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Kainic acid-induced generalized seizures alter the regional hippocampal expression of the rat m1 and m3 muscarinic acetylcholine receptor genes
Epilepsy Research 29 (1997) 71 – 79
Kainic acid-induced generalized seizures alter the regional hippocampal expression of the rat m1 and m3 muscarinic acetylcholine receptor genes Nancy S. Mingo a,b,c, Georgia Cottrell a,b, Liang Zhang a,b,e, M. Christopher Wallace a,d, W. McIntyre Burnham a,b,c, James H. Eubanks a,b,d,* a Playfair Neuroscience Unit, 399 Bathurst Street, Toronto, Ontario M5T 2S8, Canada Bloor6iew Epilepsy Program, Uni6ersity of Toronto, Toronto, Ontario M5S 1A8, Canada c Department of Pharmacology, Uni6ersity of Toronto, Toronto, Ontario M5S 1A8, Canada d Department of Surgery (Neurosurgery), Uni6ersity of Toronto, Toronto, Ontario M5S 1A8, Canada e Department of Medicine (Neurology), Uni6ersity of Toronto, Toronto, Ontario M5S 1A8, Canada b
Received 30 May 1997; received in revised form 7 July 1997; accepted 4 August 1997
Abstract We investigated the gene expression responses using in situ hybridization with radiolabelled riboprobes for the m1 and m3 subtypes of muscarinic cholinergic receptors in the rat hippocampus following a brief (5-min) kainic acid-induced behavioral seizure. The kainic acid was intraperitoneally administered, and the ensuing generalized convulsive seizure terminated with diazepam. Our results demonstrate that the expression of the m1 subtype was significantly reduced in the CA1, CA3 and the dentate granule cells by 3 h after the administration of kainic acid, while no significant change was observed in any hippocampal subfield for the m3 subtype. By 6 h post challenge, the m1 subtype was still decreased in all hippocampal subfields examined, while the m3 subtype remained unchanged from vehicle injected control. At 24 h post challenge, both the m1 and m3 subtypes were significantly reduced in the CA1 and CA3 subfields; the expression of the m1 subtype in the dentate granule cells, however, had recovered to levels indistinguishable from vehicle-injected control. These results demonstrate that epileptiform activity induced by kainic acid administration promotes alterations in the expression levels for both the m1 and m3 muscarinic receptor genes, and suggest that the activity of this neuromodulatory system in the hippocampus may be altered through activity-dependent mechanisms at early times following seizures. © 1997 Elsevier Science B.V. Keywords: Muscarinic receptor; Kainic acid; In situ hybridization; Gene expression; Hippocampus; Epilepsy
* Corresponding author. Tel.: +1 416 6035800, ext. 2469; fax: +1 416 6035745; e-mail: [email protected] 0920-1211/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 0 9 2 0 - 1 2 1 1 ( 9 7 ) 0 0 0 6 7 - 3
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1. Introduction Muscarinic cholinergic receptors comprise one of the principal neuromodulatory systems in the brain. The hippocampus is highly enriched in cholinergic terminals, many of which originate from medial septal pathways [1]. These receptors exist both pre- and post-synaptically, and the post-synaptic muscarinic receptors are known to modulate several aspects of hippocampal neuron function [2]. These actions include the potentiation of N-methyl-D-apartate (NMDA) receptors, the inhibition of some types of potassium currents, and the augmentation of protein synthesis rates in hippocampal CA1 subfield dendrites ([3], for review [4,2]). The family of muscarinic receptors currently consists of five members, known molecularly as m1– m5, and each molecular subtype exhibits different agonist and antagonist binding properties. Ligand binding studies conducted on brain homogenates, as well as from reconstituted systems, have pharmacologically categorized populations of muscarinic receptors into M1–M4 subtypes [5 – 7]. The pharmacological M1 subtype has been primarily assigned to muscarinic receptors that display a ‘high’ affinity to the ligand pirenzipine, however, this pharmacological class may not be unique to a single molecular subtype [6]. Recent immunocytochemical and gene expression studies have demonstrated that two of the principal muscarinic receptors in the hippocampus are the m1 and the m3 subtypes [7 – 9]. Previous studies have investigated the pharmacological profile of brain muscarinic receptor populations after challenge in several seizure models. A change in pharmacological M1 class affinity for pirenzepine has been reported in adult rats that had been administered kainic acid at a prepubescent stage [10]. Additionally, the pharmacological M1 class was found to be altered in the brain after seizures in models of electrical kindling and electroconvulsive shock [11,12]. Because the pharmacological M1 class may contain more than one molecular subtype in brain membranes [6], the molecular mechanism for these alterations has not been resolved. In the present study, we employed the kainic acid seizure model to examine
the gene expression responses of the m1 and m3 muscarinic receptor subtypes in the hippocampus following a brief episode of generalized convulsive seizure activity. We report here that both the m1 and m3 subtypes demonstrate significant changes in expression by 24 h after the seizure, and that the kinetics of the m1 subtype response differs from that of the m3 subtype in different subfields of the hippocampus following challenge.
2. Materials and methods
2.1. Animal model Male Wistar rats, weighing between 200 and 300 g (Charles River, Canada), were given free access to food and water prior to treatment. Subjects were housed singly and kept on a 12-h light–dark cycle. Kainic acid administration was initiated 7 days (minimum) after arrival from the breeding farm. Experimental subjects were given 12 mg/kg kainic acid (Sigma) by i.p. injection. Kainic acid was dissolved in normal saline to a concentration of 6 mg/ml, with an injection volume of 2 ml/kg body weight. Subjects were observed for up to 2 h after administration of the drug for convulsive activity. When an animal exhibited tonic-clonic seizure activity with a loss of postural control for 5 min, the convulsive seizure was terminated by administration of diazepam (Valium, Roche), 5 mg/kg i.p. Control animals were yoked to the experimental animals in pairs, and received a saline and diazepam injection at times matched to those of the appropriate experimental animal. At 3, 6, or 24 h after the injection of kainic acid, the subjects were given a deep anesthesia with sodium pentobarbital (MTC Pharmaceuticals), and perfused for in situ hybridization (see below). At each time point, a minimum of three paired sections from at least three experimental and control animals were studied.
2.2. Histology For histological evaluation, two pairs of kainic-
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acid and vehicle treated animals were sacrificed at 24 h. They were perfused with 4% formalin, and the brains removed for paraffin embedding. Sections of 7 mm were obtained by cryostat sectioning from each brain, and stained with hematoxylin and eosin as described by Clark et al. [13]. The hippocampal region of each section was evaluated by light microscopy in a blinded manner, and the percentage of pyknotic neurons determined. These values were statistically compared using the paired Student’s t-test, and no significant difference in neuronal viability was observed between the kainic acid administered and sham-injected subjects.
2.3. Isolation of fragments and probe synthesis The m1 fragment corresponding to the predicted third intracellular domain used for this study was obtained as a gift from Dr E. Hess (Pennsylvania State University, Hershey, PA), which we have employed in previous investigations [14]. A region of the predicted third intracellular domain of the m3 muscarinic receptor subtype was isolated using the polymerase chain reaction, and subcloned into the pCR-II vector (In Vitrogen, San Diego, CA). A similar fragment has been used in previous investigations of the m3 subtype by in situ hybridization and Northern blot analysis [5,15,9]. The following primers were used in the reactions: m3-sense 5%-tac caa aga gct ggc tgg cct-3% m3-antisense 5%-gca aac ctc tta gcc agc gtg-3% The identity of subclones containing the m3 fragment was confirmed by dideoxy sequencing (Amersham). Antisense riboprobes were generated from gel purified, polylinker linearized, subclones to avoid vector sequence inclusion in the probe. Probes were purified over a DEPC treated G-50 column, and used at a final concentration of 5×106 to 1 × 107 cpm/ml of hybridization solution as described by Simmons et al. [16]. This concentration was found to be in excess of target sequences, and did not produce high non-specific background levels.
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2.4. In situ hybridization
Three different times of recovery following challenge were examined: 3, 6, and 24 h. At each time, a minimum of three experimental and sham-control animals were examined. Animals were anesthetized and sacrificed by transcardiac perfusion with a 4% paraformaldehyde solution (JBS Scientific) as described by Simmons et al. [16]. Following perfusion, the brains were removed and post fixed at 4°C overnight in a 4% paraformaldehyde solution supplemented with 15% sucrose. Brain sections of 20 mm were obtained by cryostat sectioning, and mounted onto subbed, poly-L lysine (MW \ 150 000, Sigma) coated slides. Each slide contained a section from a sham-control and an experimental animal, which were matched for planes of the hippocampus. Sections were then prehybridized, hybridized, and posthybridized as described by Simmons et al. [16]. Control hybridizations consisted of ‘sense’ probes hybridized at the same specific activities, and ‘antisense’ probes hybridized to RNAse-pretreated sections. None of these control conditions generated specific hybridization.
2.5. Data analysis
Following posthybridization, slides were exposed to XAR-5 film for 3–10 days. Films were developed, and analyzed using the quantitative densitometry system (MCID-3.0, St. Catherines, Ontario). Pixel densities were recorded for each region of the hippocampus, and the film background was subtracted. The resulting values did not exceed the linear values of the film, as determined by included 14C standard strips. Statistical evaluation was done using a paired two-tailed t-test of the pooled differences between experimental density and control autoradiographic density. Alterations in expression were considered significant if the observed P value was less than 0.025.
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3. Results
3.1. m1 muscarinic receptor expression following challenge The expression levels of the m1 gene changed rapidly after challenge (Figs. 1 and 2). At 3 h post challenge, mRNA levels were found to be significantly decreased in all hippocampal subfields examined (P B 0.025). Expression of the m1 gene was decreased to the greatest extent in the dentate granule cells, to levels of 22% of control (Table 1). Expression in the CA1 and CA3 subfields was also decreased, although to a lesser extent, at 3 h post seizure, to 80 and 84% of control, respectively. At 6 h, the significant decrease in m1 mRNA level throughout the hippocampus was maintained, with the expression level in the dentate granule cells being 28% that of control. Expression of m1 was further diminished at 6 h than at 3 h in the CA1 and CA3 subfields, to 51 and 59%, respectively (Table 1). By 24 h, however, m1 gene expression in the dentate granule cells had recovered to levels indistinguishable from control, although
the m1 expression levels in both the CA1 and CA3 subfields remained significantly diminished, at 44 and 38% of control, respectively.
3.2. m3 Muscarinic receptor expression following challenge The expression levels of the m3 gene changed with slower kinetics than the m1 subtype after challenge. At 3 and at 6 h after challenge, m3 mRNA levels in the experimental subjects were indistinguishable from control values in all regions evaluated (Figs. 3 and 4). At 24 h, however, m3 gene expression in the CA1 and CA3 subfields was significantly decreased to 64 and 43% of control values, respectively (Figs. 3 and 4, Table 1). Considerable variation in the expression levels of the m3 gene were observed in the dentate granule cells at all times examined, yielding large standard errors. This may be attributed to the normally low levels of m3 gene expression in the control dentate granule cells, such that small fluctuations in the overall m3 mRNA levels would produce dramatic differences in autoradiographic density. Thus, the dentate granule cells were not included in the m3 subtype evaluation.
3.3. E6aluation of CA3 neuronal integrity at 24 h post seizure
Fig. 1. Dynamics of m1 gene expression following kainic acid-seizure in rat brain. Antisense in situ hybridization was conducted as described in Section 2. For each panel, the top brain section represents the time matched, sham control, while the bottom section originates from a kainic acid-challenged test animal.
Severe seizures resulting from kainic acid administration have been demonstrated to promote neuronal death, with the CA3 subfield exhibiting particular sensitivity to the challenge [17–19]. We examined the CA3 subfield of rats that had experienced 5 min of seizure to determine if neuronal dropout was present at our most delayed evaluation time point of 24 h post challenge. As may be seen in Fig. 5, the CA3 subfield in animals subjected to 5 min of seizure does not exhibit an elevated number of pyknotic neurons at 24 h compared to control. This result indicates that the changes in the expression levels of the m1 and m3 gene that occur in the CA3 subfield at 24 h post challenge are not due to a diminished number of neurons.
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Fig. 2. Histogram illustrating the mean and standard error of the densitometric analysis of the antisense in situ hybridization for the m1 muscarinic receptor. A minimum of four animals for each time point was included. Control levels obtained from sham-control sections are represented as a value of 1.0, and changes in expression from control are presented. Statistical significance is denoted by an asterisk, at levels PB 0.01 (Student’s paired two-tailed t-test). Times post seizure are shown below the histograms.
4. Discussion The present study was designed to examine changes in gene expression of muscarinic cholinergic receptors in the hippocampus resulting from a brief episode kainic acid-induced seizure activity. Previous pharmacological binding studies have demonstrated that seizure-induced alterations in the pharmacological M1 receptor class occurs in
several models [20,11,10,12], but these investigations did not distinguish which of the molecularly defined subtypes were altered by the seizure. The present study, which employed antisense in situ
Table 1 Percentage change in expression levels for the m1 and m3 muscarinic receptor subtypes following kainic acid-induced generalized seizures m1 expression
3h 6h 24 h
m3 expression
CA1
CA3
DG
CA1
CA3
−19.9* −48.7* −56.4*
−16.4* −40.4* −62.0*
−77.2* −72.1* +9.9
−21.0 +3.8 −36.0*
−3.4 −3.3 −56.7*
Negative values represent decreases in mRNA expression, while positive numbers represent increases in mRNA expression with respect to control levels. * changes in expression that were found to be statistically significant (paired t-test, PB0.025).
Fig. 3. Representative example of m3 gene expression following kainic acid-seizure in rat brain. For each panel, the top brain section represents the time matched, sham-control, while the bottom section originates from an kainic acid-challenged test animal.
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Fig. 4. Histogram illustrating the mean and standard error of the densitometric analysis of the antisense in situ hybridization for the m3 muscarinic receptor. Due to the low m3 expression signal observed in the dentate granule cells, only the CA1 and CA3 subfields were evaluated for this gene. Statistical significance is denoted by an asterisk, at levels P B 0.025 (Student’s paired two-tailed t-test). Times post seizure are shown below the histograms.
hybridization, concentrated on defining the expression responses of the molecular m1 and m3 subtypes of muscarinic receptors in the hippocampus after seizure. Two principal findings emerge from this work. (i) Both the m1 and m3 muscarinic receptor subtypes exhibit time dependent decreases in gene expression in the hippocampus following kainic acid-induced seizures; and (ii) the kinetics of the change in expression for the m1 subtype differs from that of the m3 subtype. We observed significant differences in the levels of expression as a function of time following challenge within the different subfields of the hippocampus for the m1 gene. At both 3 and 6 h following seizure, the expression levels for the m1 gene were significantly reduced from control in every region of the hippocampus examined. This is in contrast to the expression of the m3 gene, where no significant change in expression from control was observed at either 3 or 6 h. The difference in response between the m1 and m3 genes at these early times following challenge likely reflect the complex regulation of gene expression for these two muscarinic receptor subtypes in the brain, and how each may be
influenced differently by seizure activity. Such diverse expression responses have been reported for other genes examined in this seizure model, such as members of the GABA receptor family [19], the glutamate receptor family [19], G-proteins [21] synaptotagmins [22,23], neuropeptides and neurotrophins [24–28] and several immediate early genes [29]. From these previous investigations, it is clear that the response of any individual gene is determined by its own pattern of regulation, and that different genes respond to the kainic acid-induced seizure activity in different manners. The reductions in muscarinic mRNA expression presented in this study do not result from the loss of muscarinic receptor expressing neurons. While prolonged kainic acid-induced seizures are known to promote cell loss in widespread areas of the limbic system [17,19,30], histological examination of the CA3 subfield in a subgroup of our own subjects showed no significant cell loss at 24 h post seizure. We purposefully terminated the generalized seizure after 5 min by diazepam injection, and examined test subjects at or before 24 h to minimize the degree of neuronal dropout that would be associated with the challenge. A recent
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report evaluated the degree of cell loss in mice subjected to the same duration of kainic acidinduced seizures that we employed at 3 days post challenge, and found that roughly 20% of the hippocampal CA3 neurons had expired [18]. Whether our subjects would display similar a degree of neuronal dropout at 3 days has not been examined. However, because no significant neuronal loss was observed at our most delayed examination time of 24 h, the declines in m1 and m3 expression reported represent genuine changes in gene expression. These changes, moreover, appear to relate to the neuronal ac-
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tivity associated with the behavoral seizure, as subjects administered kainic acid that did not exhibit generalized seizures showed no changes from control in either m1 or m3 expression levels (Eubanks, unpublished). The findings presented in this study are consistent with previous observations reported from muscarinic receptor pharmacological binding studies in several models of seizures. GuzmanGodinez and Schliebs [10] reported that if prepubescent rats are subjected to a similar duration of kainic acid-induced seizure, an alteration in the pharmacological M1 receptor profile is observed in the hippocampus of the treated rats when they reach adulthood. In an adult model, Churchill et al. [20] reported that at 3 days after kainic acid-induced seizures, a decrease in the prevalence of the pharmacological M1 receptor exists in the CA4 subfield of the hippocampus. Our findings that decreased mRNA levels for the m1 subtype at and before 24 h post challenge—prior to any discernible neuronal loss in the vulnerable CA3 subfield— would be consistent with a loss of muscarinic M1 receptor binding at these early times following challenge, should the change in expression lead to similar changes in the amount of translated protein. Furthermore, the pharmacological M1 receptor has been shown to be decreased in the hippocampus following amygdala kindling [12], entorhinal cortex kindling [11], and electroshock seizures [11]. These studies suggest that a reduction in muscarinic cholinergic receptor prevalence results from many types of seizures. Studies of the molecular subtypes involved in the changes seen in these other models are currently underway.
Acknowledgements
Fig. 5. Histological assessment of the dorsal CA3 subfield viability at 24 h post seizure. Top field originates from a control subject, and the bottom field is taken from an animal 24 h post seizure. No significant changes in cell viability were observed (blinded examination, Student’s two-paired t-test).
We thank Lucy Teves, Antonio Mendonca and Jerome Cheng for technical assistance. This work was supported by grants from the Bloorview Children’s Hospital Foundation and The Heart and Stroke Foundation of Canada,
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and the Medical Research Council of Canada. NM is a Fellow of the Bloorview Epilepsy Program, and LZ is a Heart and Stroke Foundation of Canada Scholar.
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Kainic acid-induced generalized seizures alter the regional hippocampal expression of the rat m1 and m3 muscarinic acetylcholine receptor genes Nancy S. Mingo a,b,c, Georgia Cottrell a,b, Liang Zhang a,b,e, M. Christopher Wallace a,d, W. McIntyre Burnham a,b,c, James H. Eubanks a,b,d,* a Playfair Neuroscience Unit, 399 Bathurst Street, Toronto, Ontario M5T 2S8, Canada Bloor6iew Epilepsy Program, Uni6ersity of Toronto, Toronto, Ontario M5S 1A8, Canada c Department of Pharmacology, Uni6ersity of Toronto, Toronto, Ontario M5S 1A8, Canada d Department of Surgery (Neurosurgery), Uni6ersity of Toronto, Toronto, Ontario M5S 1A8, Canada e Department of Medicine (Neurology), Uni6ersity of Toronto, Toronto, Ontario M5S 1A8, Canada b
Received 30 May 1997; received in revised form 7 July 1997; accepted 4 August 1997
Abstract We investigated the gene expression responses using in situ hybridization with radiolabelled riboprobes for the m1 and m3 subtypes of muscarinic cholinergic receptors in the rat hippocampus following a brief (5-min) kainic acid-induced behavioral seizure. The kainic acid was intraperitoneally administered, and the ensuing generalized convulsive seizure terminated with diazepam. Our results demonstrate that the expression of the m1 subtype was significantly reduced in the CA1, CA3 and the dentate granule cells by 3 h after the administration of kainic acid, while no significant change was observed in any hippocampal subfield for the m3 subtype. By 6 h post challenge, the m1 subtype was still decreased in all hippocampal subfields examined, while the m3 subtype remained unchanged from vehicle injected control. At 24 h post challenge, both the m1 and m3 subtypes were significantly reduced in the CA1 and CA3 subfields; the expression of the m1 subtype in the dentate granule cells, however, had recovered to levels indistinguishable from vehicle-injected control. These results demonstrate that epileptiform activity induced by kainic acid administration promotes alterations in the expression levels for both the m1 and m3 muscarinic receptor genes, and suggest that the activity of this neuromodulatory system in the hippocampus may be altered through activity-dependent mechanisms at early times following seizures. © 1997 Elsevier Science B.V. Keywords: Muscarinic receptor; Kainic acid; In situ hybridization; Gene expression; Hippocampus; Epilepsy
* Corresponding author. Tel.: +1 416 6035800, ext. 2469; fax: +1 416 6035745; e-mail: [email protected] 0920-1211/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 0 9 2 0 - 1 2 1 1 ( 9 7 ) 0 0 0 6 7 - 3
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1. Introduction Muscarinic cholinergic receptors comprise one of the principal neuromodulatory systems in the brain. The hippocampus is highly enriched in cholinergic terminals, many of which originate from medial septal pathways [1]. These receptors exist both pre- and post-synaptically, and the post-synaptic muscarinic receptors are known to modulate several aspects of hippocampal neuron function [2]. These actions include the potentiation of N-methyl-D-apartate (NMDA) receptors, the inhibition of some types of potassium currents, and the augmentation of protein synthesis rates in hippocampal CA1 subfield dendrites ([3], for review [4,2]). The family of muscarinic receptors currently consists of five members, known molecularly as m1– m5, and each molecular subtype exhibits different agonist and antagonist binding properties. Ligand binding studies conducted on brain homogenates, as well as from reconstituted systems, have pharmacologically categorized populations of muscarinic receptors into M1–M4 subtypes [5 – 7]. The pharmacological M1 subtype has been primarily assigned to muscarinic receptors that display a ‘high’ affinity to the ligand pirenzipine, however, this pharmacological class may not be unique to a single molecular subtype [6]. Recent immunocytochemical and gene expression studies have demonstrated that two of the principal muscarinic receptors in the hippocampus are the m1 and the m3 subtypes [7 – 9]. Previous studies have investigated the pharmacological profile of brain muscarinic receptor populations after challenge in several seizure models. A change in pharmacological M1 class affinity for pirenzepine has been reported in adult rats that had been administered kainic acid at a prepubescent stage [10]. Additionally, the pharmacological M1 class was found to be altered in the brain after seizures in models of electrical kindling and electroconvulsive shock [11,12]. Because the pharmacological M1 class may contain more than one molecular subtype in brain membranes [6], the molecular mechanism for these alterations has not been resolved. In the present study, we employed the kainic acid seizure model to examine
the gene expression responses of the m1 and m3 muscarinic receptor subtypes in the hippocampus following a brief episode of generalized convulsive seizure activity. We report here that both the m1 and m3 subtypes demonstrate significant changes in expression by 24 h after the seizure, and that the kinetics of the m1 subtype response differs from that of the m3 subtype in different subfields of the hippocampus following challenge.
2. Materials and methods
2.1. Animal model Male Wistar rats, weighing between 200 and 300 g (Charles River, Canada), were given free access to food and water prior to treatment. Subjects were housed singly and kept on a 12-h light–dark cycle. Kainic acid administration was initiated 7 days (minimum) after arrival from the breeding farm. Experimental subjects were given 12 mg/kg kainic acid (Sigma) by i.p. injection. Kainic acid was dissolved in normal saline to a concentration of 6 mg/ml, with an injection volume of 2 ml/kg body weight. Subjects were observed for up to 2 h after administration of the drug for convulsive activity. When an animal exhibited tonic-clonic seizure activity with a loss of postural control for 5 min, the convulsive seizure was terminated by administration of diazepam (Valium, Roche), 5 mg/kg i.p. Control animals were yoked to the experimental animals in pairs, and received a saline and diazepam injection at times matched to those of the appropriate experimental animal. At 3, 6, or 24 h after the injection of kainic acid, the subjects were given a deep anesthesia with sodium pentobarbital (MTC Pharmaceuticals), and perfused for in situ hybridization (see below). At each time point, a minimum of three paired sections from at least three experimental and control animals were studied.
2.2. Histology For histological evaluation, two pairs of kainic-
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acid and vehicle treated animals were sacrificed at 24 h. They were perfused with 4% formalin, and the brains removed for paraffin embedding. Sections of 7 mm were obtained by cryostat sectioning from each brain, and stained with hematoxylin and eosin as described by Clark et al. [13]. The hippocampal region of each section was evaluated by light microscopy in a blinded manner, and the percentage of pyknotic neurons determined. These values were statistically compared using the paired Student’s t-test, and no significant difference in neuronal viability was observed between the kainic acid administered and sham-injected subjects.
2.3. Isolation of fragments and probe synthesis The m1 fragment corresponding to the predicted third intracellular domain used for this study was obtained as a gift from Dr E. Hess (Pennsylvania State University, Hershey, PA), which we have employed in previous investigations [14]. A region of the predicted third intracellular domain of the m3 muscarinic receptor subtype was isolated using the polymerase chain reaction, and subcloned into the pCR-II vector (In Vitrogen, San Diego, CA). A similar fragment has been used in previous investigations of the m3 subtype by in situ hybridization and Northern blot analysis [5,15,9]. The following primers were used in the reactions: m3-sense 5%-tac caa aga gct ggc tgg cct-3% m3-antisense 5%-gca aac ctc tta gcc agc gtg-3% The identity of subclones containing the m3 fragment was confirmed by dideoxy sequencing (Amersham). Antisense riboprobes were generated from gel purified, polylinker linearized, subclones to avoid vector sequence inclusion in the probe. Probes were purified over a DEPC treated G-50 column, and used at a final concentration of 5×106 to 1 × 107 cpm/ml of hybridization solution as described by Simmons et al. [16]. This concentration was found to be in excess of target sequences, and did not produce high non-specific background levels.
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2.4. In situ hybridization
Three different times of recovery following challenge were examined: 3, 6, and 24 h. At each time, a minimum of three experimental and sham-control animals were examined. Animals were anesthetized and sacrificed by transcardiac perfusion with a 4% paraformaldehyde solution (JBS Scientific) as described by Simmons et al. [16]. Following perfusion, the brains were removed and post fixed at 4°C overnight in a 4% paraformaldehyde solution supplemented with 15% sucrose. Brain sections of 20 mm were obtained by cryostat sectioning, and mounted onto subbed, poly-L lysine (MW \ 150 000, Sigma) coated slides. Each slide contained a section from a sham-control and an experimental animal, which were matched for planes of the hippocampus. Sections were then prehybridized, hybridized, and posthybridized as described by Simmons et al. [16]. Control hybridizations consisted of ‘sense’ probes hybridized at the same specific activities, and ‘antisense’ probes hybridized to RNAse-pretreated sections. None of these control conditions generated specific hybridization.
2.5. Data analysis
Following posthybridization, slides were exposed to XAR-5 film for 3–10 days. Films were developed, and analyzed using the quantitative densitometry system (MCID-3.0, St. Catherines, Ontario). Pixel densities were recorded for each region of the hippocampus, and the film background was subtracted. The resulting values did not exceed the linear values of the film, as determined by included 14C standard strips. Statistical evaluation was done using a paired two-tailed t-test of the pooled differences between experimental density and control autoradiographic density. Alterations in expression were considered significant if the observed P value was less than 0.025.
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3. Results
3.1. m1 muscarinic receptor expression following challenge The expression levels of the m1 gene changed rapidly after challenge (Figs. 1 and 2). At 3 h post challenge, mRNA levels were found to be significantly decreased in all hippocampal subfields examined (P B 0.025). Expression of the m1 gene was decreased to the greatest extent in the dentate granule cells, to levels of 22% of control (Table 1). Expression in the CA1 and CA3 subfields was also decreased, although to a lesser extent, at 3 h post seizure, to 80 and 84% of control, respectively. At 6 h, the significant decrease in m1 mRNA level throughout the hippocampus was maintained, with the expression level in the dentate granule cells being 28% that of control. Expression of m1 was further diminished at 6 h than at 3 h in the CA1 and CA3 subfields, to 51 and 59%, respectively (Table 1). By 24 h, however, m1 gene expression in the dentate granule cells had recovered to levels indistinguishable from control, although
the m1 expression levels in both the CA1 and CA3 subfields remained significantly diminished, at 44 and 38% of control, respectively.
3.2. m3 Muscarinic receptor expression following challenge The expression levels of the m3 gene changed with slower kinetics than the m1 subtype after challenge. At 3 and at 6 h after challenge, m3 mRNA levels in the experimental subjects were indistinguishable from control values in all regions evaluated (Figs. 3 and 4). At 24 h, however, m3 gene expression in the CA1 and CA3 subfields was significantly decreased to 64 and 43% of control values, respectively (Figs. 3 and 4, Table 1). Considerable variation in the expression levels of the m3 gene were observed in the dentate granule cells at all times examined, yielding large standard errors. This may be attributed to the normally low levels of m3 gene expression in the control dentate granule cells, such that small fluctuations in the overall m3 mRNA levels would produce dramatic differences in autoradiographic density. Thus, the dentate granule cells were not included in the m3 subtype evaluation.
3.3. E6aluation of CA3 neuronal integrity at 24 h post seizure
Fig. 1. Dynamics of m1 gene expression following kainic acid-seizure in rat brain. Antisense in situ hybridization was conducted as described in Section 2. For each panel, the top brain section represents the time matched, sham control, while the bottom section originates from a kainic acid-challenged test animal.
Severe seizures resulting from kainic acid administration have been demonstrated to promote neuronal death, with the CA3 subfield exhibiting particular sensitivity to the challenge [17–19]. We examined the CA3 subfield of rats that had experienced 5 min of seizure to determine if neuronal dropout was present at our most delayed evaluation time point of 24 h post challenge. As may be seen in Fig. 5, the CA3 subfield in animals subjected to 5 min of seizure does not exhibit an elevated number of pyknotic neurons at 24 h compared to control. This result indicates that the changes in the expression levels of the m1 and m3 gene that occur in the CA3 subfield at 24 h post challenge are not due to a diminished number of neurons.
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Fig. 2. Histogram illustrating the mean and standard error of the densitometric analysis of the antisense in situ hybridization for the m1 muscarinic receptor. A minimum of four animals for each time point was included. Control levels obtained from sham-control sections are represented as a value of 1.0, and changes in expression from control are presented. Statistical significance is denoted by an asterisk, at levels PB 0.01 (Student’s paired two-tailed t-test). Times post seizure are shown below the histograms.
4. Discussion The present study was designed to examine changes in gene expression of muscarinic cholinergic receptors in the hippocampus resulting from a brief episode kainic acid-induced seizure activity. Previous pharmacological binding studies have demonstrated that seizure-induced alterations in the pharmacological M1 receptor class occurs in
several models [20,11,10,12], but these investigations did not distinguish which of the molecularly defined subtypes were altered by the seizure. The present study, which employed antisense in situ
Table 1 Percentage change in expression levels for the m1 and m3 muscarinic receptor subtypes following kainic acid-induced generalized seizures m1 expression
3h 6h 24 h
m3 expression
CA1
CA3
DG
CA1
CA3
−19.9* −48.7* −56.4*
−16.4* −40.4* −62.0*
−77.2* −72.1* +9.9
−21.0 +3.8 −36.0*
−3.4 −3.3 −56.7*
Negative values represent decreases in mRNA expression, while positive numbers represent increases in mRNA expression with respect to control levels. * changes in expression that were found to be statistically significant (paired t-test, PB0.025).
Fig. 3. Representative example of m3 gene expression following kainic acid-seizure in rat brain. For each panel, the top brain section represents the time matched, sham-control, while the bottom section originates from an kainic acid-challenged test animal.
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Fig. 4. Histogram illustrating the mean and standard error of the densitometric analysis of the antisense in situ hybridization for the m3 muscarinic receptor. Due to the low m3 expression signal observed in the dentate granule cells, only the CA1 and CA3 subfields were evaluated for this gene. Statistical significance is denoted by an asterisk, at levels P B 0.025 (Student’s paired two-tailed t-test). Times post seizure are shown below the histograms.
hybridization, concentrated on defining the expression responses of the molecular m1 and m3 subtypes of muscarinic receptors in the hippocampus after seizure. Two principal findings emerge from this work. (i) Both the m1 and m3 muscarinic receptor subtypes exhibit time dependent decreases in gene expression in the hippocampus following kainic acid-induced seizures; and (ii) the kinetics of the change in expression for the m1 subtype differs from that of the m3 subtype. We observed significant differences in the levels of expression as a function of time following challenge within the different subfields of the hippocampus for the m1 gene. At both 3 and 6 h following seizure, the expression levels for the m1 gene were significantly reduced from control in every region of the hippocampus examined. This is in contrast to the expression of the m3 gene, where no significant change in expression from control was observed at either 3 or 6 h. The difference in response between the m1 and m3 genes at these early times following challenge likely reflect the complex regulation of gene expression for these two muscarinic receptor subtypes in the brain, and how each may be
influenced differently by seizure activity. Such diverse expression responses have been reported for other genes examined in this seizure model, such as members of the GABA receptor family [19], the glutamate receptor family [19], G-proteins [21] synaptotagmins [22,23], neuropeptides and neurotrophins [24–28] and several immediate early genes [29]. From these previous investigations, it is clear that the response of any individual gene is determined by its own pattern of regulation, and that different genes respond to the kainic acid-induced seizure activity in different manners. The reductions in muscarinic mRNA expression presented in this study do not result from the loss of muscarinic receptor expressing neurons. While prolonged kainic acid-induced seizures are known to promote cell loss in widespread areas of the limbic system [17,19,30], histological examination of the CA3 subfield in a subgroup of our own subjects showed no significant cell loss at 24 h post seizure. We purposefully terminated the generalized seizure after 5 min by diazepam injection, and examined test subjects at or before 24 h to minimize the degree of neuronal dropout that would be associated with the challenge. A recent
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report evaluated the degree of cell loss in mice subjected to the same duration of kainic acidinduced seizures that we employed at 3 days post challenge, and found that roughly 20% of the hippocampal CA3 neurons had expired [18]. Whether our subjects would display similar a degree of neuronal dropout at 3 days has not been examined. However, because no significant neuronal loss was observed at our most delayed examination time of 24 h, the declines in m1 and m3 expression reported represent genuine changes in gene expression. These changes, moreover, appear to relate to the neuronal ac-
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tivity associated with the behavoral seizure, as subjects administered kainic acid that did not exhibit generalized seizures showed no changes from control in either m1 or m3 expression levels (Eubanks, unpublished). The findings presented in this study are consistent with previous observations reported from muscarinic receptor pharmacological binding studies in several models of seizures. GuzmanGodinez and Schliebs [10] reported that if prepubescent rats are subjected to a similar duration of kainic acid-induced seizure, an alteration in the pharmacological M1 receptor profile is observed in the hippocampus of the treated rats when they reach adulthood. In an adult model, Churchill et al. [20] reported that at 3 days after kainic acid-induced seizures, a decrease in the prevalence of the pharmacological M1 receptor exists in the CA4 subfield of the hippocampus. Our findings that decreased mRNA levels for the m1 subtype at and before 24 h post challenge—prior to any discernible neuronal loss in the vulnerable CA3 subfield— would be consistent with a loss of muscarinic M1 receptor binding at these early times following challenge, should the change in expression lead to similar changes in the amount of translated protein. Furthermore, the pharmacological M1 receptor has been shown to be decreased in the hippocampus following amygdala kindling [12], entorhinal cortex kindling [11], and electroshock seizures [11]. These studies suggest that a reduction in muscarinic cholinergic receptor prevalence results from many types of seizures. Studies of the molecular subtypes involved in the changes seen in these other models are currently underway.
Acknowledgements
Fig. 5. Histological assessment of the dorsal CA3 subfield viability at 24 h post seizure. Top field originates from a control subject, and the bottom field is taken from an animal 24 h post seizure. No significant changes in cell viability were observed (blinded examination, Student’s two-paired t-test).
We thank Lucy Teves, Antonio Mendonca and Jerome Cheng for technical assistance. This work was supported by grants from the Bloorview Children’s Hospital Foundation and The Heart and Stroke Foundation of Canada,
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and the Medical Research Council of Canada. NM is a Fellow of the Bloorview Epilepsy Program, and LZ is a Heart and Stroke Foundation of Canada Scholar.
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