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Effect of locus ceruleus stimulation on the development of kindled seizures
EXPERIMENTAL
NEUROLOGY
95,13-20 (1987)
Effect of Locus Ceruleus Stimulation on the Development of Kindled Seizures CARLOSJIMENEZ-RIVERA, ANNA VOLTURA,
AND GERALD K. WEISS’
Department of Physiology, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131 Received February 4, 1986; revision received August 1, 1986 The effects of locus ceruleus stimulation on amygdala kindling development were investigated. Twenty to thirty minutes of locus ceruleus stimulation was delivered prior to each amygdala kindling session. Locus ceruleus-stimulated animals spent a significantly longer time in stage 1 than did nonstimulated or reticular formation stimulated controls. However, the total number of stimulations to produce the first stage 5 did not differ between groups. The afterdischarge duration in locus ceruleusstimulated animals increased even though the behavioral stages did not progress. There appeared to be an uncoupling of the electrographic and behavioral manifestations of the kindling process in the locus ceruleus-stimulated animals. These data support the view ofan inhibitory role ofthe central noradrenergic system in the development of kindled seizures. o 1987 Academic PMS, hc.
INTRODUCTION The kindling model of epilepsy has provided the most convincing evidence concerning the question of the role of brain catecholamines in epileptogenesis. This model consists of electrographic afterdischarges (AD), behavior automatisms, and eventually, generalized motor convulsions all induced by repeated application of low-intensity electrical stimulation to specific forebrain structures (8). The depletion of brain norepinephrine (NE) using 6hydroxydopamine or surgical lesions in NE pathways (2, 3) facilitates kindling development by accelerating the progression toward full seizures but Abbreviations: AD-afterdischarge, LC--Locus ceruleus. NE-norepinephrine, RF-reticular formation. ’ The authors thank Anthony Vigil and Craig Laubert for their technical assistance. This work was supported by grant DHHS 506 RR08 I39- 12 from the National Institutes of Health. 13 0014-4886/87 $3.00 Copyright 0 1987 by Academic Press, Inc. All rights ofrepreduction in any form reserved.
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JIMENEZ-RIVERA,
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AND WEISS
does not affect either the threshold for inducing the kindling event or the motor convulsion when it has developed (2 1). The majority of the cell bodies of the noradrenergic neurons are situated in the locus ceruleus (LC), a region of the pons that projects widely to the cerebellum, spinal cord, and forebrain structures (6,9). Brain noradrenergic function can be altered by stimulating or damaging the LC (14,20). Electrical stimulation of the LC suppresses the epileptiform activity produced by the systemic administration of Metrazol (13) or focal application of either cobalt (5) or penicillin ( 16). However, the physiologic significance of an increased LC activity in the development of kindled seizures has not been studied. We hypothesize that LC activation will retard amygdala kindling development. The following report presents data that are consistent with this hypothesis. MATERIALS
AND
METHODS
Subjects were male Sprague-Dawley rats weighing 280 to 425 g at the time of surgery. They were housed in separate cages in temperature-regulated quarters with food and water ad libitum on a 12-h light: 12-h dark cycle. Stress, both physical and psychological (novelty), was kept at a minimum by fully adapting the animals to all new environments and by frequent handling. Weight gain was followed closely and was required to be at the same rate following surgery for the animal to be included in the kindling experiments. Animals were anesthetized with sodium pentobarbital(50 mg/kg, i.p.). A bipolar electrode made from twisted Nichrome insulated wire (0.125 mm diameter) with 0.5 mm tip separation was stereotaxically implanted in the right amygdala using coordinates from the Pellegrino and Cushman atlas (AP 4.8, lateral 5.1, ventral -3.0) (18). One group of rats received in the LC bilateral implants consisting of unipolar electrodes made from stainless-steel entymological pins (Ward Natural Science, Rochester, New York) insulated with Epoxilite and having a tip exposure of 0.5 mm. These were placed in the LC either on an angle of 23.5” from midline [AP -2.4, vertical -2.5 as extrapolated from ( 18)] or from a perpendicular approach using target coordinates of 1.1 mm lateral to the midline, 1.2 mm posterior to lambda, and 5.8 mm below the dura surface (incisor bar at -2.4 mm). The reference electrode was a screw placed over the frontal sinus. The electrode leads were fitted to an Amphenol plug that was attached to the skull with dental acrylic. After postoperative recovery, the animals were randomly assigned to two groups. One group of 18 animals was kindled after a 20- to 30-min stimulation of the LC. The other group of 18 animals was kindled after 20 to 30 min
THE LC AND KINDLING
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15
in the experimental chamber without stimulation to the LC electrodes. All rats were stimulated in the amygdala with a current value that produced an afterdischarge of 5 to 15 s. This was carried out three times per day with a minimum of 90 min between each stimulation. The amygdala-kindling stimulation consisted of constant current, biphasic square wave pulses of 1 ms duration at 60 Hz for I s. The threshold strength of the amygdala stimulation was determined using the methods of limits (7). Beginning at 50 PA, the current was increased every 2 min by 10 PA until an AD of 5 to 15 s was produced in the amygdala. The experimental animals received the LC stimulation for 20 to 30 min before these determinations and continuously during the 2 min between each increment of current. Parameters for LC stimulation were 0.2 ms duration, at 100 Hz using interrupted trains of stimulus pulses consisting of 1 s on, followed by 1 s stimulus off. The amount of LC stimulus current for each rat was determined by increasing the current intensity (to a maximum of 300 PA) until jaw and facial movements were elicited, which signified spreading to the motor nucleus of the trigeminal nerve; the current was then decreased until no motor behavior was observed. In addition to the control group that did not receive LC stimulation, a third group of eight animals was kindled after applying stimulation through electrodes placed ventral to the LC in the tegmental reticular formation (RF). That location was not intended to always be in exactly the same position but rather to be in the vicinity of the LC. The stimulus strength was the average of that obtained for the LC stimulations of the experimental group. This group provided a control for the possibility that current spread into other regions of the RF near the LC produced an effect that would be attributed to the LC stimulation. The behavioral responses to the electrical stimulation of the amygdala were graded according to the five stages defined by Racine (19): l-mouth and facial movements, 2-head nodding, 3-forelimb clonus, 4-rearing, 5-rearing and falling. Placement of the LC, RF, and amygdala electrodes was verified histologically after producing a lesion at the electrode tip by passing 25 PA direct anodal current for 1 min. Coronal sections (32 pm) were cut from frozen brains and stained using a methyl-green pyronine stain. Animals were included in the study only if the LC electrodes were within or slightly medial to the LC. A medial locus was acceptable because the jaw movement produced by LC stimulation must have occurred by passage of current through the LC. The position of the RF electrode was not intended to be at a specific site but had to be in the same AP plane and ventral to the locus of the LC electrodes. Some rats were eliminated from the study due to electrode position outside the desired region.
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AND WEISS
LC STIMULATION RF STIMULATION
STAGE FIG. 1. Number of stimulations in each stage for locus ceruleus (LC)-stimulated (N = 15), control (N = 13), and reticular formation (RF)-stimulated (N = 6) animals. LC-stimulated animals exhibited a significantly longer time in stage I but the number of stimulations in stage 2 was significantly less than the other two groups. The time spent in stages 3 and 4 did not differ between groups. Means + SE are indicated; asterisk denotes a significance of P < 0.05 using the Schefi test.
The data of interest were analyzed using an analysis of variance. Post-hoc analysis using a Sche& test compared the various groups using a significance level of 0.05. Statistical results are represented only for the comparison of the control unstimulated group to the other two groups, LC- and RF-stimulated. RESULTS Stimulation of the LC prior to amygdala kindling increased the time spent in stage 1 compared with controls. As shown in Fig. 1, the total number of stimulations that the animal received in stage 1, before it advance to the next stage was significantly greater in LC-stimulated animals. There was an average of 8.2 stimulations in comparison to 3.9 for the controls. The number of stimulations in stage 2 was significantly less in the LC-stimulated animals. However, no significant difference was observed in the total number of stimulations in stages 3 or 4 or the number of stimulations to attain stage 5 (Fig. 2). The RF group did not differ from the nonstimulated controls in either the total number of stimulations in each individual stage nor in the number of stimulations to reach the first stage 5 (Figs. 1 and 2). When the number of stimulations for stages 2, 3, and 4 were combined the LC-stimulated animals had significantly fewer than the control, unstimulated animals. This was due primarily to the rapid progression through stage 2. It was ob-
THE LC AND KINDLING
17
DEVELOPMENT
* I 4
11
iii3
0
LC STIMULATION
3 J = e
6 7
RF STIMULATION
P g
6 4
2
3
5 c/J l
2
10
6
1
STAGE5 FIG. 2. Number of stimulations to reach the first stage 5. No significant differences were found for the total number of stimulations required to reach the first stage 5 seizure. Means + SE are indicated for the LC-stimulated group (N = 14), control (IV = 13), and RF-stimulated animals (N= 6).
served that only in the LC-stimulated group, four animals advanced from stage 1 to stage 5 without any intervening stagesand three animals went directly to stage4. Table 1 presents data showing the duration of afterdischarges (ADS) recorded from the amygdala during kindling. The initial AD at the threshold
TABLE 1 Afierdischarge (AD) Duration in Amygdala-Kindled Rats during Stimulation of the Locus Ceruleus (LC) or Reticular Formation (RF)” Amygdala kindled group (N) LC-stimulated (N = 15) Control (N = 13) RF-stimulated (N = 6)
Initial AD at threshold 10.7 f 0.8 (6.0-15.0) 9.9 k 1.1 (5.0-17.0) 7.8 f 0.9 (6.0-I 1.0)
Pooled AD of last 3 in stage 1 33.1 +4.7* (9.3-70.7) 19.2 + 2.6 (8.741.0) 16.6 % 3.8 (7.0-33.0)
First AD of
First AD of
stage2
stage 5
61.9+5.2** (28.4-86.0) 39.1 % 5.8 (9.0-71.0) 35.0 + 9.0 (13.0-68.0)
69.0 f 3.6** (46.0-89.0) 88.2 + 3.8 (68.0-l 13.0) 83.5 + 5.3 (67.5-100.4)
a Values are X+- SE duration in seconds of ADS; range in parentheses. * Different from the control group, P-c 0.05. ** Different from the control group, P -c0.01.
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AND WEISS
stimulation for the LC- and the RF-stimulated groups were not significantly different from the control. The average duration of pooled ADS for the last three occurring in stage 1 for LC-stimulated animals were significantly longer as was the duration of the first AD in stage 2. However, the AD duration of the first stage 5 stimulation was significantly shorter for the LC-stimulated animals. Similar comparisons of AD duration between the control and the RF group were not significantly different. Comparison of the threshold currents for producing the initial AD of 5 to 15 s did not differ across the three groups (control 129 f 43; LC-stimulated 162 k 52; RF stimulated 158 f 47). DISCUSSION The results reported here indicate that locus ceruleus activation by electrical stimulation retards the development of kindled seizures. The retardation was produced by a longer time spent in stage 1. This observation is consistent with other reports in which facilitation of the kindling process with drug treatments (I, 12) or by lesions of the stria terminalis (4) shortens selectively the time spent in the early stages. When the kindling process is slowed, as in bilaterally amygdalectomized rats, the delay is due to an extension of the early stages (11). Kindling in structures that require a longer time to kindle, such as in the hippocampus, also have a lengthening of the early stages (10). Le Gal La Salle (10) has emphasized that there are two phases in the kindling process. The first phase is represented by the early stages which are characterized by a “progressive buildup” of activation in the primary focus. This has an increasingly greater influence on structures that will eventually propogate the AD away from the primary site. It may be that activation of the LC prior to amygdala kindling produces a delay in the ability of this early buildup of ADS to incorporate other structures and thereby prevent the rapid progression toward full blown seizures. Increased LC activity may raise the threshold for the transition from this first phase to the second phase of the kindling process. Consistent with these observations are the data on AD duration. It is well documented that the AD duration of the kindled site increases as the behavioral stages progress ( 19). The first amygdala AD duration of stage 2 and the average duration of the last few in stage 1 are significantly longer in the LCstimulated animals (Table 1). It appears that the electrographic manifestation of the kindling process in the amygdala continues to progress even though the behavioral manifestation usually associated with the longer ADS is retarded by LC stimulation. One possibility is that the LC activation is preventing the progressive involvement of the motor system which accompanies the spread of the AD away from the primary site of kindling. The appar-
THE LC AND KINDLING
DEVELOPMENT
19
ent lack of a direct effect of the LC on the amygdala AD is also evident in the observation that neither the thresholds for producing the initial AD nor the length of the initial AD are different in the two groups. The same conclusion was reached from other studies in which brain catecholamines have been altered during the kindling process (2,3, 15). Even though the AD duration at the end of stage 1 is longer in the LCstimulated animals, the rapid behavioral transition to stage 5 seizures is not accompanied by a significant lengthening of the AD (Table 1). It must be remembered that some animals advanced from stage 1 to stage 5. Consequently, the first stage 5 seizure had a shorter AD compared with the control (Table 1). Apparently this AD is of sufficient strength and duration to invade the motor regions that produce the behavior of a stage 5 seizure and may constitute the critical factor that is necessary to overcome the increased threshold produced by the LC stimulation for the transition out of stage 1. In addition to the extension of the time in stage 1, the experimental animals showed an acceleration of progression through stage 2 to the extent that there was no significant difference for the number of stimulations to produce the first stage 5 seizure. In fact, many experimental animals advanced from stage 1 to either stage 4 or 5 and did not display the normal progression through the intermediate stages. Previous work has shown that the rate of kindling depends on the length of the initial AD (10). All initial ADS were short for the experimental animals but this was not the case when they finally left stage 1 and went on to the next stage. At that time the ADS were much longer. It has been observed that longer ADS should facilitate AD propagation and accelerate development of the stage 5 seizure (10). Thus, the AD propagation in the experimental rats would be facilitated greatly when the elevated threshold was reached for the activation of other structures. The mechanism for the LC inhibitory effect on the development of kindled seizures remains to be investigated. However, a direct LC inhibitory action upon the complex neuronal circuits that are involved in the spread of seizure may not be the only possible consideration. Locus ceruleus activation or NE release induces a bias that alters the postsynaptic response to other neurotransmitters, either excitatory or inhibitory (22). It has been suggested that the inhibitory GABAergic transmission in the substantia innominata plays an important role in regulating the propagation of amygdala kindled seizures in fully kindled animals ( 17). It may be that increased LC activity potentiates an inhibitory GABAergic process and thereby retards the early stages of seizure development. In this regard, it is of interest to note that an augmentation of the GABAergic system in the substantia innominata blocks the motor convulsions of kindled animals while the amygdala ADS continue ( 17). This is similar to the results obtained when the LC inhibits seizure development.
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REFERENCES 1. ARNOLD, R. S., R. J. RACINE, AND R. A. WISE. 1973. Effects of atropine, reserpine, 6hydroxydopamine and handling on seizure development in the rat. Exp. Neurol. 40: 457470. 2. CORCORAN, M. E., AND S. T. MASON. 1980. Role of forebrain catecholamines in amygdaloid kindling. Brain Res. 190: 473-484. 3. EHLERS, C. L., D. K. CLIFTON, ANDC. H. SAWYER. 1980. Facilitation of amygdala kindling in the rat by transecting ascending noradrenergic pathways. Brain Res. 189: 274-278. 4. ENGEL, J., JR., AND R. KATZMAN. 1977. Facilitation of amygdaloid kindling by lesions of the stria terminalis. Brain Res. 122: 137-142. 5. FISCHER,W., I. KASTNER, R. LASEK, AND M. MULLER. 1983. Wirkung der Reizung des Locus Coeruleus auf Kobalt-induzierte eoukeotufime aktivitat bei der Ratte. Biomed. Biochim. Acta 42: 1179-l 187. 6. FOOTE, S., F. BLOOM, AND G. ASTON-JONES. 1983. Nucleus locus coeruleus: new evidence of anatomical and physiological specificity. Physiol. Rev. 63: 844-9 14. 7. FREEMAN, G. F., AND M. F. JARVIS. (1981). The effect of interstimulation interval on the assessment and stability of kindled seizure thresholds. Brain Res. Bull. 7: 629-633. 8. GODDARD, G. D., MCINTYRE, AND C. LEECH. 1969. A permanent change in brain function resulting from daily electrical stimulation. Exp. Neurol. 25: 295-330. 9. GRANT, S. J., AND D. E. REDMOND, JR. 1981. The neuroanatomy and pharmacology of the nucleus locus coeruleus. Pages 5-27 in H. LAL AND S. FIELDING, Ed., Psychopharmacology of Clonidine. Liss, New York. 10. LEGAL LA SALLE, G. 198 1. Amygdaloid kindling in the rat: regional differences and general properties. Pages 3 1-47 in J. A. WADA, Ed., Kindling2. Raven Press, New York. 11. LEGAL LA SALLE, G., AND J. LAGOWSKIA. 1980. Amygdaloid kindling procedure reduces severity of morphine withdrawal syndrome in rats. Brain Res. 184: 239-242. 12. LEVIEL, V., AND R. NAQUET. 1977. A study of the action of vaproic acid on the kindling e&t. Epilepsia 18: 229-234. 13. LIBET, B., C. A. GLEASON, E. W. WRIGHT, AND B. FEINSTEIN. 1977. Suppression of an epileptiform type of electrocortical activity in rat by stimulation in the vicinity of locus coeruleus. Epilepsia 18: 45 l-46 1. 14. LOGAN, W. J., AND S. I. HARIK. 1982. Specific uptake of norepinephrine and dopamine by homogenates of rat cerebral cortex after locus coeruleus lesion. J. Neurosci. 2: 394-398. 15. MCINTYRE, D. C. 198 1. Catecholamine involvement in amygdala kindling of the rat. Pages 67-85 in J. A, WADA, Ed., Kindling2. Raven Press, New York. 16. NEWMAN, R. S. 1984. Suppression of penicillin induced epileptiform activity by locus coeruleus (LC) or noxious stimulation. Sot. Neurosci. Abstr. 10: 406. 17. OKAMOTO, M., AND J. A. WADA. 1984. Reversible suppression of amygdaloid kindled convulsion following unilateral gabaculine injection into the substantia innominata. Brain Res. 305: 389-392. 18. PELLEGFUNO,L. J., AND A. J. CUSHMAN. 1967. A Stereotaxic Atlas of the Rat Brain. Appleton-Century-Crofts, New York. 19. RACINE, R. J. 1972. Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroenceph. Clin. Neurophysiol. 32: 28 l-294. 20. WALTER, D. S., ANDD. ECCLESTON. 1973. Increase ofnomdrenaline metabolism following electrical stimulation of the locus coeruleus in the rat. J. Neurochem. 21: 28 l-289. 21. WESTERBERG, V., J. LEWIS, AND M. E. CORCORAN. 1984. Depletion of noradrenaline fails to affect kindled seizures. Exp. Neurol. 841237-240. 22. WOODWARD, D. J., H. C. MOISES, B. D. WATERHOUSE, B. J. HOFFER,AND R. FREEDMAN. 1979. Modulatory actions of norepinephrine in the central nervous system. Fed. Proc. 38~2109-2116.
NEUROLOGY
95,13-20 (1987)
Effect of Locus Ceruleus Stimulation on the Development of Kindled Seizures CARLOSJIMENEZ-RIVERA, ANNA VOLTURA,
AND GERALD K. WEISS’
Department of Physiology, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131 Received February 4, 1986; revision received August 1, 1986 The effects of locus ceruleus stimulation on amygdala kindling development were investigated. Twenty to thirty minutes of locus ceruleus stimulation was delivered prior to each amygdala kindling session. Locus ceruleus-stimulated animals spent a significantly longer time in stage 1 than did nonstimulated or reticular formation stimulated controls. However, the total number of stimulations to produce the first stage 5 did not differ between groups. The afterdischarge duration in locus ceruleusstimulated animals increased even though the behavioral stages did not progress. There appeared to be an uncoupling of the electrographic and behavioral manifestations of the kindling process in the locus ceruleus-stimulated animals. These data support the view ofan inhibitory role ofthe central noradrenergic system in the development of kindled seizures. o 1987 Academic PMS, hc.
INTRODUCTION The kindling model of epilepsy has provided the most convincing evidence concerning the question of the role of brain catecholamines in epileptogenesis. This model consists of electrographic afterdischarges (AD), behavior automatisms, and eventually, generalized motor convulsions all induced by repeated application of low-intensity electrical stimulation to specific forebrain structures (8). The depletion of brain norepinephrine (NE) using 6hydroxydopamine or surgical lesions in NE pathways (2, 3) facilitates kindling development by accelerating the progression toward full seizures but Abbreviations: AD-afterdischarge, LC--Locus ceruleus. NE-norepinephrine, RF-reticular formation. ’ The authors thank Anthony Vigil and Craig Laubert for their technical assistance. This work was supported by grant DHHS 506 RR08 I39- 12 from the National Institutes of Health. 13 0014-4886/87 $3.00 Copyright 0 1987 by Academic Press, Inc. All rights ofrepreduction in any form reserved.
14
JIMENEZ-RIVERA,
VOLTURA,
AND WEISS
does not affect either the threshold for inducing the kindling event or the motor convulsion when it has developed (2 1). The majority of the cell bodies of the noradrenergic neurons are situated in the locus ceruleus (LC), a region of the pons that projects widely to the cerebellum, spinal cord, and forebrain structures (6,9). Brain noradrenergic function can be altered by stimulating or damaging the LC (14,20). Electrical stimulation of the LC suppresses the epileptiform activity produced by the systemic administration of Metrazol (13) or focal application of either cobalt (5) or penicillin ( 16). However, the physiologic significance of an increased LC activity in the development of kindled seizures has not been studied. We hypothesize that LC activation will retard amygdala kindling development. The following report presents data that are consistent with this hypothesis. MATERIALS
AND
METHODS
Subjects were male Sprague-Dawley rats weighing 280 to 425 g at the time of surgery. They were housed in separate cages in temperature-regulated quarters with food and water ad libitum on a 12-h light: 12-h dark cycle. Stress, both physical and psychological (novelty), was kept at a minimum by fully adapting the animals to all new environments and by frequent handling. Weight gain was followed closely and was required to be at the same rate following surgery for the animal to be included in the kindling experiments. Animals were anesthetized with sodium pentobarbital(50 mg/kg, i.p.). A bipolar electrode made from twisted Nichrome insulated wire (0.125 mm diameter) with 0.5 mm tip separation was stereotaxically implanted in the right amygdala using coordinates from the Pellegrino and Cushman atlas (AP 4.8, lateral 5.1, ventral -3.0) (18). One group of rats received in the LC bilateral implants consisting of unipolar electrodes made from stainless-steel entymological pins (Ward Natural Science, Rochester, New York) insulated with Epoxilite and having a tip exposure of 0.5 mm. These were placed in the LC either on an angle of 23.5” from midline [AP -2.4, vertical -2.5 as extrapolated from ( 18)] or from a perpendicular approach using target coordinates of 1.1 mm lateral to the midline, 1.2 mm posterior to lambda, and 5.8 mm below the dura surface (incisor bar at -2.4 mm). The reference electrode was a screw placed over the frontal sinus. The electrode leads were fitted to an Amphenol plug that was attached to the skull with dental acrylic. After postoperative recovery, the animals were randomly assigned to two groups. One group of 18 animals was kindled after a 20- to 30-min stimulation of the LC. The other group of 18 animals was kindled after 20 to 30 min
THE LC AND KINDLING
DEVELOPMENT
15
in the experimental chamber without stimulation to the LC electrodes. All rats were stimulated in the amygdala with a current value that produced an afterdischarge of 5 to 15 s. This was carried out three times per day with a minimum of 90 min between each stimulation. The amygdala-kindling stimulation consisted of constant current, biphasic square wave pulses of 1 ms duration at 60 Hz for I s. The threshold strength of the amygdala stimulation was determined using the methods of limits (7). Beginning at 50 PA, the current was increased every 2 min by 10 PA until an AD of 5 to 15 s was produced in the amygdala. The experimental animals received the LC stimulation for 20 to 30 min before these determinations and continuously during the 2 min between each increment of current. Parameters for LC stimulation were 0.2 ms duration, at 100 Hz using interrupted trains of stimulus pulses consisting of 1 s on, followed by 1 s stimulus off. The amount of LC stimulus current for each rat was determined by increasing the current intensity (to a maximum of 300 PA) until jaw and facial movements were elicited, which signified spreading to the motor nucleus of the trigeminal nerve; the current was then decreased until no motor behavior was observed. In addition to the control group that did not receive LC stimulation, a third group of eight animals was kindled after applying stimulation through electrodes placed ventral to the LC in the tegmental reticular formation (RF). That location was not intended to always be in exactly the same position but rather to be in the vicinity of the LC. The stimulus strength was the average of that obtained for the LC stimulations of the experimental group. This group provided a control for the possibility that current spread into other regions of the RF near the LC produced an effect that would be attributed to the LC stimulation. The behavioral responses to the electrical stimulation of the amygdala were graded according to the five stages defined by Racine (19): l-mouth and facial movements, 2-head nodding, 3-forelimb clonus, 4-rearing, 5-rearing and falling. Placement of the LC, RF, and amygdala electrodes was verified histologically after producing a lesion at the electrode tip by passing 25 PA direct anodal current for 1 min. Coronal sections (32 pm) were cut from frozen brains and stained using a methyl-green pyronine stain. Animals were included in the study only if the LC electrodes were within or slightly medial to the LC. A medial locus was acceptable because the jaw movement produced by LC stimulation must have occurred by passage of current through the LC. The position of the RF electrode was not intended to be at a specific site but had to be in the same AP plane and ventral to the locus of the LC electrodes. Some rats were eliminated from the study due to electrode position outside the desired region.
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JIMENEZ-RIVERA,
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LC STIMULATION RF STIMULATION
STAGE FIG. 1. Number of stimulations in each stage for locus ceruleus (LC)-stimulated (N = 15), control (N = 13), and reticular formation (RF)-stimulated (N = 6) animals. LC-stimulated animals exhibited a significantly longer time in stage I but the number of stimulations in stage 2 was significantly less than the other two groups. The time spent in stages 3 and 4 did not differ between groups. Means + SE are indicated; asterisk denotes a significance of P < 0.05 using the Schefi test.
The data of interest were analyzed using an analysis of variance. Post-hoc analysis using a Sche& test compared the various groups using a significance level of 0.05. Statistical results are represented only for the comparison of the control unstimulated group to the other two groups, LC- and RF-stimulated. RESULTS Stimulation of the LC prior to amygdala kindling increased the time spent in stage 1 compared with controls. As shown in Fig. 1, the total number of stimulations that the animal received in stage 1, before it advance to the next stage was significantly greater in LC-stimulated animals. There was an average of 8.2 stimulations in comparison to 3.9 for the controls. The number of stimulations in stage 2 was significantly less in the LC-stimulated animals. However, no significant difference was observed in the total number of stimulations in stages 3 or 4 or the number of stimulations to attain stage 5 (Fig. 2). The RF group did not differ from the nonstimulated controls in either the total number of stimulations in each individual stage nor in the number of stimulations to reach the first stage 5 (Figs. 1 and 2). When the number of stimulations for stages 2, 3, and 4 were combined the LC-stimulated animals had significantly fewer than the control, unstimulated animals. This was due primarily to the rapid progression through stage 2. It was ob-
THE LC AND KINDLING
17
DEVELOPMENT
* I 4
11
iii3
0
LC STIMULATION
3 J = e
6 7
RF STIMULATION
P g
6 4
2
3
5 c/J l
2
10
6
1
STAGE5 FIG. 2. Number of stimulations to reach the first stage 5. No significant differences were found for the total number of stimulations required to reach the first stage 5 seizure. Means + SE are indicated for the LC-stimulated group (N = 14), control (IV = 13), and RF-stimulated animals (N= 6).
served that only in the LC-stimulated group, four animals advanced from stage 1 to stage 5 without any intervening stagesand three animals went directly to stage4. Table 1 presents data showing the duration of afterdischarges (ADS) recorded from the amygdala during kindling. The initial AD at the threshold
TABLE 1 Afierdischarge (AD) Duration in Amygdala-Kindled Rats during Stimulation of the Locus Ceruleus (LC) or Reticular Formation (RF)” Amygdala kindled group (N) LC-stimulated (N = 15) Control (N = 13) RF-stimulated (N = 6)
Initial AD at threshold 10.7 f 0.8 (6.0-15.0) 9.9 k 1.1 (5.0-17.0) 7.8 f 0.9 (6.0-I 1.0)
Pooled AD of last 3 in stage 1 33.1 +4.7* (9.3-70.7) 19.2 + 2.6 (8.741.0) 16.6 % 3.8 (7.0-33.0)
First AD of
First AD of
stage2
stage 5
61.9+5.2** (28.4-86.0) 39.1 % 5.8 (9.0-71.0) 35.0 + 9.0 (13.0-68.0)
69.0 f 3.6** (46.0-89.0) 88.2 + 3.8 (68.0-l 13.0) 83.5 + 5.3 (67.5-100.4)
a Values are X+- SE duration in seconds of ADS; range in parentheses. * Different from the control group, P-c 0.05. ** Different from the control group, P -c0.01.
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JIMENEZ-RIVERA,
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stimulation for the LC- and the RF-stimulated groups were not significantly different from the control. The average duration of pooled ADS for the last three occurring in stage 1 for LC-stimulated animals were significantly longer as was the duration of the first AD in stage 2. However, the AD duration of the first stage 5 stimulation was significantly shorter for the LC-stimulated animals. Similar comparisons of AD duration between the control and the RF group were not significantly different. Comparison of the threshold currents for producing the initial AD of 5 to 15 s did not differ across the three groups (control 129 f 43; LC-stimulated 162 k 52; RF stimulated 158 f 47). DISCUSSION The results reported here indicate that locus ceruleus activation by electrical stimulation retards the development of kindled seizures. The retardation was produced by a longer time spent in stage 1. This observation is consistent with other reports in which facilitation of the kindling process with drug treatments (I, 12) or by lesions of the stria terminalis (4) shortens selectively the time spent in the early stages. When the kindling process is slowed, as in bilaterally amygdalectomized rats, the delay is due to an extension of the early stages (11). Kindling in structures that require a longer time to kindle, such as in the hippocampus, also have a lengthening of the early stages (10). Le Gal La Salle (10) has emphasized that there are two phases in the kindling process. The first phase is represented by the early stages which are characterized by a “progressive buildup” of activation in the primary focus. This has an increasingly greater influence on structures that will eventually propogate the AD away from the primary site. It may be that activation of the LC prior to amygdala kindling produces a delay in the ability of this early buildup of ADS to incorporate other structures and thereby prevent the rapid progression toward full blown seizures. Increased LC activity may raise the threshold for the transition from this first phase to the second phase of the kindling process. Consistent with these observations are the data on AD duration. It is well documented that the AD duration of the kindled site increases as the behavioral stages progress ( 19). The first amygdala AD duration of stage 2 and the average duration of the last few in stage 1 are significantly longer in the LCstimulated animals (Table 1). It appears that the electrographic manifestation of the kindling process in the amygdala continues to progress even though the behavioral manifestation usually associated with the longer ADS is retarded by LC stimulation. One possibility is that the LC activation is preventing the progressive involvement of the motor system which accompanies the spread of the AD away from the primary site of kindling. The appar-
THE LC AND KINDLING
DEVELOPMENT
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ent lack of a direct effect of the LC on the amygdala AD is also evident in the observation that neither the thresholds for producing the initial AD nor the length of the initial AD are different in the two groups. The same conclusion was reached from other studies in which brain catecholamines have been altered during the kindling process (2,3, 15). Even though the AD duration at the end of stage 1 is longer in the LCstimulated animals, the rapid behavioral transition to stage 5 seizures is not accompanied by a significant lengthening of the AD (Table 1). It must be remembered that some animals advanced from stage 1 to stage 5. Consequently, the first stage 5 seizure had a shorter AD compared with the control (Table 1). Apparently this AD is of sufficient strength and duration to invade the motor regions that produce the behavior of a stage 5 seizure and may constitute the critical factor that is necessary to overcome the increased threshold produced by the LC stimulation for the transition out of stage 1. In addition to the extension of the time in stage 1, the experimental animals showed an acceleration of progression through stage 2 to the extent that there was no significant difference for the number of stimulations to produce the first stage 5 seizure. In fact, many experimental animals advanced from stage 1 to either stage 4 or 5 and did not display the normal progression through the intermediate stages. Previous work has shown that the rate of kindling depends on the length of the initial AD (10). All initial ADS were short for the experimental animals but this was not the case when they finally left stage 1 and went on to the next stage. At that time the ADS were much longer. It has been observed that longer ADS should facilitate AD propagation and accelerate development of the stage 5 seizure (10). Thus, the AD propagation in the experimental rats would be facilitated greatly when the elevated threshold was reached for the activation of other structures. The mechanism for the LC inhibitory effect on the development of kindled seizures remains to be investigated. However, a direct LC inhibitory action upon the complex neuronal circuits that are involved in the spread of seizure may not be the only possible consideration. Locus ceruleus activation or NE release induces a bias that alters the postsynaptic response to other neurotransmitters, either excitatory or inhibitory (22). It has been suggested that the inhibitory GABAergic transmission in the substantia innominata plays an important role in regulating the propagation of amygdala kindled seizures in fully kindled animals ( 17). It may be that increased LC activity potentiates an inhibitory GABAergic process and thereby retards the early stages of seizure development. In this regard, it is of interest to note that an augmentation of the GABAergic system in the substantia innominata blocks the motor convulsions of kindled animals while the amygdala ADS continue ( 17). This is similar to the results obtained when the LC inhibits seizure development.
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