Non convulsive spike-wave discharges do not induce Fos in cerebro-cortical neurons

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Moh, culur Brain Research, 18 (1993) 178-180 O 1993 Elsevier Science Publishers B.V. All rights reserved 0169-328x/93/$06.00

BRESM 80161

Short Communications

Non convulsive spike-wave discharges do not induce Fos in cerebro-cortical neurons John O. Willoughby

a, L o r r a i n e

Mackenzie

a, J e n n i f e r J. H i s c o c k ~' a n d S t e p h e n

Sagar b

" Centre for Neuroscience and Department of Medicine, Flinders Unicersity and Medical Centre, Adelaide, SA (Australia) and hNeurology Sert,ice. Veterans Affairs Medical Centre, Unicersity of California, San Fransisco, CA (USA) (Accepted 9 December 1992)

Key words: Absence epilepsy; Spike-wave; Arousal; Cerebral cortex; Fos immunohistochemistry; Animal model

lmmunohistochemical localisation of Fos was used as a marker of neuronal activity to demonstrate neurons active during non-convulsive spike-wave epilepsy. Fos-positive neurons in cortex and several subcortical areas were counted. In undisturbed animals Fos counts were not related to spike-wave in any region. With the electroencephalographic (EEG) recording procedure, Fos induction occurred in all regions, even after habituation. However, in central cortex, counts were found to be inversely related to spike-wave duration. This suggests that neuronal activity is not increased during spike-wave and that the central cortex in these animals is less responsive to arousal than in non-epileptic animals.

Numerous inbred rat strains exhibit spontaneous brief periods of behavioural immobility with bilateral, synchronous discharges of spike and slow-wave activity in the electroencephalogram ( E E G ) 1'3'4's'1°'15'16. The phenomenon is regarded as a counterpart of human absence epilepsy. An immunohistochemically identifiable marker of activated neurons, Fos protein 7'11, was used to determine the distribution of activated neurons in such animals. Wistar K y o t o / N o r m o t e n s i v e ( W K y / N ) rats which express spike-wave approximately 12% of the time 16 were implanted under anaesthesia with extradural electrodes (_+ 2.5 mm lateral, 2 mm anterior and 2 and 6 mm posterior to bregma) as well as indifferent and earth electrodes. They were wired to a microconnector embedded in dental cement. Venous catheters were implanted in some animals to enable sampling for prolactin, a hormone responsive to environmental stimuli 13. E E G recordings were made using a Beckman E E G machine via a cable and preamplifier 1 attached to the connector. Spike-wave duration in each animal was expressed as a percentage of total recording time 16 and was defined by E E G appearances of bilateral, synchronous spike-wave activity.

For immunohistochemistry, animals were anaesthetised and perfused with formaldehyde/picric acid fixative. Brains were sectioned and processed for Fos using either Oncogene Science Fos antibody (1 : 1000, 3 day incubation, experiment I) raised in rabbit, or Cambridge Research Biochemicals Fos antibody (1:2000, overnight incubation, experiment It) raised in sheep. The secondary antibodies were localised using the avidin-biotin-horseradish peroxidase complex technique (Amersham, UK) and a nickel enhanced diaminobenzidine method. Counts of immunoreactive nuclei or perikarya were undertaken at 100 × magnification on an Olympus BH-2 Microscope using Magellan programme (P. Halasz, University of New South Wales) on a computer interfaced to the microscope via a drawing tube. Experiment I: Fos counts were compared in unhandled (quiescent) and handled W K y / N rats (handled). Quiescent animals were anaesthesed in their home cages and then perfused. The amount of spike-wave expressed by these animals was estimated with E E G recordings made before the day of death. Handled animals were those anaesthetised and killed 1 h after an EEG. Blood for prolactin was drawn from quiescent

Correspondence: J.O. Willoughby, Department of Medicine, Flinders Medical Centre, Bedford Park, SA 5042, Australia. Fax: (61) 8-204-5450.

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BRAIN REGION Fig. 1. Counts (mean _+S.E.M.) of Fos positive neurons in 4 brain areas in quiescent and handled rats defined in text. The m e a n count of 3 unilateral coronal sections were used with each animal. The procedure induced Fos in all regions. Dorsolateral quadrant of central cortex: 5.86 anterior to the interaural line (AP), (CORTEX); hypothalamic paraventricular nucleus: A P 7.2, (PVN, known to respond to stress with Fos induction2); endopyriform area: A P 11.2, (ENDO); central grey: AP 2.96, (PAG). Regressions between counts and spike-wave duration in each group not significant, P > 0.05.

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animals in their home cages and, in handled animals, 10 and 20 min after transfer to the E E G recording chamber. EEGs were recorded for 30 min, commencing 10 min after placement in the recording chamber. Prolactin was measured by radioimmunoassay. In quiescent rats, there were few Fos immunopositive neurons in cerebral cortex or endopyriform area (Fig. 1) indicating that spike-wave does not induce Fos. In handled rats, Fos was induced in all areas and prolactin concentrations were elevated (quiescent 10 _+ 2 vs handled 46 _ 7 n g / m l , P = 0.0064). These findings suggest that animals were aroused by the procedure. Experiment II: Animals were exposed to the recording procedure on more than 9 occasions, perfused on the last day of recording and processed as above. There was an inverse correlation between spike-wave duration and Fos-positive counts in central cortex (Fig. 2, top left panel). Combining Brown Norway and W K y / N cortical counts, the regression was also significant for anterior cortex ( P < 0.05). Striking Fos-induction occurred with picrotoxin (data not shown), indicatx 4000 w

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Fig. 2. Regressions of Fos immuno-positive cell counts against spike-wave duration in W K y / N rats (n = 7, e). Mid-cortex here includes all neurons unilaterally in a coronal section between the interhemispheric fissure and the rhinal fissure. There is an inverse correlation for mid-cortex (r = 0.95, y = - 1 4 0 x + 4796, P < 0.0002, other regressions not significant) (a). Counts from 2 Brown Norway rats ( • ) without spike-wave are shown but are not included in regression. There were few Fos-positive neurons in the substantia nigra (data not shown).

180 ing c o r t i c a l n e u r o n s c a n s y n t h e s i z e Fos. B e c a u s e o f t h e l o n g half-life o f d i s a p p e a r a n c e o f F o s 7,jl, it w o u l d n o t b e p o s s i b l e for t h e i n v e r s e c o r r e l a t i o n to h a v e b e e n an i n h i b i t o r y e f f e c t o f s p i k e - w a v e o n F o s i n d u c e d by t h e E E G p r o c e d u r e . R a t h e r , c e n t r a l c o r t e x in a n i m a l s w i t h s p i k e - w a v e m a y be less s e n s i t i v e t h a n o t h e r a r e a s o f c o r t e x to a c t i v a t i o n by e n v i r o n m e n t a l

stimuli. T h u s ,

s p i k e - w a v e a n d r e d u c e d c o r t i c a l r e s p o n s i v e n e s s m a y be two i n d e p e n d e n t m e a s u r e s o f a l t e r e d c o r t i c a l circuitry. Electrical recordings of spike-wave indicate that cortical cells fire in p h a s e w i t h t h e s p i k e a n d t h a t all cells a r e i n h i b i t e d d u r i n g t h e w a v e ~. I n h i b i t i o n is i m p o r t a n t for s p i k e - w a v e g e n e r a t i o n 4,6,10,12. O u r f i n d i n g t h a t F o s is n o t i n d u c e d by s p i k e - w a v e s u p p o r t s t h e v i e w t h a t t h e r e is little c h a n g e to t h e a v e r a g e activity o f c o r t i c a l neurons

and that spike-wave might constitute repat-

t e r n i n g 9. T h i s s t u d y will assist i n t e r p r e t a t i o n o f findings

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creased 9 metabolic rates with bilateral non-convulsive s p i k e - w a v e attacks. The authors thank Dr Glenda Halliday for advice and Dr AF Parlow and the National Pituitary Program, NIDDK, for prolactin radioimmunoassay materials. S.S. was Merk, Sharp and Dohme Visiting Professor to the Centre for Neuroscience. The studies were supported by Australian Brain Foundation, Flinders Medical Centre Research Foundation and Glenside Research Foundation. 1 Buzsaki, G., Bickford, R.G., Ponomareff, G., Thal, L.J., Mandel, R. and Gage, F.H., Nucleus basalis and thalamic control of neocortical activity in the freely moving rat, ,L Neurosci., 8 (1988) 4007-4026. 2 Ceccatelli, S., Villar, M.J., Goldstein, M. and Hokfelt, T., Expression of c-los immunoreactivity in transmitter-characterised neurons after stress, Proc. Nat. Acad. Sci. USA, 86 (1989) 9569-9573. 3 Coenen, A.M.E. and Van Luijtelaar, E.L.J.M., The WAG/Rij rat model for absence epilepsy: age and sex factors, Epilepsy Res., 1 (1987) 297-301.

4 Depaulis, A., Vergnes, M., Marescaux, C., Lannes, B. and Warte., J.-M., Evidence that activation of GABA receptors in the substantia nigra suppresses spontaneous spike-and-wave discharges in the rat, Brain Res., 448 (1988) 20-29. 5 Engel, J., Lubens, P., Kuhl, D.E. and Phelps, M.E., Local cerebral metabolic rate for glucose during petit mal absences, Ann. Neurol., 17 (1985) 121-128. 6 Fromm, G.H., Glass, J.D., Chattha, A.S., Martinez, A.J. and Silverman, M., Antiabsence drugs and inhibitory pathways, Neurology, 30 (1980) 126-131. 7 Morgan, J.I., Cohen, D.R., Hempstead, J.L. and Curran, T.. Mapping patterns of c-fos expression in the central nervous system after seizure, Science, 237 (19871 192-197. 8 Nehlig, A., Vergnes, M., Marescaux, C., Boyet, S. and Lannes, B., Local cerebral glucose utilization in rats with petit real-like seizures, Ann. Neurol., 29 (1991) 72-77. 9 0 c h s , R.F., GIoor, P., Tyler, J.L., Wolfson, T., Worsley, K.. Andermann, F., Diksic, M., Meyer, E. and Evans, A., Effect of generalized spike-and-wave discharge on glucose metabolism measured by positron emission tomography, Ann. Neurol., 21 (1987) 458-464. 10 Peeters, B.W.M.M., van Rijn, C.M., Vossen, J.M.H. and Coenen, A.M.L., Effects of gaba-ergic agents on spontaneous non-convulsive epilepsy, EEG and behaviour, in the WAG/Rij inbred strain of rats, Life Sci., 45 (1989) 1171-1176. 11 Sagar, S.M., Sharp, F.R. and Curran, T., Expression of c-los protein in brain: metabolic mapping at the cellular level, Science, 240 (1988) 1328-1331. 12 Snead, O.C., Gamma-hydroxybutyrate model of generalized absence seizures: further characterisation and comparison with other absence models, Epilepsia, 29 (19881 361-368. 13 Terry, L.C., Saunders, A., Audet, J., Willoughby, J.O. Brazeau, P. and Martin, J.B., Physiologic secretion of growth hormone and prolactin in male and female rats, Clin. Endocrinol., (Suppl.) 6 (1977) 19-28. 14 Theodore, W.H., Brooks, R., Sato, S., Patronas, N., Magolin, R., Di Chiro, G. and Porter, R.J., The role of positron emission tomography in the evaluation of seizure disorders, Ann. Neurol., 15 (suppl.) (1984) s176-s179. 15 Vergnes, M., Marescaux. C., Micheletli, G., Reis, J., Depaulis, A., Rumbach, I. and Warter, J.M., Spontaneous paroxysmal electroclinical patterns in rat: a model of generalized nonconvulsive epilepsy, Neurosci. Lett., 33 (1982) 97-1(11. 16 Willoughby, J.O. and Mackenzie, L., Nonconvulsive electrocorticographic paroxysms (absence epilepsy) in rat strains. Lab. Anim. Sci., in press.