Brain Research, 514 (1990) 167-170

167

Elsevier BRES 24048

Granule cells in the ventral, but not dorsal, dentate gyrus are essential for kainic acid-induced wet dog shakes Laura M. Grimes 1, Todd S. Earnhardt 2, Clifford L. Mitchell 3, Hugh A. Tilson 3 and Jau-Shyong Hong 3 JCurriculum in Toxicology and 2Biological Sciences Research Center, University of North Carolina, Chapel Hill, NC 27514 (U.S.A.) and 3Laboratory of Molecular and Integrative Neuroscience, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709 (U.S.A.)

(Accepted 26 December 1989) Key words: Dentate granule cell; Dorsal hippocampus; Ventral hippocampus; Wet dog shakes; Kainate; Colchicine

Intrahippocampal injections of colchicine selectively destroy dentate granule cells. Wet dog shaking elicited by systemic administration of kainic acid is eliminated by bilateral destruction of ventral dentate granule cells but unaffected by bilateral destruction of dorsal dentate granule cells. This implies that ventral dentate granule cells are essential for the generation of kainic acid-induced wet dog shakes.

A stereotypic behavior exemplified by paroxysmal shaking of the head, neck and trunks in rats and referred to as 'wet dog shakes' (WDS) can be induced by electrical stimulation of limbic structures [cf. refs. 6, 8, 20, 23]. WDS can also be induced by kainic acid (KA), a cyclic analog of the excitatory amino acid glutamate, which has been used to produce limbic seizures in experimental animals 24. Destruction of dentate granule cells (DGC) results in a dramatic suppression of WDS induced by electrical stimulation of the entorhinal cortex 8 or by systemic injection of K A 13. In these studies, D G C in both the dorsal and ventral portions of the dentate gyrus were destroyed. Recently, it has been reported that injection of mu opioid receptor agonists into the ventral, but not dorsal, hippocampal formation induced WDS 18. We were interested, therefore, in determining the relative contribution of the D G C in the ventral vs dorsal dentate gyrus on the WDS elicited by KA. Male, Fischer-344 rats were obtained from Charles River Breeding Co. They were housed (4/cage) in plastic cages with cedar chip bedding and maintained on a 12 h light/dark cycle in a temperature and humidity controlled room with access to N I H diet 31 and water ad libitum. At the time of surgery, the animals weighed 210-237 g. They were anesthetized with pentobarbital, 50 mg/kg, i.p., and placed in a stereotaxic instrument. Injection coordinates, taken from Paxinos and Watson 25 were as follows: dorsal hippocampus - - 3.8 mm posterior, 2.5

mm lateral and 3.3 mm deep; ventral hippocampus - - 5.8 mm posterior, 4.5 mm lateral, and 7.0 mm deep. Anterior-posterior measurements were made with reference to bregma, lateral measurements were made with reference to the sagittal suture, and depth was measured from the skull at the point of placement. All measurements were made with the skull fiat. Each animal was injected bilaterally (into the dorsal or ventral hippocampi) with 0.5 /~1 of physiological saline or 2.0 /~g colchicine (Sigma, St. Louis, MO) in 0.5/~1 physiological saline per site. Injections were made at a rate of 0.1 /A/min using a 1.0/~1 Hamilton syringe and a manually operated microinjector (David Kopf Instruments). Cannulae were left in place for 2.0 min to allow diffusion of fluid away from the site. Four weeks later the animals were injected with KA, 8 mg/kg, s.c., and observed for 2 h for the number of WDS and the onset of behavioral seizures. Seizure onset was defined as the first occurrence of facial and forelimb clonus with rearing. Seizures were terminated with pentobarbital sodium, 50 mg/kg, i.p., in order to prevent death. At the time of KA injection, the rats weighed 265-297 g. One week later the animals were again anesthetized with pentobarbital sodium and perfused intracardially with 0.37% sulfide solution 28 followed b y 4% paraformaldehyde in 0.1 M phosphate buffer. The brains were removed, blocked and mounted in paraffin. They were subsequently sectioned at 10 gm. Coronal sections (one of every 10) were taken in the dorsal

Correspondence: J.S. Hong, NIEHS, P.O. Box 12233, Research Triangle Park, NC 27709, U.S.A.

168 h i p p o c a m p u s f r o m 1.0 m m a n t e r i o r to 1.0 m m p o s t e r i o r

site. E v e r y o t h e r section was stained with Cresyl v i o l e t o r

to the i n j e c t i o n site (as j u d g e d f r o m the surface of the

m o d i f i e d T i m m s stain 28 in o r d e r to assess the e x t e n t of

brain). T h e block was t h e n d e p a r a f f i n a t e d , r e - e m b e d d e d

d a m a g e to d e n t a t e g r a n u l e cells. A n i m a l s w e r e d i s c a r d e d

and r o t a t e d so that h o r i z o n t a l sections could be t a k e n

f r o m f u r t h e r analysis if t h e i r lesions w e r e u n i l a t e r a l or

f r o m 1.0 m m dorsal to 1.0 m m v e n t r a l to t h e i n j e c t i o n

involved another

structure.

Fig.

1 illustrates

typical

Fig. 1. The intactness of the granule cell-mossy fiber system was determined with Cresyl violet and a modified Timms stain for mossy fiber zinc28. In rats injected with saline in dorsal (A) or ventral (B) hippocampus, only the remnant of a needle track could be detected (arrow, A). In rats injected with 2.0 pg colchicine in dorsal (C) or ventral (F) hippocampus, destruction of granule cells (single arrow) and their mossy fiber terminals (double arrows) was observed. This destruction extended 1 mm on either side of the injection site, but did not extend as far as the opposite pole of the hippocampus (D and E). For colchicine lesions, dorsal and ventral sections were taken from the same rat.

169 TABLE I

both dorsal and ventral D G C does not affect the latency

Number of wet dog shakes and onset of seizures following kainic acid injection

to onset of KA-induced behavioral seizures. In this context, it is important to point out that dentate granule cells are essential in the WDS induced by other means such as electrical kindling 8. The abolition of WDS by destruction of ventral D G C is consistent with reports demonstrating the importance of ventral DGC in WDS induced by mu-opioid receptor agonists 18"19, perforant path I and intrahippocampal stimulation 2. Thus, it appears that ventral D G C may be crucial for expressing limbic system-induced WDS irrespective of the manner in which the limbic system is activated. Although this study shows a clear effect of colchicine on WDS, it is !mportant to acknowledge the fact that we cannot rule out the possibility that colchicine may affect the other hippocampal cells other than granule cells which may participate in the expression of WDS. Morphological examination from our previous study 13, however, shows selected degeneration of dentate granule cells after colchicine injection. The results of this study further support a growing body of evidence that the dorsal and ventral hippocampus subserve different behavioral functions. Studies of learning and memory suggest that although both the dorsal and ventral hippocampus are involved in information processing, the dorsal portion appears to be related to behavioral inhibition whereas the ventral portion appears to be related to behavioral arousal 27. Such a finding is in keeping with studies that suggest that the ventral hippocampus may play a pacemaker role in the generation of limbic seizures 3'7'12. At the present time, it is not clear whether the pacemaker properties of the ventral hippocampus are due to intrinsic or extrinsic factors. There are several marked differences in the neuroanatomical or neurochemical make up of the dorsal and ventral hippocampus which could enhance the excitability of the ventral hippocampus 5'9'11"16"22"26'32. On

Mean number of wet dog shakes -+ S.E.M. occurringduring the first 2 h following subcutaneous injection of kainic acid (8 mg/kg) and mean onset of seizures (chewing and forelimb clonuswith rearing) + S.E.M. (in minutes). Groups received saline, 0.5 gl or colchicine, 2.0 gg in 0.5/zl saline into the dorsal or ventral dentate gyrus 4 weeks prior to kainic acid injection. Statistical differences were determined by Student's t-test with Bonferroni's correctionfor multiple comparisons following a significant F ratio for treatments in an analysis of variance, n, number of animals per group. Saline injection

No. of wet dog shakes 96 + 9 Seizure onset 76 + 6 n (7)

Colchicineinjection Dorsal

Ventral

65 + 19 59 + 10 (7)

0 + 0* 69 + 8 (8)

*P < 0.01 from both the saline and dorsal colchicinegroups.

histological effects seen in saline- and colchicine-treated animals. Upon completion of histological analysis, a total of 22 animals remained for statistical analysis: 7 dorsal colchicine, 8 ventral colchicine, and 7 animals which received either dorsal saline (4) or ventral saline (3). For statistical analysis, the saline animals were combined into one group since none of these animals exhibited any histological damage and a previous study ~3 demonstrated no effect of intrahippocampal saline injections on responsiveness to KA. Thus, the data were analyzed as 2 separate analyses of variance (ANOVA), one each for number of WDS and time of onset of seizures. If there was a significant F ratio for treatment, the means were compared by Student's t-tests using Bonferroni's correction for multiple comparisons3~, with P ~< 0.05 as the level of significance. The data for the mean number of wet dog shakes and the mean onset of seizures are shown in Table I. Colchicine lesions in the ventral dentate gyrus eliminated KA-induced WDS. Although colchicine lesions of the dorsal dentate gyrus tended to reduce WDS, this was not statistically significant. The A N O V A for these data showed a significant F ratio for treatments (F2,I9 = 19.73, P < 0.001). This was due to the lack of WDS in the ventral colchicine group. In contrast, neither dorsal nor ventral colchicine injections significantly altered the onset of seizures (F2,19 = 1.04, P > 0.37). Moreover, all the animals exhibited seizures. Two major conclusions can be drawn from this study. First, granule ceils in the ventral, but not dorsal dentate gyrus appear to be essential for the expression of KA-induced WDS. Second, they are not essential for KA-induced behavioral seizures. This latter finding is consistent with our previous report 13 that destruction of

the other hand, the efferent and afferent connections of the dorsal and ventral hippocampus differ 14"15'17'29"3°. In particular, ventral hippocampus and subiculum have intimate connections with structures that have been shown to be involved in limbic seizure 4'21. In summary, our studies provide evidence that lesions of the granule cells of ventral but not dorsal dentate gyrus abolish KA-induced WDS, an early behavioral manifestation of limbic seizures. These findings suggest that the release of neurotransmitters or peptides from ventral granule cells, whose mossy fiber axons proceed in a transverse direction to synapse with CA3 pyramidal cells of a given lamella 1°, are essential for the expression of WDS. The precise manner in which release of granule cell contents modulates the activity of pyramidal cells to produce WDS requires further investigation. Our studies

170 also provide support for the growing body of evidence that granule cells are not essential for every animal model of limbic convulsions13'19.

This research was supported in part by training Grant 5T32-ES-07126 (L.M.G.). We wish to thank Loretta Moore for manuscript preparation.

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Granule cells in the ventral, but not dorsal, dentate gyrus are essential for kainic acid-induced wet dog shakes.

Intrahippocampal injections of colchicine selectively destroy dentate granule cells. Wet dog shaking elicited by systemic administration of kainic aci...
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