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Monoaminergic correlates of kindling
Brain Research, 403 (1987) 205-212 Elsevier
205
BRE 12334
Research Reports
Monoaminergic correlates of kindling Johnnye Lewis*, Verner Westerberg* and Michael E. Corcoran Departmentof Psychology, Universityof Victoria, Victoria, B. C. (Canada) (Accepted 24 June 1986) Key words: Kindling; Experimental epilepsy; Amygdala; Noradrenaline; Dopamine; Serotonin
High-performance liquid chromatography with electrochemical detection was used to measure the regional concentrations of monoamines and metabolites in the brains of rats killed 2 or 4 weeks after kindling of generalized seizures with amygdaloid stimulation. Each kindled rat was compared to a yoked control that received brief trains of non-convulsive low-frequency stimulation of the amygdala. Two weeks after kindling we found a significant depletion of noradrenaline (NA) in the ipsilateral frontal cortex, a significant depletion of serotonin (5-HT) in the stimulated amygdala and contralateral hypothalamus, and no significant changes in concentration of dopamine (DA). Four weeks after kindling we found significant depletions of NA in the stimulated amygdala and ipsilateral hypothalamus, a significant depletion of 5-HT in the ipsilateral hippocampus, and no significant changes in DA. These findings generally fail to replicate previous reports of monoaminergic correlates of kindling. Furthermore, the alterations in monoamines produced by kindling do not fall into a simple and readily interpretable pattern. INTRODUCTION A considerable body of evidence points to a link between central catecholamines and the mechanisms of kindling 1°a9. Perhaps the strongest evidence is the finding that depletion of central noradrenaline (NA) or destruction of noradrenergic fibers produced by lesions or by injection of 6-hydroxydopamine (6O H D A ) results in a dramatic acceleration in the rate of kindling with electrical stimulation of limbic or neocortical sites 1-3,12-14,21-23. However, depletion of N A does not affect established seizures 32, suggesting that N A ' s suppressive action is limited to seizure development per se. 6 - O H D A - i n d u c e d depletion of forebrain dopamine ( D A ) appears to have no effect on rate of kindling 13,22. There is also some evidence that serotonin (5-HT) might modulate kindling. The 5-HT antagonists metergoline and mianserin have been reported to attenuate forepaw clonus in established seizures 4. The 5-
H T uptake inhibitor fluoxetine increased seizure thresholds, as did low frequency stimulation of the raphe 29. Depletion of stored 5-HT by p-chloroamphetamine failed, however, to lower thresholds 19. Electrical stimulation of the median raphe has also been shown to antagonize amygdaloid kindled seizures 19. There have been a number of studies of monoaminergic correlates of kindling. However, attempts to correlate short-term changes in monoamine concentrations with kindling have yielded quite inconsistent results. In some studies forebrain concentrations of N A and D A were unchanged after kindling 29'3°'32, whereas in other experiments localized depletion of N A 9 or widespread depletion of both N A and D A 27 was observed. Other investigators have described depletion of 5-HT restricted to the midbrain shortly after completion of amygdaloid kindling 23. Inconsistent results have also been found when monoamine concentrations have been measured at
* Present address: Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Manitoba, Winnipeg, Man., Canada, R3E 0W3. Correspondence: M.E. Corcoran. Present address (from 1 March 1987): Department of Psychology, University of Victoria, P.O. Box 1700, Victoria, B.C. Canada, V8W 2Y2. 0006-8993/87/$03,50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
206 long intervals after the last seizure. At one month postseizure a selective depletion of D A in the kindled amygdala was observed in one study 15, whereas other studies have reported no changes in NA and D A 6 or increases in hypothalamic D A and NA but no changes in striatal or limbic amines in amygdaloid kindled rats tested under similar conditions 7. In perhaps the most comprehensive study to date, Okazaki 25 found no changes in concentrations of N A or D A in any of 13 regions examined in the brains of amygdaloid kindled rats killed two months after their last seizure. Finally, Kant et al.17 found no change in in vitro release of N A and D A in tissue taken from amygdaloid kindled rats killed 3 weeks after their last seizure. The reported changes in turnover of monoamines after kindling have also been inconsistent. In one experiment 33 an increase in D A turnover in the ipsilateral forebrain but no change in NA turnover was found in amygdaloid kindled rats killed 7 days postseizure. In contrast, other investigators found no changes in turnover of catecholamines in amygdaloid kindled rats at one month postseizure 6 or at two months postseizure zS. The correlative data are hard to assess due to inconsistencies in both procedures and results in the relevant studies. First, two different species have been used in different experiments. Second, many of the earlier studies used the radioenzymatic assay for catecholamines, whereas the later studies employed high performance liquid chromatography (HPLC). Third, kindling stimulation was applied once daily in some studies and twice daily in others. Fourth, the endpoint for kindling varied considerably, from 3 to 20 generalized seizures (stage 5 in the terminology of Racine27). Finally, perhaps the most important inconsistency is that analyses were done at different intervals postseizure, from 24 h to two months. We have readdressed the question of monoaminergic correlates of kindling by examining alterations of monoamine concentrations in amygdaloid kindled rats killed at two and 4 weeks postseizure, using identical procedures for kindling and neurochemical analysis at both intervals. In addition, our study of the short-term correlates of kindling was performed in two replications to allow us to confirm positive results within the same laboratory.
MATERIALS AND METHODS We first examined possible short-term effects of kindling on concentrations of monoamines and their metabolites in rats killed two weeks after their last kindled seizure. This was done in two replications performed six months apart. We then examined ~'ossible long-term effects of kindling on monoam; ~es in rats killed 4 weeks after their last seizure. ~I*~is :~econd study was carried out 6 months after the second replication of short-term effects.
Subjects and surgery Male Long-Evans hooded rats (Charles River, Canada) weighing 300-350 g were used. They were handled every day after arrival in the colony. The rats were anesthetized with sodium pentobarbital, and bipolar electrodes were surgically implanted bilaterally in the basolateral amygdala, using the following coordinates from the atlas of Pellegrino et a1.26:0.8 mm posterior to bregma, 4.5 mm lateral, and 8.5 mm below the superior surface of the skull, with the incisor bar at +5.0 mm. Electrodes were constructed of twisted pairs of enameled nichrome wire (127/~m). Two weeks of recovery were allowed after surgery. For the short-term study, rats were randomly assigned to either the experimental group (n = 4 in the first replication; n = 5 in the second replication) or the stimulated control group (n = 4 in the first replication; n = 5 in the second replication). Rats from the two groups were yoked, and each yoked pair was thereafter treated identically in all respects but frequency of stimulation. In the long-term study, there were 8 such pairs of kindled rats and yoked controls, tested in one replication.
Kindling procedure Monopolar E E G was recorded from each amygdaloid electrode referenced to a surgical screw in the frontal bone. Electrical stimulation was in the form of balanced biphasic constant-current square-wave pulses, 1.0 ms in duration and delivered at 60 pulse pairs per s for 1 s daily. Thresholds were determined by initial stimulation of experimental animals at 30 /*A (base to peak) on the left side. The intensity was raised in increments of 10 # A until afterdischarge
207 (AD) was evoked, and the lowest such intensity was arbitrarily designated the A D threshold. No more than 4 stimulations per day at 2-min intervals were given during threshold determination. Experimental rats thereafter received 1 s of electrical stimulation at threshold intensity once daily. Yoked controls received 1 s of 3-Hz stimG,mon at the same intensity immediately after their experimental partner both during threshold determination and kindling trials. Although AD can be induced with trains of 3-Hz stimultion applied for 20 or more s and at higher intensities n, A D is not observed with 1-s trains applied at the intensities used here 11,17. By routinely monitoring E E G in the controls, we confirmed that no AD was evoked by the 3-Hz stimulation. The subjects were stimulated until experimental rats developed 3 stage-5 seizures 27. During the interval between the last seizure and sacrifice, all rats were handled at least twice weekly.
Dissection and chemistry At the appropriate interval after the third stage-5 seizure (two weeks for the short-term group; 4 weeks for the long-term group) rats were killed by cervical fracture, and their brains were removed and rapidly dissected over ice by the procedure of Heffner et al. 16. Right and left samples of frontal cortex, amygdala/pyriform, hypothalamus, and hippocampus were each homogenized on ice in 500#1 of 0.05 N hydrochloric acid/0.1 M cysteine (with D H B A added as an internal standard) by a Brinkman polytron at setting 4 for 20 s. The homogenate was centrifuged at 15,000 g for 20 min at 4 °C. Supernatant w a s then centrifugally filtered at 3000 g through 0.2/~m filters. The filtrate was frozen in liquid nitrogen and stored at-196 °C until analysis. Monoamines and metabolites were quantified by reverse-phase HPLC with electrochemical detection. We used a BAS LC-4B amperometric detector equipped with a glassy carbon working electrode and a Ag/AgCI 2 reference electrode. The chromatographic column was a Biophase ODS 0.5-pm reversephase column (250 mm x 4.6 mm i.d.), protected by a Brownlee Labs RP-18 spheri-5 guard column. Twenty pl of thawed sample was injected directly, and monoamines and metabolites were eluted by a 6% methanol-water mobile phase (v/v), pH 3.1, containing 0.15 M chloroacetate buffer, 0.03 M
Na2HPO 4 (anhydrous), 0.1 mM E D T A , and 200 mg/ liter SOS. The flow rate was 1.0-1.5 ml/min, at a temperature of 37 °C. Single-electrode detection was performed at an oxidation potential of +0.8 V. This system produced good separation for NA, adrenaline (A), D H B A (internal standard), DA, dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindole acetic acid (5-HIAA), homovanillic acid (HVA) and 5-HT. Mean regional concentrations of amines determined with our technique are shown in Fig. 1. During dissection and chemistry each kindled rat was always treated in parallel with its yoked control.
Statistics Data in the present studies were collected over a period of two years. The unavoidable changes in chemicals, columns, and other conditions resulted in high within-cell variability, although the yoking of experimental and control rats controlled for this within the pairs that were directly compared. This factor, along with the relatively small numbers of animals (9 pairs in the short-term study and 8 pairs in the long-term study), made analysis by non-parametric techniques the most appropriate method. To take full advantage of the matched groups design and examine the question of direction of change, a Wilcoxon test for matched pairs was used to analyze percentage difference scores, defined as follows: score = ((E-C)/C) × 100, where E represents the regional concentration of the relevant amine in a kindled rat and C represents comparable data from its yoked control. Since we were attempting to replicate previous studies, an a level of 0.05 was used. In those cases where our results failed to replicate previous findings, we also report t-tests of the significance of the differences between control rats and kindled rats, both in the raw data and in percentage change scores, in an effort to ensure that the method of analysis was not biasing the results. These results are reported from all data in each test (untrimmed) and for data with extreme scores excluded (trimmed), to ensure that replication was not either occurring because of, or being suppressed by, one or two extreme scores. In cases where significant changes in concentration of the parent monoamine were obtained, we also analyzed data for major metabolites.
208 crease = 12.9%, z = 1.836, P = 0.033). The differ-
RESULTS
Short-term effects Noradrenaline. As shown in Table I, a significant
ence was consistent across both replications at this interval, with 4 out of 4 kindled rats in the first replication and 4 out of 5 in the second replication displaying a decrease. A 5.3% decrease in N A in the contralateral frontal cortex was not significant (z = 1.126, P
decrease in concentrations of N A was found in the ip-
= 0.132), and there were no other significant regio-
silateral frontal cortex of kindled rats (mean de-
nal differences between kindled and control rats in
The results are summarized in Tables I and II.
A 2800-
/
B [ ] control • kindled
NA
~z 24001-
2000~
~
1~ IO00~ r ~ 60?-
1~oop 0/1~I
C FC
1400
z~
AM
[
r~ ~c
HYPO
o/1~I
HIPP
, FC
[ ] control • kindled
DA
[ ] control • kindled
1400[- NA ~: ~200~-I
700
1200
60C
1000
'~ 50C
800
4oc
6OO
300
AM
HYPO
HIPP
[ ] control • kindled
DA
oot
400 200
1oo
r/rti
0
t
c
o,
I
FC
c
AM
I
c
c
[ ] control • kindled
28o0/- 5 - HT
I
FC
HYPO
/
140C
c AM
l c HYPO
[ ] control • kindled
5-HT
120C
2400 I
.-~ IOQQ
~ 16001-
~" 80C 60C
~i
oollI
~ 4oc 20C 0
I
c
FC
I
c
AM
I
c
HYPO
[
c
C
HIPP
FC
I
c AM
I
c
HYPO
l
c
HIPP
Fig. 1. Mean absolute concentrations of NA, DA, and 5-HT in the brains of kindled and control rats killed two weeks (A) or 4 weeks (B) after the last kindled seizure. The vertical bars indicate S.E.M. AM, amygdala; FC, frontal cortex; HIPP, hippocampus; HYPO, hypothalamus.
209 TABLE I
Differences in regional concentrations of monoamines in kindled and control rats Rats were killed two weeks after the last seizure; differences are expressed as percentage difference scores derived according to the formula E-C× i00. Data are displayed as mean percentages _+ S.E.M. for the stimulated hemisphere (Ipsi) and the contraC lateral hemisphere (Contra).
Amygdala NA DA 5-HT
Frontal cortex
Hypothalamus
Hippocampus
lpsi
Contra
Ipsi
Contra
lpsi
Contra
lpsi
- 7.5-+6.3 -12.3+_14.0 - 8.7-+5.0*
+16.4+12.9 - 3.1+24.7 - 0.9-+4.6
-12.9+7.6 * +68.6+72.8 - 1.8+10.9
- 5.3-+7.3 +66.0-+29.4 - 2.1+9.0
+12.3+8.4 + 1.2-+4.4 -11.8-+6.3
+ 3.8-+12.4 -3.0_+9.0 - 4.6+9.2 -19.3+10.1" -2.1+__9.0
Contra + 6.0-+14.9 -13.4+_5.3"
* P < 0.05.
but a similar d i f f e r e n c e was n o t e v i d e n t in the ipsilat-
c o n c e n t r a t i o n s of N A .
Dopamine. N o significant changes in c o n c e n t r a -
eral h i p p o c a m p u s
(mean decrease
= 2.1%,
z =
0.700, P = 0.250). F o r the c o n t r a l a t e r a l h i p p o c a m -
tions of D A w e r e f o u n d ( T a b l e I).
Serotonin. A s s h o w n in T a b l e I, t h e r e was a signifi-
pus, h o w e v e r , t h e d e c r e a s e was again consistent
cant d e c r e a s e in c o n c e n t r a t i o n s of 5 - H T in t h e stimu-
across b o t h replications, a p p e a r i n g in 3 out of 3
lated a m y g d a l a of k i n d l e d rats (z = 1.68, P = 0.047),
k i n d l e d rats in t h e first r e p l i c a t i o n and in 4 o u t of 5 in
which averaged 8.7%. The lowered concentration of
the second. A significant d e c r e a s e in c o n c e n t r a t i o n s
5 - H T was o b s e r v e d in two of 3 k i n d l e d rats in t h e first
of 5 - H I A A was also e v i d e n t in t h e c o n t r a l a t e r a l hip-
r e p l i c a t i o n and in 4 of 5 in t h e second. C o n c e n t r a -
p o c a m p u s of k i n d l e d rats ( m e a n = 1 2 . 7 % , z = 1.820,
tions of 5 - H T in t h e c o n t r a l a t e r a l a m y g d a l a w e r e n o t
P = 0.035), w h e r e a s w e d e t e c t e d no o t h e r significant
affected by kindling. A significant d e p l e t i o n of 5 - H T
a l t e r a t i o n s of 5 - H I A A .
was also f o u n d in t h e c o n t r a l a t e r a l h y p o t h a l a m u s of k i n d l e d rats ( m e a n d e c r e a s e = 1 9 . 3 % , z = 1.720, P f o u n d in the ipsilateral h y p o t h a l a m u s but did n o t
Long-te)m effects Noradrenaline. T h e d e c r e a s e in c o n c e n t r a t i o n s of
r e a c h statistical significance (z = 1.600, P = 0.055).
N A in the frontal c o r t e x seen two w e e k s after kind-
= 0.043). A d e c r e a s e in 5 - H T a v e r a g i n g 11.8% was
N o significant c h a n g e was f o u n d in a m y g d a l o i d or hy-
ling did not persist at the l o n g e r i n t e r v a l ( T a b l e II):
p o t h a l a m i c 5 - H I A A in e i t h e r h e m i s p h e r e .
the m e a n d e c r e a s e in N A in t h e ipsilateral c o r t e x of
T h e r e was also a significant d e c r e a s e in 5 - H T con-
k i n d l e d rats was 1 . 3 % at 4 w e e k s (z = 1.64, P =
of
0.101), w h e r e a s t h e r e was an i n c r e a s e of 0 . 8 % in
k i n d l e d rats ( m e a n = 1 3 . 4 % , z = 1.960, P = 0.025),
c o n c e n t r a t i o n s of N A in the c o n t r a l a t e r a l c o r t e x , also
centrations
in the
contralateral
hippocampus
TABLE II
Differences in regional concentrations of monoamines in kindled and control rats Rats were killed 4 weeks after the last seizure; differences are expressed as percentage difference scores derived according to the formula of Table I. Data are displayed as mean percentages _+ S.E.M. for the stimulated hemisphere (Ipsi) and the contralateral hemisphere (Contra).
Amygdala NA DA 5-HT *P < 0.05.
Frontal cortex
Hypothalamus
Hippocampus
Ipsi
Contra
Ipsi
Contra
Ipsi
Contra
lpsi
Contra
-11.9+4.1 * + 6.5+15.8 - 4.2+5.8
-4.8+4.1 +7.4-+13.0 -2.6-+4.7
- 1.3+4.1 +43.2+21.3 - 9.5+7.8
+ 0.8+6.2 +12.8-+47.6 +13.5-+26.5
-10.1+3.4" +40.4+17.9 +10.2-+5.0
-3.2-+6.3 +7.4+13.0 +4.2+5.2
- 5.5+7.7 -29.2+8.6 *
-13.5+6.5 -17.9+9.2
210 not significant. However, concentrations of NA in the amygdala of kindled rats were significantly lower than in controls (mean difference = 11.9%, z = 2.31, P = 0.021), and this decrease was observed in 8 of the 9 pairs compared. Concentrations of NA in the ipsilateral hypothalamus were also significantly lower in kindled rats (mean difference = 10.1%, z = 2.1, P = 0.035), a difference that again was observed in 8 of the 9 pairs. There were no other significant regional variations in concentrations of N A after kindling. Dopamine. No significant changes in concentrations of D A were found (Table II). Serotonin. As shown in Table II, hypothalamic and amygdaloid concentrations of 5-HT had recovered to control levels by 4 weeks. There was, however, a significant decrease in hippocampal concentrations of 5HT in the ipsilateral hemisphere (mean decrease = 29.2%, z = 2.24, P = 0.025) that was observed in 8 of 9 kindled rats. A smaller and non-significant decrease was observed in the contralateral hippocampus (mean decrease = 17.9%, z = 1.54, P = 0.124), occurring in 7 of the 9 pairs compared. Concentrations of 5-HIAA were increased non-significantly in the ipsilateral hippocampus of kindled rats (mean = 16.1%, z = 1.541, P = 0.062).
Attempts to replicate previous effects For data obtained at the two-week interval, t-test analyses of NA concentrations in amygdala, hippocampus, and hypothalamus indicated that there were no significant differences between kindled and control rats in either the ipsilateral or contralateral hemispheres. This applied to both raw data and percentage difference scores and to both total and trimmed scores. For hypothalamic and hippocampal data probabilities ranged from 0.639 to 0.970. Changes in concentrations of NA in the amygdala most closely approached significance, but even here probability levels were in the range of 0.15-0.30. For data obtained at the 4-week interval, the only significant effect was in a percentage difference score, the 10% decrease in NA in the ipsilateral hypothalamus (t -2.98, P = 0.021, untrimmed; t = 2.82, P = 0.037, trimmed). This decrease was not found when raw data were analyzed, and no similar tendency occurred in the contralateral hypothalamus. t-Test analyses of the changes in the concentrations of D A in the amygdala of kindled rats were not
significant for either raw data or percentage difference scores, with probabilities ranging from 0.670 to 0.902. Finally, no significant increase in hypothalamic D A was found, although analysis of the percentage difference scores approached significance (t = -2.26, P = 0.058) in the ipsilateral hypothalamus. This probability increased to 0.122 when extreme scores were trimmed. Analyses of the raw data gave probabilities of 0.120 untrimmed and 0.160 trimmed. D A in the contralateral hypothalamus also failed to vary significantly (P = 0.41-0.76). DISCUSSION In the present experiment we found that amygdaloid kindling results in a short-lasting depletion of NA in the ipsilateral frontal cortex, followed by a late-appearing depletion of amygdaloid and hypothalamic NA; a short-lasting depletion of amygdaloid and hypothalamic 5-HT, followed by a late-appearing depletion of hippocampal 5-HT; and no significant changes in DA. In contrast to the reports of other investigators 7'28, we failed to find any significant variation in concentrations of D A after kindling. Indeed, the only finding of previous studies that was replicated in the present report was the short-lasting decrease in concentrations of NA in the ipsilateral frontal cortex. The 12.9% depletion reported here is considerably less than the 43% depletion reported by Callaghan and Schwark 9, but it was consistent across the two replications of the present study. The fact that this depletion of cortical NA was not observed at the longer interval and has not been reported in any other longterm studies suggests that it is an after-effect of seizures that recovers with time. Depletion of amygdaloid and hippocampal NA detectable at short intervals after kindling 9 was also not replicated here, nor did we find evidence of a decrease in hypothalamic NA s. However, we did observe a late-appearing decrease in NA in the stimulated amygdala and contralateral hippocampus. Although this perhaps is reminiscent of Callaghan and Schwark's finding 9, we are hesistant to attribute too much significance to it since there was no similar effect observed at two weeks and it has not been reported in any other studies of longlasting correlates of kindling. In any case, the func-
211 tional consequences of the variations in NA that we observed are unclear in view of our preliminary finding (unpublished) that they are not accompanied by variations in MHPG sulfate, a major metabolite of NA in rat brain whose concentrations are correlated with physiological activity of noradrenergic neurons 18. These results may have some bearing on the 'noradrenergic hypothesis' of kindling 1°,2°. If kindling is due in part to a progressive erosion of noradrenergic inhibitory influences, one could reasonably expect to find persisting alterations in concentrations of NA or metabolites in kindled brain. That such alterations were not found might be taken as evidence incompatible with the NA hypothesis. We note, however, that interference with the synaptic action of NA consistently affects only seizure development and not established seizures 5,31,32, suggesting that NA is involved in the processes modulating kindling itself but does not regulate the maintenance of kindled seizures. Thus alterations in the presynaptic or postsynaptic actions of N A could be important for kindling but might not be evident, either physiologically or biochemically, after the development of generalized seizures. From this perspective, therefore, it might be more appropriate to seek noradrenergic correlates during the early stages of kindling. It is of course also possible that kindling produces changes in concentrations of NA or its metabolites in regions of the nervous system other than those sampled here, such as the lower brainstem or spinal cord. The effects of kindling on concentrations of 5-HT reported here may be more consistent with the limited data previously reported. The short-term depletion of hypothalamic 5-HT that we observed is similar to the tendency in Munkenbeck and Schwark's
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data 24, and it also was replicated across each of the two short-term studies here. Furthermore, the significant depletion of 5-HT in the hippocampus contralateral to the stimulated amygdala at the shorter interval was consistent across both replications. However, no decrease was seen in the ipsilateral hippocampus. Yet at the longer interval the opposite pattern was seen, with a significant depletion of 5-HT occurring in the ipsilateral but not contralateral hippocampus. It is moot whether these changes in 5-HT are part of the mechanism of kindling or are merely an aftereffect of seizures, and they will have to be replicated independently before their significance can be assessed. In summary, amygdaloid kindling is correlated with small but significant decreases in regional concentrations of NA and 5-HT but not DA. The temporal and anatomical characteristics of these changes are complex, however, and do not fall into a simple and readily interpretable pattern. The monoaminergic correlates of kindling thus remain unresolved at present. Although a number of such correlates have been reported previously, we have been largely unable to replicate them. It is entirely possible that the changes in monoamines that we and others have described after kindling are only noise, not signal. ACKNOWLEDGEMENTS Supported by grants awarded to M.E.C. by the Natural Sciences and Engineering Research Council and the Medical Research Council and by an equipment grant from the British Columbia Health Care Research Foundation. We thank David Greeley and James Plant for capable technical assistance and Morag MacNeil for exceptional secretarial help.
mitters and receptor binding in amygdaloidkindled rats: serotonergic and noradrenergic modulatory effects, Life Sci., 27 (1980) 1547-1556. 5 Babington, R.G. and Wedeking, P.W., The pharmacology of seizures induced by sensitization with low intensity brain stimulation, Pharmacol. Biochem. Behav., 1 (1973) 461-467. 6 Blackwood,D., The role of noradrenaline and dopamine in amygdaloid kindling. In P.L. Morselli, K.G. Lloyd, W. Loscher, B. Meldrum and E.H. Reynolds (Eds.), Neurotransmitters, Seizures and Epilepsy, Raven, New York, 1981, pp. 203-211. 7 Burnham, W.M., King, G.A. and Lloyd, K.G., Extra-focal
212 catecholamine levels in 'kindled' rat forebrains, Prog. Neuropsychopharmacol., 5 (1981) 537-541. 8 Callaghan, D.A. and Schwark, W.S., Neurochemical changes and drug effects in a model of epilepsy in the rat, Soc. Neurosci. Abstr., 2 (1976) 257. 9 Callaghan, D.A. and Schwark, W.S., Involvement of catecholamines in kindled amygdaloid convulsions in the rat, Neuropharmacology, 18 (1979) 541-545. 10 Corcoran, M.E., Catecholamines and kindling. In J.A. Wada (Ed.), Kindling Two, Raven, New York, 1981, pp. 87-100. 11 Corcoran, M.E. and Cain, D.P., Kindling of seizures with low-frequency electrical stimulation, Brain Research, 196 (1980) 262-265. 12 Corcoran, M.E., Fibiger, H.C., McCaughran, J.A., Jr. and Wada, J.A., Potentiation of amygdaloid kindling and metrazol-induced seizures by 6-hydroxydopamine in rats, Exp. Neurol., 45 (1974) 118-133. 13 Corcoran, M.E. and Mason, S.T., Role of forebrain catecholamines in amygdaloid kindling, Brain Research, 190 (1980) 473-484. 14 Ehlers, C.L., Clifton, D.K. and Sawyer, C.H., Facilitation of amygdala kindling in the rat by transecting ascending noradrenergic pathways, Brain Research, 189 (1980) 274-278. 15 Engel, J. and Sharpless, N.S., Long-lasting depletion ofdopamine in the rat amygdala induced by kindling stimulation, Brain Research, 136 (1977) 381-386. 16 Heffner, T.G., Hartman, J.A. and Seiden, L.S., A rapid method for the regional dissection of the rat brain, Pharmacol. Biochem. Behav., 13 (1980) 453-456. 17 Kant, J.G., Meyerhoff, J.L. and Corcoran, M.E., Release of norepinephrine and dopamine from brain regions of amygdaloid-kindled rats, Exp. Neurol., 70 (1980) 701-705. 18 Korf, J., Aghajanian, G.K. and Roth, R.H., Stimulation and destruction of the locus coeruleus: opposite effects on 3-methoxy-4-hydroxyphenylglycol sulfate levels in the rat cerebral cortex, Eur. J. Pharmacol., 21 (1973) 305-310. 19 Kovacs, D.A. and Zoll, J.G., Seizure inhibition by median raphe nucleus stimulation in rat, Brain Research, 70 (1974) 165-169.
20 McIntyre, D.C., Catecholamine involvement in amygdala kindling of the rat. In J.A. Wada (Ed.), Kindling Two, Raven, New York, 1981, pp. 67-80. 21 McIntyre, D.C. and Edson, N., Facilitation of amygdala kindling after norepinephrine depletion with 6-hydroxydopamine in rats, Exp. Neurol., 74 (1981) 748-757. 22 McIntyre, D.C., Saari, M. and Pappas, B.A., Potentiation of amygdala kindling in adult or infant rats by injections of 6-hydroxydopamine, Exp. Neurol., 63 (1979) 527-544. 23 Mohr, E. and Corcoran, M.E., Depletion of noradrenaline and amygdaloid kindling, Exp. Neurol., 72 (1981) 507-511. 24 Munkenbeck, K.E. and Schwark, W.S., Serotonergic mechanisms in amygdaloid-kindled seizures in the rat, Exp. Neurol., 76 (1982) 246-253. 25 Okazaki, M.M., An Investigation of the Noradrenaline Hypothesis of Amygdala Kindling, Ph.D. Dissertation, University of Toronto, 1984. 26 Pellegrino, L.J., Pellegrino, A.S. and Cushman, A.J., A Stereotaxic Atlas of the Rat Brain, Plenum, New York, 1979. 27 Racine, R.J., Modification of seizure activity by electrical stimulation II. Motor seizure, Electroenceph. Clin. Neurophysiol., 32 (1972) 281-294. 28 Sato, M. and Nakashima, T., Kindling: secondary epileptogenesis, sleep and catecholamines, Can. J. Neurol. Sci., 2 (1975) 439-446. 29 Siegel, J. and Murphy, G.J., Serotonergic inhibition of amygdala-kindled seizures in cats, Brain Research, 174 (1979) 337-340. 30 Stock, G., Klummer, P., Stumpf, H., Zenner, K. and Sturm, V., Involvement of dopamine in amygdaloid kindling, Exp. NeuroL, 80 (1983) 439-450. 31 Wada, J.A., Pharmacological prophylaxis in the kindling model of epilepsy, Arch. Neurol., 34 (1977) 389-395. 32 Westerberg, V.S., Lewis, J. and Corcoran, M.E., Depletion of noradrenaline fails to affect kindled seizures, Exp. Neurol., 84 (1984) 237-240. 33 Wilkison, D.M. and Halpern, L.M., Turnover kinetics of dopamine and norepinephrine in forebrain after kindling in rats, Neuropharmacology, 18 (1979) 219-222.
205
BRE 12334
Research Reports
Monoaminergic correlates of kindling Johnnye Lewis*, Verner Westerberg* and Michael E. Corcoran Departmentof Psychology, Universityof Victoria, Victoria, B. C. (Canada) (Accepted 24 June 1986) Key words: Kindling; Experimental epilepsy; Amygdala; Noradrenaline; Dopamine; Serotonin
High-performance liquid chromatography with electrochemical detection was used to measure the regional concentrations of monoamines and metabolites in the brains of rats killed 2 or 4 weeks after kindling of generalized seizures with amygdaloid stimulation. Each kindled rat was compared to a yoked control that received brief trains of non-convulsive low-frequency stimulation of the amygdala. Two weeks after kindling we found a significant depletion of noradrenaline (NA) in the ipsilateral frontal cortex, a significant depletion of serotonin (5-HT) in the stimulated amygdala and contralateral hypothalamus, and no significant changes in concentration of dopamine (DA). Four weeks after kindling we found significant depletions of NA in the stimulated amygdala and ipsilateral hypothalamus, a significant depletion of 5-HT in the ipsilateral hippocampus, and no significant changes in DA. These findings generally fail to replicate previous reports of monoaminergic correlates of kindling. Furthermore, the alterations in monoamines produced by kindling do not fall into a simple and readily interpretable pattern. INTRODUCTION A considerable body of evidence points to a link between central catecholamines and the mechanisms of kindling 1°a9. Perhaps the strongest evidence is the finding that depletion of central noradrenaline (NA) or destruction of noradrenergic fibers produced by lesions or by injection of 6-hydroxydopamine (6O H D A ) results in a dramatic acceleration in the rate of kindling with electrical stimulation of limbic or neocortical sites 1-3,12-14,21-23. However, depletion of N A does not affect established seizures 32, suggesting that N A ' s suppressive action is limited to seizure development per se. 6 - O H D A - i n d u c e d depletion of forebrain dopamine ( D A ) appears to have no effect on rate of kindling 13,22. There is also some evidence that serotonin (5-HT) might modulate kindling. The 5-HT antagonists metergoline and mianserin have been reported to attenuate forepaw clonus in established seizures 4. The 5-
H T uptake inhibitor fluoxetine increased seizure thresholds, as did low frequency stimulation of the raphe 29. Depletion of stored 5-HT by p-chloroamphetamine failed, however, to lower thresholds 19. Electrical stimulation of the median raphe has also been shown to antagonize amygdaloid kindled seizures 19. There have been a number of studies of monoaminergic correlates of kindling. However, attempts to correlate short-term changes in monoamine concentrations with kindling have yielded quite inconsistent results. In some studies forebrain concentrations of N A and D A were unchanged after kindling 29'3°'32, whereas in other experiments localized depletion of N A 9 or widespread depletion of both N A and D A 27 was observed. Other investigators have described depletion of 5-HT restricted to the midbrain shortly after completion of amygdaloid kindling 23. Inconsistent results have also been found when monoamine concentrations have been measured at
* Present address: Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Manitoba, Winnipeg, Man., Canada, R3E 0W3. Correspondence: M.E. Corcoran. Present address (from 1 March 1987): Department of Psychology, University of Victoria, P.O. Box 1700, Victoria, B.C. Canada, V8W 2Y2. 0006-8993/87/$03,50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
206 long intervals after the last seizure. At one month postseizure a selective depletion of D A in the kindled amygdala was observed in one study 15, whereas other studies have reported no changes in NA and D A 6 or increases in hypothalamic D A and NA but no changes in striatal or limbic amines in amygdaloid kindled rats tested under similar conditions 7. In perhaps the most comprehensive study to date, Okazaki 25 found no changes in concentrations of N A or D A in any of 13 regions examined in the brains of amygdaloid kindled rats killed two months after their last seizure. Finally, Kant et al.17 found no change in in vitro release of N A and D A in tissue taken from amygdaloid kindled rats killed 3 weeks after their last seizure. The reported changes in turnover of monoamines after kindling have also been inconsistent. In one experiment 33 an increase in D A turnover in the ipsilateral forebrain but no change in NA turnover was found in amygdaloid kindled rats killed 7 days postseizure. In contrast, other investigators found no changes in turnover of catecholamines in amygdaloid kindled rats at one month postseizure 6 or at two months postseizure zS. The correlative data are hard to assess due to inconsistencies in both procedures and results in the relevant studies. First, two different species have been used in different experiments. Second, many of the earlier studies used the radioenzymatic assay for catecholamines, whereas the later studies employed high performance liquid chromatography (HPLC). Third, kindling stimulation was applied once daily in some studies and twice daily in others. Fourth, the endpoint for kindling varied considerably, from 3 to 20 generalized seizures (stage 5 in the terminology of Racine27). Finally, perhaps the most important inconsistency is that analyses were done at different intervals postseizure, from 24 h to two months. We have readdressed the question of monoaminergic correlates of kindling by examining alterations of monoamine concentrations in amygdaloid kindled rats killed at two and 4 weeks postseizure, using identical procedures for kindling and neurochemical analysis at both intervals. In addition, our study of the short-term correlates of kindling was performed in two replications to allow us to confirm positive results within the same laboratory.
MATERIALS AND METHODS We first examined possible short-term effects of kindling on concentrations of monoamines and their metabolites in rats killed two weeks after their last kindled seizure. This was done in two replications performed six months apart. We then examined ~'ossible long-term effects of kindling on monoam; ~es in rats killed 4 weeks after their last seizure. ~I*~is :~econd study was carried out 6 months after the second replication of short-term effects.
Subjects and surgery Male Long-Evans hooded rats (Charles River, Canada) weighing 300-350 g were used. They were handled every day after arrival in the colony. The rats were anesthetized with sodium pentobarbital, and bipolar electrodes were surgically implanted bilaterally in the basolateral amygdala, using the following coordinates from the atlas of Pellegrino et a1.26:0.8 mm posterior to bregma, 4.5 mm lateral, and 8.5 mm below the superior surface of the skull, with the incisor bar at +5.0 mm. Electrodes were constructed of twisted pairs of enameled nichrome wire (127/~m). Two weeks of recovery were allowed after surgery. For the short-term study, rats were randomly assigned to either the experimental group (n = 4 in the first replication; n = 5 in the second replication) or the stimulated control group (n = 4 in the first replication; n = 5 in the second replication). Rats from the two groups were yoked, and each yoked pair was thereafter treated identically in all respects but frequency of stimulation. In the long-term study, there were 8 such pairs of kindled rats and yoked controls, tested in one replication.
Kindling procedure Monopolar E E G was recorded from each amygdaloid electrode referenced to a surgical screw in the frontal bone. Electrical stimulation was in the form of balanced biphasic constant-current square-wave pulses, 1.0 ms in duration and delivered at 60 pulse pairs per s for 1 s daily. Thresholds were determined by initial stimulation of experimental animals at 30 /*A (base to peak) on the left side. The intensity was raised in increments of 10 # A until afterdischarge
207 (AD) was evoked, and the lowest such intensity was arbitrarily designated the A D threshold. No more than 4 stimulations per day at 2-min intervals were given during threshold determination. Experimental rats thereafter received 1 s of electrical stimulation at threshold intensity once daily. Yoked controls received 1 s of 3-Hz stimG,mon at the same intensity immediately after their experimental partner both during threshold determination and kindling trials. Although AD can be induced with trains of 3-Hz stimultion applied for 20 or more s and at higher intensities n, A D is not observed with 1-s trains applied at the intensities used here 11,17. By routinely monitoring E E G in the controls, we confirmed that no AD was evoked by the 3-Hz stimulation. The subjects were stimulated until experimental rats developed 3 stage-5 seizures 27. During the interval between the last seizure and sacrifice, all rats were handled at least twice weekly.
Dissection and chemistry At the appropriate interval after the third stage-5 seizure (two weeks for the short-term group; 4 weeks for the long-term group) rats were killed by cervical fracture, and their brains were removed and rapidly dissected over ice by the procedure of Heffner et al. 16. Right and left samples of frontal cortex, amygdala/pyriform, hypothalamus, and hippocampus were each homogenized on ice in 500#1 of 0.05 N hydrochloric acid/0.1 M cysteine (with D H B A added as an internal standard) by a Brinkman polytron at setting 4 for 20 s. The homogenate was centrifuged at 15,000 g for 20 min at 4 °C. Supernatant w a s then centrifugally filtered at 3000 g through 0.2/~m filters. The filtrate was frozen in liquid nitrogen and stored at-196 °C until analysis. Monoamines and metabolites were quantified by reverse-phase HPLC with electrochemical detection. We used a BAS LC-4B amperometric detector equipped with a glassy carbon working electrode and a Ag/AgCI 2 reference electrode. The chromatographic column was a Biophase ODS 0.5-pm reversephase column (250 mm x 4.6 mm i.d.), protected by a Brownlee Labs RP-18 spheri-5 guard column. Twenty pl of thawed sample was injected directly, and monoamines and metabolites were eluted by a 6% methanol-water mobile phase (v/v), pH 3.1, containing 0.15 M chloroacetate buffer, 0.03 M
Na2HPO 4 (anhydrous), 0.1 mM E D T A , and 200 mg/ liter SOS. The flow rate was 1.0-1.5 ml/min, at a temperature of 37 °C. Single-electrode detection was performed at an oxidation potential of +0.8 V. This system produced good separation for NA, adrenaline (A), D H B A (internal standard), DA, dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindole acetic acid (5-HIAA), homovanillic acid (HVA) and 5-HT. Mean regional concentrations of amines determined with our technique are shown in Fig. 1. During dissection and chemistry each kindled rat was always treated in parallel with its yoked control.
Statistics Data in the present studies were collected over a period of two years. The unavoidable changes in chemicals, columns, and other conditions resulted in high within-cell variability, although the yoking of experimental and control rats controlled for this within the pairs that were directly compared. This factor, along with the relatively small numbers of animals (9 pairs in the short-term study and 8 pairs in the long-term study), made analysis by non-parametric techniques the most appropriate method. To take full advantage of the matched groups design and examine the question of direction of change, a Wilcoxon test for matched pairs was used to analyze percentage difference scores, defined as follows: score = ((E-C)/C) × 100, where E represents the regional concentration of the relevant amine in a kindled rat and C represents comparable data from its yoked control. Since we were attempting to replicate previous studies, an a level of 0.05 was used. In those cases where our results failed to replicate previous findings, we also report t-tests of the significance of the differences between control rats and kindled rats, both in the raw data and in percentage change scores, in an effort to ensure that the method of analysis was not biasing the results. These results are reported from all data in each test (untrimmed) and for data with extreme scores excluded (trimmed), to ensure that replication was not either occurring because of, or being suppressed by, one or two extreme scores. In cases where significant changes in concentration of the parent monoamine were obtained, we also analyzed data for major metabolites.
208 crease = 12.9%, z = 1.836, P = 0.033). The differ-
RESULTS
Short-term effects Noradrenaline. As shown in Table I, a significant
ence was consistent across both replications at this interval, with 4 out of 4 kindled rats in the first replication and 4 out of 5 in the second replication displaying a decrease. A 5.3% decrease in N A in the contralateral frontal cortex was not significant (z = 1.126, P
decrease in concentrations of N A was found in the ip-
= 0.132), and there were no other significant regio-
silateral frontal cortex of kindled rats (mean de-
nal differences between kindled and control rats in
The results are summarized in Tables I and II.
A 2800-
/
B [ ] control • kindled
NA
~z 24001-
2000~
~
1~ IO00~ r ~ 60?-
1~oop 0/1~I
C FC
1400
z~
AM
[
r~ ~c
HYPO
o/1~I
HIPP
, FC
[ ] control • kindled
DA
[ ] control • kindled
1400[- NA ~: ~200~-I
700
1200
60C
1000
'~ 50C
800
4oc
6OO
300
AM
HYPO
HIPP
[ ] control • kindled
DA
oot
400 200
1oo
r/rti
0
t
c
o,
I
FC
c
AM
I
c
c
[ ] control • kindled
28o0/- 5 - HT
I
FC
HYPO
/
140C
c AM
l c HYPO
[ ] control • kindled
5-HT
120C
2400 I
.-~ IOQQ
~ 16001-
~" 80C 60C
~i
oollI
~ 4oc 20C 0
I
c
FC
I
c
AM
I
c
HYPO
[
c
C
HIPP
FC
I
c AM
I
c
HYPO
l
c
HIPP
Fig. 1. Mean absolute concentrations of NA, DA, and 5-HT in the brains of kindled and control rats killed two weeks (A) or 4 weeks (B) after the last kindled seizure. The vertical bars indicate S.E.M. AM, amygdala; FC, frontal cortex; HIPP, hippocampus; HYPO, hypothalamus.
209 TABLE I
Differences in regional concentrations of monoamines in kindled and control rats Rats were killed two weeks after the last seizure; differences are expressed as percentage difference scores derived according to the formula E-C× i00. Data are displayed as mean percentages _+ S.E.M. for the stimulated hemisphere (Ipsi) and the contraC lateral hemisphere (Contra).
Amygdala NA DA 5-HT
Frontal cortex
Hypothalamus
Hippocampus
lpsi
Contra
Ipsi
Contra
lpsi
Contra
lpsi
- 7.5-+6.3 -12.3+_14.0 - 8.7-+5.0*
+16.4+12.9 - 3.1+24.7 - 0.9-+4.6
-12.9+7.6 * +68.6+72.8 - 1.8+10.9
- 5.3-+7.3 +66.0-+29.4 - 2.1+9.0
+12.3+8.4 + 1.2-+4.4 -11.8-+6.3
+ 3.8-+12.4 -3.0_+9.0 - 4.6+9.2 -19.3+10.1" -2.1+__9.0
Contra + 6.0-+14.9 -13.4+_5.3"
* P < 0.05.
but a similar d i f f e r e n c e was n o t e v i d e n t in the ipsilat-
c o n c e n t r a t i o n s of N A .
Dopamine. N o significant changes in c o n c e n t r a -
eral h i p p o c a m p u s
(mean decrease
= 2.1%,
z =
0.700, P = 0.250). F o r the c o n t r a l a t e r a l h i p p o c a m -
tions of D A w e r e f o u n d ( T a b l e I).
Serotonin. A s s h o w n in T a b l e I, t h e r e was a signifi-
pus, h o w e v e r , t h e d e c r e a s e was again consistent
cant d e c r e a s e in c o n c e n t r a t i o n s of 5 - H T in t h e stimu-
across b o t h replications, a p p e a r i n g in 3 out of 3
lated a m y g d a l a of k i n d l e d rats (z = 1.68, P = 0.047),
k i n d l e d rats in t h e first r e p l i c a t i o n and in 4 o u t of 5 in
which averaged 8.7%. The lowered concentration of
the second. A significant d e c r e a s e in c o n c e n t r a t i o n s
5 - H T was o b s e r v e d in two of 3 k i n d l e d rats in t h e first
of 5 - H I A A was also e v i d e n t in t h e c o n t r a l a t e r a l hip-
r e p l i c a t i o n and in 4 of 5 in t h e second. C o n c e n t r a -
p o c a m p u s of k i n d l e d rats ( m e a n = 1 2 . 7 % , z = 1.820,
tions of 5 - H T in t h e c o n t r a l a t e r a l a m y g d a l a w e r e n o t
P = 0.035), w h e r e a s w e d e t e c t e d no o t h e r significant
affected by kindling. A significant d e p l e t i o n of 5 - H T
a l t e r a t i o n s of 5 - H I A A .
was also f o u n d in t h e c o n t r a l a t e r a l h y p o t h a l a m u s of k i n d l e d rats ( m e a n d e c r e a s e = 1 9 . 3 % , z = 1.720, P f o u n d in the ipsilateral h y p o t h a l a m u s but did n o t
Long-te)m effects Noradrenaline. T h e d e c r e a s e in c o n c e n t r a t i o n s of
r e a c h statistical significance (z = 1.600, P = 0.055).
N A in the frontal c o r t e x seen two w e e k s after kind-
= 0.043). A d e c r e a s e in 5 - H T a v e r a g i n g 11.8% was
N o significant c h a n g e was f o u n d in a m y g d a l o i d or hy-
ling did not persist at the l o n g e r i n t e r v a l ( T a b l e II):
p o t h a l a m i c 5 - H I A A in e i t h e r h e m i s p h e r e .
the m e a n d e c r e a s e in N A in t h e ipsilateral c o r t e x of
T h e r e was also a significant d e c r e a s e in 5 - H T con-
k i n d l e d rats was 1 . 3 % at 4 w e e k s (z = 1.64, P =
of
0.101), w h e r e a s t h e r e was an i n c r e a s e of 0 . 8 % in
k i n d l e d rats ( m e a n = 1 3 . 4 % , z = 1.960, P = 0.025),
c o n c e n t r a t i o n s of N A in the c o n t r a l a t e r a l c o r t e x , also
centrations
in the
contralateral
hippocampus
TABLE II
Differences in regional concentrations of monoamines in kindled and control rats Rats were killed 4 weeks after the last seizure; differences are expressed as percentage difference scores derived according to the formula of Table I. Data are displayed as mean percentages _+ S.E.M. for the stimulated hemisphere (Ipsi) and the contralateral hemisphere (Contra).
Amygdala NA DA 5-HT *P < 0.05.
Frontal cortex
Hypothalamus
Hippocampus
Ipsi
Contra
Ipsi
Contra
Ipsi
Contra
lpsi
Contra
-11.9+4.1 * + 6.5+15.8 - 4.2+5.8
-4.8+4.1 +7.4-+13.0 -2.6-+4.7
- 1.3+4.1 +43.2+21.3 - 9.5+7.8
+ 0.8+6.2 +12.8-+47.6 +13.5-+26.5
-10.1+3.4" +40.4+17.9 +10.2-+5.0
-3.2-+6.3 +7.4+13.0 +4.2+5.2
- 5.5+7.7 -29.2+8.6 *
-13.5+6.5 -17.9+9.2
210 not significant. However, concentrations of NA in the amygdala of kindled rats were significantly lower than in controls (mean difference = 11.9%, z = 2.31, P = 0.021), and this decrease was observed in 8 of the 9 pairs compared. Concentrations of NA in the ipsilateral hypothalamus were also significantly lower in kindled rats (mean difference = 10.1%, z = 2.1, P = 0.035), a difference that again was observed in 8 of the 9 pairs. There were no other significant regional variations in concentrations of N A after kindling. Dopamine. No significant changes in concentrations of D A were found (Table II). Serotonin. As shown in Table II, hypothalamic and amygdaloid concentrations of 5-HT had recovered to control levels by 4 weeks. There was, however, a significant decrease in hippocampal concentrations of 5HT in the ipsilateral hemisphere (mean decrease = 29.2%, z = 2.24, P = 0.025) that was observed in 8 of 9 kindled rats. A smaller and non-significant decrease was observed in the contralateral hippocampus (mean decrease = 17.9%, z = 1.54, P = 0.124), occurring in 7 of the 9 pairs compared. Concentrations of 5-HIAA were increased non-significantly in the ipsilateral hippocampus of kindled rats (mean = 16.1%, z = 1.541, P = 0.062).
Attempts to replicate previous effects For data obtained at the two-week interval, t-test analyses of NA concentrations in amygdala, hippocampus, and hypothalamus indicated that there were no significant differences between kindled and control rats in either the ipsilateral or contralateral hemispheres. This applied to both raw data and percentage difference scores and to both total and trimmed scores. For hypothalamic and hippocampal data probabilities ranged from 0.639 to 0.970. Changes in concentrations of NA in the amygdala most closely approached significance, but even here probability levels were in the range of 0.15-0.30. For data obtained at the 4-week interval, the only significant effect was in a percentage difference score, the 10% decrease in NA in the ipsilateral hypothalamus (t -2.98, P = 0.021, untrimmed; t = 2.82, P = 0.037, trimmed). This decrease was not found when raw data were analyzed, and no similar tendency occurred in the contralateral hypothalamus. t-Test analyses of the changes in the concentrations of D A in the amygdala of kindled rats were not
significant for either raw data or percentage difference scores, with probabilities ranging from 0.670 to 0.902. Finally, no significant increase in hypothalamic D A was found, although analysis of the percentage difference scores approached significance (t = -2.26, P = 0.058) in the ipsilateral hypothalamus. This probability increased to 0.122 when extreme scores were trimmed. Analyses of the raw data gave probabilities of 0.120 untrimmed and 0.160 trimmed. D A in the contralateral hypothalamus also failed to vary significantly (P = 0.41-0.76). DISCUSSION In the present experiment we found that amygdaloid kindling results in a short-lasting depletion of NA in the ipsilateral frontal cortex, followed by a late-appearing depletion of amygdaloid and hypothalamic NA; a short-lasting depletion of amygdaloid and hypothalamic 5-HT, followed by a late-appearing depletion of hippocampal 5-HT; and no significant changes in DA. In contrast to the reports of other investigators 7'28, we failed to find any significant variation in concentrations of D A after kindling. Indeed, the only finding of previous studies that was replicated in the present report was the short-lasting decrease in concentrations of NA in the ipsilateral frontal cortex. The 12.9% depletion reported here is considerably less than the 43% depletion reported by Callaghan and Schwark 9, but it was consistent across the two replications of the present study. The fact that this depletion of cortical NA was not observed at the longer interval and has not been reported in any other longterm studies suggests that it is an after-effect of seizures that recovers with time. Depletion of amygdaloid and hippocampal NA detectable at short intervals after kindling 9 was also not replicated here, nor did we find evidence of a decrease in hypothalamic NA s. However, we did observe a late-appearing decrease in NA in the stimulated amygdala and contralateral hippocampus. Although this perhaps is reminiscent of Callaghan and Schwark's finding 9, we are hesistant to attribute too much significance to it since there was no similar effect observed at two weeks and it has not been reported in any other studies of longlasting correlates of kindling. In any case, the func-
211 tional consequences of the variations in NA that we observed are unclear in view of our preliminary finding (unpublished) that they are not accompanied by variations in MHPG sulfate, a major metabolite of NA in rat brain whose concentrations are correlated with physiological activity of noradrenergic neurons 18. These results may have some bearing on the 'noradrenergic hypothesis' of kindling 1°,2°. If kindling is due in part to a progressive erosion of noradrenergic inhibitory influences, one could reasonably expect to find persisting alterations in concentrations of NA or metabolites in kindled brain. That such alterations were not found might be taken as evidence incompatible with the NA hypothesis. We note, however, that interference with the synaptic action of NA consistently affects only seizure development and not established seizures 5,31,32, suggesting that NA is involved in the processes modulating kindling itself but does not regulate the maintenance of kindled seizures. Thus alterations in the presynaptic or postsynaptic actions of N A could be important for kindling but might not be evident, either physiologically or biochemically, after the development of generalized seizures. From this perspective, therefore, it might be more appropriate to seek noradrenergic correlates during the early stages of kindling. It is of course also possible that kindling produces changes in concentrations of NA or its metabolites in regions of the nervous system other than those sampled here, such as the lower brainstem or spinal cord. The effects of kindling on concentrations of 5-HT reported here may be more consistent with the limited data previously reported. The short-term depletion of hypothalamic 5-HT that we observed is similar to the tendency in Munkenbeck and Schwark's
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data 24, and it also was replicated across each of the two short-term studies here. Furthermore, the significant depletion of 5-HT in the hippocampus contralateral to the stimulated amygdala at the shorter interval was consistent across both replications. However, no decrease was seen in the ipsilateral hippocampus. Yet at the longer interval the opposite pattern was seen, with a significant depletion of 5-HT occurring in the ipsilateral but not contralateral hippocampus. It is moot whether these changes in 5-HT are part of the mechanism of kindling or are merely an aftereffect of seizures, and they will have to be replicated independently before their significance can be assessed. In summary, amygdaloid kindling is correlated with small but significant decreases in regional concentrations of NA and 5-HT but not DA. The temporal and anatomical characteristics of these changes are complex, however, and do not fall into a simple and readily interpretable pattern. The monoaminergic correlates of kindling thus remain unresolved at present. Although a number of such correlates have been reported previously, we have been largely unable to replicate them. It is entirely possible that the changes in monoamines that we and others have described after kindling are only noise, not signal. ACKNOWLEDGEMENTS Supported by grants awarded to M.E.C. by the Natural Sciences and Engineering Research Council and the Medical Research Council and by an equipment grant from the British Columbia Health Care Research Foundation. We thank David Greeley and James Plant for capable technical assistance and Morag MacNeil for exceptional secretarial help.
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