Diversity and excitability of deep-layer entorhinal cortical neurons in a model of temporal lobe epilepsy

The entorhinal cortex (ERC) is critically implicated in temporal lobe epileptogenesis-the most common type of adult epilepsy. Previous studies have suggested that epileptiform discharges likely initiate in seizure-sensitive deep layers (V-VI) of the medial entorhinal area (MEA) and propagate into seizure-resistant superficial layers (II-III) and hippocampus, establishing a lamina-specific distinction between activities of deep- versus superficial-layer neurons and their seizure susceptibilities. While layer II stellate cells in MEA have been shown to be hyperexcitable and hypersynchronous in patients and animal models of temporal lobe epilepsy (TLE), the fate of neurons in the deep layers under epileptic conditions and their overall contribution to epileptogenicity of this region have remained unclear. We used whole cell recordings from slices of the ERC in normal and pilocarpine-treated epileptic rats to characterize the electrophysiological properties of neurons in this region and directly assess changes in their excitatory and inhibitory synaptic drive under epileptic conditions. We found a surprising heterogeneity with at least three major types and two subtypes of functionally distinct excitatory neurons. However, contrary to expectation, none of the major neuron types characterized showed any significant changes in their excitability, barring loss of excitatory and inhibitory inputs in a subtype of neurons whose dendrite extended into layer III, where neurons are preferentially lost during TLE. We confirmed hyperexcitability of layer II neurons in the same slices, suggesting minimal influence of deep-layer input on superficial-layer neuron excitability under epileptic conditions. These data show that deep layers of ERC contain a more diverse population of excitatory neurons than previously envisaged that appear to belie their seizure-sensitive reputation.     

Disruption of inhibition in area CA1 of the hippocampus in a rat model of temporal lobe epilepsy.

Previous studies have revealed a loss of neurons in layer III of the entorhinal cortex (EC) in patients with temporal lobe epilepsy. These neurons project to the hippocampus and may activate inhibitory interneurons, so that their loss could disrupt inhibitory function in the hippocampus. The present study evaluates this hypothesis in a rat model in which layer III neurons were selectively destroyed by focal injections of the indirect excitotoxin, aminooxyacetic acid (AOAA). Inhibitory function in the hippocampus was assessed by evaluating the discharge of CA1 neurons in response to stimulation of afferent pathways in vivo. In control animals, stimulation of the temporo-ammonic pathway leads to heterosynaptic inhibition of population spikes generated by subsequent stimulation of the commissural projection to CA1. This heterosynaptic inhibition was substantially reduced in animals that had received AOAA injections 1 mo previously. Stimulation of the commissural projection also elicited multiple population spikes in CA1 in AOAA-injected animals, and homosynaptic inhibition in response to paired-pulse stimulation of the commissural projection was dramatically diminished. These results suggest a disruption of inhibitory function in CA1 in AOAA-injected animals. To determine whether the disruption of inhibition occurred selectively in CA1, we assessed paired-pulse inhibition in the dentate gyrus. Both homosynaptic inhibition generated by paired-pulse stimulation of the perforant path, and heterosynaptic inhibition produced by activation of the commissural projection to the dentate gyrus appeared largely comparable in AOAA-injected and control animals; thus abnormalities in inhibitory function following AOAA injections occurred relatively selectively in CA1. Electrolytic lesions of the EC did not cause the same loss of inhibition as seen in animals with AOAA injections, indicating that the loss of inhibition in CA1 is not due to the loss of excitatory driving of inhibitory interneurons. Also, electrolytic lesions of the EC in animals that had been injected previously with AOAA had little effect on the abnormal physiological responses in CA1, suggesting that most alterations in inhibition in CA1 are not due to circuit abnormalities within the EC. Comparisons of control and AOAA-injected animals in a hippocampal kindling paradigm revealed that the duration of afterdischarges elicited by high-frequency stimulation of CA3, and the number of stimulations required to elicit kindled seizures were comparable. Taken together, our results reveal that the selective loss of layer III neurons induced by AOAA disrupts inhibitory function in CA1, but this does not create a circuit that is more prone to at least one form of kindling.     

Differential contribution of kainate receptors to excitatory postsynaptic currents in superficial layer neurons of the rat medial entorhinal cortex

Although in situ hybridization studies have revealed the presence of kainate receptor (KAR) mRNA in neurons of the rat medial entorhinal cortex (mEC), the functional presence and roles of these receptors are only beginning to be examined. To address this deficiency, whole cell voltage clamp recordings of locally evoked excitatory postsynaptic currents (EPSCs) were made from mEC layer II and III neurons in combined entorhinal cortex-hippocampal brain slices. Three types of neurons were identified by their electroresponsive membrane properties, locations, and morphologies: stellate-like "Sag" neurons in layer II (S), pyramidal-like "No Sag" neurons in layer III (NS), and "Intermediate Sag" neurons with varied morphologies and locations (IS). Non-NMDA EPSCs in these neurons were composed of two components, and the slow decay component in NS neurons had larger amplitudes and contributed more to the combined EPSC than did those observed in S and IS neurons. This slow component was mediated by KARs and was characterized by its resistance to either 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466, 100 microM) or 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[lsqb]f[rsqb]quinoxaline-7-sulfonamide (NBQX, 1 microM), relatively slow decay kinetics, and sensitivity to 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10-50 microM). KAR-mediated EPSCs in pyramidal-like NS neurons contributed significantly more to the combined non-NMDA EPSC than did those from S and IS neurons. Layer III neurons of the mEC are selectively susceptible to degeneration in human temporal lobe epilepsy (TLE) and animal models of TLE such as kainate-induced status epilepticus. Characterizing differences in the complement of postsynaptic receptors expressed in injury prone versus injury resistant mEC neurons represents an important step toward understanding the vulnerability of layer III neurons seen in TLE.     

Afferents to the seizure-sensitive neurons in layer III of the medial entorhinal area: a tracing study in the rat

Neurons in layer III of the medial entorhinal area (MEA) in the rat are extremely vulnerable to local injections of amino-oxyacetic acid and to exprimentally induced limbic seizures. A comparable specific pathology has been noted in surgical specimens from patients with temporal lobe epilepsy. Efforts to understand this preferential neuronal vulnerability led us to study the neural input to this layer in the rat. Iontophoretic injection of the retrograde tracer fast blue, aimed at layer III of the MEA, resulted in retrogradely labeled neurons in the presubiculum in all the injected hemispheres. The nucleus reuniens thalami, the anteromedial thalamic nucleus, the ventral portion of the claustrum (endopiriform nucleus), the dorsomedial parts of the anteroventral thalamic nucleus, and the septum-diagonal band complex were labeled less frequently. In only one experiment, retrogradely labeled neurons were observed in the ventrolateral hypothalamus and in the brainstem nucleus raphe dorsalis. Since projections from claustrum to the entorhinal cortex has not been studied in the rat with modern sensitive anterograde tracing techniques, iontophoretic injections of the anterograde tracer Phaseolus vulgaris-leucoagglutinin were placed into the ventral portion of the claustrum. Anterogradely labeled fibers in the entorhinal area proved not to be confined to the MEA, since a prominent projection distributed to the lateral entorhinal area as well. In both areas, the densest terminal labeling was present in layers IV–VI, whereas layer III appeared to be only sparsely labeled. The present data indicate that of all potential afferents only those from the presubiculum distribute preferentially to layer III of the MEA. This, in turn, suggests a potentially important role of the presubiculum in the seizure-related degeneration of neurons in layer III of the MEA.     

Neuropeptide-Y immunoreactivity in the pilocarpine model of temporal lobe epilepsy

 Neuropeptide-Y (NPY) is expressed by granule cells and mossy fibres of the hippocampal dentate gyrus during experimental temporal lobe epilepsy (TLE). This expression may represent an endogenous damping mechanism since NPY has been shown to block seizure-like events following high-frequency stimulation in hippocampal slices. The pilocarpine (PILO) model of epilepsy is characterized by an acute period of status epilepticus followed by spontaneous recurrent seizures and related brain damage. We report peroxidase-antiperoxidase immunostaining for NPY in several brain regions in this model. PILO-injected animals exhibited NPY immunoreactivity in the region of the mossy fibre terminals, in the dentate gyrus inner molecular layer and, in a few cases, within presumed granule cells. NPY immunoreactivity was also dramatically changed in the entorhinal cortex, amygdala and sensorimotor areas. In addition, PILO injected animals exhibited a reduction in the number of NPY-immunoreactive interneurons compared with controls. The results demonstrate that changes in NPY expression, including expression in the granule cells and mossy fibres and the loss of vulnerable NPY neurons, are present in the PILO model of TLE. However, the significance of this changed synthesis of NPY remains to be determined. Received: 19 August 1996 / Accepted: 21 March 1997     

Sprouting and synaptic reorganization in the subiculum and CA1 region of the hippocampus in acute and chronic models of partial-onset epilepsy

Repeated seizures induce permanent alterations in the hippocampal circuitry in experimental models and patients with intractable temporal lobe epilepsy (TLE). Most studies have concentrated their attention on seizure-induced reorganization of the mossy fiber pathway. The present study examined the projection pathway of the CA1 pyramidal neurons to the subiculum, which is the output of the hippocampal formation in five models of TLE. We examined the laminar pattern of Timm's histochemistry in the stratum lacunosum-moleculare of CA1 in three acute and two chronic models of TLE: intraventricular kainic acid (KA), systemic KA, systemic pilocarpine, chronic electric kindling and chronic i.p. pentylenetetrazol. The laminar pattern of Timm histochemistry in the stratum moleculare of CA1 was permanently remodeled in epileptic models suggesting sprouting of Timm containing terminals from the adjacent stratum lacunosum. Ultrastructural examination confirmed that Timm granules were localized in synaptic terminals. As the source of Timm-labeled terminals in this region was not known, sodium selenite, a selective retrograde tracer for zinc-containing terminals, was iontophoretically injected in vivo in rats exposed to systemic pilocarpine, systemic KA or chronic pentylenetetrazol. The normal projection of CA1 pyramidal neurons to the subiculum is topographically organized in a lamellar fashion. In normal rats, the extent of the injection site (terminals) and the retrogradely labeled pyramidal neurons (cell soma) corresponded to the same number of lamellas. In epileptic rats, the retrograde labeling extended 42-67% farther than the normal dorso-ventral extent including lamellas above and below the expected. This is direct evidence for sprouting of CA1 pyramidal axons into the subiculum and stratum lacunosum-moleculare of the CA1 region confirming the alterations of the laminar pattern of Timm's histochemistry. Sprouting of the CA1 projection to subiculum across hippocampal lamellas might lead to translamellar hyperexcitability, and to amplification and synchronization of epileptic discharges in the output gate of the hippocampal formation.     

Temporal lobe epilepsy is a disease of faulty neuronal resonators rather than oscillators, and all seizures are provoked, usually by stress

Temporal lobe epilepsy (TLE) is the most common cause of intractable adult epilepsy. It is proposed that different kinds of epilepsy be classified into one of two categories, which correspond to the two basic kinds of neurons in the brain, that is, as diseases of oscillators or as diseases of resonators. Oscillator (or pacemacker) neurons are endowed with intrinsic conductances that permit periodic spontaneous generation of action potentials; in contrast, resonators are neurons which process information coming from sensory stimuli or from other neurons. A literature review supports the idea that TLE is a disease of faulty resonators. This means that seizures do not arise de novo in the seizure focus. The seizure focus responds to normal input with an abnormally large discharge that causes seizures. The most frequent trigger for TLE is psychological stress. A previously published theory of stress is reviewed. The stress circuit runs from the hippocampus to the amygdala to the dorsal raphe nucleus to the entorhinal cortex and back to the hippocampus. Cell loss in the dentate is central to the pathophysiology of both chronic stress and TLE, which establish a "vicious circle" relationship with one another. Once it is grasped that TLE is a disease of resonators and that all seizures in TLE are triggered, then it makes sense to address the major recognized trigger, which is stress. New therapeutic ideas for decreasing seizure frequency in TLE include the use of anti-depressants, ethosuximide (which blocks firing in the dorsal raphe nucleus), and mood-stabilizers (which block firing in the entorhinal cortex). The latter category includes several recognized anti-epileptic drugs. Drugs from all three categories should be used simultaneously and on an empirical basis in each patient.     

Parvalbumin-immunoreactive neurons in the entorhinal cortex of the rat: localization, morphology, connectivity and ultrastructure

Summary We studied the distribution, morphology, ultrastructure and connectivity of parvalbumin-immunoreactive neurons in the entorhinal cortex of the rat. Immunoreactive cell bodies were found in all layers of the entorhinal cortex except layer I. The highest numbers were observed in layers II and III of the dorsal division of the lateral entorhinal area whereas the lowest numbers occurred in the ventral division of the lateral entorhinal area, Most such neurons displayed multipolar configurations with smooth dendrites. We distinguished a type with long dendrites and a type with short dendrites. We also observed pyramidal immunoreactive neurons. A dense plexus of immunoreactive dendrites and axons was prominent in layers II and III of the dorsal division of the lateral entorhinal area and the medial entorhinal area. None of the parvalbuminimmunoreactive cells became retrogradely labelled after injection of horseradish peroxidase into the hippocampal formation. By electron microscopy, immunoreactivity was observed in cell bodies, dendrites, myelinated and unmyelinated axons and axon terminals. Immunoreactive dendrites and axons occurred in all cortical layers. We noted many myelinated immunoreactive axons. Immunoreactive axon terminals were medium sized, contained pleomorphic synaptic vesicles, and established symmetrical synapses. Both horseradish peroxidase labelled and unlabelled immunonegative cell bodies often received synapses from immunopositive axon terminals arranged in baskets. Synapses between immunoreactive axon terminals and unlabelled dendritic shafts and spines were abundant. Synapses with initial axon segments occurred less frequently. In addition, synaptic contacts were present between immunopositive axon terminals and cell bodies and dendrites. Thus, the several types of parvalbumin-containing neuron in the entorhinal cortex are interneurons, connected to one another and to immunonegative neurons through a network of synaptic contacts. Immunonegative cells projecting to the hippocampal formation receive axo-somatic basket synapses from immunopositive terminals. This connectivity may form the morphological substrate underlying the reported strong inhibition of cells in layers II and III of the entorhinal cortex projecting to the hippocampal formation.     

Concise review: prospects of stem cell therapy for temporal lobe epilepsy

Certain regions of the adult brain have the ability for partial self-repair after injury through production of new neurons via activation of neural stem/progenitor cells (NSCs). Nonetheless, there is no evidence yet for pervasive spontaneous replacement of dead neurons by newly formed neurons leading to functional recovery in the injured brain. Consequently, there is enormous interest for stimulating endogenous NSCs in the brain to produce new neurons or for grafting of NSCs isolated and expanded from different brain regions or embryonic stem cells into the injured brain. Temporal lobe epilepsy (TLE), characterized by hyperexcitability in the hippocampus and spontaneous seizures, is a possible clinical target for stem cell-based therapies. This is because these approaches have the potential to curb epileptogenesis and prevent chronic epilepsy development and learning and memory dysfunction after hippocampal damage related to status epilepticus or head injury. Grafting of NSCs may also be useful for restraining seizures during chronic epilepsy. The aim of this review is to evaluate current knowledge and outlook pertaining to stem cell-based therapies for TLE. The first section discusses the behavior of endogenous hippocampal NSCs in human TLE and animal models of TLE and evaluates the role of hippocampal neurogenesis in the pathophysiology and treatment of TLE. The second segment considers the prospects for preventing or suppressing seizures in TLE using exogenously applied stem cells. The final part analyzes problems that remain to be resolved before initiating clinical application of stem cell-based therapies for TLE. Disclosure of potential conflicts of interest is found at the end of this article.     

An anatomical study of cholinergic innervation in rat cerebral cortex

The cholinergic innervation of rat cerebral cortex was studied by immunohistochemical localization of choline acetyltransferase. Stained bipolar cells, fibers and terminals were found in all areas of cortex. The density of cholinergic terminals was similar in all cortical areas with the exception of entorhinal and olfactory cortex, which showed a marked increase in the number of stained terminals. A laminar distribution of cholinergic terminals was found in many cortical areas. In motor and most sensory areas, terminal density was high in layer 1 and upper layer 5, and lowest in layer 4. Visual cortex, in contrast to other cortical areas, was characterized by a dense band of innervation in layer 4. It has been known that the majority of cortical cholinergic structures derive from a projection to cortex from large, multipolar neurons in the basal forebrain, which stain heavily for choline acetyltransferase. In this study, stained fibers were observed to take three different pathways from basal forebrain to cortex. The first, confined to medial aspects of forebrain and cortex, was observed to originate in the septal area, from where fibers formed a discrete bundle, swinging forward around the rostral end of the corpus callosum, then travelling caudally in the cingulate bundle. The second was found to consist of fibers fanning out laterally from the area of the globus pallidus, travelling through the caudate, then continuing for various distances in the corpus callosum before finally turning into the cortex. A third pathway appeared to innervate olfactory and entorhinal cortex. Ibotenic acid injections were made in the area of the globus pallidus to study the effect of lesioning the lateral pathway on the cholinergic innervation in cortex. A major loss of choline acetyltransferase positive terminals was observed in neocortex, but retrosplenial, cingulate, entorhinal and olfactory cortex showed a normal density of cholinergic innervation. The borders separating areas with lesioned cholinergic input from non-lesioned areas were precise. The distribution of stained terminals remaining in cortical areas with lesioned basal forebrain innervation suggests that the basal forebrain projection to cerebral cortex, and not the intrinsic cortical cholinergic neurons, give rise to the laminar distribution of cholinergic terminals observed in normal cortex. To compare the relative densities of different cholinergic cortical systems, the distribution of choline acetyltransferase staining was compared with that of vasoactive intestinal polypeptide and substance P, which are co-localized in some choline acetyltransferase-positive neurons innervating cortex.     

Organization of projections to temporal cortex originating in the thalamic posterior intralaminar nucleus of the rat

Thalamic nuclei surrounding the medial geniculate body, among which the posterior intralaminar nucleus (PIN) is one of the largest, have great importance in fear-potentiated emotional behavior. Due to limited knowledge of the distribution of the cortical projections of the PIN, the connections between the temporal neocortex and the PIN were investigated by means of axonal transport of Phaseolus vulgaris leucoagglutinin or Miniruby. After iontophoretic injections of either tracer, anterogradely labeled terminals showed a broad, but not a diffuse, distribution in temporal and adjacent cortices (perirhinal, secondary auditory, visceral, secondary somatosensory, agranular insular cortices). A common projection to all areas was found in the upper layer I except for perirhinal cortex, where this projection was confined to the basal layer I. In selected cortical fields (ectorhinal, perirhinal, visceral cortices), an additional projection to layers III/IV was found. The corticofugal projection to the PIN originated from pyramidal neurons in layer V and – in some regions – in layer VI. The present results demonstrate a distinct and selective projection of the PIN to several areas of the temporal neocortex, which may activate inter- and intra-areal cortical circuits during processing of auditory stimuli. Received: 04 December 1998 / Accepted: 24 March 1999     

Projections from the hippocampal and parahippocampal regions to the entorhinal cortex. An anterograde and retrograde tract-tracing study in the cat

Projections from the hippocampal and parahippocampal regions to the entorhinal cortex (EC) were examined in the cat by anterograde and retrograde tract-tracing with Phaseolus vulgaris leucoagglutinin and cholera toxin B subunit. CA1 fibers to EC were distributed more densely in the medial EC than in the lateral EC; these were seen in all EC layers, but most densely in layers II and III. The septotemporal axis of the area of origin of CA1-EC fibers corresponded to a caudal-to-rostral axis of the area of their termination in the EC. CA2 and CA4 also sent a small number of fibers to the EC. The subiculum sent fibers mainly to the lateral EC; more densely to layers IV-VI than to layers I-III. The septotemporal axis of the area of origin of subiculum-EC fibers corresponded to a caudolateral-to-rostromedial axis of their termination in the EC. Distribution pattern of fibers from the prosubiculum regions close to CA1 or from prosubiculum regions close to the subiculum was similar to that of CA1 fibers or subiculum fibers, respectively. The presubiculum sent fibers mainly to the medial EC; most densely to layers I and III. The parasubiculum sent fibers mainly to the medial EC; most densely to layer II. Fibers to the contralateral EC were detected only from the presubiculum; they originated from the superficial layers and terminated in layer III of the medial entorhinal area.     

Effects of glutamate uptake blockers on stimulus-induced field potentials in rat entorhinal cortex in vitro

L-Glutamic acid (Glu) is a key excitatory transmitter in the central nervous system. Excessive amounts of Glu are highly toxic to neurons and particularly the entorhinal cortex (EC) exhibits a remarkable loss of cells in the superficial layers in acute brain injury. The accumulation of Glu is limited by a family of high-affinity Glu transporters. Using extracellular potential recordings in rat brain slices we tested whether application of the Glu uptake blockers dihydrokainate and L-trans-pyrrolidine-2,4-dicarboxylate (L-trans-2,4-PDC) affect stimulus-induced field potentials (FPs) in superficial layer III and deep layer V of the medial EC. We found that a high concentration (400 microM) of the uptake blockers significantly reduces stimulus-induced FPs in both layers. At lower concentration (200 microM), only dihydrokainate is efficient. The data show that Glu uptake is involved in the control of extracellular Glu levels during synaptic excitation of layers III and V of the medial EC.     

Septal GABAergic neurons are selectively vulnerable to pilocarpine-induced status epilepticus and chronic spontaneous seizures

The septal region of the basal forebrain plays a critical role modulating hippocampal excitability and functional states. Septal circuits may also play a role in controlling abnormal hippocampal hyperexcitability in epilepsy. Both lateral and medial septal neurons are targets of hippocampal axons. Since the hippocampus is an important epileptogenic area in temporal lobe epilepsy, we hypothesize that excessive excitatory output will promote sustained neurodegeneration of septal region neurons. Pilocarpine-induced status epilepticus (SE) was chosen as a model to generate chronic epileptic animals. To determine whether septal neuronal populations are affected by hippocampal seizures, immunohistochemical assays were performed in brain sections obtained from age-matched control, latent period (7 days post-SE) and chronically epileptic (more than one month post-SE survival) rats. An anti-NeuN (neuronal nuclei) antibody was used to study total neuronal numbers. Anti-ChAT (choline acetyltransferase), anti-GAD (glutamic acid decarboxylase) isoenzymes (65 and 67), and anti-glutamate antibodies were used to reveal cholinergic, GABAergic and glutamatergic neurons, respectively. Our results revealed a significant atrophy of medial and lateral septal areas in all chronically epileptic rats. Overall neuronal density in the septum (medial and lateral septum), assessed by NeuN immunoreactivity, was significantly reduced by approximately 40% in chronically epileptic rats. The lessening of neuronal numbers in both regions was mainly due to the loss of GABAergic neurons (80-97% reduction in medial and lateral septum). In contrast, populations of cholinergic and glutamatergic neurons were spared. Overall, these data indicate that septal GABAergic neurons are selectively vulnerable to hippocampal hyperexcitability, and suggest that the processing of information in septohippocampal networks may be altered in chronic epilepsy.     

SYMPOSIUM REPORT

The entorhinal cortex (EC) is a key brain area controlling both hippocampal input and output via neurones in layer II and layer V, respectively. It is also a pivotal area in the generation and propagation of epilepsies involving the temporal lobe. We have previously shown that within the network of the EC, neurones in layer V are subject to powerful synaptic excitation but weak inhibition, whereas the reverse is true in layer II. The deep layers are also highly susceptible to acutely provoked epileptogenesis. Considerable evidence now points to a role of spontaneous background synaptic activity in control of neuronal, and hence network, excitability. In the present article we describe results of studies where we have compared background release of the excitatory transmitter, glutamate, and the inhibitory transmitter, GABA, in the two layers, the role of this background release in the balance of excitability, and its control by presynaptic auto- and heteroreceptors on presynaptic terminals.     

Type 1 metalloproteinase is selectively expressed in adult rat brain and can be rapidly up-regulated by kainate

The expression of metalloproteinase MMP-1 was traced in frontal sections of the rat brain in normal conditions and 4 h after an intraperitoneal injection of kainate. In the olfactory lobe, immunoreactivity was normally detected in the lateral olfactory tract. Kainate treatment led to the appearance of additional immunoreactivity in the neuropilar tracts. In the hippocampal part of brain, immunoreactive neurons were found exclusively after the kainate treatment in several hypothalamic and amygdalar nuclei, and in the restricted cortex areas (clusters of neurons in layers 3–4 of cortex, and a stripe of cells in layer 6). In the area between the hippocampus and cerebellum, MMP-1-like immunoreactivity was normally present in the entorhinal cortex, in the lateral periaqueductal gray, and in the pontine nucleus. After kainate treatment, the immunoreactive neurons were also found in the medial entorhinal cortex and in the dorsal raphe nucleus. In the brain stem, the immunoreactive cells were normally found in six nuclei. After kainate treatment, additional immunoreactivity appeared in the inferior olive neurons and in tracts supplying the cerebellar cortex. Thus, MMP-1 is present in several brain areas in normal conditions at a detectable level, and its expression increases after kainate-induced seizures.     

Contacts between medial and lateral perforant pathway fibers and parvalbumin expressing neurons in the subiculum of the rat

The entorhinal cortex (EC) projects via the perforant pathway to all subfields in the hippocampal formation. One can distinguish medial and lateral components in the pathway, originating in corresponding medial and lateral subdivisions of EC. We analyzed the innervation by medial and lateral perforant pathway fibers of parvalbumin-expressing neurons in the subiculum. A neuroanatomical tracer (biotinylated dextran amine, BDA) was stereotaxically injected in the medial or lateral entorhinal cortex, thus selectively labeling either perforant pathway component. Transport was allowed for 1 week. Transported BDA was detected with streptavidin-Alexa Fluor 488. Parvalbumin neurons were visualized via immunofluorescence histochemistry, using the fluorochrome Alexa Fluor 594. Via a random systematic sampling scheme using a two-channel, sequential-mode confocal laser scanning procedure, we obtained image series at high magnification from the molecular layer of the subiculum. Labeled entorhinal fibers and parvalbumin-expressing structures were three dimensionally (3D) reconstructed using computer software. Further computer analysis revealed that approximately 16% of the 3D objects ('boutons') of BDA-labeled fibers was engaged in contacts with parvalbumin-immunostained dendrites in the subiculum. Both medial and lateral perforant pathway fibers and their boutons formed such appositions. Contacts are suggestive for synapses. We found no significant differences between the medial and lateral components in the relative numbers of contacts. Thus, the medial and lateral subdivisions of the entorhinal cortex similarly tune the firing of principal neurons in the subiculum by way of parvalbumin positive interneurons in their respective terminal zones.     

Effects of phencyclidines on signal transfer from the entorhinal cortex to the hippocampus in rats

The information transfer from the superficial layers of the entorhinal cortex (EC) to the hippocampus is regulated in a frequency dependent manner. Phencyclidine and related compounds such as MK-801 produce psychotic symptoms that closely resemble schizophrenia. We studied the effects of systemic administration of MK-801 on the signal transfer from the EC layer III to the hippocampal area CA1. High frequency (above 10 Hz) activation of the bi-synaptic entorhinal input in control animals results in a strong suppression of the field potentials in the stratum lacunosum-moleculare of the area CA1. In contrast, in MK-801 pretreated rats the field response was less reduced. The field potential responses evoked in these two groups of animals by high-frequency activation of the monosynaptic input were similar suggesting selective alterations in layer III of the medial EC. We suggest, that MK-801 causes disinhibition of layer III projection cells and, therefore, may cause strong, pathological activation of direct layer III-CA1 pathway.     

Network and pharmacological mechanisms leading to epileptiform synchronization in the limbic system in vitro

Seizures in patients presenting with mesial temporal lobe epilepsy result from the interaction among neuronal networks in limbic structures such as the hippocampus, amygdala and entorhinal cortex. Mesial temporal lobe epilepsy, one of the most common forms of partial epilepsy in adulthood, is generally accompanied by a pattern of brain damage known as mesial temporal sclerosis. Limbic seizures can be mimicked in vitro using preparations of combined hippocampus-entorhinal cortex slices perfused with artificial cerebrospinal fluid containing convulsants or nominally zero Mg(2+), in order to produce epileptiform synchronization. Here, we summarize experimental evidence obtained in such slices from rodents. These data indicate that in control animals: (i) prolonged, NMDA receptor-dependent epileptiform discharges, resembling electrographic limbic seizures, originate in the entorhinal cortex from where they propagate to the hippocampus via the perforant path-dentate gyrus route; (ii) the initiation and maintenance of these ictal discharges is paradoxically contributed by GABA (mainly type A) receptor-mediated mechanisms; and (iii) CA3 outputs, which relay a continuous pattern of interictal discharge at approximately 1Hz, control rather than sustain ictal discharge generation in entorhinal cortex. Recent work indicates that such a control is weakened in the pilocarpine model of epilepsy (presumably as a result of CA3 cell damage). In addition, in these experiments electrographic seizure activity spreads directly to the CA1-subiculum regions through the temporoammonic pathway. Studies reviewed here indicate that these changes in network interactions, along with other mechanisms of synaptic plasticity (e.g. axonal sprouting, decreased activation of interneurons, upregulation of bursting neurons) can confer to the epileptic, damaged limbic system, the ability to produce recurrent limbic seizures as seen in patients with mesial temporal lobe epilepsy.