Atropine slows olfactory bulb kindling while diminished cholinergic innervation does not

Brain Research

Bulkin,

0361-9230188 $3.00 + .OO

Vol. 20, pp. 203-209. o Pergamon Press plc, 1988. Printed in the U.S.A

Atropine Slows Olfactory Bulb Kindling While Diminished Cholinergic Innervation Does Not CARL R. LUPICA Wayne St&e University, Biopsychology

Laborutory,

Received LUPICA, does not.

AND R. F. BERMAN’ Department

8 October

of Psychology,

Detroit, MI 48202

1987

C. R. AND R. F. BERMAN. Atropine YIOH~Sdfuctory bulb kindling u,hile diminished cholinergic innervation BRAIN RES BULL 20(2) 203-209, 1988.-The development of kindled seizures elicited through electrical

stimulation of the rat olfactory bulb (OB) was examined under two conditions which decrease cholinergic neurotransmission. Atropine sulfate (25 mg/kg, IP) administered 1 hr prior to stimulation of the OB was found to significantly delay the acquisition of the fully kindled state. In a second experiment, diminished cholinergic innervation of the OB was established using chemical lesions of the basal forebrain cholinergic system. Despite the depletion of acetylcholine (Ach), as determined by acetylcholinesterase (AchE) and choline acetyltransferase (ChAt) assays, no significant alterations in kindling parameters were observed. Based upon these findings we suggest that Ach is not critical to the establishment of an OB kindled focus but is important for the propogation and generalization of epileptiform activity initiated through OB stimulation. Acetylcholine Olfactory bulb

Acetylcholinesterase Seizures

Basal forebrain

Kindling

development of kindled seizures has shown that Ach agonists injected directly into the amygdala can result either in the development of seizures alone or facilitate the subsequent acquisition of electrically kindled seizures [6, 27, 311. A recent approach to investigating the contribution of Ach to seizure development has been to utilize lesions of the basal forebrain cholinergic system [28]. Recent anatomical studies indicate that several neural regions receive their major extrinsic cholinergic input from magnocellular cholinergic neurons located in the basal forebrain. These regions include large areas of neocortex, the amygdala, and the olfactory bulb (OB) [24]. Cholinergic innervation of the OB in particular, is derived mostly or entirely from extrinsic sources [IS] originating ipsilaterally in the horizontal nucleus of Broca’s diagonal band (nHDB) [9, 10, 15, 321. Walker, Hirsch and Crawford [28] employing unilateral electrolytic lesions of the rat lateral preoptic area (LPO), reported a 50% loss of choline acetyltransferase (ChAt) levels in ipsilateral amygdala. Despite this large decrease in the Ach marker, no significant alterations in kindling rates were observed. In contrast, a significant decrease in the rate of amygdala kindling following bilateral electrolytic lesions of the LPO in cats has been reported [26]. Although levels of cholinergic markers were not determined in the latter experiment, it is likely that these basal forebrain lesions also diminished Ach activity in a variety of limbic structures.

THE kindling phenomenon is a process through which behavioral and electrographic manifestations of epileptiform seizures develop following brief, low intensity electrical stimulation of one of several brain regions [ 121. Initially, such stimulation results in a brief electrophysiological afterdischarge (AD) limited to the focal site of stimulation. Following repeated daily stimulations, the AD duration increases, the behavioral aspects of the seizures emerge, change in form, and secondary foci become established. The behavioral components of the seizure (i.e., motoric) progress through several stages which have been delineated by Racine [20]. The search for the neural mechanisms which support kindled seizures has led many investigators to manipulate neurotransmitter systems and to examine the subsequent modifications in either seizure development or maintenance. The neurotransmitter acetylcholine (Ach) has attracted much attention in the elucidation of possible mechanisms underlying the kindling process. Arnold, Racine and Wise [2] were the first to demonstrate that intraperitoneal injections of atropine one hour prior to amygdala kindling could slow but not eliminate the development of seizures. This atropine induced delay in the acquisition of fully generalized seizures has been shown with amygdala [ 1,2,33] and cortical kindling [22]. However, not all studies have supported these findings [3,71. Other evidence implicating cholinergic processes in the

‘Requests for reprints should be addressed Detroit, MI 48202.

Choline acetyltransferase

to Robert F. Berman, Wayne State University,

203

Department

of Psychology,

71 W. Warren,

204

LUPICA AND BERMAN

In the present investigations, we attempted to elucidate the contribution of Ach to the acquisition of kindled seizures using the OB as the site of focal simulation. The OB was chosen because it is a sensory-limbic structure from which seizures are readily kindled [5]. In addition, it represents an area of the mammalian brain which lends itself to cholinergic denervation due to the well described pattern of cholinergic innervation discussed above. The first experiment examined the effects of systemically administered atropine sulfate on the rate of OB kindling. In the second experiment, the effects of reducing cholinergic activity in the OB on the rate of seizure development were determined. In this study, lesions of the LPOnHDB area were made unilaterally by injection of the excitotoxin ibotenic acid. This second study was performed to further clarify the results obtained by other researchers who used electrolytic lesions of the LPO. The possibility remains that the results obtained by these researchers were influenced by the disruption of fibers coursing through and surrounding the lateral preoptic area. We reasoned that the use of ibotenic acid would lessen the possibility of damage to fibers of passage and thereby provide a more discrete lesion of the cell bodies which exist in the LPO-nHDB region. EXPERIMENT

1

TABLE

I

THE EFFECT OF ATROPINE SULFATE (25 m&kg), GIVEN 1 HR PRIOR TO ELECTRICAL STiMULA~ON, ON SELECTED MEASURES OF OLFACTORY BULB KINDLING

Group

n

Saline Atropine

6 6

AD Threshold (PA) 177.7 t 24.5 168.3 h 17.2

AD’s to Criterion?

AD Duration (see)

13.7 2 1.0 21.2 t 0.9*

39.5 t 6.0 35.7 -+ 6.4

*p
Stage 5 seizures.

Drug profocol. Six of the twelve animals received a single intraperitoneal injection of atropine sulfate (25 mglkg), one hr prior to each electrical stimulation of the OB at the AD threshold. The remaining six animals each received a single IP injection of isotonic saline one hr prior to elect&a1 stimulation of the OB. Injections and subsequent stimulations were given every 48 hours until 2 consecutive stage 5 generalized motor seizures had been elicited. RESULTS

METHOD

Histology

Seventeen male Long Evans rats purchased from Charles Rivers Laboratories and weighing between 295 and 370 grams were used in this experiment. All animals were housed separately, had free access to food and water, and were maintained on a twelve hour light-dark cycle. Surgery All surgery was performed while animals were surgically anesthetized with sodium pentobarbital(65 mg/kg, IP). Each subject received twisted, nichrome wire, bipolar electrodes (Plastic Products MS-303/2) stereotaxically implanted into the right olfactory bulb. These electrodes were located 6.2 mm anterior to Bregma, 0.7 mm lateral to the skull midline suture, 5.5 mm ventral to the skull surface, and were used for electrical stimulation and electrographic recording. All coordinates were determined with the cranium level. A two week postsurgical recovery period was allowed prior to beginning kindling procedures. Frocedure

The procedures for electrode construction and a description of the apparatus have been previously reported [23]. The dependent measures, analyzed by the Student’s independent groups t-test, were the AD threshold, AD duration and the rate of kindling. The AD thresholds were determined for each subject by initially stimulating the OB at 10 @A. The stimulation current was raised in 10 &A increments every two minutes until a brief (>5 set) AD was recorded. This current level was used for all subsequent stimulations. The AD duration was recorded from the onset of the electrical stimulus until the cessation of the electrographic AD. The rate of kindling was defmed as the number of AD elicitations required to result in two consecutive stage 5 seizures according to the scale described by Racine [ZOI.

In Experiments 1 and 2, electrodes were aimed for the right OB of each animal. All subjects with electrode tips located in the internal granule cell, internal plexiform, external plexiform or mitral cell layers developed kindled seizures. Five subjects, two from the drug injection group and three from the control group, with electrode tips inadvertently placed in the olfactory nerve did not exhibit AD’s or develop kindled seizures. The data derived from these animals were not included in the analyses. Kindling

Table 1 displays the effects of atropine sulfate (25 mg/kg, IP) on the AD threshoId, AD duration and the rate of kindling. These data indicate that animals receiving atropine sulfate injections one hr prior to electrical stimuiation of the OB required an average of approximately 7.5 additional AD elicitations to reach the criterion of two consecutive stage 5 seizures. This difference between the two groups was statistically significant, t(.5)=5.9,p<0.001. Although these groups differed with respect to the rate of kindhng acquisition, the mean AD duration did not differ s~n~cantly between the groups. Figure 1 shows that the atropine sulfate induced delay in reaching stage 5 seizures was most apparent during the progression from stage 3 to stages 4 and 5. DISCUSSlON

The results from Experiment 1 demonstrate that peripheral administration of the cholinergic antagonist atropine sulfate can delay the development of kindled seizures elicited by electrical stimulation of the rat olfactory bulb. This finding supports and extends to OB kindling the findings of other researchers who have previously reported that systemically administered atropine can delay the development of amygdaloid and cortically kindled seizures [l, 2, 22, 337. The ob-

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BULB KINDLING Procedure

1

I

I

I

I

I

3

6

9

12

15

18

Number

of

-W-

Saline

+

AtroDine Sulfate

I

21

/

24

I

27

30

Afterdischarges

FIG. 1. The effect of atropine sulfate on the rate of olfactory bulb kindling. Each point represents the mean seizure stage (?SEM) following a given number of AD elicitations.

servation that a muscarinic antagonist can delay the course of kindling from several sites suggests that endogenous Ach must play an important role in the development of kindled seizures. However, the precise role that cholinergic neurons play remains poorly understood. EXPERIMENT

2

METHOD

The purpose of Experiment 2 was to determine if a reduction in Ach neurotransmission through the destruction of cholinergic afferents to the OB would result in the protraction of the kindling process. Subjects

Thirty-two male Long Evans maintained as described above.

rats were purchased

and

Surgery

Twenty-six animals received stimulating/recording electrodes implanted as described above in Experiment 1 methods. In addition to the OB electrode, fifteen animals each received three 0.5 ~1 unilateral injections of the excitotoxin ibotenic acid (5.0 pg/pl). The injections were ipsilateral to the OB electrodes and aimed for the horizontal nucleus of the diagonal band of Broca. The injection coordinates were 0.2 mm anterior to Bregma, 0.3 mm and 0.6 mm posterior to Bregma, 2.5 mm lateral to the skull midline, and 9.0 mm ventral to the cranium surface. The ibotenic acid (Sigma, St. Louis) was made up in cold phosphate buffered saline at pH 7.7. Eleven control animals were given intracranial injections of isotonic saline using the same coordinates described above.

Following a two week postsurgical recovery period, afterdischarge threshold determination and kindling procedures were commenced in the same manner as described in Experiment 1 except that these animals were stimulated at the AD threshold once every 24 hours. Histology. Electrode placement, lesion extent and relative levels of Ach innervation of OB and in other neural areas of the 26 kindled animals were assessed by staining for acetylcholinesterase (AchE). One day following the second consecutive stage 5 seizure, each electrode implanted animal was given a lethal dose of pentobarbital. Subjects were then decapitated, the brains quickly removed and frozen in isopentane cooled with dry ice and acetone. The cerebellum and brainstem were removed, and the brain sectioned in a cryostat at 18 microns. Starting with the OBs, every twentieth coronal section was collected and mounted on a chrome-alum subbed slide. All of the sections were stored frozen at -70°C until stained for AchE. Staining was conducted within three days of sectioning according to the procedures of Koelle, Hossaini, Akbarzaded and Koelle [13]. Non-specific acetylthiocholine iodide binding was inhibited with promethazine hydrochloride (Sigma, St. Louis). Acetylcholinesterase depletion rankings. Three independent observers ranked the level of AchE depletion seen under microscopic examination (25 x). Comparisons were made between coronal sections of right olfactory bulb (ROB) and left olfactory bulb (LOB) of the same neural section. The contralateral OB was chosen as the appropriate control based upon previous anatomical studies showing that the cholinergic projections to the main OB are almost entirely ipsilateral [9,15]. The comparison between ROB and LOB was done to provide an indication of the degree of diminished AchE staining for further correlation with the kindling measures. Rankings were scaled from zero to three: (0) No difference between ROB and LOB in terms of darkness of AchE staining; (1) Minimal difference between ROB and LOB; (2) Moderate difference between ROB and LOB (i.e., greater than 0 and 1, less than 3); and (3) Marked difference between ROB and LOB. Choline acetyltransferase (ChAt) assays. An additional group of six animals received unilateral ibotenic acid injections using the same injection coordinates and procedures described above. This group was used to assess the degree of cholinergic depletion in the OB of the lesioned hemisphere as compared to the OB in the unlesioned hemisphere at the start of kindling procedures. This group did not receive OB electrodes and was therefore not kindled. Two weeks following the ibotenic acid injections, these animals were sacrificed by decaptitation, the brains rapidly removed, and the right and left OB separated on ice. The tissues were rapidly frozen in isopentane and stored as described above. The tissues were then homogenized in ice-cold 0.5% Triton X-100, 10 mM EDTA, pH 7.4, and assayed for ChAt activity according to the methods of Fonnum [ll]. A selective inhibitor of ChAt, hydroxyethyl-1-4-napthylvinyl pyridium (NVP) was included in some of the assays to verify that the measured enzyme activity was due to the presence of choline acetyltransferase. Protein concentration was measured according to the methods of Lowry, Rosenbrough, Farr and Randall [ 141. Scoring and statistical analysis. The AD threshold, the AD duration, and the rate of kindling and were measured as described in Experiment 1. Differences between groups on these dependent measures were analyzed using the inde-

LUPICA AND BERMAN largest and the smallest ibotenic acid lesions and the extent of those lesions in the rostral-caudal dimension. The ibotenic acid injections resulted in the subsequent loss of AchE activity and neuronal degeneration in large areas of the basal forebrain. These areas included most of the nHDB, the ventral paIl~dum, substantia innominata, and nucleus basalis; lateral portions of the vertical diagonal band and LPO; small medial portions of piriform cortex; and most of anterior amygdala. Also, due to the spread of ibotenic acid along the length of the injection cannula tract, a cylinder (1 .O-2.0 mm) of reduced AchE reactivity was observed in the globus pallidus, striatum, and very small portions of anterior parietal cortex in the medioventral to mediodorsal aspect. Photographs of representative neural sections of OB, which show diminished AchE staining in the lesioned hemisphere, and the lesion site are shown in Figs. 3 and 4. Several neural areas ipsilateral to the basal forebrain lesion site displayed large reductions in AchE staining. These structures were OB, piriform and frontal cortex, ventral pallidum, the basolateral and central amygdaIoid nuclei and lateral portions of CA2, CA3 and dentate gyrus of the hippocampal formation. Neural structures displaying a moderate degree of AchE depletion were the lateral olfactory tract, anterior amygdala, the amygdalohippocampal area and medial portions of striate and entorhinal cortex. Regions with small levels of AchE stain reduction were subiculum and cingulate cortex.

FIG. 2. A schematic reconstruction of ibotenic acid induced lesions of the basal forebrain. The stippled area represents the largest lesion, the darkened area represents the smallest lesion in the group. The upper coronal section corresponds to 0.2 mm anterior to Bregma, the bottom section corresponds to I.3 mm posterior to Bregma. Figures and coordinates are from Paxinos and Watson [191. pendent group Student’s t-test. The Kendall’s tau rank order correlation statistic was used to determine the degree of correspondence between the amount of AchE depletion and the dependent variables in the lesion group, and to assess rater agreement about depletion rankings. Differences in ChAt concentrations between the right and left OB’s were analyzed using a dependent samples t-test. RESULTS

All kindled animals used in this study which received ibotenic acid injections in the region of the nHDB-LPO, demonstrated diminished AchE reactivity in the OB ipsilateral to the lesion. Figure 2 schematically shows the

As shown in Table 2, the mean (+-SEM) threshold current required to elicit an AD for the ibotenic acid lesioned animals (141.8216.6 PA) did not differ significantly, t(25)=0.87, ~~0.39, from the saline injected control group (161223.4 @A). An examination of the first-trial AD durations was performed to determine if any differences between the groups in initial seizure susceptibility existed. The ibotenic acid injected group demonstrated an initial AD duration of 9.920.72 set, while that for the saline injected group was 9.820.43 set (meanrtSEM). This difference was not statistically signi~~ant, t(2.5)=0.63, p>O.OS. The rate of kindling, defined as the number of ADS required to reach a second consecutive Stage 5 seizure, also did not differ between the ibotenic acid injected and the saline injected groups, t(25)=0.33, ~~0.74. The meanGEM number of AD elicitations required to kindle the ibotenic acid group was 12.6+0.8, while that for the saline injected group was 13.020.9. Similarly, the mean AD duration did not differ between the two groups, t(25)=0.72, ~~0.47. The meantSEM for the lesioned group was 38.422.7 seconds, and 35.1 r3.9 seconds for the saline group. Correlations between the degree of AchE depletion, AD duration, AD threshold and the rate of OB kindling in the lesioned group, were performed using the Kendall’s tau rank order correlation statistic. The correspondence between AchE depletion rank and AD duration yielded a moderate coefftcient of Tau=O.#. This correlation was not significantly different from a zero correlation @>O.OS). Similarly, the correlations for AD threshold (Tau=O.lO, p>O.OS), and the rate of kindling (Tau=O.40, p ~0.05) were not significant. In an effort to ensure that raters were judging AchE depletions in a similar manner, the Kendall’s tau statistic was again utilized. The correlation among raters was high (Tau==0.86) and was significantly different from zero @
ACETYLCHOLINE

AND OLFACTORY

BULB KINDLING

FIG. 4. A representative photograph of an ibotenic acid induced lesion of the rat basal forebrain. Diminished acetylcholinesterase activity can be seen in the lower left portion of the section. This area corresponds to the horizontal diagonal band area of the basal forebrain.

207

208

LUPICA TABLE 2 THE EFFECT OF IBOTENIC ACID LESIONS OF THE BASAL FOREBRAIN ON SELECTED MEASURES OF OLFACTORY BULB KINDLING

Group

n

Saline Ibotenic

11 15

Threshold &A) 166.1 k 23.4 141.8 ” 16.6

AD’s to Criterion*

AD Duration (see)

13.0 + 0.9 12.6 -+ 0.8

35.1 2 3.9 38.4 k 2.7

*The number of ADS to reach 2 consecutive All values represent the mean 2 SEM.

Stage 5 seizures.

Neurochemistry

The ChAt assay revealed that ibotenic acid lesions of the nHDB-LPO area resulted in a mean 5223.6% decrease in ChAt activity in the right OB, which was ipsilateral to the lesions. The range of ChAt depletion was from 22% to 70% in the OB located in the lesioned hemisphere when compared to the OB in the non-lesioned side of the brain. The dependent groups t-test revealed this depletion to be statistically significant, t(S)=21, p
DISCUSSION

The neurotoxic lesions produced in Experiment 2 were extensive. Yet, despite widespread cell destruction within the basal forebrain (e.g., LPO, ventral pallidum, substantia innominata) and the marked depletion of ChAt activity (i.e., 52%) in the ipsilateral OB, no significant alterations in kindling parameters were seen. In a similar study it was reported that unilateral electrolytic lesions of the LPG reduced ChAt levels to 50% of control values in the ipsilateral amygdala but failed to alter amygdala kindling rates [28]. These results which employed electrolytic lesions, are consistent with the results of the present investigation. In contrast, Shouse rt al. 1261recently reported that bilateral electrolytic lesions of the LPG in cats can facilitate the acquisition of basolateral amygdala kindling. Cholinergic markers were not examined in their study, but such lesions would be likely to result in some depletion of Ach in basolatera1 amygdala. Their findings are surprising in view of the facilitory effect Ach agonists have on kindled seizures [f&27, 29-311, and the an~gonistic effect Ach receptor blocking agents have on kindling [I, 2, 22, 331. It is possible that the electrolytic lesions may have damaged axons in the medial forebrain bundle (MFB) which lies immediately adjacent to the LPO-basal forebrain area. The MFB contains many catecholaminergic fibers coursing to the forebrain, including the amygdala and olfactory bulb. Since catecholamine depletion can s~~cantly facilitate amygdala kindling [2,16], it is possible that the facilitation of kindling observed by Shouse er al. 1261 was due to the disruption of catecholaminergic projections to the amygdala, rather than to disruption of cholinergic projections. The observation in Experiment 1 that atropine SU)fate influences the rate of OB kindling but not the AD threshold or the AD duration suggests that the level of cholinergic activity is important for the spread of seizures, but may not be critically involved in other aspects of kindling. This assertion

AND BERMAN

may explain the inability of cholinergic antagonists to modify ictal events in fully kindled animals [21]. Studies in which Ach antagonists were injected directly into the kindled focus prior to electrical stimulation suggest that the retardin effect atropine exerts against seizure development is not mediated at the focal point of electrical stimulation [31]. This does not, however, rule out the possibility that cholinergic neurons may participate directly in the establishment of seizures in some neural areas [6, 27, 29, 301. More generally, it is possible that the process of kindling involves the participation of several neural systems each of which may contribute differentially to particular aspects of kindling. For example it has recently been reported that the AD threshold can be modulated by GABAergic activity in the substantia nigra [ 17, IS]. It is therefore tenable that cholinergic systems may contribute primarily to seizure spread, and not to the establishment of the initial focus itself. One possible site for the mediation of the atropineinduced delay of kindling may be within the hippocampus. The blockade of muscarinic receptors within the hippocampus may prevent the prolonged hypersensitivity to Ach seen in vitro in pyramidal cells following an AD [4]. Hence, the recruitment of neural elements within the hippocampus and associated structures would be blocked with Ach antagonists, possibly delaying generalization of the kindled seizure. There is also evidence that kindling results in a reduction in the number of cholinergic receptors found in the hippocampus 18,251. Atropine may delay the development of kindling by contributing to the diminished responsiveness of neurons to Ach following a seizure, thereby decreasing the recruitment of critical neural elements. While the present results suggest only an indirect link between kindling and cholinergic activity (i.e., the modulation of seizure spread) there are alternative explanations. It remains possible that unilateral lesions of the basal forebrain area may not influence kindling in the amygdala or the OB because the decrease in cholinergic innervation of these structures may not have been sufficient. Alternatively, these lesions may not have affected OB kindling because Ach is not critically involved in the establishment of the focus for OB kindled seizures. It is also possible that ibotenic acid, as an excitotoxin, could have directly increased seizure susceptibility in lesioned animals. If so, this could have partially offset any protraction of kindling which may otherwise have been seen in the lesioned animals. This seems unlikely, however, because the lesioned group did not differ from the shamlesioned group on measures of initial AD threshold or AD duration. Also, none of the ibotenate lesioned animals displayed behavioral signs of seizure activity during or following surgery. In summary, the results from these experiments show that a peripherally administered cholinergic antagonist can delay the acquisition of fully generalized 03 seizures, while marked cholinergic depletion at the focus prior to kindling does not affect kindling. It is speculated that Ach may facilitate seizure propagation and generalization to a number of possible brain sites, including the hippocampus, and that atropine may delay kindling by inhibiting seizure spread. These data also suggest that kindling is not a unitary phenomenon, and that several neurotrans~tter systems each may contribute differentially to the collection of neural events which constitute kindling.

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ACKNOWLEDGEMENTS This research was supported by NIH research grant no. RR08167 and by a grant from the Wayne State University Institute of Chemical Toxicology. We wish to thank Dr. Gary Wenk for his assistance with the ChAt assays.

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