Tore Eid

Sleep/waking effects of a selective 5-HT1A receptor agonist given systemically as well as perfused in the dorsal raphe nucleus in rats.

Sleep/waking stages and behavior were studied following the selective 5-HT1A agonist 8-OH-DPAT given subcutaneously (s.c.) (0.010-0.375 mg/kg) as well as perfused continuously (10 microM) for 6 h into the dorsal raphe nucleus (DRN) using microdialysis. Given systemically, 8-OH-DPAT at 0.375 mg/kg s.c. induced 5-HT behavioral syndrome, increased waking to 149% and reduced slow wave sleep (SWS) to 86%, transition to 76% and rapid eye movement (REM) sleep to 73%. The effect on deep SWS (SWS-2) was biphasic, with an increase after 2 h. 8-OH-DPAT at 0.010 mg/kg did not have any vigilance effects. 8-OH-DPAT perfusion in DRN produced a fourfold increase in REM sleep compared to perfusion of artificial cerebrospinal fluid. This is consistent with the hypothesis that reduced 5-HT neurotransmission following 5-HT1A autoreceptor stimulation will disinhibit cholinergic REM-promoting mesopontine neurons and thereby lead to a REM sleep increase. The other sleep/waking stages were not significantly affected by 8-OH-DPAT perfusion in DRN.

Glutamate receptor subunits GluR1 and GluR2/3 distribution shows reorganization in the human epileptogenic hippocampus

The AMPA‐type glutamate receptor subunits GluR1 and GluR2/3 were localized by immunohistochemistry with subunit‐specific antibodies in hippocampi removed surgically from patients with temporal lobe epilepsy for the control of seizures. The flip and flop splice variants of the subunits were localized by in situ hybridization histochemistry with specific oligoprobes. In patient hippocampi that were not the seizure focus, the GluR1 subunit proteins were diffusely expressed on the dendrites of neurons in all regions. In contrast, in these same hippocampi, the GluR2/3 subunit proteins were expressed strongly on the soma and proximal dendrites of principal neurons in all regions. The flip variant of these subunits was localized in the hilus and fields of Ammons Horn (CA), while the flop variants were prominent on the dentate granule cells. In the epileptogenic hippocampus, while immunoreactivity was decreased in all fields that showed neuronal loss, there was an increased expression of GluR1 on the dendritic excrescences on the proximal dendrites of hilar neurons and CA3 pyramidal neurons, as well as expression of GluR2/3 in hilar neuron excrescences. Electron microscopic examination confirmed that the GluR1 immunoreactivity was only in dendritic processes, particularly dense at the postsynaptic membranes. Such expression of GluR1 may provide for an enhanced glutamatergic response by these neurons. GluR2/3 was also significantly increased on the dendrites of dentate granule cells in the epileptogenic hippocampus and may provide some protection against excitotoxic injury by reducing calcium flux into neurons.

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.

Neurons in layer III of the entorhinal cortex. A role in epileptogenesis and epilepsy

Abstract: A preferential lesion of neurons in layer III of the entorhinal cortex (EC) is often observed in patients suffering from temporal lobe epilepsy and in several animal models of the disease. This lesion is duplicated in rats by a focal, intra‐entorhinal injection of the “indirect” excitotoxin aminooxyacetic acid (AOAA), providing a model that can be used to study the mechanisms underlying seizure‐induced cell death and epilepsy. Doomed neurons in the EC and in several associated limbic structures show pathological changes within hours after the AOAA injection, but GABAergic neurons in layer III of the EC are quite resistant. This pattern of neuron loss eventually results in hippocampal and entorhinal hyperexcitability. Notably, the seizure‐induced death of layer III neurons in the EC can be attenuated by eliminating the prominent excitatory input from the presubiculum. Taken together, these results suggest opportunities to target parahippocampal structures for the treatment of temporal lobe epilepsy.

Neuronal damage after the injection of aminooxyacetic acid into the rat entorhinal cortex: a silver impregnation study.

In rats, most neurons in layer III of the medial entorhinal cortex are exquisitely vulnerable to prolonged seizure activity. These neurons have also been shown to die preferentially in the entorhinal cortex of patients with temporal lobe epilepsy. This lesion can be duplicated in rats by a focal injection of the indirect excitotoxin aminooxyacetic acid into the entorhinal cortex. The present study was designed to examine the neuropathological consequences of an intra-entorhinal aminooxyacetic acid injection at various time-points with a sensitive silver staining method for the visualization of damaged neurons. After 3 h, affected cells with prominently stained processes were readily observed in the transition zone of the hippocampal CA1 field and the subiculum, but no silver-stained neurons were seen in the entorhinal cortex. Less consistently, damaged neurons were observed in the presubiculum, in the temporal and perirhinal cortices and in the lateral amygdaloid nucleus. At 6 h after an aminooxyacetic acid injection, numerous silver-stained neurons, which were typically devoid of processes, were also seen in layer III of the medial entorhinal cortex. This pattern of neurodegeneration remained similar at 12 and 24 h following the aminooxyacetic acid injection, though many silver-stained neurons were noted in layer II of the lateral entorhinal cortex as well. Notably, at five days, silver-stained neurons had disappeared. Instead, dendritic arbors, debris of degenerated neurons and reactive glial cells were present in lesioned brain regions. These data demonstrate the chronology and the extent of neuronal damage following an intra-entorhinal injection of aminooxyacetic acid. The results suggest that a detailed examination of the temporal sequence of neuronal death in the entorhinal cortex and in extra-entorhinal areas is likely to benefit our understanding of the pathophysiology of temporal lobe epilepsy.

Ultrastructure and immunocytochemical distribution of GABA in layer III of the rat medial entorhinal cortex following aminooxyacetic acid-induced seizures.

Abstractu2002Layer III of the entorhinal cortex (EC) is lesioned in patients with temporal lobe epilepsy (TLE). A similar neuropathology is also present in different animal models of TLE. For example, injection of the ”indirect” excitotoxin aminooxyacetic acid (AOAA) into the EC of rats causes behavioral seizures and preferential loss of neurons in layer III of the medial EC. The animals also develop hyperexcitability of the EC and the hippocampal region CA1. To further explore the neuropathological changes within the EC, the ultrastructure and distribution of GABA-like immunoreactivity were assessed in layer III, 28 days after an intraentorhinal AOAA injection. At this time point, light microscopic preparations revealed that a large proportion of pyramidal (putative excitatory) neurons in layer III of the medial EC had degenerated, whereas GABA-immunoreactive neurons had survived. In immunogold-labeled ultrathin sections, the lesioned neuropil was found to contain morphologically intact GABA-containing neurons and nerve terminals. Pathologically swollen dendrites and electron-dense neuronal profiles were present in the lesioned sector as well. The majority of the electron-dense profiles was identified as degenerating dendritic spines that were closely apposed to strongly glutamate-immunopositive axon terminals. Thus, the entorhinal chemoarchitecture is dramatically altered following an episode of AOAA-induced epileptic seizures. One possible consequence of this pathology is a reduced ”drive” of the surviving layer III GABA neurons, which in turn may cause hyperexcitability of the EC and the hippocampus. These findings may be of relevance for the genesis and spread of temporal lobe seizures.

Differential neuronal vulnerability to amino-oxyacetate and quinolinate in the rat parahippocampal region.

Injection of the indirect excitotoxin amino-oxyacetate into the entorhinal area causes acute behavioral seizures and preferential neuronal loss in layer III of the medial entorhinal cortex in rats. We examined here whether the effects of amino-oxyacetate could be duplicated by local injections of the endogenous N-methyl-D-aspartate receptor agonist and direct excitotoxin, quinolinate. Amino-oxyacetate (685 nmol) or quinolinate (30, 45 or 60 nmol) were injected into the entorhinal cortex of rats anesthetized with choral hydrate (360 mg/kg). Separate groups of animals were co-treated with the N-methyl-D-aspartate receptor antagonist dizocilpine maleate (2 mg/kg) or given a higher dose of chloral hydrate (500 mg/kg). Rats that received amino-oxyacetate and a low anesthetic dose consistently displayed acute behavioral seizures and showed preferential loss of neurons in layer III of the medial entorhinal cortex. Animals that were given quinolinate did not display behavioral seizures, and showed preferential degeneration of neurons in layer V of the entorhinal cortex. Moreover, quinolinate-injected rats frequently exhibited neuronal loss in the superficial layers of the dorsal perirhinal cortex. The behavioral and neuropathological sequelae of amino-oxyacetate, but not quinolinate-induced neurotoxicity, were abolished by prolonged chloral hydrate anesthesia. In spite of these apparent qualitative differences between the two toxins, neurodegeneration induced by either amino-oxyacetate or quinolinate was completely prevented by dizocilpine maleate. These data suggest that a heterogeneous distribution of pharmacologically distinct N-methyl-D-aspartate receptor subtypes in the parahippocampal region may underlie the distinct neurodegenerative properties of the two toxins. Since the lesion caused by amino-oxyacetate bears remarkable similarities to neuropathological changes which have been described in this structure in temporal lobe epilepsy, further elucidation of the mechanisms of cellular toxicity of amino-oxyacetate may hold clues for the pathogenesis of this disease.

Ibotenate Injections into the Pre- and Parasubiculum Provide Partial Protection against Kainate-Induced Epileptic Damage in Layer III of Rat Entorhinal Cortex

Summary: u2002Purpose: A loss of neurons in layer III of the entorhinal cortex (EC) is often observed in patients with temporal lobe epilepsy and in animal models of the disorder. We hypothesized that the susceptibility of layer III of the EC to prolonged seizure activity might be mediated by excitatory afferents originating in the presubiculum.

The use of hydrophobic adhesive tape to produce miniature wells on microscope slides

Hydrophobic adhesive tape was used to produce miniature wells on microscope slides for staining several sections of tissue with minimal amounts of cytochemical reagents. The wells could be tailored to individual specifications and the method allowed coverslips to be mounted close to the sections using either aqueous or xylene based mounting media. This method was especially useful for multiple immunolabelling of serial semithin cryosections.