- Email: [email protected]
Colchicine lesions of ventral, bur not dorsal, dentate granule cells attenuate wet dog shakes elicited by perforant path stimulation
Brain Research, 512 (1990) 159-163
159
Elsevier
BRES 24006
Colchicine lesions of ventral, but not dorsal, dentate granule cells attenuate wet dog shakes elicited by perforant path stimulation Martha I. Barnes and Clifford L. Mitchell Laboratory of Molecular and Integrative Neuroscience, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 (U.S.A.)
(Accepted 28 November 1989) Key words: Colchicine; Dentate granule cell; Ventral hippocampus; Dorsal hippocampus; Wet dog shake; Perforant path stimulation
Intrahippocampal injections of colchicine selectivelydestroy dentate granule cells. Wet dog shaking elicited by perforant path stimulation is unaffected by bilateral destruction of dorsal dentate granule cells but virtually eliminated by bilateral destruction of ventral dentate granule cells. This implies that ventral dentate granule cells are essential for the generation of perforant path stimulation-induced wet dog shakes.
The major input to the hippocampal formation (dentate gyrus and Ammon's horn) from the neocortex is through the entorhinal cortex via the perforant path 5. Electrical stimulation of the entorhinal c o r t e x 6 o r the perforant path 3, under proper conditions, produces a stereotypic behavior exemplified by paroxysmal shaking of the head, neck and trunk in rats. This behavior has been referred to as 'wet dog shakes' (WDS) because it resembles the behavior observed in a dog shaking itself while wet 14. Destruction of dentate granule cells (DGC) results in a dramatic suppression of WDS induced by electrical stimulation of the entorhinal cortex6 or by systemic injection of kainic acid 9. In these studies, DGC in both the dorsal and the ventral portions of the hippocampal formation were destroyed. Recently, it was reported that injection of mu opioid receptor agonists into the ventral, but not dorsal, hippocampus induced WDS 12. Moreover, destruction of DGC in the ventral hippocampal formation markedly reduced these WDS 11. We were interested, therefore, in determining the relative contribution of the DGC in the ventral vs. dorsal hippocampal formation in the WDS elicited by perforant path stimulation. Male, Fischer-344 rats weighing 250-300 g were obtained from Charles River Breeding Co. (Raleigh, NC). They were housed individually 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 NIH Diet 31 and water ad lib. For surgery, each animal received atropine sulfate, 2
mg/kg, s.c., followed approximately 10 min later with pentobarbital sodium, 50 mg/kg, i.p. Methoxyflurane was supplemented as needed. In each animal, a bipolar stimulating electrode aimed at the perforant path in the region of the angular bundle was chronically implanted under stereotaxic guidance. The electrodes were made from twisted 0.25 mm nichrome wire with a tip separation of approximately 0.5 mm. Stainless steel guide cannulae (22 gauge) were bilaterally implanted in order to allow injection cannulae (stainless steel, 28 gauge) to be aimed at the right and left dorsal hippocampus only (one-half of the animals) or the right and left ventral hippocampus only (the other half of the animals). The guide cannulae extended approximately 1 mm below the skull and were permanently attached to the skull. They were sealed with dummy injection cannulae until the time of colchicine administration. Coordinates were taken from the atlas of Paxinos and Watson 15 and were as follows: perforant path - - 7.8 mm posterior, 4.4 mm lateral and 4.0 mm deep; dorsal hippocampus - - 3.8 mm posterior, 1.7 mm lateral, and 3.2 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 flat. Two or three days after surgery, all animals were stimulated to determine the threshold for eliciting WDS. Stimulus parameters consisted of an 8-s train of biphasic
Correspondence: C.L. Mitchell, Lab. of Molecular and Integrative Neuroscience, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709, U.S.A.
160 paired pulses, duration of 0.1 ms/phase, and a train rate of 5 Hz with a 20-ms interval between the first and second pulse pair. The initial stimulus intensity was 0.1 mA. Every 2 min, the intensity was increased by 0.1 m A until W D S were observed. The threshold was defined as that current which elicited at least 2 WDS within 2 min following stimulation. Animals were matched according to thresholds and assigned to 1 of 4 groups: dorsal artificial cerebrospinal fluid (ACSF), dorsal colchicine, ventral ACSF, or ventral colchicine. Each animal was then injected bilaterally with 0.5 ~tl ACSF or 1 ~tg colchicine (Sigma, St. Louis, MO) in 0.5 ~1 A C S F per site over a 11/2 to 2 min time period. Two weeks later, all animals were tested for WDS using the parameters described above. The initial stimulus intensity was 0.2 m A below that previously determined to elicit WDS. As before, the intensity was increased by 0.1 m A every 2 min until WDS were observed or a current level of 1.0 m A was attained. The number of W D S occurring within 3 min post stimulation was recorded. This procedure was repeated 3 more times with a 5 min interval between replications. Subsequently,
the animals were anesthetized with pentobarbital sodium and perfused intracardially with 0.85% saline followed by 10% neutral buffered formalin (NBF) and a supersaturated solution of potassium ferrocyanide in 10% N B E The brains were removed from the brain case, blocked and mounted in paraffin. They were subsequently sliced at 10 /~m. One of every 10 slices was mounted, stained with Cresyl violet, and counterstained with luxol fast blue for analysis of lesion damage. Animals receiving dorsal injections of A C S F showed no visible cell damage in any hippocampal cell layer from the injection. Any cell damage was the result of the injection cannula and was restricted primarily to CA1, though in one animal the cannula went into the upper dentate granule cell layer on one side. There was minimal cell damage as a result of the ventral A C S F injection. When it occurred, it was limited to the immediate area surrounding the injection cannula in the rostral cell layers. There was no damage to dentate granule cell layers in the caudal region from either ACSF or the cannulae. In the dorsal dentate, colchicine damage ranged from moderate, limited primarily to the medial portion of the
Fig. 1. Low magnification photomicrographs of dorsal and ventral hippocampus two weeks after treatment. Dorsal (A) and ventral (B) hippocampal sections taken from a dorsal ACSF-treated animal vs dorsal (C) and ventral (D) sections from a dorsal colchicine-treated animal illustrate the dramatic loss of dorsal dentate granule cells (arrow) resulting from the application of colchicine. Note that ventral dentate granule cells remain intact.
161 upper blade, to more extensive damage which essentially destroyed all of the dentate granule cell layer of the upper blade and the medial portions of the lower blade. There was thinning of the CA1 layer in the immediate vicinity of the injection cannulae in some animals. The lesions never extended to the rostral portion of the ventral hippocampus (V-HPC). Ventral colchicine damage was centered mainly in the caudal dentate granule cells with damage extended to the rostral dentate in 4 of the 7 animals. The general impression was that the degree of D G C damage in the ventral HPC (V-HPC) was more variable than in the D-HPC. This may be due to the nature of the injection sites. In D-HPC, the injection site is relatively shallow, thus yielding a very localized injection site. In contrast, the V-HPC injection site is very deep. The cannulae frequently tended to pass through the edge of the lateral ventricle. Thus, in V-HPC colchicine may have diffused more than in D-HPC, resulting in more variable damage to the D G C of the V-HPC than the D-HPC. Damage to the other cell layers was due primarily to the cannula tract, although there was some thinning of caudal CA1 and medial CA3 in two animals. Figs. 1 and 2 illustrate typical histological effects seen in ACSF and colchicine treated animals. Animals
were discarded from further analysis if their lesions were unilateral or in another structure (e.g. thalamus or medial geniculate). Thus, upon completion of the histological analysis, a total of 35 animals remained for statistical analysis: 12 dorsal ACSF, 7 dorsal colchicine, 9 ventral ACSF, and 7 ventral colchicine. Data were analyzed for the initial threshold for WDS, for the 1st post-injection trial and for the total number of WDS by separate analyses of variance (ANOVA) for a 2 (substance injected) by 2 (injection site) factorial experiment. If the F ratio for substance by injection site interaction was significant then comparisons between groups were made by Student's t-test using Bonferroni's correction for multiple comparisons. In all cases, P ~< 0.05 was chosen as the level of significance. The mean values _+ S.E.M. for the thresholds for WDS obtained 2-3 days post surgery (pre-injection threshold) and the 1st threshold determination 2 weeks post ACSF or colchicine (first post injection) are shown in Table I. Since the animals were grouped according to their initial threshold, there i.s no statistically significant difference at this time period. The first post injection threshold for the ventral colchicine group is misleading since this mean was determined from truncated data. Four of the 7 animals
Fig. 2. Low magnification photomicrographs of dorsal and ventral hippocampus two weeks after treatment. Dorsal (A) and ventral (B) hippocampal sections taken from a ventral ACSF-treated animal vs dorsal (C) and ventral (D) sections from a ventral colchicine-treated animal illustrate the dramatic loss of ventral dentate granule cells (arrow) resulting from the application of colchicine. Note that the dorsal dentate granule cells remain intact.
162 TABLE I 80
Thresholds for wet dog shakes
Numbers represent mean + S.E.M. in mA; the number of animals per group is shown in parentheses. Threshold
Dorsal
]-
60
Ventral 40
Preinjection Postinjection
A CSF
Colchicine
A CSF
Colchicine
(12)
(7)
(9)
(7)
0.38+0.06 0.37+0.06 0.41+0.08 0.43+0.09 0.32+0.03 0.38+0.09 0.44+0.06 >1.00+0.00 *#
* Statistically different from all other post injection groups by Student's t-test at P < 0.001 (with Bonferroni correction for multiple comparisons). # Maximum value tested was 1.00 mA.
exhibited 0-1 WDS at the cutoff of 1 mA. Thus, the actual threshold, if one exists, is even higher than that shown in Table I. In fact, only 3 of 7 animals exhibited any WDS 2 days later when tested at much higher current strengths, for the appearance of overt seizures. These three animals had only 2 WDS at any time during seizure testing, whereas all the animals in the other 3 groups exhibited many WDS. The A N O V A for the postinjection threshold data showed significant F ratios for substance (F1.31 = 61.82, P < 0.001), for injection site (F1.31 = 87.25, P < 0.001) and for substance by injection site interaction (Fi,31 = 37.5, P < 0.001). With respect to the number of WDS, the A N O V A revealed significant F-ratios for substance (F1,31 --- 60.89, P < 0.001), for injection site (F1,31 = 28.38, P < 0.001) and for substance by injection site interaction (F1,31 = 49.02, P < 0.001). The means + S.E.M. are shown in Fig. 3. These data clearly show that the ability of colchicine to reduce WDS was restricted to the ventral injection site. The most plausible explanation of the effect of colchicine on WDS is destruction of DGC, since intrahippocampal injection of colchicine is known to preferentially destroy DGC, with little effect on other cell populations 16J7. Evidence implicating the hippocampal formation as a critical component of the network underlying WDS induced by electrical stimulation of the limbic system has been summarized by Frush and McNamara 6. Suffice it to say that, 'taken together, the data suggest that activation of D G C results in activation of the CA3 pyramidal cells, which in turn leads to activation of extrahippocampal structures '6. Our data specifically implicate the D G C in the ventral portion of the hippocampal formation. Lee et al. cite several lines of evidence indicating that the ventral hippocampal formation may be more important than its dorsal component for the expression of
20
D-ACSF
D- COLCHICINE V-ACSF
I I V-COLCHICINE
TREATMENT
Fig. 3. Mean number of wet dog shakes elicited by perforant path stimulation in animals receiving artificial cerebrospinal fluid (ACSF) or colchicine bilaterally into the dorsal (D) or ventral (V) dentate gyrus. The vertical lines represent S.E.M. The mean of the V-colchicine group is significantly different from all other groups at P < 0.001 by Student's t-test with Bonferroni's correction for multiple comparisons.
seizures and WDS in rats 12. Among these are the facts that (1) CA3 pyramidal cells in the ventral hippocampus are more prone to potassium-induced bursting than those from the dorsal end 1'8 and (2) the ventral, but not the dorsal, hippocampus plays an important role in opioidinduced WDS 12. Moreover, these opioid-induced WDS can be attenuated by destruction of the ventral D G C 11. Of interest also are the reports that the ventral hippocampus contains a greater abundance of opioid peptides than the dorsal portion 1° and a higher density of mu opioid receptors 2'13. This may be particularly relevant to our findings since the region of the ventral hippocampus which contains this high density of mu opioid receptors is also the region where enkephalin-containing perforant path fibers from the entorhinal cortex terminate 4'7. Thus, release of enkephalins in the ventral hippocampal formation by perforant path stimulation may contribute to the importance of the ventral, relative to the dorsal, D G C in the generation of WDS. In support of this notion is the fact that injection of naltrexone, an opioid antagonist, into this region of the ventral hippocampus elevates the threshold for perforant path stimulationinduced WDS (Xie et al., unpublished observations). Other factors, both intrinsic and extrinsic to the hippocampal formation may also play an important role in the septotemporal difference observed in this study. Whatever the reason, our results clearly demonstrate the importance of the D G C in the ventral hippocampal formation for the generation of perforant path stimulation-induced WDS. We thank Mrs. Loretta Moore for secretarial assistance.
163
1 Bragdon, A.C., Taylor, D.M. and Wilson, W.A., Potassiuminduced epileptiform activity in area CA3 varies along the septotemporal axis of the rat hippocampus, Brain Research, 378 (1986) 169-173. 2 Crain, B.J., Chang, K.J. and McNamara, J.O., Quantitative autoradiographic analysis of mu and delta opioid binding sites in the rat hippocampal formation, J. Comp. Neurol., 246 (1986) 170-180. 3 Damiano, B.P. and Connor, J.D., Hippocampal mediation of shaking behavior induced by electrical stimulation of the perforant path in the rat, Brain Research, 309 (1984) 383-386. 4 Fredens, K., Stengaard-Peterson, K. and Larsson, L.I., Localization of enkephalin and cholecystokinin immunoreactivities in the perforant path terminal fields of the rat hippocampal formation, Brain Research, 304 (1984) 255-263. 5 Frotscher, M., Neuronal elements in the hippocampus and their synaptic connections, Adv. Anat. Embryol. Cell Biol., 111 (1988) 2-19. 6 Frush, D.P. and McNamara, J.D., Evidence implicating dentate granule cells in wet dog shakes produced by kindling stimulations of entorhinal cortex, Exp. Neurol., 92 (1986) 102-113, 7 Gall, C., Brecha, N., Karten, H.J. and Chang, K.J., Localization of enkephalin-like immunoreactivity to identified axonal and neuronal populations of the rat hippocampus, J. Comp. Neurol., 198 (1981) 335-350. 8 Gilbert, M., Racine, R.J. and Smith, G.K., Epileptiform burst responses in ventral vs. dorsal hippocampal slices, Brain Research, 361 (1985) 389-391. 9 Grimes, L., McGinty, J., McLain, E, Mitchell, C., Tilson, H. and Hong, J., Dentate granule cells are essential for kainic acid-induced wet dog shakes but not seizures or hippocampal cell
loss, J. Neurosci., 8 (1988) 256-264. 10 Kanamatsu, T., Obie, J., Grimes, L., McGinty, J., Yoshikawa, K., Sabol, S. and Hong, J.S., Kainic acid alters the metabolism of metS-enkephalin and the level of dynorphin A in the rat hippocampus, J. Neurosci., 6 (1986) 3094-3102. 11 Lee, P.H.K. and Hong, J.S., Ventral hippocampal dentate granule cell lesions enhance motor seizures but reduce wet dog shakes induced by mu opioid receptor agonist, Neuroscience, in press. 12 Lee, P.H.K., Obie, J. and Hong, J.S., Opioids induce convulsions and wet dog shakes in rats: mediation by hippocampal mu, but not delta or kappa opioid receptors, J. Neurosci., 9 (1989) 692-697. 13 Mansour, A., Khachaturian, H., Lewis, M.E,, Akil, H. and Watson, S.J., Autoradiographic differentiation of mu, delta and kappa opioid receptors in the rat forebrain and midbrain, J. Neurosci., 7 (1987) 2445-2464. 14 Martin, W.R., Wikler, A., Eades, C.G. and Pescor, ET., Tolerance to and physical dependence on morphine in rats, Psychopharmacologia, 4 (1963) 247-260. 15 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic Press, New York, 1986, Plates 33, 41 and 49. 16 Steward, O., Goidschmidt, R.B. and Sutula, T., IV. Neurotoxicity of colchicine and other tubulin-bindingagents: a selective vulnerability of certain neurons to the disruption of microtubules, Life Sci., 35 (1984) 43-51. 17 Walsh, T.J., Schulz, D.W., Tilson, H.A. and Schmechel, D.E., Colchicine-induced granule cell loss in rat hippocampus: selective behavioral and histological alterations, Brain Research, 398 (1986) 23-36.
159
Elsevier
BRES 24006
Colchicine lesions of ventral, but not dorsal, dentate granule cells attenuate wet dog shakes elicited by perforant path stimulation Martha I. Barnes and Clifford L. Mitchell Laboratory of Molecular and Integrative Neuroscience, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 (U.S.A.)
(Accepted 28 November 1989) Key words: Colchicine; Dentate granule cell; Ventral hippocampus; Dorsal hippocampus; Wet dog shake; Perforant path stimulation
Intrahippocampal injections of colchicine selectivelydestroy dentate granule cells. Wet dog shaking elicited by perforant path stimulation is unaffected by bilateral destruction of dorsal dentate granule cells but virtually eliminated by bilateral destruction of ventral dentate granule cells. This implies that ventral dentate granule cells are essential for the generation of perforant path stimulation-induced wet dog shakes.
The major input to the hippocampal formation (dentate gyrus and Ammon's horn) from the neocortex is through the entorhinal cortex via the perforant path 5. Electrical stimulation of the entorhinal c o r t e x 6 o r the perforant path 3, under proper conditions, produces a stereotypic behavior exemplified by paroxysmal shaking of the head, neck and trunk in rats. This behavior has been referred to as 'wet dog shakes' (WDS) because it resembles the behavior observed in a dog shaking itself while wet 14. Destruction of dentate granule cells (DGC) results in a dramatic suppression of WDS induced by electrical stimulation of the entorhinal cortex6 or by systemic injection of kainic acid 9. In these studies, DGC in both the dorsal and the ventral portions of the hippocampal formation were destroyed. Recently, it was reported that injection of mu opioid receptor agonists into the ventral, but not dorsal, hippocampus induced WDS 12. Moreover, destruction of DGC in the ventral hippocampal formation markedly reduced these WDS 11. We were interested, therefore, in determining the relative contribution of the DGC in the ventral vs. dorsal hippocampal formation in the WDS elicited by perforant path stimulation. Male, Fischer-344 rats weighing 250-300 g were obtained from Charles River Breeding Co. (Raleigh, NC). They were housed individually 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 NIH Diet 31 and water ad lib. For surgery, each animal received atropine sulfate, 2
mg/kg, s.c., followed approximately 10 min later with pentobarbital sodium, 50 mg/kg, i.p. Methoxyflurane was supplemented as needed. In each animal, a bipolar stimulating electrode aimed at the perforant path in the region of the angular bundle was chronically implanted under stereotaxic guidance. The electrodes were made from twisted 0.25 mm nichrome wire with a tip separation of approximately 0.5 mm. Stainless steel guide cannulae (22 gauge) were bilaterally implanted in order to allow injection cannulae (stainless steel, 28 gauge) to be aimed at the right and left dorsal hippocampus only (one-half of the animals) or the right and left ventral hippocampus only (the other half of the animals). The guide cannulae extended approximately 1 mm below the skull and were permanently attached to the skull. They were sealed with dummy injection cannulae until the time of colchicine administration. Coordinates were taken from the atlas of Paxinos and Watson 15 and were as follows: perforant path - - 7.8 mm posterior, 4.4 mm lateral and 4.0 mm deep; dorsal hippocampus - - 3.8 mm posterior, 1.7 mm lateral, and 3.2 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 flat. Two or three days after surgery, all animals were stimulated to determine the threshold for eliciting WDS. Stimulus parameters consisted of an 8-s train of biphasic
Correspondence: C.L. Mitchell, Lab. of Molecular and Integrative Neuroscience, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709, U.S.A.
160 paired pulses, duration of 0.1 ms/phase, and a train rate of 5 Hz with a 20-ms interval between the first and second pulse pair. The initial stimulus intensity was 0.1 mA. Every 2 min, the intensity was increased by 0.1 m A until W D S were observed. The threshold was defined as that current which elicited at least 2 WDS within 2 min following stimulation. Animals were matched according to thresholds and assigned to 1 of 4 groups: dorsal artificial cerebrospinal fluid (ACSF), dorsal colchicine, ventral ACSF, or ventral colchicine. Each animal was then injected bilaterally with 0.5 ~tl ACSF or 1 ~tg colchicine (Sigma, St. Louis, MO) in 0.5 ~1 A C S F per site over a 11/2 to 2 min time period. Two weeks later, all animals were tested for WDS using the parameters described above. The initial stimulus intensity was 0.2 m A below that previously determined to elicit WDS. As before, the intensity was increased by 0.1 m A every 2 min until WDS were observed or a current level of 1.0 m A was attained. The number of W D S occurring within 3 min post stimulation was recorded. This procedure was repeated 3 more times with a 5 min interval between replications. Subsequently,
the animals were anesthetized with pentobarbital sodium and perfused intracardially with 0.85% saline followed by 10% neutral buffered formalin (NBF) and a supersaturated solution of potassium ferrocyanide in 10% N B E The brains were removed from the brain case, blocked and mounted in paraffin. They were subsequently sliced at 10 /~m. One of every 10 slices was mounted, stained with Cresyl violet, and counterstained with luxol fast blue for analysis of lesion damage. Animals receiving dorsal injections of A C S F showed no visible cell damage in any hippocampal cell layer from the injection. Any cell damage was the result of the injection cannula and was restricted primarily to CA1, though in one animal the cannula went into the upper dentate granule cell layer on one side. There was minimal cell damage as a result of the ventral A C S F injection. When it occurred, it was limited to the immediate area surrounding the injection cannula in the rostral cell layers. There was no damage to dentate granule cell layers in the caudal region from either ACSF or the cannulae. In the dorsal dentate, colchicine damage ranged from moderate, limited primarily to the medial portion of the
Fig. 1. Low magnification photomicrographs of dorsal and ventral hippocampus two weeks after treatment. Dorsal (A) and ventral (B) hippocampal sections taken from a dorsal ACSF-treated animal vs dorsal (C) and ventral (D) sections from a dorsal colchicine-treated animal illustrate the dramatic loss of dorsal dentate granule cells (arrow) resulting from the application of colchicine. Note that ventral dentate granule cells remain intact.
161 upper blade, to more extensive damage which essentially destroyed all of the dentate granule cell layer of the upper blade and the medial portions of the lower blade. There was thinning of the CA1 layer in the immediate vicinity of the injection cannulae in some animals. The lesions never extended to the rostral portion of the ventral hippocampus (V-HPC). Ventral colchicine damage was centered mainly in the caudal dentate granule cells with damage extended to the rostral dentate in 4 of the 7 animals. The general impression was that the degree of D G C damage in the ventral HPC (V-HPC) was more variable than in the D-HPC. This may be due to the nature of the injection sites. In D-HPC, the injection site is relatively shallow, thus yielding a very localized injection site. In contrast, the V-HPC injection site is very deep. The cannulae frequently tended to pass through the edge of the lateral ventricle. Thus, in V-HPC colchicine may have diffused more than in D-HPC, resulting in more variable damage to the D G C of the V-HPC than the D-HPC. Damage to the other cell layers was due primarily to the cannula tract, although there was some thinning of caudal CA1 and medial CA3 in two animals. Figs. 1 and 2 illustrate typical histological effects seen in ACSF and colchicine treated animals. Animals
were discarded from further analysis if their lesions were unilateral or in another structure (e.g. thalamus or medial geniculate). Thus, upon completion of the histological analysis, a total of 35 animals remained for statistical analysis: 12 dorsal ACSF, 7 dorsal colchicine, 9 ventral ACSF, and 7 ventral colchicine. Data were analyzed for the initial threshold for WDS, for the 1st post-injection trial and for the total number of WDS by separate analyses of variance (ANOVA) for a 2 (substance injected) by 2 (injection site) factorial experiment. If the F ratio for substance by injection site interaction was significant then comparisons between groups were made by Student's t-test using Bonferroni's correction for multiple comparisons. In all cases, P ~< 0.05 was chosen as the level of significance. The mean values _+ S.E.M. for the thresholds for WDS obtained 2-3 days post surgery (pre-injection threshold) and the 1st threshold determination 2 weeks post ACSF or colchicine (first post injection) are shown in Table I. Since the animals were grouped according to their initial threshold, there i.s no statistically significant difference at this time period. The first post injection threshold for the ventral colchicine group is misleading since this mean was determined from truncated data. Four of the 7 animals
Fig. 2. Low magnification photomicrographs of dorsal and ventral hippocampus two weeks after treatment. Dorsal (A) and ventral (B) hippocampal sections taken from a ventral ACSF-treated animal vs dorsal (C) and ventral (D) sections from a ventral colchicine-treated animal illustrate the dramatic loss of ventral dentate granule cells (arrow) resulting from the application of colchicine. Note that the dorsal dentate granule cells remain intact.
162 TABLE I 80
Thresholds for wet dog shakes
Numbers represent mean + S.E.M. in mA; the number of animals per group is shown in parentheses. Threshold
Dorsal
]-
60
Ventral 40
Preinjection Postinjection
A CSF
Colchicine
A CSF
Colchicine
(12)
(7)
(9)
(7)
0.38+0.06 0.37+0.06 0.41+0.08 0.43+0.09 0.32+0.03 0.38+0.09 0.44+0.06 >1.00+0.00 *#
* Statistically different from all other post injection groups by Student's t-test at P < 0.001 (with Bonferroni correction for multiple comparisons). # Maximum value tested was 1.00 mA.
exhibited 0-1 WDS at the cutoff of 1 mA. Thus, the actual threshold, if one exists, is even higher than that shown in Table I. In fact, only 3 of 7 animals exhibited any WDS 2 days later when tested at much higher current strengths, for the appearance of overt seizures. These three animals had only 2 WDS at any time during seizure testing, whereas all the animals in the other 3 groups exhibited many WDS. The A N O V A for the postinjection threshold data showed significant F ratios for substance (F1.31 = 61.82, P < 0.001), for injection site (F1.31 = 87.25, P < 0.001) and for substance by injection site interaction (Fi,31 = 37.5, P < 0.001). With respect to the number of WDS, the A N O V A revealed significant F-ratios for substance (F1,31 --- 60.89, P < 0.001), for injection site (F1,31 = 28.38, P < 0.001) and for substance by injection site interaction (F1,31 = 49.02, P < 0.001). The means + S.E.M. are shown in Fig. 3. These data clearly show that the ability of colchicine to reduce WDS was restricted to the ventral injection site. The most plausible explanation of the effect of colchicine on WDS is destruction of DGC, since intrahippocampal injection of colchicine is known to preferentially destroy DGC, with little effect on other cell populations 16J7. Evidence implicating the hippocampal formation as a critical component of the network underlying WDS induced by electrical stimulation of the limbic system has been summarized by Frush and McNamara 6. Suffice it to say that, 'taken together, the data suggest that activation of D G C results in activation of the CA3 pyramidal cells, which in turn leads to activation of extrahippocampal structures '6. Our data specifically implicate the D G C in the ventral portion of the hippocampal formation. Lee et al. cite several lines of evidence indicating that the ventral hippocampal formation may be more important than its dorsal component for the expression of
20
D-ACSF
D- COLCHICINE V-ACSF
I I V-COLCHICINE
TREATMENT
Fig. 3. Mean number of wet dog shakes elicited by perforant path stimulation in animals receiving artificial cerebrospinal fluid (ACSF) or colchicine bilaterally into the dorsal (D) or ventral (V) dentate gyrus. The vertical lines represent S.E.M. The mean of the V-colchicine group is significantly different from all other groups at P < 0.001 by Student's t-test with Bonferroni's correction for multiple comparisons.
seizures and WDS in rats 12. Among these are the facts that (1) CA3 pyramidal cells in the ventral hippocampus are more prone to potassium-induced bursting than those from the dorsal end 1'8 and (2) the ventral, but not the dorsal, hippocampus plays an important role in opioidinduced WDS 12. Moreover, these opioid-induced WDS can be attenuated by destruction of the ventral D G C 11. Of interest also are the reports that the ventral hippocampus contains a greater abundance of opioid peptides than the dorsal portion 1° and a higher density of mu opioid receptors 2'13. This may be particularly relevant to our findings since the region of the ventral hippocampus which contains this high density of mu opioid receptors is also the region where enkephalin-containing perforant path fibers from the entorhinal cortex terminate 4'7. Thus, release of enkephalins in the ventral hippocampal formation by perforant path stimulation may contribute to the importance of the ventral, relative to the dorsal, D G C in the generation of WDS. In support of this notion is the fact that injection of naltrexone, an opioid antagonist, into this region of the ventral hippocampus elevates the threshold for perforant path stimulationinduced WDS (Xie et al., unpublished observations). Other factors, both intrinsic and extrinsic to the hippocampal formation may also play an important role in the septotemporal difference observed in this study. Whatever the reason, our results clearly demonstrate the importance of the D G C in the ventral hippocampal formation for the generation of perforant path stimulation-induced WDS. We thank Mrs. Loretta Moore for secretarial assistance.
163
1 Bragdon, A.C., Taylor, D.M. and Wilson, W.A., Potassiuminduced epileptiform activity in area CA3 varies along the septotemporal axis of the rat hippocampus, Brain Research, 378 (1986) 169-173. 2 Crain, B.J., Chang, K.J. and McNamara, J.O., Quantitative autoradiographic analysis of mu and delta opioid binding sites in the rat hippocampal formation, J. Comp. Neurol., 246 (1986) 170-180. 3 Damiano, B.P. and Connor, J.D., Hippocampal mediation of shaking behavior induced by electrical stimulation of the perforant path in the rat, Brain Research, 309 (1984) 383-386. 4 Fredens, K., Stengaard-Peterson, K. and Larsson, L.I., Localization of enkephalin and cholecystokinin immunoreactivities in the perforant path terminal fields of the rat hippocampal formation, Brain Research, 304 (1984) 255-263. 5 Frotscher, M., Neuronal elements in the hippocampus and their synaptic connections, Adv. Anat. Embryol. Cell Biol., 111 (1988) 2-19. 6 Frush, D.P. and McNamara, J.D., Evidence implicating dentate granule cells in wet dog shakes produced by kindling stimulations of entorhinal cortex, Exp. Neurol., 92 (1986) 102-113, 7 Gall, C., Brecha, N., Karten, H.J. and Chang, K.J., Localization of enkephalin-like immunoreactivity to identified axonal and neuronal populations of the rat hippocampus, J. Comp. Neurol., 198 (1981) 335-350. 8 Gilbert, M., Racine, R.J. and Smith, G.K., Epileptiform burst responses in ventral vs. dorsal hippocampal slices, Brain Research, 361 (1985) 389-391. 9 Grimes, L., McGinty, J., McLain, E, Mitchell, C., Tilson, H. and Hong, J., Dentate granule cells are essential for kainic acid-induced wet dog shakes but not seizures or hippocampal cell
loss, J. Neurosci., 8 (1988) 256-264. 10 Kanamatsu, T., Obie, J., Grimes, L., McGinty, J., Yoshikawa, K., Sabol, S. and Hong, J.S., Kainic acid alters the metabolism of metS-enkephalin and the level of dynorphin A in the rat hippocampus, J. Neurosci., 6 (1986) 3094-3102. 11 Lee, P.H.K. and Hong, J.S., Ventral hippocampal dentate granule cell lesions enhance motor seizures but reduce wet dog shakes induced by mu opioid receptor agonist, Neuroscience, in press. 12 Lee, P.H.K., Obie, J. and Hong, J.S., Opioids induce convulsions and wet dog shakes in rats: mediation by hippocampal mu, but not delta or kappa opioid receptors, J. Neurosci., 9 (1989) 692-697. 13 Mansour, A., Khachaturian, H., Lewis, M.E,, Akil, H. and Watson, S.J., Autoradiographic differentiation of mu, delta and kappa opioid receptors in the rat forebrain and midbrain, J. Neurosci., 7 (1987) 2445-2464. 14 Martin, W.R., Wikler, A., Eades, C.G. and Pescor, ET., Tolerance to and physical dependence on morphine in rats, Psychopharmacologia, 4 (1963) 247-260. 15 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic Press, New York, 1986, Plates 33, 41 and 49. 16 Steward, O., Goidschmidt, R.B. and Sutula, T., IV. Neurotoxicity of colchicine and other tubulin-bindingagents: a selective vulnerability of certain neurons to the disruption of microtubules, Life Sci., 35 (1984) 43-51. 17 Walsh, T.J., Schulz, D.W., Tilson, H.A. and Schmechel, D.E., Colchicine-induced granule cell loss in rat hippocampus: selective behavioral and histological alterations, Brain Research, 398 (1986) 23-36.