Dentate Granule Cells Form Novel Basal Dendrites in a Rat Model of Temporal Lobe Epilepsy
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Neuroscience Vol. 86, No. 1, pp. 109–120, 1998 Copyright ? 1998 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved Pergamon PII: S0306-4522(98)00028-1 0306–4522/98 $19.00+0.00 DENTATE GRANULE CELLS FORM NOVEL BASAL DENDRITES IN A RAT MODEL OF TEMPORAL LOBE EPILEPSY I. SPIGELMAN,*†** X.-X. YAN,‡ A. OBENAUS,§ E. Y.-S. LEE,‡ C. G. WASTERLAIN†¶Q and C. E. RIBAK‡ *Section of Oral Biology, UCLA School of Dentistry, Los Angeles, CA 90095-1668, U.S.A. †UCLA Brain Research Institute, Los Angeles, California, U.S.A. ‡Department of Anatomy and Neurobiology, College of Medicine, UC, Irvine, CA 92697-1275, U.S.A. §University of Saskatchewan, Department of Medical Imaging, Saskatoon, Saskatchewan, Canada, S7N 0W8 ¶Epilepsy Research Laboratory, VA Medical Center (127), Sepulveda, CA 91343-2099, U.S.A. QDepartment of Neurology, UCLA School of Medicine, Los Angeles, California, U.S.A. Abstract––Mossy fibre sprouting and re-organization in the inner molecular layer of the dentate gyrus is a characteristic of many models of temporal lobe epilepsy including that induced by perforant-path stimulation. However, neuroplastic changes on the dendrites of granule cells have been less-well studied. Basal dendrites are a transient morphological feature of rodent granule cells during development. The goal of the present study was to examine whether granule cell basal dendrites are generated in rats with epilepsy induced by perforant-path stimulation. Adult Wistar rats were stimulated for 24 h at 2 Hz and with intermittent (1/min) trains (10 s duration) of single stimuli at 20 Hz (20 V, 0.1 ms) delivered 1/min via an electrode placed in the angular bundle. The brains of these experimental rats and age- and litter-matched control animals were processed for the rapid Golgi method. All rats with perforant-path stimulation displayed basal dendrites on many Golgi-impregnated granule cells. These basal dendrites mainly originated from their somata at the hilar side and then extended into the hilus. Quantitative analysis of more than 800 granule cells in the experimental and matched control brains showed that 6–15% (mean=8.7%) of the impregnated granule cells have spiny basal dendrites on the stimulated side, as well as the contralateral side (mean=3.1%, range=2.9–3.9%) of experimental rats, whereas no basal dendrites were observed in the dentate gyrus from control animals. The formation of basal dendrites appears to be an adaptive morphological change for granule cells in addition to the previously described mossy fibre sprouting, as well as dendritic and somatic spine formation observed in the dentate gyrus of animal and human epileptic brains. The presence of these dendrites in the subgranular region of the hilus suggests that they may be postsynaptic targets of the mossy fibre collaterals. ? 1998 IBRO. Published by Elsevier Science Ltd. Key words: perforant-path stimulation, basal dendrites, mossy fibre, sprouting, dentate gyrus, rat. The kainate and pilocarpine animal models of status ably similar to that seen in human patients with epilepticus (SE) produce widespread brain damage temporal lobe epilepsy.11,15,19 Spontaneous seizures and the delayed occurrence of limbic and general- have been tentatively explained either by the sprout- ized convulsions.8,9 Electrical stimulation of the ing and re-organization of recurrent excitatory perforant-path under urethane anaesthesia in adult connections1,9,10,49,50,53 or by the loss26 or deaffer- rats produces neuronal injury which is restricted to entation4,43 of inhibitory interneurons in the the hippocampus, has an excitotoxic appearance hippocampus. similar to that induced by kainic acid27,29,42,45 and The sprouting of granule cell axons, the mossy is associated with a loss of frequency-dependent, fibres, is commonly inferred in the dentate gyrus of paired-pulse inhibition in the dentate gyrus,43 as humans and experimental animals with epilepsy. In well as the progressive development of spontaneous the affected hippocampal formation, sprouted mossy recurrent seizures.41 The type of hippocampal dam- fibres form an aberrant band in the inner molecular age that is seen in animal models of SE is remark- layer of the dentate gyrus where they are in position to synapse with the proximal apical dendrites of granule cells.11,15,31,50,53 The elements postsynaptic **To whom correspondence should be addressed at the UCLA School of Dentistry. to the mossy fibres also display changes in temporal Abbreviations: ISI, interstimulus interval; PB, phosphate lobe epilepsy. For example, kainic acid treatment in buffer; PS, population spike; SE, status epilepticus. adult mice results in dendritic hypertrophy with 109 110 I. Spigelman et al. increased numbers of dendritic spines that is separation <1 mm for recording electrodes and <0.5 mm correlated with the sprouting of mossy fibres.52 In for the stimulating electrodes) were cemented to the skull by human studies, a greater density of dendritic spines dental acrylate. Final electrode depth (2.5–3.5 mm from cortical surface) was determined by maximizing the ampli- was noted in granule cells that exhibited aberrant tude of the characteristic potential recorded in the dentate mossy fibre collaterals as compared to cells without gyrus in response to ipsilateral perforant-path stimulation. such collaterals.16 In another study, longer and more Chromium non-coated wires (Consolidated Wire and branched granule cell apical dendrites were found in Associated Co.) were connected to the screws on the skull and used as ground electrodes. The skin was cleaned tissue from patients with mesial temporal lobe scler- with Povidine solution (Rugby Laboratories) and 10 mg of 54 osis as compared to those from a control group. penicillin (Sigma) was injected subcutaneously once per day Associated with the seizure-induced changes in for three days after surgery. granule cells are molecular responses that include increases in mRNAs encoding various neuro- In vivo recordings trophic substances.13 These latter changes are often Input/output (I/O) responses were obtained at 125, 200, reminiscent of developmental processes. 300, 500, 700, 1000, 2000 and 3000 ìA (0.1 Hz, 0.2 ms duration, biphasic stimulation). Interstimulus interval (ISI)- The granule cells of adult rats lack basal dendrites, dependent paired-pulse responses were studied at intervals although they are transiently expressed in rats during of 40 and 400 ms (0.1 Hz, 0.1 ms duration, monophasic postnatal development.37,38,40,57 This transient ex- stimulation). Population spike (PS) amplitudes were pression is also observed in several other species, measured from the average of five consecutive evoked including mouse, rabbit, guinea-pigs and cats (see potentials. PS amplitude was calculated as [(field potential at the beginning of PS)+(field potential at the end of Ref. 37). In contrast, basal dendrites are normal PS)]/2"(field potential at the peak of PS). In multiple PS features of granule cells in the developing and adult discharges, we used the first PS to measure PS amplitude. dentate gyrus of monkeys and humans.37 The bio- The inhibition score in paired-pulse examinations was logical significance of the developmental appearance calculated as [100"(PS amplitude of second pulse)/(PS amplitude of the 1st pulse) 100]. and subsequent attrition of granule cell basal den- # drites in rats is unknown. In a recent study, Franck Perforant-path stimulation et al.12 remarked on the apparent high spine density One to two weeks after surgery, rats were anaesthetized on the basal dendrites of granule cells in the brains with a loading dose of urethane (1000 mg/kg) via the tail of adult epileptic humans and suggested that their vein followed by a continuous infusion at 0.08 ml/min occurrence may provide an additional basis for recur- (1200 mg/kg). Once anaesthetized, rats were then stimulated rent excitation. However, no comparison was made for 22–24 h with single stimuli (20 V, 0.1 ms duration) at 2 Hz and with intermittent trains (10 s duration) of stimuli with the basal dendrites of age-matched humans and at 20 Hz delivered once per minute to the implanted stimu- therefore it is unclear whether these basal dendrites lating electrode. Paired-pulse stimulation was carried out are within the normal range observed in humans.37 before, during and at various times after stimulation. One The present study aimed to determine whether hour after the end of stimulation animals were injected with basal dendrites appear on granule cells of adult rats furosemide (10 mg/kg, i.m.). At various times rats were also injected subcutaneously with small volumes of dextrose as a consequence of SE induced by perforant-path solution. stimulation. Such an analysis conducted in epileptic rats would provide novel information about the Neurobiotin detection in granule cells formation of dendrites in the epileptic brain because, Intracellular recordings were made in hippocampal slices unlike humans, normal adult rats lack basal den- (400 ìm-thick) from stimulated and control rats according 56 drites. A rapid Golgi method was used to assess to previously described methods. Intracellular micro- the dendritic arbors of granule cells. A preliminary electrodes contained 1.5% Neurobiotin in 3 M potassium acetate (pH=7.25). Neurobiotin was injected into granule report of the data has been published in abstract cells (one cell/slice) using square-wave current pulses form.33 (1–3 nA, 100 ms, 3 Hz) for 15 to 25 min. Slices were im- mersed overnight in 4% paraformaldehyde in 0.1 M phos- EXPERIMENTAL PROCEDURES phate buffer (PB, pH=7.4). Slices were then incubated in 0.1 M PB containing 1% avidin–biotin–peroxidase complex Surgery (ABC; Vector) and 0.2% Triton X-100 at 4)C for 48 h. The Neurobiotin labelling was visualized in 0.05% diaminoben- Male adult Wistar rats (400–500 g; Simonsen Labs) were zidine and 0.005% H O in PB.