Proepileptic influence of a focal vascular lesion affecting entorhinal cortex-CA3 connections after status epilepticus

In limbic seizures, neuronal excitation is conveyed from the entorhinal cortex directly to CA1 and subicular regions. This phenomenon is associated with a reduced ability of CA3 to respond to entorhinal cortex inputs. Here, we describe a lesion that destroys the perforant path in CA3 after status epilepticus (SE) induced by pilocarpine injection in 8-week-old rats. Using magnetic resonance imaging, immunohistochemical, and ultrastructural analyses, we determined that this lesion develops after 30 minutes of SE and is characterized by microhemorrhages and ischemia. After a longer period of SE, the lesion invariably involves the upper blade of the dentate gyrus. Adult rats treated with subcutaneous diazepam (20 mg kg for 3 days) did not develop the dentate gyrus lesion and had less frequent spontaneous recurrent seizures (p < 0.01). Young (3-week-old) rats rarely (20%) developed the CA3 lesion, and their spontaneous seizures were delayed (p < 0.01). To investigate the role of the damaged CA3 in seizure activity, we reinduced SE in adult and young epileptic rats. Using FosB/DeltaFosB markers, we found induction of FosB/DeltaFosB immunopositivity in CA3 neurons of young but not in adult rats. These experiments indicate that SE can produce a focal lesion in the perforant path that may affect the roles of the hippocampus in epileptic rats.     

Subiculum network excitability is increased in a rodent model of temporal lobe epilepsy

In this study, we used in vitro electrophysiology along with immunohistochemistry and molecular techniques to study the subiculum--a limbic structure that gates the information flow from and to the hippocampus--in pilocarpine-treated epileptic rats. Comparative data were obtained from age-matched nonepileptic controls (NEC). Subicular neurons in hippocampal-entorhinal cortex (EC) slices of epileptic rats were: (i) hyperexcitable when activated by CA1 or EC inputs; and (ii) generated spontaneous postsynaptic potentials at higher frequencies than NEC cells. Analysis of pharmacologically isolated, GABA(A) receptor-mediated inhibitory postsynaptic potentials revealed more positive reversal potentials in epileptic tissue (-67.8 +/- 6.3 mV, n = 16 vs. -74.8 +/- 3.6 mV in NEC, n = 13; P < 0.001) combined with a reduction in peak conductance (17.6 +/- 11.3 nS vs. 41.1 +/- 26.7 nS in NEC; P < 0.003). These electrophysiological data correlated in the epileptic subiculum with (i) reduced levels of mRNA expression and immunoreactivity of the neuron-specific potassium-chloride cotransporter 2; (ii) decreased number of parvalbumin-positive cells; and (iii) increased synaptophysin (a putative marker of sprouting) immunoreactivity. These findings identify an increase in network excitability within the subiculum of pilocarpine-treated, epileptic rats and point at a reduction in inhibition as an underlying mechanism.     

Repetitive low-frequency stimulation reduces epileptiform synchronization in limbic neuronal networks

Deep-brain electrical or transcranial magnetic stimulation may represent a therapeutic tool for controlling seizures in patients presenting with epileptic disorders resistant to antiepileptic drugs. In keeping with this clinical evidence, we have reported that repetitive electrical stimuli delivered at approximately 1 Hz in mouse hippocampus-entorhinal cortex (EC) slices depress the EC ability to generate ictal activity induced by the application of 4-aminopyridine (4AP) or Mg(2+)-free medium (Barbarosie, M., Avoli, M., 1997. CA3-driven hippocampal-entorhinal loop controls rather than sustains in vitro limbic seizures. J. Neurosci. 17, 9308-9314.). Here, we confirmed a similar control mechanism in rat brain slices analyzed with field potential recordings during 4AP (50 microM) treatment. In addition, we used intrinsic optical signal (IOS) recordings to quantify the intensity and spatial characteristics of this inhibitory influence. IOSs reflect the changes in light transmittance throughout the entire extent of the slice, and are thus reliable markers of limbic network epileptiform synchronization. First, we found that in the presence of 4AP, the IOS increases, induced by a train of electrical stimuli (10 Hz for 1 s) or by recurrent, single-shock stimulation delivered at 0.05 Hz in the deep EC layers, are reduced in intensity and area size by low-frequency (1 Hz), repetitive stimulation of the subiculum; these effects were observed in all limbic areas contained in the slice. Second, by testing the effects induced by repetitive subicular stimulation at 0.2-10 Hz, we identified maximal efficacy when repetitive stimuli are delivered at 1 Hz. Finally, we discovered that similar, but slightly less pronounced, inhibitory effects occur when repetitive stimuli at 1 Hz are delivered in the EC, suggesting that the reduction of IOSs seen during repetitive stimulation is pathway dependent as well as activity dependent. Thus, the activation of limbic networks at low frequency reduces the intensity and spatial extent of the IOS changes that accompany ictal synchronization in an in vitro slice preparation. This conclusion supports the view that repetitive stimulation may represent a potential therapeutic tool for controlling seizures in patients with pharmaco-resistant epileptic disorders.     

The entorhinal cortex of the mouse: organization of the projection to the hippocampal formation

The origin and the terminations of the projections from the entorhinal cortex to the hippocampal formation of the mouse (C57BL/6J strain) have been studied using anterogradely and retrogradely transported tracers. The entorhinal cortex is principally divided into two areas, the lateral entorhinal area (LEA) and the medial entorhinal area (MEA). LEA is the origin of the lateral perforant path that terminates in the outer one-third of the molecular layer of the dentate gyrus, and MEA is the origin of the medial perforant path that ends in the middle one-third of the molecular layer of the dentate gyrus. This projection is mostly to the ispsilateral dentate gyrus; only a few labeled axons and terminals are found in the contralateral dentate gyrus. The projection to the dentate gyrus originates predominantly from neurons in layer II of the entorhinal cortex. The entorhinal cortex also projects to CA3 and CA1 and to subiculum; in both CA3 and CA1, the terminals are present in stratum lacunosum-moleculare, whereas in the subiculum the terminals are in the outer part of the molecular layer. The projection from the entorhinal cortex to CA3, CA1, and subiculum is bilateral, and it originates predominantly from neurons in layer III, but a small number of neurons in the deeper layers of the entorhinal cortex contributes to this projection. The projection of entorhinal cortex to the hippocampus is topographically organized, neurons in the lateral part of both LEA and MEA project to the dorsal part (i.e., septal pole) of the hippocampus, whereas the projection to the ventral (i.e., temporal pole) hippocampus originates from neurons in medial parts of the entorhinal cortex.     

Fluorescent tracer in pilocarpine-treated rats shows widespread aberrant hippocampal neuronal connectivity

Neuronal fibres of the hippocampal formation of normal and chronic epileptic rats were investigated by fluorescent tracing methods using the pilocarpine model of limbic epilepsy. Two months after onset of spontaneous limbic seizures, hippocampal slices were prepared and maintained in vitro for 10 h. Small crystals of fluorescent dye [fluorescein (fluoro-emerald) and tetramethylrhodamine (fluoro-ruby)] were applied to different hippocampal regions. The main findings were: (i) in control rats there was no supragranular labelling when the mossy fibre tract was stained in stratum radiatum of area CA3. However, in epileptic rats a fibre network in the inner molecular layer of the dentate gyrus was retrogradely labelled; (ii) a retrograde innervation of area CA3 by CA1 pyramidal cells was disclosed by labelling remote CA1 neurons after dye injection into the stratum radiatum of area CA3 in chronic epileptic rats; (iii) labelling of CA1 neurons apart from the injection site within area CA1 was observed in epileptic rats but not in control animals; and (iv), a subicular-hippocampal projection was present in pilocarpine-treated rats when the tracer was injected just below the stratum pyramidale of area CA1. The findings show that fibre rearrangement in distinct regions of the epileptic hippocampal formation can occur as an aftermath of pilocarpine-induced status epilepticus.     

Entorhinal cortex of the monkey: V. Projections to the dentate gyrus, hippocampus, and subicular complex.

The topographic and laminar organization of entorhinal projections to the dentate gyrus, hippocampus, and subicular complex was investigated in the Macaca fascicularis monkey. Injections of 3H-amino acids were placed at various positions within the entorhinal cortex and the distribution of anterogradely labeled fibers and terminals within the other fields of the hippocampal formation was determined. Injections of the retrograde tracers Fast blue, Diamidino yellow, and wheat germ agglutinin-horseradish peroxidase (WGA-HRP) were also placed into the dentate gyrus, hippocampus, and subicular complex, and the distribution of retrogradely labeled cells in the entorhinal cortex was plotted using a computer-aided digitizing system. The entorhinal cortex gave rise to projections that terminated in the subiculum, in the CA1, CA2, and CA3 fields of the hippocampus, and in the dentate gyrus. Projections to the dentate gyrus, and fields CA3 and CA2 of the hippocampus, originated preferentially in layers II and VI of the entorhinal cortex whereas projections to CA1 and to the subiculum originated mainly in layers III and V. Anterograde tracing experiments demonstrated that all regions of the entorhinal cortex project to the outer two-thirds of the molecular layer of the dentate gyrus and to much of the radial extent of the stratum lacunosum-moleculare of CA3 and CA2. While the terminal distributions of entorhinal projections to the dentate gyrus, CA3, and CA2 were not as clearly laminated as in the rat, projections from rostral levels of the entorhinal cortex preferentially innervated the outer portion of the molecular layer and stratum lacunosum-moleculare, whereas more caudal levels of the entorhinal cortex projected relatively more heavily to the deeper portions of the entorhinal terminal zones. The entorhinal projection to the CA1 field of the hippocampus and to the subiculum followed a transverse rather than radial gradient of distribution. Rostral levels of the entorhinal cortex terminated most heavily at the border of CA1 and the subiculum. More caudal levels of the entorhinal cortex projected to progressively more distal portions of the subiculum (towards the presubiculum) and more proximal portions of CA1 (towards CA2). Lateral portions of the entorhinal cortex projected to caudal levels of the recipient fields and more medial parts of the entorhinal cortex projected to progressively more rostral portions of the fields.     

Intrinsic Optical Signals and Electrographic Seizures in the Rat Limbic System

We measured the intrinsic optical signals (IOSs) generated by rat hippocampus-entorhinal cortex (EC) slices in response to single shock electrical stimuli delivered in the EC deep layers during application of the convulsant drug 4-aminopyridine (50 microM). With field potential recordings the stimulus-induced responses had duration = 35 +/- 6.3 s mean +/- SEM, n = 7 slices) and characteristics resembling electrographic seizures. IOS changes reflecting an increase in light transmittance occurred in the EC and hippocampus following similar stimuli (n = 45). IOSs increased progressively to reach peak values 20-30 s after the stimulus and returned slowly to prestimulus values within 100 s, thus outlasting the field potential discharge. IOS changes initiated in the medial EC, near to the stimulation site, and spread to the lateral EC, the dentate, and the CA3/CA1 areas. IOS spread from EC to hippocampus was not seen after perforant path cut (n = 5). Moreover, field potential and IOS responses were markedly decreased by excitatory amino acid receptor antagonists (n = 12). The antiepileptic drugs topiramate (10-100 microM, n = 16) or lamotrigine (100-400 microM, n = 12) reduced the IOS changes in the EC and their spread to distant areas. These effects were reversible and dose-dependent (IC50 = 48 microM and 210 microM for topiramate and lamotrigine, respectively). Thus, in 4AP-treated hippocampus-EC slices, IOS changes accompany and outlast the field potential epileptiform responses, depend on glutamatergic transmission and are characterized by temporal and spatial distributions consistent with propagation through established anatomical pathways. We also propose that IOSs may represent a reliable tool for screening the effects of neuroactive compounds such as antiepileptic drugs.     

Species differences in the projections from the entorhinal cortex to the hippocampus

Both differences and similarities exist between mammalian species in the projections from entorhinal cortex to the hippocampal formation. In most species, layer II cells of the entorhinal cortex project to the dentate gyrus, and they terminate in the outer two-thirds of the molecular layer of the dentate gyrus. The axons from layer III cells project bilaterally to areas CA(1) and CA(3) of the hippocampus, terminating in the stratum lacunosum moleculare. We have analyzed these projections in mice, and in general, the entorhinal cortex-to-hippocampus projections are similar to those in rats. Axons from layer II neurons terminate in the outer and middle thirds of the molecular layer of the dentate gyrus, and axons from layer III neurons terminate bilaterally in the stratum lacunosum moleculare of areas CA(1) and CA(3), and in the molecular layer of the subiculum. However, in contrast to rat, mouse entorhinal cortex neurons do not appreciably project to the contralateral dentate gyrus. Most species, including mice, show a similar topographical organization of the entorhinal-hippocampal projections, with neurons in the lateral part of both the lateral and medial entorhinal cortex projecting to the dorsal part or septal pole of the hippocampus, whereas the projection to the ventral hippocampus originates primarily from neurons in medial parts of the entorhinal cortex.     

Limbic system-associated membrane protein (LAMP) in primate amygdala and hippocampus

The distribution of the limbic system-associated membrane protein in the amygdaloid complex and hippocampal formation of cynomolgus monkeys (Macaca fascicularis) was studied with immunohistochemical procedures. A highly complex and heterogeneous staining pattern is encountered in the macaque amygdala. The basal, lateral, and accessory basal nuclei display the most intense immunostaining with local heterogeneities. The lateral division of the central nucleus also stains intensely, whereas the medial division of the central nucleus and the medial nucleus are more weakly stained. The dorsal division of the bed nucleus-amygdala continuum (extended amygdala) is strongly immunoreactive. The hippocampus displays the strongest immunoreactivity encountered so far in the primate brain. The intensity of the immunostaining is highest in the cornu Ammonis (Ammon's horn; CA1-CA3 fields) and gradually decreases toward the dentate gyrus or the subicular area. In the hippocampus proper, the stratum radiatum, the pyramidal layer, the stratum oriens, and the alveus all display intense immunoreactivity. The immunostaining is much less prominent in the dentate gyrus, whose granule cell layer is completely devoid of labeling. In the subicular area, there is a lateromedial decreasing gradient in immunostaining intensity, the subiculum being moderately stained and the parasubiculum weakly stained. These results reveal that the limbic system-associated membrane protein labels structures that form the core of the limbic system in primates. Within each of these structures, however, the labeling is highly heterogeneous and appears to be confined to specific functional domains. © 1996 Wiley-Liss, Inc.     

Spatiotemporal analyses of interactions between entorhinal and CA1 projections to the subiculum in rat brain slices

The subiculum and the entorhinal cortex (EC) are important structures in processing and transmitting information between the neocortex and the hippocampus. The subiculum potentially receives information from the EC through two routes. In addition to a direct projection from EC to the subiculum, there is an indirect polysynaptic connection. The latter uses a number of possible pathways, which all converge onto the final projection from the hippocampal field CA1 to the subiculum. In this series of experiments we investigated to what extent activity in both pathways influences population activity of subicular neurons. We used voltage sensitive dyes in combined hippocampal-EC slices of the rat to measure the spatio-temporal activity patterns. To activate the two inputs to the subiculum, stimulation electrodes were placed in the stratum oriens/alveus of CA1 and in layer III of the medial EC. The response patterns evoked in the subiculum after electrical stimulation of each of these input pathways separately were compared with the response patterns after simultaneous stimulation of both areas (medial EC + CA1). A comparison of the computed added responses of the two individual stimulations with the measured responses after simultaneous stimulation suggests that both inputs are linearly added in the subiculum with very little nonlinear interactions. This strongly suggests that in the subiculum interaction at a single cell level of the direct and the indirect pathways from the EC is an unlikely scenario.     

Neurotransmitter receptor sites in human hippocampus: a quantitative autoradiographic study

The distribution of serotonin₁, cholinergic-muscarinic, α-adrenergic and opiate-type receptors was studied in the human hippocampus by quantitative autoradiography. Each receptor type exhibited a unique distribution. Serotonin₁ receptors were found to predominate in the subiculum, whereas α₁-adrenergic receptor are absent in that area and present at high levels in the dentate and the CA fields. High densities of muscarinic receptors appear in the subiculum and dentate gyrus. Opiate receptors are restricted to a medial aspect of the subiculum, with lower levels in the dentate and CA fields. The high anatomical resolution and quantitative character of the data may make it useful in the investigation of hippocampal pathology in humans.     

Interictal-ictal interactions and limbic seizure generation.

Interictal discharges are used in clinical practice to localize the epileptogenic focus in patients with partial epilepsy. However, the interaction between interictal and ictal discharges remains debatable. For instance, interictal events may lead to seizure onset in some models of epileptiform discharge. By contrast, in other models, disappearance of interictal activity (for example by activation of GABAB receptors) induces or potentiates ictal events. We have recently obtained new evidence for a control exerted by interictal discharges on ictal activity in rodent combined slices of hippocampus-entorhinal cortex. In this preparation continuous application of 4-aminopyridine induces: (i) interictal activity which initiates in CA3 and propagates via CA1 and subiculum to the entorhinal cortex, and return to the hippocampus through the dentate gyrus; and (ii) ictal discharges, which originate in the entorhinal cortex and propagate via the dentate gyrus to the hippocampus. Ictal discharges disappear over time, while synchronous interictal discharges continue to occur. Lesioning the Schaffer collaterals abolishes interictal discharges in CA1, entorhinal cortex and dentate gyrus and discloses entorhinal ictal discharges that propagate, via the dentate gyrus, to the CA3 subfield. Interictal activity of CA3 origin also prevents the occurrence of ictal events recorded in the entorhinal cortex in Mg(2+)-free medium. Moreover, in both models, ictal discharge generation in the entorhinal cortex after Schaffer collateral cut is prevented by mimicking CA3 activity through rhythmic electrical stimulation of CA1 hippocampal outputs. Hence, our data demonstrate that hippocampus interictal discharges control the expression of electrographic seizures in entorhinal cortex. Sectioning the Schaffer collaterals may model the epileptic condition in which cell damage in the CA3 subfield results in loss of CA3 control over the entorhinal cortex. Hence, the functional integrity of hippocampal CA3 neurons may represent a critical control point in temporal lobe epilepsy.     

Increased GABA(A)-receptor alpha1-subunit expression in hippocampal dentate gyrus after early-life status epilepticus

PURPOSE: Previous studies in neonatal (postnatal day 10) and adult rats suggest that status epilepticus (SE) induces changes in the alpha1 subunit of the GABA(A) receptor (GABRA1) in dentate granule neurons (DGNs) that are age dependent and vary inversely with the likelihood of epilepsy development. In the present study, we examined GABRA1 expression after SE at postnatal day 20 (P20), an intermediate age when only a subset of SE-exposed animals develop epilepsy. METHODS: SE was induced with lithium-pilocarpine or kainate at P20. Animals were video-EEG monitored after SE to determine the presence or absence of spontaneous seizures. GABRA1 mRNA and protein levels were determined 7 days or 3 months later in SE-exposed and control animals by using a combination of aRNA amplification, Western blotting, and immunohistochemistry techniques. RESULTS: GABRA1 mRNA levels in DGNs of SE-exposed rats that did not become epileptic were higher than those in control rats, but were not different from DGNs in epileptic SE-exposed rats. GABRA1 protein levels in dentate gyrus were significantly increased in both epileptic and nonepileptic SE-exposed rats compared with controls. GABRA1 mRNA changes were region specific and did not occur in CA1 or CA3 areas of hippocampus. GABRA1 alterations were present by 1 week after P20 SE and were similar whether pilocarpine or kainate was used to induced SE. CONCLUSIONS: P20 SE results in persistent increases in GABRA1 levels selectively in dentate gyrus. These changes preceded the onset of epilepsy, were not model specific, and occurred in both epileptic and nonepileptic animals.     

High-frequency (80-500 Hz) oscillations and epileptogenesis in temporal lobe epilepsy

High-frequency oscillations (HFOs), termed ripples (80-200 Hz) and fast ripples (250-600 Hz), are recorded in the EEG of epileptic patients and in animal epilepsy models; HFOs are thought to reflect pathological activity and seizure onset zones. Here, we analyzed the temporal and spatial evolution of interictal spikes with and without HFOs in the rat pilocarpine model of temporal lobe epilepsy. Depth electrode recordings from dentate gyrus (DG), CA3 region, subiculum and entorhinal cortex (EC), were obtained from rats between the 4th and 15th day after a status epilepticus (SE) induced by i.p. injection of pilocarpine. The first seizure occurred 6.1 ± 2.5 days after SE (n = 7 rats). Five of 7 animals exhibited interictal spikes that co-occurred with fast ripples accounting for 4.9 ± 4.6% of all analyzed interictal spikes (n = 12,886) while all rats showed interictal spikes co-occurring with ripples, accounting for 14.3 ± 3.4% of all events. Increased rates of interictal spikes without HFOs in the EC predicted upcoming seizures on the following day, while rates of interictal spikes with fast ripples in CA3 reflected periods of high seizure occurrence. Finally, interictal spikes co-occurring with ripples did not show any specific relation to seizure occurrence. Our findings identify different temporal and spatial developmental patterns for the rates of interictal spikes with or without HFOs in relation with seizure occurrence. These distinct categories of interictal spikes point at dynamic processes that should bring neuronal networks close to seizure generation.     

Axons Regenerate with Correct Specificity in Horizontal Slice Culture of the Postnatal Rat Entorhino-hippocampal System

We have used slice culture of the entorhino-hippocampal system to investigate (1) whether nerve fibres which are cut postnatally are able to regenerate and (2) whether the regenerating fibres are able to establish correct selective target specificity in the formation of their terminal fields. Slices of tissue were taken in the horizontal plane through the caudo-ventral pole of the cerebral hemisphere of 9- to 10-day-old rats. Such slices maintain the entorhinal cortex in continuity with the hippocampus and intervening retrohippocampal areas. However, because of the dorsal inclination of the entorhino-hippocampal projection fibres in situ, the segments of the entorhinal cortex and hippocampus contained within each individual horizontal slice were disconnected from each other. During subsequent culture, the formation of fibre connections between the entorhinal area and the hippocampal complex was studied by the extracellular and intracellular anterograde transport of biocytin or biotin dextran, the retrograde transport of biotin dextran or carbocyanine dyes, and by electrical stimulation and recording. For the first 24 h after taking the slice, there were no entorhinal projections beyond the deep white matter, and no fibres reached the hippocampus or dentate gyrus. After 3 days in culture a small number of growing fibres had perforated the subiculum and entered the target areas. Between 6 and 14 days these projections increased and matured. As in the normal adult brain, entorhinal layer II stellate cells projected correctly to the dentate gyrus and hippocampal field CA3, whereas layer III pyramidal cells projected to hippocampal field CA1 and the subiculum. The new fibres grew along both alvear and perforant pathways. Anterograde and retrograde labelling showed that the reciprocal projections from the pyramidal cells of the subiculum and CA1 to the entorhinal area had also been severed at the time of taking the slices, and had similarly regenerated. Our results demonstrate that by taking tissue slices in appropriate planes it is possible to study the regeneration of axons in the tissue environment through which they normally run. This approach avoids the use of coculture and the concomitant difficulties associated with the need for fibres to cross a coculture interface. In horizontal slices of postnatal tissue, severed fibre projections between the entorhinal cortex and the hippocampal complex can regenerate in both directions and re-establish their correct laminar, pathway and target specificity.     

Visualization of specific angiotensin II binding sites in the rat limbic system

The present study examined the distribution of angiotensin-binding cells by using a fluorescence-coupled angiotensin II in fixed horizontal sections that contained several limbic structures. In normal female rats, dense staining was found in the CA3 and CA1 regions and the dentate gyrus of the hippocampus--in the subiculum as well as in the entorhinal cortex and piriform cortex. Moderate staining was found in the CA2 region, in the central and medial nuclei of the amygdala. Low-level staining was obtained in the basolateral and lateral nucleus of the amygdala as well as in the bed nucleus of the stria terminalis. The co-incubation of fluorescence-coupled angiotensin II together with angiotensin II in excess and with saralasin, respectively, suppressed the angiotensin staining in structures investigated.     

Induction of prolonged electrographic seizures in vitro has a defined threshold and is all or none: implications for diagnosis of status epilepticus

PURPOSE: To study whether induction of prolonged (>30-min duration) in vitro electrographic seizure discharges resembling status epilepticus (SE) is graded or all-or-none, and to determine the critical factors mediating SE induction. METHODS: Prolonged electrographic seizure discharges were induced in combined hippocampal-entorhinal cortical (HEC) brain slices by electrical stimulation of the Schaeffer collaterals. Discharges were recorded by using field-potential electrodes in the dentate gyrus, CA3, CA1, and entorhinal cortex. Slices were prepared from rats that were (a). 21- to 30-day-old naive, (b). 60- to 120-day old naive, (c). epileptic, and (d). status post a prior traumatic brain injury. RESULTS: Induction of SE discharges was dependent on the duration, but not amplitude of the preceding stimulus train-induced afterdischarge in HEC slices from 21- to 30-day-old control, brain-injured, and epileptic animals, but not from 60- to 120-day-old animals. In slices from 21- to 30-day-old control animals, once afterdischarges exceeded 4 min in duration, SE was induced in 50% of slices, and after >or=6 min 37 s seizure activity; SE was induced in 95% of slices. A defined SE threshold also was evident in brain-damaged rats, including rats in which an epileptic condition was induced by pilocarpine injection 4-16 weeks before recording, and rats subjected to a fluid percussive head trauma 1-8 weeks before recording. However, in these brain-damaged animals, mean SE threshold was considerably lower (24 and 44 s, respectively). HEC slices from 60- to 120-day-old controls for the brain-injured and epileptic animals did not develop SE even after 20 stimulations, demonstrating the pronounced effect of brain injury and epilepsy on the development of SE in the HEC slice preparation compared with that in age-matched controls. CONCLUSIONS: In vitro, SE discharges have a defined temporal threshold for initiation. Once a seizure exceeds 6-7 min in duration in control animals, and 30-55 s in brain-damaged animals, the probability of SE induction is greatly increased. This demonstrates that brain injury lowers the afterdischarge duration required to produce SE and suggests that brains injured from trauma or SE are more susceptible to develop status epilepticus.     

Acetylcholinesterase fiber staining in the human hippocampus and parahippocampal gyrus

The AChE fiber distribution within the human hippocampus and parahippocampal gyrus was studied in order to provide normative data for the examination of cholinergic fiberarchitecture in human pathology and to clarify the cytoarchitectonic organization of these structures. A modification of the Koelle method was used to stain temporal lobe serial sections from 6 neurologically normal human brains collected at autopsy. The hippocampal formation contains some of the densest staining of any cortical area. Regions with the heaviest concentrations of AChE fibers include a thin band along the inner edge of the molecular layer of the dentate gyrus (ml-DG) and parts of the CA2, CA3, and CA4 sectors of Ammon's horn. Staining is of intermediate intensity in the CA1 region. The subiculum (S) is more lightly stained than the CA fields. Staining in the parahippocampal gyrus is generally less dense than in the hippocampal formation. The most conspicuous feature of the human entorhinal cortex (EC) is the AChE-rich fiber patches seen overlapping the stellate cell islands in layer II. An additional band of relatively dense AChE staining is identified in layers IV-V. Prominent AChE-rich polymorphic neurons are present within the hilum of the dentate gyrus. The CA1/subiculum transition in Nissl preparation is characterized by an oblique interdigitation of CA1 cells. The transition from EC to prorhinal cortex occurs along the medial bank of the rhinal sulcus and is characterized by a band of AChE staining, which slopes obliquely away from layer II until it joins an intermediate pyramidal cell layer. Some comparisons with AChE staining in the monkey were made. The monkey has a similar pattern except in DG, where the intensely AChE staining band along the inner ml-DG is thicker and much more prominent. In comparison to the human, the monkey has more conspicuous AChE staining in the parasubicular region.     

Visualization of muscarinic receptor-mediated phosphoinositide turnover in the hippocampus of young and aged, learning-impaired Long Evans rats.

Hippocampal receptor-mediated phosphoinositide (PI) turnover is severely blunted in aged rats that demonstrate cognitive deficits in the Morris water maze. To further examine the anatomical localization of this deficit, we examined the topography of muscarinic receptor-mediated PI turnover in young and aged-learning impaired rats by taking advantage of an autoradiographic method that visualizes PI turnover by measuring the diacylglycerol (DAG) branch of the PI turnover signal transduction system. Using this method, muscarinic cholinergic receptors were stimulated in hippocampal slices with agonist, and the receptor-mediated incorporation of [3H] cytidine into [3H]CDP-DAG was subsequently quantified in subregions of the hippocampus using film autoradiography. Our results show a significant decrease in basal incorporation of [ 3H]CDP-DAG in the subiculum and in the dentate gyrus in the aged rats. The muscarinic receptor-mediated [3H]CDP-DAG response was significantly blunted in the aged rats in subiculum, CA3, and CA1. In contrast, the receptor-mediated response was maintained in the dentate gyrus and hilus. These results indicate that the age-associated impairment in receptor-mediated PI turnover differs regionally, with a reduction in the subiculum and hippocampus proper that is pronounced relative to the hilus and dentate gyrus.     

Spread of Excitation in Chronically Lesioned Mouse Hippocampus Determined by Laser Scanning Microscopy

Fast optical recordings by means of laser scanning microscopy in conjunction with a voltage-sensitive dye (RH 414) were performed to monitor the spatio-temporal spread of neuronal activity in CA3/CA4-lesioned C57BL6 mouse hippocampal slices prepared approximately 3 months after intracerebroventricular kainic acid (KA) injection. The aim of our study was to assess the effects of a circumscribed neuronal loss on the propagation of electrical activity along the trisynaptic hippocampal circuit. Both in physiological bathing solution and in bicuculline (10 μM), hilar stimulation failed to activate the downstream pathway, so that, under these conditions, the chronically disinhibited CA1 region appeared to be effectively isolated from burst activity arising upstream; however, epileptiform discharges evoked in zero Mg²⁺solution were reliably transmitted from the dentate gyrus to the CA1 region. That these bursts were indeed spreading across the lesion, and not along newly formed connections (e.g., between dentate gyrus and CA1), was confirmed by acute transection experiments of the Schaffer collateral/commissural pathway, which completely abolished translesional burst propagation. The fact that the surviving CA3–CA1 connections are unable to trigger epileptiform bursts after suppression of GABAergic inhibition suggests that the lesioned region might serve as a filter that shields hyperexcitable CA1 neurons from epileptic activity arising upstream, in particular from chronically disinhibited granule cells of the dentate gyrus. An impaired GABAergic inhibition will thus only have minor facilitating effects on seizure propagation in the hippocampus of CA3-lesioned animals.