reticular formation (PRF) is implicated in the neuronal network for AGS in the GEPR-9. The present study examined PRF neuronal firing and convulsive behavior simultaneously in the GEPR-9. Chronically implanted microwire electrodes in PRF allowed single neuronal responses and behavior to be examined in freely-moving rats. PRF neurons in the GEPR-9 exhibit precipitous intensity-evoked increases at a significantly lower (approx. 15 dB SPL) intensity than normal Sprague-Dawley rats. PRF neurons in the GEPR-9 also exhibit increased auditory response latencies. At the onset of AGS (wild running) the firing rate of PRF neurons increased, and the rate of PRF firing increased dramatically as the tonic phase of the seizure began. During post-ictal depression the rate of PRF neuronal firing slowed, gradually returning to normal. This pattern of PRF periseizural neuronal firing changes differ dramatically in pattern and temporal characteristics from those previously observed in inferior colliculus (IC). The IC serves as the AGS initiation site. IC neurons show extensive firing increases prior to and during the initial wild running, silence during the tonic and post-ictal phases, and gradual recovery of responses thereafter. The changes in PRF neuronal firing pattern suggest that the PRF may play a major role in the generation of the tonic phase of AGS. The premature onset of the precipitous rise in PRF neuronal firing suggests that the influence of the IC on PRF neurons may be magnified in association with AGS susceptibility. The PRF neuronal firing increases observed in the present study coupled with previous observation of AGS blockade by PRF microinjections in the GEPR-9 further support an important role of the PRF in the propagation of AGS in the GEPR-9. The mechanisms of PRF firing elevation may also be relevant in other seizure models in which the brain-stem reticular formation is implicated.
Previous work showed that bilateral lesions made between the inferior and superior colliculi reduced the severity of audiogenic seizures in genetically epilepsy-prone rats (GEPR-9s), and indicated that the connections between these two structures are vital for the propagation of seizure activity. To determine the involvement of the superior colliculus (SC) in seizure propagation, GEPR-9s were given four audiogenic seizures within 1 h by ringing a loud bell, and their brains were processed 30 min later for in situ hybridization for c-fos mRNA. Brain sections from such rats showed dense labeling in both the dorsal cortex and external nucleus of the inferior colliculus. Labeling continued rostrally into the intermediate and deep layers of the SC and the periaqueductal gray region. In addition, other brain regions such as the amygdala, piriform cortex and dorsal endopiriform nucleus showed dense labeling for c-fos mRNA. Comparable increases were not observed in the brains of Sprague-Dawley (SD) rats receiving auditory stimulation or in unstimulated GEPR-9s and SD rats, thereby indicating that increases in stimulated GEPR-9s are seizure-specific. This study provides further evidence that the SC is involved in the propagation of seizure activity in GEPR-9s, and also demonstrates the activation of other brain regions by audiogenic seizures.
Frequent repetition of audiogenic seizure (AGS) ('AGS kindling') in the severe substrain of genetically epilepsy-prone rats (GEPR-9s) results in the appearance of cortical epileptiform electrographic activity, increases of seizure duration and additional convulsive behaviors. These findings suggest that the initial AGS network, which is located primarily in the brainstem, has undergone expansion to the forebrain. The medial geniculate body (MGB) is a thalamic structure that is the first major auditory nucleus efferent to the AGS-initiating site in the inferior colliculus. The MGB is not required for AGS induction, but it has been implicated in the expanded AGS network in GEPR-9s based on focal, pharmacological blockade experiments. The present study examined changes in acoustically evoked MGB neuronal responses in awake and behaving GEPR-9s and in anesthetized GEPR-9s after 14 repetitive AGS-inducing stimuli given daily. An elevated number of action potentials was observed in the MGB neuronal responses after AGS kindling in GEPR-9s. This increase of MGB neuronal responses was associated with a loss of habituation and lasted for at least 28 days after the 14th AGS. An increase in the incidence of sustained acoustic responses in MGB neurons was observed after repetitive AGS in GEPR-9s. Increases in the peak latency and threshold of MGB neuronal responses were also observed after AGS kindling. MGB neurons exhibited a rapid tonic firing during tonic seizures in behaving GEPR-9s, suggesting that the MGB may be implicated in the propagation of seizure activity. However, MGB neuronal firing was silent during post-tonic clonus, a behavior seen in GEPR-9s only after AGS repetition, suggesting that MGB does not play a direct role in the generation of this convulsive behavior. Thus, changes in neuronal firing in nuclei efferent to the MGB, in the expanded neuronal network for repetitive AGS, may be responsible for the generation of post-tonic clonus in GEPR-9s.
Projections from the medial geniculate body (MGB) to the lateral nucleus of the amygdala (LA) have been implicated in the conditioning of emotional reactions to acoustic stimuli. Anatomical and physiological studies indicate that this pathway uses the excitatory amino acid L-glutamate as a transmitter. Recent physiological studies have demonstrated that synaptic transmission in the thalamo-amygdala pathway requires the activation of both N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors, two of the major classes of ionotrophic glutamate receptors. In order to characterize the nature of thalamoamygdala interactions, we examined the synaptic associations between thalamic afferents and amygdala neurons that contain at least one glutamate receptor subtype. Thalamic afferents to the amygdala were identified by lesion-induced anterograde degeneration and anterograde transport of biotinylated dextran-amine, while postsynaptic glutamate receptors were labeled immunocytochemically using antisera directed the R1 subunit of the NMDA receptor and the GluR1 and GluR2/3 subunits of the AMPA receptors. Both methods demonstrated that the majority (77%) of thalamic afferents contact dendritic spines, and most (60%) of these spines express at least one glutamate receptor subtype. To a lesser extent, identified afferents also contacted small and large dendritic shafts, and many of these were immunoreactive. Thalamic afferents terminated on approximately the same proportion (60%) of immunoreactive targets for each glutamate receptor studied. These data provide morphological evidence that thalamic afferents directly synapse onto amygdala neurons that express glutamate receptors and suggest ways in which thalamic afferents activate and influence amygdala circuitry.
Seizures in genetically epilepsy-prone rats (GEPRs) may result from hypoactivity of locus coeruleus (LC) neurons during seizures. This study examined Fos-like-immunoreactivity (FLI) in the LC following audiogenic seizures in two strains of GEPRs (GEPR-9s and -3s), and following pentylenetetrazol (PTZ) or maximal electroshock seizures (MES) in normal rats. After tonic seizure, GEPR-9s showed an identical LC-FLI response to that of normal rats following tonic seizures induced by either PTZ or MES. GEPR-3s, having clonic seizures, had less FLI in the LC. Therefore, stimulus-transcription coupling in the GEPR LC is apparently normo-typic in its FLI response to seizure and thus is not likely the root cause of NE abnormalities in this seizure model.
Generalized tonic-clonic seizures of brain stem origin in rats are associated with acute induction of neuronal Fos in several discrete regions of the brain. One particular site in the dorsal pons shows remarkable Fos induction following generalized tonic seizures induced by maximal electroshock in normal rats or by audiogenic stimulation in genetically epilepsy-prone rats (GEPRs). Although this area shows the most intense Fos induction of any brain area following generalized tonic seizures, its identity has been uncertain. Based on its general location, we hypothesized that this nucleus was either 1) a component of the pedunculopontine tegmentum nucleus-pars compacta (PPTn-pc) or 2) the superior lateral subnucleus of lateral parabrachial area (LPBsl). The present study used Fos-protein immunocytochemistry in combination with the reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry, cholecystokinin (CCK) immunocytochemistry, and neuronal tract-tracing to determine the identity of this cluster of Fos-immunoreactive neurons in the dorsal pons. Following maximal electroshock seizure (MES), Fos labeling was compared to NADPH diaphorase staining (a marker for cholinergic neurons of the PPTn-pc); retrograde transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) injected into the ventromedial nucleus of the hypothalamus (VMH; to identify the LPBsl) or CCK immunoreactivity (also a marker for LPBsl neurons). Results showed this cluster of Fos immunoreactive (FI) neurons to be closely associated, but not overlapping, with the lateral and most caudal aspect of the PPTn-pc. Alternatively, WGA-HRP retrograde-labeled neurons corresponded precisely with the seizure-induced FI neurons. Additionally, the location of CCK immunoreactive neurons directly overlapped with the FI neurons, although they were not nearly as prevalent. These results demonstrate that the seizure-induced FI neurons in this area are neurons of the LPBsl and not cholinergic neurons of the PPTn-pc. This is the first report of seizure-induced Fos expression specifically localized to the superior lateral subnucleus of the lateral parabrachial area.
Recent investigations suggest that the deep layers of superior colliculus (DLSC) play a role in the neuronal network for audiogenic seizures (AGS). The present study examined DLSC neuronal firing and convulsive behavior simultaneously in freely-moving genetically epilepsy-prone rats (GEPR-9s) using chronically implanted microwire electrodes. An abrupt onset of acoustically-evoked firing at approximately 80-90 dB was observed in DLSC neurons of GEPR-9s, which was significantly above the normal threshold. DLSC neurons began to exhibit rapid tonic burst firing 1-2 s prior to the onset of the wild running behavior at the beginning of AGS. As the tonic phase of the seizure began, DLSC firing ceased, and only returned towards normal following post-ictal depression. These neuronal mechanisms may be relevant to other seizure models in which the DLSC is implicated. The temporal pattern of neuronal firing during AGS is specific to DLSC and differs markedly from those observed elsewhere in the AGS neuronal network. The temporal firing pattern suggests that the DLSC plays a primary role in the generation of the wild running phase of AGS. Previous studies indicate that the inferior colliculus is dominant during AGS initiation, and the pontine reticular formation is dominant during the tonic extension phase of AGS. Taken together these data suggest that the neurons in the neuronal network undergo a dominance shift as each specific convulsive behavior of AGS is elaborated.
Recent studies suggest that the deep layers of superior colliculus (DLSC) play a role in the network for audiogenic seizures (AGS) in genetically epilepsy-prone rats (GEPR-9s). The present study examined the role of glutamatergic and noradrenergic receptors in DLSC in modulation of AGS susceptibility. The study examined effects of a competitive NMDA receptor antagonist [dl-2-amino-7-phosphonoheptanoic acid (AP7)] or an alpha1 noradrenergic agonist (phenylephrine) focally microinjected into DLSC as compared to effects in the inferior colliculus (IC) and pontine reticular formation (PRF), which are major established components of the AGS network. The results demonstrated that blockade of NMDA receptors in DLSC suppressed AGS susceptibility. AP7 microinjection was effective at relatively low doses in IC, but required higher doses in DLSC and PRF. The DLSC was relatively more sensitive to seizure reduction by the alpha1 noradrenergic agonist as compared to the IC and PRF. The anticonvulsant effect of AP7 was longer-lasting than phenylephrine in the DLSC and IC but not in the PRF. These data suggest that neurons in the DLSC are a requisite component for the neuronal network for AGS in GEPR-9s and that NMDA and alpha1 adrenoreceptors in this site may play important roles in the modulation of AGS propagation. The relatively greater sensitivity of DLSC to phenylephrine as compared to IC and PRF indicates that norepinephrine may be more important in the modulation of AGS in DLSC, which contrasts to the role of glutamate modulation. These data support recent neuronal recording data, which indicate that DLSC neurons play a critical role in AGS.
Previous studies have demonstrated that generalized tonic-clonic seizures (GTCS) consisting of running/bouncing clonic and tonic extension can still be elicited in rats after brain transections which separate forebrain from brain stem, showing that forebrain circuitry is not required for GTCS. Inasmuch as sound-induced generalized tonic-clonic seizures in rodents are characterized by running-bouncing clonic and tonic convulsions, we have hypothesized that these are brain stem seizures that can occur independently of the forebrain. To test this hypothesis, we examined the response of two strains of genetically epilepsy-prone rats (GEPR-3s and GEPR-9s) to seizure-evoking auditory stimuli 3 h after a precollicular transection or sham surgery performed under ether anesthesia. In addition, the effect of a precollicular transection on audiogenic seizures was evaluated in normal rats made susceptible to such seizures by infusing NMDA into the inferior colliculus. Following the transection 58% of GEPR-9s displayed a sound-induced tonic-clonic convulsion and the remaining 42% exhibited a sound-induced seizure when subjected to stimulation 5 min after a subconvulsant dose of pentylenetetrazol (PTZ). While sham surgery and the precollicular transection both reduced sound-induced seizure severity in GEPR-3s, the full seizure response could be elicited by sound stimulation following a subconvulsant dose of PTZ. Moreover, the audiogenic seizures in normal rats rendered susceptible by NMDA were unaltered by the precollicular transection. These findings show that the anatomical circuitry required for generalized tonic-clonic seizures evoked by sound stimulation in rodents resides within the brain stem.
The inferior colliculus (IC) is established as the initiation site within the neuronal network for audiogenic seizures (AGS), but the relative importance of the IC subnuclei in AGS is controversial. The lateral and basolateral subdivisions of the amygdala are implicated in the expansion of the AGS network that occurs during AGS kindling. However, the role of the amygdala in the AGS network in nonkindled AGS is unknown. NMDA receptors are implicated in modulation of AGS and in neurotransmission in both the IC and amygdala. Therefore, changes in AGS severity in genetically epilepsy-prone rats (GEPR-9s) were examined after bilateral focal microinjection into IC subnuclei or lateral/basolateral subdivisions of the amygdala of a competitive NMDA receptor antagonist, 3-((+)-2-carboxypiperazine-4-yl)propyl-1-phosphonic acid (CPP). Blockade of AGS in IC central nucleus (ICc) and external cortex (ICx) was observed at identical doses of CPP, but these doses were ineffective in IC dorsal cortex (ICd). Microinjection of CPP into the amygdala did not produce significant changes in AGS severity except at doses 20 times those effective in IC. The latter data contrast with the anticonvulsant effects of amygdala microinjections on seizure severity in kindled AGS reported previously. The present data in concord with neuronal recording studies of these nuclei suggest that the ICc is the most critical site in AGS initiation, the ICx in propagation, and that the ICd plays a lesser role in the AGS network. The amygdala does not appear to play a requisite role in the neuronal network for AGS in animals that have not been subjected to AGS kindling.
In a strain of Wistar rats selected in our laboratory, audiogenic seizures (AS), characterized by a wild running phase followed by a tonic seizure, can be elicited by exposure to sound. In these animals repeated daily stimulations induce permanent changes which reflect the extension of seizure activity from the brainstem to the forebrain. C-Fos immunoreactivity was used to further characterize the sound-susceptibility of the strain and to specify the spatiotemporal relationships between c-Fos expression and development of AS kindling. AS susceptible rats appeared to be more sensitive to a subthreshold sound as compared to controls. Sound-evoked wild running induced a similar pattern of c-Fos as a full AS in naive rats, confirming the epileptic nature of this early component. AS-induced c-Fos labeling in the auditory pathways of the brainstem extended to the forebrain with repetition of AS and marked increases in c-Fos expression sequentially occurred in the amygdala and perirhinal cortex, followed by the frontoparietal cortex, the piriform cortex, and finally the hippocampus and entorhinal cortex. These results show that the kindled AS preferentially propagate from the brainstem, through the amygdala and the perirhinal cortex, to the motor cortex, with the piriform cortex and hippocampus as secondary targets. No more c-Fos expression was detected 24 h after an AS. A down-regulation of cortical c-Fos induction was observed 1 and 2 days after daily exposure to kindled AS, with full recovery of c-Fos expression after a 5-day seizure-free period. This suggests a regulatory function of c-Fos expression in development of kindling.
The nuclei comprising the neuronal network for audiogenic seizures (AGS) are located primarily in the brainstem. Previous studies suggested a role for the periaqueductal grey (PAG) in the AGS network. The present study evaluated this possibility in genetically-epilepsy prone rats (GEPR-9s) by examining the effects of bilateral focal microinjection of a competitive NMDA receptor antagonist (DL-2-amino-7-phosphonoheptanoic acid (AP7), 1 and 5 nmol/side), a GABA(A) agonist (gaboxedol (THIP), 10 and 15 nmol) or an opioid peptide receptor antagonist (naloxone, 5 nmol) into PAG, based on the proposed role of these receptors in PAG neurotransmission. Blockade of NMDA receptors by AP7 (both doses) or activation of GABA(A) receptors with THIP (15 nmol/side) in the PAG suppressed AGS susceptibility. Naloxone displayed a seizure-suppressant effect that was delayed and incomplete. The seizure suppressant effect of AP7 or naloxone, unlike THIP, was observed at doses that did not produce motor quiescence. These data suggest that the PAG is a requisite nucleus in the neuronal network for AGS in GEPR-9s and that GABA(A), opioid peptide and NMDA receptors in the PAG modulate AGS propagation.
It is now possible to develop a dynamic neuronal network model for generalized convulsive seizures because of in vivo data recently obtained in a naturally occurring epilepsy model--the genetically epilepsy-prone rats (GEPR-9s). GEPR-9s exhibit audiogenic seizures (AGS) that consist of a sequence of discrete behavioral phases (i.e., wild running, clonus-tonus, and post-ictal depression). The neuronal firing changes in most nuclei implicated in the network during each phase of AGS in behaving GEPR-9s have been examined. The inferior colliculus is critical in AGS initiation, because extensive firing increases in inferior colliculus are observed preceding seizure initiation. The deep layers of superior colliculus (DLSC) are crucial to wild running, based on the emergence of tonic firing of DLSC neurons just preceding this phase. The pontine reticular nucleus (PRF) and periaqueductal gray (PAG) are critical to the clonic-tonic phase, because tonic firing patterns appear in these neurons just prior to this phase. During post-ictal depression all areas except the PRF are quiescent. These temporal relationships suggest that each nucleus plays a specific hierarchic role in each discrete convulsive behavior. Generalized tonic-clonic seizure behavior observed in human epilepsy, in GEPR-9s, and in other seizure models is likely to involve similar neuronal network components. The neurotransmitter mechanisms subserving the abnormal neuronal responses in the GEPR-9 neuronal network involve an increased availability of glutamate and a decrease in the effectiveness of gamma-aminobutyric acid (GABA) in many brain regions. Focal modification of the effects of GABA, glutamate, norepinephrine, or serotonin also modulates the nuclei of the network differentially. Together, these data reveal the anatomic, neurotransmitter, and neurophysiologic mechanisms of the neuronal network hierarchy in GEPR-9s, which is currently the most completely developed of any generalized convulsive model. Differential effects of anticonvulsants on the AGS phases and concomitant differential modifications of neuronal firing are observed on neurons in these network nuclei. With nearly complete identification of the network nuclei, the differential effects of these anticonvulsant drugs on different aspects of neuronal firing in different brain sites indicate that this experimental approach can likely identify the most sensitive therapeutic target for these agents. This concept is potentially vital to developing the most selective treatment of different convulsive behaviors occurring in human epilepsy. The neuronal network for AGS does not require brain structures rostral to the midbrain for seizure expression. However, the forebrain is recruited into an expanded seizure network through AGS repetition ("kindling"), resulting in prolonged AGS, post-tonic clonus, and epileptiform electrographic cortical abnormalities. AGS kindling produces network expansion into medial geniculate body (MGB) and amygdala and involves neuronal firing increases in MGB.