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Kindling with stimulation of the dentate gyrus. I. Characterization of electrographic and behavioral events
Brain Research, 509 (1990) 249-256 Elsevier
249
BRES 15202
Kindling with stimulation of the dentate gyrus. I. Characterization of electrographic and behavioral events Gloria M. Grace, Michael E. Corcoran and Ronald W. Skelton Department of Psychology, University of Victoria, Victoria, B.C. (Canada) (Accepted 18 July 1989)
Key words: Kindling; Dentate gyrus; Hippocampal formation
Once daily for 60 days, hooded rats received unilateral high-frequency stimulation in the hilus of the dentate gyrus (DG), at an intensity sufficient to evoke epileptiform afterdischarge (AD). Although most rats eventually developed generalized stage-5 seizures (Generalized group), some did not progress beyond partial stage-1 or stage-2 seizures (Partial group). Hilar kindling also displayed several other characteristics that distinguished it from typical limbic kindling, including low rate of development, marked instability of the seizures, and little or no growth in duration of AD.
INTRODUCTION In the present study we describe the electrographic and behavioral characteristics of kindling with stimulation in the hilus of the d e n t a t e gyrus ( D G ) . T h e hilus was of interest because it is the area of the h i p p o c a m p a l f o r m a t i o n through which the mossy fibers course on their way to h i p p o c a m p a l field CA3. The mossy fibers are the axons of the granule cells of the D G , neurons that contain both an amino acid n e u r o t r a n s m i t t e r and several different opioid p e p t i d e s ~3. Some of the behavioral effects of electrical stimulation of the hilus can be reversed by t r e a t m e n t with the opiate antagonist naloxone 9`t°, suggesting that electrical stimulation of the mossy fibers can result in the release of opioid peptides. Because r e p e a t e d infusions of low concentrations of opioid p e p t i d e s into the D G and h i p p o c a m p u s can kindle seizures 5"6, it would be of interest to assess the susceptibility of the D G to kindling with electrical stimulation. Several previous studies have described kindling in different regions of the h i p p o c a m p a l formation. Differences have been noted in the rate and pattern of kindling in the p y r a m i d a l cell fields of the dorsal and ventral h i p p o c a m p u s lx'26. Kindling with stimulation of the perforant path (PP), the m a j o r projection from the entorhinal cortex to the granule cells of the D G , has been described 12"19"2°'2x~31"32. H o w e v e r , only limited information is available ~2'14 concerning kindling of the hilus of the D G , the region between the u p p e r and lower blades
of granule cells. We therefore characterized hilar kindling in terms of rate and p a t t e r n of d e v e l o p m e n t of clinical seizures; threshold of afterdischarge ( A D ) ; frequency, amplitude, and duration of A D ; n u m b e r of days off (i.e. days in which stimulation failed to e v o k e A D after it had previously been elicited); and behavioral effects. We also m o n i t o r e d field potentials (population spikes and population excitatory postsynaptic potentials (EPSPs)) in the hilus e v o k e d by stimulation of the PP during kindling. These electrophysiological correlates of hilar kindling are r e p o r t e d in the accompanying p a p e r ~5'1. MATERIALS AND METHODS
Animals Male hooded rats of the Long-Evans strain were used, weighing between 300 and 375 g at the time of surgery. The rats were housed in individual stainless steel cages with food and water available ad libitum. A 12 h light-dark cycle was in effect throughout the study, and all testing was conducted during the light portion of the cycle. Surgery Using pentobarbital anesthesia (60 mg/kg), chronic electrodes were implanted in the hilus of the DG and in the PP. The hilar electrode was used for delivery of kindling stimulation and recording of EEG activity. The PP electrode was used for recording EEG during kindling sessions and for evoking field potentials in the DG, as described separately~5~'. Each electrode consisted of a single strand of stainless-steel wire, 761~m in diameter, coated with Teflon to a total diameter of 114 urn. Cemented to each electrode, approximately 5 mm above the tip, was a small plastic bead that served to anchor the electrode in the dental acrylic. Between 0.25 and (I.5 mm of insulation on the tips of the electrodes was removed. Two skull screws, connected to uninsulated stainless-steel wire,
Correspondence. R.W. Skelton, Department of Psychology, University of Victoria, P.O. Box 1700, Victoria, B.C., Canada V8W 2Y2, 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
25(I served as current-return and ground-reference electrodes. Goldplated pins (Amphenol 220-S02) were soldered to the ends of the 4 wires, inserted into a small connector, and affixed to the skull using dental acrylic and two additional anchoring skull screws. Electrodes were implanted in the left side of the brain under electrophysiological guidance. With the skull level between bregma and lambda, coordinates for the hilus were 4.0 mm posterior to bregma, 2.0 mm lateral to the midline, and 3.3 mm ventral to the surface of the cortex; coordinates for the PP were 8.1 mm posterior to bregma, 4.3 mm lateral to the midline, and 2.4 mm ventral to the surface of the cortex. As both electrodes were lowered, a storage oscilloscope displayed field potentials recorded from the electrode in the hilus, evoked by single square-wave pulses delivered to the PP electrode (0.1 ms, 0.2 Hz, 400/xA). The positions and depths of the electrodes were adjusted to produce a hilar evoked potential with a population spike of maximum amplitude and minimum threshold. After a 12 day recovery period, population spike amplitudes were measured in response to a stimulus intensity of 400 flA. Rats with amplitudes of less than 6 mV were excluded from the study. Field potentials were monitored on a regular basis throughout kindling, and rats that exhibited unstable field potentials were also excluded.
on each day, and spike frequency was measured from the 4 s block containing the highest frequency spikes recorded from that electrode for each rat on each day.
Histology At the end of the experiment, rats were given an overdose of pentobarbital, and were perfused with normal saline followed by 10% formalin. Brains were sectioned into 40 um slices, mounted on slides, and stained with thionin.
Statistical analysis The data were analyzed with independent t-tests, with two-way analysis of variance (ANOVA; group x kindling period) with repeated measures, and, where appropriate, with multivariate analysis of variance. A rejection criterion of P < 0.05 was used.
RESULTS A f t e r r e c o v e r y f r o m s u r g e r y , 20 r a t s d i s p l a y e d field potentials with spike amplitudes above 6 mV (range 9-20
Determination of A D threshold
m V ) in r e s p o n s e to a s i n g l e 4 0 0 / ~ A p u l s e . D u r i n g t h e
Electrical stimulation for kindling consisted of a 1 s train of constant current balanced biphasic square-wave pulses, with a pulse width of 1.0 ms and a frequency of 60/s. On the first day, a series of stimulations was delivered, once every 3 min, starting at 30 ~tA (base-to-peak) and increasing in 20/~A steps until AD was elicited. On the following day, the stimulation intensity was decreased by steps of 10/xA from this first 'threshold' until the AD was no longer evoked. Threshold was defined as the minimum intensity sufficient to evoke AD on this second day.
study, 4 rats were rejected due to problems with their e l e c t r o d e s so t h a t u l t i m a t e l y a t o t a l o f 16 r a t s u n d e r w e n t the entire kindling procedure.
Kindling rates Ten
of the
16 r a t s
developed
generalized
s e i z u r e s , w i t h i n a m e a n o f 18.2 ( + 1 2 . 3
stage-5
S.D.) stimula-
tions. T h e r e m a i n i n g 6 r a t s p r o g r e s s e d o n l y t o s t a g e 1
Kindling
(n = 1), s t a g e 2 (n = 4), o r s t a g e 3 ( n = 1) s e i z u r e s ,
Rats were stimulated once daily at their threshold intensities, and the intensity and duration of the seizures were noted (see ref. 25 for behavioral classification system used). When stage-5 generalized seizures were noted on 3 consecutive stimulation days, stimulation was discontinued for 3 weeks. After this rest period, rats were rekindled by administering stimulation once daily until a stage-5 seizure was displayed, in order to test the permanency of the kindling. Rats that did not exhibit any stage-5 seizures were stimulated for a maximum of 60 days. Before and after each stimulation, referential EEG was recorded from both the hilus and PP, and evoked AD was subsequently analysed for total duration of all episodes, and for spike frequency and spike amplitude. Mean amplitude of AD was measured from the 4 s block containing the largest amplitude spikes recorded from that electrode for each rat
a l t h o u g h t h e y w e r e s t i m u l a t e d f o r 60 d a y s (i.e. 3.4 S . D . l o n g e r ) . B e c a u s e o f t h i s s t r i k i n g d i f f e r e n c e in k i n d l i n g , we
considered
Generalized
the
former
10 r a t s
kindling group
and
as c o n s t i t u t i n g
the
l a t t e r 6 r a t s as
c o n s t i t u t i n g a P a r t i a l k i n d l i n g g r o u p . T a b l e I lists t h e number of ADs to each stage of seizure and number of regressions for both groups. between
the
Partial
and
There
was no difference
Generalized
groups
in t h e
n u m b e r o f A D s t o t h e first stage-1 s e i z u r e (/14 = 0~t0, P = 0 . 9 2 ) , b u t r a t s in t h e G e n e r a l i z e d g r o u p r e q u i r e d f e w e r
TABLE I
Number of A D s to each stage of seizure and number of regressions Criterion: 3 consecutive stage-5 seizures. Regression: prior to criterion, n = number of rats displaying this seizure stage.
Criterion
Regressions
18.2 3.9 10
28.8 3.7 10
6.0 1.3 10
-
-
-
-
Stageofseizure
Generalized group Mean S.E.M. n Partial group Mean S.E.M. n
a
0
1
2
3
4
10
4.4 0.7 10
13.8 1.8 8
22.0 3.0 2
16.8 2.6 4
6
4.5 0.6 6
38.8 5.4 5
46.0 . l
.
. -
.
8.8 1.7 6
251 become prostrate and exhibited generalized clonic jerking; they did not display the rearing behavior commonly seen in an amygdaloid stage-5 seizure, or the usual progression through the seizure stages (1-5). As is commonly observed with limbic kindling, the rats would typically remain still after the clonic component of the convulsion and appeared to be unresponsive to the environment. This was followed by several minutes of automatisms, during which the rats would appear to be trying to climb the walls of the recording box. For several minutes after the automatisms, the rats showed signs of hyperreactivity to sensory stimuli.
ADs to develop stage-2 seizures than rats in the Partial group (t H = 5.24, P < 0.01). Development of stage-3 and stage-4 seizures was rare, and occurred only once or twice in a few rats. Regression to less severe behavioral seizure stages was equally common in both groups (t14 = 1.31, P = 0.21). The Generalized group displayed large variability in the number of ADs to the first stage-5 seizure (range = 5-41; mean --- 18.2) and in the number of ADs to 3 consecutive stage-5 seizures (range = 14-47; mean = 28.8). Most rats in this group developed seizures abruptly and progressed from stage-1 seizures directly to stage-5 seizures, without developing the intermediate stages of seizure characteristic of limbic kindling s'15"25. After the 3-week rest period, all rats in the Generalized group displayed a stage-5 seizure after only one or two ADs. However, one rat demonstrated 4 days off (i.e. no AD in response to stimulation) in the process. In addition to the unusual pattern of seizure development, the Generalized group also displayed unusual stage-5 seizure topography, During AD, the rats often
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A D threshold The mean threshold for A D in all 16 rats was 42/~A (S.E.M. = 8, range -- 15-130). Rats in the Generalized group had slightly lower thresholds (mean = 37.5, S.E.M. -- 7.27, range = 15-80) than rats in the Partial group (mean = 49, S.D. = 18.4, range -- 15-130), but the difference was not significant (t14 -0.70, P = 0.50).
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Fig. 1. Sample electroencephalogram (EEG) illustrating the changes in afterdischarge (AD) that occurred during kindling. The lower trace is a record from the stimulation/recording site in the hilus of the dentate gyrus (DG), and the upper trace is a record from the recording electrode in the ipsilateral perforant path (PP). A: the 1st A D induced by hilar stimulation; there was no behavioral seizure. B: the 8th A D from the same rat, during a partial seizure. Note the increase in A D frequency from the hilus relative to the first A D . C - E : continuous record of the 26th A D from the same rat, during a generalized seizure. Note the increased duration and complexity of the A D from both the DG and PP electrodes. Calibrations: 5 s, upper trace, 600 ~V; lower trace, 3.0 mV.
i
252
A
100r
b 12
DG-Gen~
A D
eo~
D U R A T
s° i
I
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..1~ -~t~
4o ~ "-O
°f N
2 L~
i Fltat
o
Flnll
Middle AD E P I S O D E
~
5
D
L FIrM
8eoond
Third
KINDLING PERIOD
Fig. 3. Changes in the duration of afterdischarge (AD) recorded from the dentate gyrus (DG) and perforant path (PP) in the Generalized (Gen) and Partial (Part) groups. Duration of AD (s) was the sum of primary and secondary ADs for each rat on each day, averaged over each of three equal phases of kindling (1/3 total number of kindling stimulations).
B A
20
~ 0
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A M P L
4
T U D E
a
a
DO-Gen -<) DG-Psr t -I- PP-GBn -C} PP-.Part []
I 0
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FIrM
Mid(lie AD E P I S O D E
Final
Fig. 2. Changes in afterdischarge (AD) recorded from the dentate gyrus (DG) and perforant path (PP) electrodes over kindling in the Generalized (Gen) and Partial (Part) groups. A: spike frequency (spikes/s), measured in the 4 s block of AD containing the highest frequency spikes of the first, middle, and final AD of kindling. B: spike amplitude (uV peak-to-peak), measured in the 4 s block of AD containing the largest spikes of the first, middle, and final AD of kindling.
General morphology of AD The A D recorded from both PP and hilar electrodes typically began 2-4 s after the kindling stimulation ended. Hilar A D usually subsided first, and the AD recorded from the PP electrode continued for several more seconds (see Fig. 1). The AD in the hilus was almost always followed by a period of flat E E G and then gradual recovery of E E G amplitude to pre-stimulation levels. Although the amplitude of E E G recorded from the PP was lower than baseline after the AD, it was never as flat as that recorded from the hilus. There was usually a secondary episode of AD, briefer and less intense than the first in terms of both frequency and intensity, which was generally recorded from both electrode locations. Occasionally, the secondary episodes were limited to the PP region. The waveform of the A D changed over the course of kindling. A D was initially composed of regularly spaced simple biphasic spikes. However, with further stimulation
the waveform became more complex and variable, with periods of notched or double spike configurations or very high-frequency bursts. This progression of waveform complexity has also been noted in amygdaloid A D 25. The frequency of the epileptiform spikes in the A D increased significantly over the course of kindling, in both the hilar and the PP regions (hilus: F2,13 = 31.0, P < 0.01; PP: F2,13 = 12.62, P < 0.01) (see Fig. 2A). The AD frequency in the Generalized group was greater than that in the Partial group, but the difference was significant only in the PP region (F1:4 = 10.12, P < 0.01) and not in the hilus (F~: 4 = 3.59, P = 0.08). There was no significant group by phase interaction for either electrode location. The amplitude of A D was much higher in the hilus than in the PP region (see Fig. 2B). Marginal increases in amplitude were observed in both locations, a n d the increase was significant only in the PP region (F2,13 = 6.17, P < 0.02) and not in the hilus (F2,]3 = 0.30, P = 0.74). Like A D frequency, A D amplitude was greater in the Generalized group than in the Partial group, and this difference was again significant only in the PP region (Fl.14 = 9.09, P < 0.01) and not in the hilus (F1.14 = 0.73, P = 0.41). Similarly, there was no significant group by phase interaction for either electrode location. Low-frequency postictal spiking was observed in both the Generalized and Partial groups, although the proportion of rats displaying postictal spikes was higher in the Generalized group (8 of 10 rats) than in the Partial group (3 of 6 rats).
Growth of AD In order to assess changes in A D duration over kindling, and to compare rats that received different
253
1.6
W E
1.4
T
1.2
D 0 G
1
S H A K E S
I~D'- General
~-o ,.r..e,
o.e o.e 0.4 o.a Flriit
8eoond
Third
KINDLING P E R I O D
Fig. 4. Mean number of wet dog shakes per day for each rat in the Generalized (Gen) and Partial (Part) groups, within each of 3 equal phases of kindling (1/3 total number of kindling stimulations).
numbers of stimulation days, each rat's stimulation series was divided into 3 equal phases (early, middle and late). The total A D duration for each day (i.e. the sum of primary and secondary episodes) was then averaged for each rat over each phase. This analysis revealed that AD duration was greater in the PP region than in the hilus throughout the experiment (see Fig. 3). AD duration was greater in the Generalized group than in the Partial group, in both electrode locations (hilus: Fl,14 --- 20.48, P < 0.01; PP: Fl,14 = 25.26, P < 0.01). Taking both groups together, there was no main effect of phase in either electrode location (hilus: F2.~3 = 1.84, P = 0.20; PP: F2,~3 = 0.91, P = 0.43). In fact, A D duration in the first two phases was very stable in both electrode locations for both Generalized and Partial groups (see Fig. 3). However, in the third phase, AD duration in both the hilus and PP region increased in the Generalized group but decreased in the Partial group. This interaction between group and phase was significant in the hilus (F2,13 = 6.62, P = 0.01) but not in the PP (F2A 3 = 2.26, P = 0.14).
Days off Days off are defined as days when rats did not exhibit any A D in response to kindling stimulation after A D had previously been evoked. Rats in the Partial group tended to display more days off than rats in the Generalized group (mean = 3.0 + 1.3 vs mean = 2,2 +_ 0.6), but this difference was not significant (q4 = 0.63, P = 0.54).
Ictal and postictal behaviors A number of behaviors were observed during kindling, some during AD but before the behavioral seizure (ictal behaviors) and others after termination of the AD and seizure (postictal behaviors). The ictal behaviors noted most frequently in both groups of rats were grooming, stretching, and wet dog shakes (WDS). Yawns and
sneezes were also observed postictally, but much less frequently than the ictal behaviors. Grooming and stretching were observed as often in the Generalized group as in the Partial group and did not increase or decrease over the course of the experiment. In contrast, WDS were more prevalent in the Generalized group, particularly during the first third of kindling, but the incidence of WDS declined as kindling progressed (see Fig. 4). A multivariate repeated measures A N O V A revealed a significant effect of kindling phase (F2,13 = 12.20, P < 0.001) but no overall difference between groups (F1,14 = 0.99) and a nearly significant interaction between group and phase (F2,13 = 3.54, P < 0.059). An exploratory post hoc analysis of the linear trends in the two groups showed that WDS in the Generalized group declined significantly over the three phases (FI.9 = 13.63, P < 0.005) and that there was no significant change in the Partial group (F1.,s = 0.98).
Histology All electrodes but one were found to be localized to their appropriate target, either the PP or the hilus of the DG. The location of the hilar electrode of one rat in the Generalized group could not be determined. There were no obvious differences in electrode placement between the two groups. In particular, hilar electrodes from rats in the Generalized group were not located nearer to the convergence of the mossy fibers than electrodes from rats in the Partial group. DISCUSSION The present experiment demonstrated that the characteristics of kindling with stimulation of the hilus of the DG are different from those of kindling at most other limbic sites. For example, we noted that rats kindled with hilar stimulation seemed to constitute two distinct subgroups, those rats that developed stage-5 seizures (Generalized group) and those that developed only stage-1 or stage-2 seizures (Partial group) during the 60 day period of stimulation. Compared to the Partial group, rats in the Generalized group required significantly fewer ADs to reach the first stage-2 seizure. They had lower thresholds for A D and higher frequency, amplitude, and overall duration of AD during kindling. They showed an increase in duration of AD during the course of kindling. Finally, they tended to have fewer days off and were more likely to show postictai spiking. In other words, they simply were more susceptible to developing generalized seizures than were rats in the Partial group. The reasons for their greater susceptibility to kindling are not immediately apparent, particularly since there were no obvious histological differences between the groups in
254 location of their hilar electrodes. Although kindling with stimulation of the hilus was typical of limbic kindling in terms of the threshold for AD and the relatively late development of motor seizures, it differed in important ways from the prototypical form of limbic kindling, amygdaloid kindling. One of the most obvious differences was in rate of kindling. With stimulation of the amygdala in rats, stage-5 seizures typically develop after about 12 ADs and are reliably evoked by all subsequent A D s 2"7A5"25'36. In contrast, the rats in the Generalized group in the present study required about 18 ADs to develop their first stage-5 seizure and an additional 11 ADs to develop 3 consecutive stage-5 seizures. This rate of seizure development resembles kindling observed previously in other hippocampal locations 1s'26. A second important difference was that there was a subgroup of rats (the Partial group) with comparable electrode placements in the hilus that failed to develop seizures more intense than stage 1 or stage 2 during the 60 days of stimulation. Other investigators have made similar observations of these odd characteristics of hilar kindling but have not studied them systematically nor described them in detail 12~4. In the present study we found hilar kindling to be highly unstable, with rats in both Generalized and Partial groups displaying many regressions and days off (i.e. sessions in which AD and seizures were not evoked). Even after a stage-5 seizure had been kindled, regressions back to stage-1 seizures were common. Instability of kindled seizures has previously been described only with anterior neocortical stimulation 3'29"3° and with stimulation of the pp19,20,21, and has not been reported with kindling at sites in the hippocampal formation itself. The electrophysiological characteristics of hilar kindling are also somewhat unusual and, not unexpectedly, resemble those of hippocampal kindling (e.g. CA1, CA3, subiculum) more than kindling at other limbic sites (e.g. amygdala). For example, during the course of kindling the duration of AD in both the hilus and the PP increased only slightly (in the Generalized group) or not at all (in the Partial group). This is similar to the pattern sometimes reported with stimulation of the hippocampus 228. but contrasts with the rapid growth of AD typically oberved with amygdaloid kindling 2's'25. Also, the occurrence of flat E E G or a 'silent period' in the hilus after the primary A D is characteristic of hippocampal kindling 2' 18,24,26 but not of kindling at other limbic sites. Finally, we noted that hilar kindling is associated with a variety of ictal and postictal behaviors, including grooming, stretching, yawning and sneezing, and WDS. The other behaviors we observed have not previously been reported, but WDS are commonly observed during kindling of other limbic sites such as the amygdala ~7,
septum 17, and hippocampus ~2~'. In agreement with most previous reports investigating the frequency of WDS during limbic kindling ~7"Js, we found that WDS occur with greatest frequency during the early stages and decrease as kindling progresses, reaching a minimum by the time generalized seizures have developed (phase 3 in our analysis). It may be significant that, throughout the experiment, the occurrence of WDS was infrequent in the rats that failed to develop generalized seizures (i.e. the Partial group). Thus both the appearance and subsequent decline in WDS were apparently related to the development of generalized seizures. It is not surprising that epileptogenic stimulation of the hilus can evoke WDS, given that the DG appears to be critical for the occurrence of WDS after stimulation of the PP. Damiano and Connor l~ found that stimulation of the DG at nonepileptogenic levels would induce WDS but that stimulation of the PP would trigger WDS only at epileptogenic levels and only when population spikes were induced in the DG. However, the DG does not appear to be required for kindling of hippocampal afferent structures. Sutula et al. 33 found that extensive destruction of dentate granule cells by intra-dentate infusion of colchicine raised the threshold for AD but did not affect the rate of kindling in the entorhinal cortex. The causes of the peculiar characteristics of hilar kindling described here, particularly the instability and regressions in kindled seizures, can only be speculated on. Perhaps they are due to strong local inhibitory processes evoked by direct stimulation of the mossy fibers in the hilus. The existence of powerful recurrent (feedback) inhibition in the DG is well documented 16'34, and there is compelling evidence for feedforward inhibition as well 4, Activation of inhibitory interneurons directly by electrical stimulation or synaptically by granule cell discharge could presumably have contributed to the marked instability of hilar kindling and kindled seizures observed here, and to the resistance of the dentate to electrically induced kindling observed here and previously ~2. Kindling in the hippocampal formation seems to be accompanied by increased or potentiated inhibitory effects, both in the hippocampus proper ~6'23 and in the D G 12'16"21'22"34. This effect is correlated with increased densities of benzodiazepine receptors 35 and involves both early and late components of inhibition 12' 23. It seems possible that some of the effects we observed were due to a progressive potentiation of inhibition in local feedforward and feedback circuits in the DG, produced by the kindling stimulation. Although we recognize that there may be no simple relation between the resistance of the DG to kindling and the efficacy of inhibitory conduction in the hilus, we offer this idea as a speculative but plausible explanation for the present results.
255 The slow and unstable kindling p r o d u c e d by electrical stimulation of the hilus contrasts sharply with the rapid and reliable kindling induced by r e p e a t e d infusions of o p i o i d p e p t i d e s into the D G 5'6. High-frequency stimulation like that used here produces naloxone-reversible behavioral effects, p r e s u m a b l y by causing the release of e n d o g e n o u s opioid peptides 9"m, and it thus seems likely that the stimulation we used for kindling was also effective in producing the release of e n d o g e n o u s opioids. If seizure d e v e l o p m e n t in the D G involves opioid peptides, as suggested by the opioid kindling data, electrical stimulation of the hilus and intra-hilar infusions of o p i o i d p e p t i d e s should have similar epileptogenic effects. The present results indicate that they have differing epileptogenic effects. T h e r e are several plausible explanations for the app a r e n t discrepancy in the susceptibility of the D G to o p i o i d kindling and electrical kindling. O n e is based on the idea that the ability of d e n t a t e granule cells to p r o p a g a t e epileptiform activity is severely restricted by the powerful inhibitory influences discussed earlier. O p i o i d infusions and electrical stimulation would probably have different effects on such inhibition. Infusions would be e x p e c t e d to have long-lasting effects that could lead to failure of inhibition l, resulting in a progressive disinhibition of dentate granule cells. In contrast, brief REFERENCES 1 Ben-Ari, Y., Krnjevic, K. and Reinhardt, W., Hippocampal seizures and failure of inhibition, Can. J. Physiol. Pharmacol., 57 (1979) 1462-1466. 2 Burnham, W.M., Primary and 'transfer' seizure development in the kindled rat, In J.A. Wada lEd.), Kindling, Raven, New York, 1976, pp. 61-83. 3 Burnham, W.M., Cortical and limbic kindling: similarities and differences. In K.E. Livingston and O. Hornykiewicz (Eds.), Lirnbic Mechanisms, Plenum, New York, 1978, pp. 507-519. 4 Buzsaki, G., Feed-forward inhibition in the hippocampal formation, Prog. Neurobiol., 22 (1984) 131-153. 5 Cain, D.P. and Corcoran, M.E., Intracerebral fl-endorphin, met-enkephalin and morphine: kindling of seizures and handling-induced potentiation of epileptiform effects, Life Sci., 34 (1984) 2535-2542. 6 Cain, D.P. and Corcoran, M.E., Epileptiform effects of metenkephalin, fl-endorphin and morphine: kindling of generalized seizures and potentiation of epileptiform effects by handling, Brain Research, 338 (1985) 327-336. 7 Corcoran, M.E, and Mason, S.T,, Role of forebrain catecholamines in amygdaloid kindling, Brain Research, 190 (1980) 473-484. 8 Corcoran, M.E., Urstad, H., McCaughran, J.A. and Wada, J.A., Frontal lobe and kindling in the rat, Can. J. Neurol. Sci., 2 (1975) 501-508. 9 Collier, T.J. and Rounenberg, A., Electrical self-stimulation of dentate gyrus granule cells, Behav. Neurol. Biol,, 42 (1984) 85-89. 10 Collier, T.J. and Routtenberg, A., Selective impairment of declarative memory following stimulation of dentate gyrus granule cells: a naloxone-sensitive effect, Brain Research, 310 (1984) 384-387.
trains of high-frequency electrical stimulation might produce only transient activation of inhibitory mechanisms, and this would not only not lead to a failure of inhibition 34 but also might actually retard the epileptogenic effects of granule cell activation. A n alternative explanation is that the epileptiform effects of infusions of opioid p e p t i d e s are not due to direct activation of granule cells, but rather are due to inhibition of hilar inhibitory interneurons, similar to the disinhibitory effect of opioids that has been described in field CA137. Electrical stimulation of the hilus would not p r o d u c e such a specific disinhibition. A final explanation is based on evidence suggesting that opioid peptides released by the mossy fibers p r o d u c e excitatory effects on p y r a m i d a l cells in field C A 3 of the h i p p o c a m p u s 27. This finding raises the possibility that diffusion to C A 3 is responsible for the epileptiform effects of i n t r a - d e n t a t e infusions of opioid peptides. W h a t e v e r the correct explanation for the discrepancies b e t w e e n electrical and chemical kindling of the D G turns out to be, we conclude that the present results are not necessarily inconsistent with a role for opioid peptides in the seizure susceptibility of this region. Acknowledgements. This research was supported by operating grants from the Natural Sciences and Engineering Research Council of Canada awarded to M.E.C. and to R.W.S.G.M.G. was supported by a Postgraduate Scholarship from NSERC.
11 Damiano, B.P. and Connor, J.D., Hippocampal mediation of shaking behavior induced by electrical stimulation of the perforant path in the rat, Brain Research, 308 (1984) 383-386. 12 De Jonge, M. and Racine, R..I., The development and decay of kindling-induced increases in paired-pulse depression in the dentate gyrus, Brain Research, 412 (1987) 318-328. 13 Gall, C., Brecha, N., Karten, H.J. and Chang, K.-J., Localization of enkephalin-like immunoreactivity to identified axona[ and neuronal populations of the rat hippocampus, J. Comp. Neurol., 198 (1981) 335-350. 14 Goddard, G.V., Separate analysis of lasting alteration in excitatory synapses, inhibitory synapses and cellular excitability in association with kindling. In P.A. Buser, W.A. Cobb and T. Okuma (Eds.), Kyoto Symposia, Elsevier. Amsterdam, 1982, pp. 288-294. 15 Goddard, G.V., McIntyre, D.C. and Leech, C.K., A permanent change in brain function resulting from daily electrical stimulation, Exp. Neurol., 25 (1969) 295-330. 15a Grace, G.M., Corcoran, M.E. and Skelton, R.W., Kindling with stimulation of the dentate gyrus. II. Effects on evoked field potentials, Brain Research, 509 (1990) 257-265. 16 King, G.L., Dingledine, R., Giacchino, J.L. and McNamara, J.O., Abnormal neuronal excitability in hippocampal slices from kindled rats, J. Neurophysiol., 54 (1985) 1295-1304. 17 Le Gal La Salle, G. and Cavalheiro, E.A., Stimulation of septal and amygdaloid nuclei: EEG and behavioral responses during early development of kindling with special reference to wet dog shakes, Exp. Neurol., 74 (1981) 717-727. 18 Lerner-Natoli, M,, Rondouin, G. and Baldy-Moulinier, M., Hippocampal kindling in the rat: intrastructural differences, J. Neurosci. Res., 12 (1984) 101-111. 19 Maru, E. and Ooddard, G.V., Alteration in dentate neuronal activities associated with perforant path kindling. I. L0ng-term potentiation of excitatory synaptic transmission, Exp. Neurol.,
256 96 (1987) 19-32. 20 Maru, E. and Goddard, G.V., Alteration in dentate neuronal activities associated with perforant path kindling. II. Decrease in granule cell excitability, Exp. Neurol., 96 (1987) 33-45. 21 Maru, E. and Goddard, G.V., Alteration in dentate neuronal activities associated with perforant path kindling. III. Enhancement of synaptic inhibition, Exp. Neurol., 96 (1987) 46-60. 22 Maru, E., Tatsuno, J., Okamoto, J. and Ashida, H., Development and reduction of synaptic potentiation induced by perforant path kindling, Exp. Neurol., 78 (1982) 409-424. 23 Oliver, M.W. and Miller, J.J., Alterations of inhibitory processes in the dentate gyrus following kindling-induced epilepsy, Exp. Brain Res., 57 (1985) 443-447. 24 Racine, R.J., Modification of seizure activity by electrical stimulation: I. After-discharge threshold, Electroencephalogr. Clin. NeurophysioL, 32 (1972) 269-279. 25 Racine, R.J., Modification of seizure activity by electrical stimulation: II. Motor seizure, Electroencephalogr. Clin. Neurophysiol., 32 (1972) 281-294. 26 Racine, R.J., Rose, P.A. and Burnham, W.M., Afterdischarge thresholds and kindling rates in dorsal and ventral hippocampus and dentate gyrus, Can. J. Neurol. Sci., 4 (1977) 273-278. 27 Routtenberg, A., Kalkara, T. and Krnjevic, K., Hippocampal mossy fiber opioid regulation of CA3 pyramidal cell excitability: iontophoretic study in intact hippocampal formation, Fed. Proc., 43 (1984) 924. 28 Sato, M., Hippocampal seizure and secondary epileptogenesis in the 'kindled' cat preparations, Folia Psychiat. Neurol. Jpn., 29 (1975) 239-250.
29 Seidel, W.T. and Corcoran, M.E., Relations between amygdaloid and anterior neocortical kindling, Brain Research, 385 (1986) 375-378, 30 Stripling, J.S. and Russell, R.D., Effect of cocaine and pentylenetetrazol on cortical kindling, Pharmacol. Biochem. Behave. 23 (1985) 573-581. 31 Sutula, T. and Steward, O., Quantitative analysis of synaptic potentiation during kindling of the perforant path, .L Neurophysiol., 56 (1986) 732-746. 32 Sutula, ~I~ and Steward, O., Facilitation of kindling by prior induction of long-term potentiation in the perforant path, Brain Research, 420 (1987) 109-117. 33 Sutula, T., Harrison, C. and Steward, O., Chronic epileptogenesis induced by kindling of the entorhinal cortex: the role of the dentate gyrus, Brain Research, 385 (1986) 291-299. 34 Tuff, L.P., Racine, R.J. and Adamec, R., The effects of kindling on GABA-mediated inhibition in the dentate gyrus of the rat, I. Paired-pulse depression, Brain Research, 277 (1983) 79-90. 35 Tuff, L.P., Racine, R.J. and Mishra, R.K., The effects of kindling on GABA-mediated inhibition in the dentate gyrus of the rat. II. Receptor binding, Brain Research, 277 (1983) 91-98. 36 Westerberg, V.S. and Corcoran, M.E., Antagonism of central but not peripheral cholinergic receptors retards amygdala kindling in rats, Exp. Neurol.. 95 (1987) 194-206. 37 Ziegelgansberger, W., French, E.D., Siggins, G.R. and Bloom, EE., Opioid peptides may excite hippocampal pyramidal neurons by inhibiting adjacent inhibitory interneurons, Science, 205 (1979) 415-417.
249
BRES 15202
Kindling with stimulation of the dentate gyrus. I. Characterization of electrographic and behavioral events Gloria M. Grace, Michael E. Corcoran and Ronald W. Skelton Department of Psychology, University of Victoria, Victoria, B.C. (Canada) (Accepted 18 July 1989)
Key words: Kindling; Dentate gyrus; Hippocampal formation
Once daily for 60 days, hooded rats received unilateral high-frequency stimulation in the hilus of the dentate gyrus (DG), at an intensity sufficient to evoke epileptiform afterdischarge (AD). Although most rats eventually developed generalized stage-5 seizures (Generalized group), some did not progress beyond partial stage-1 or stage-2 seizures (Partial group). Hilar kindling also displayed several other characteristics that distinguished it from typical limbic kindling, including low rate of development, marked instability of the seizures, and little or no growth in duration of AD.
INTRODUCTION In the present study we describe the electrographic and behavioral characteristics of kindling with stimulation in the hilus of the d e n t a t e gyrus ( D G ) . T h e hilus was of interest because it is the area of the h i p p o c a m p a l f o r m a t i o n through which the mossy fibers course on their way to h i p p o c a m p a l field CA3. The mossy fibers are the axons of the granule cells of the D G , neurons that contain both an amino acid n e u r o t r a n s m i t t e r and several different opioid p e p t i d e s ~3. Some of the behavioral effects of electrical stimulation of the hilus can be reversed by t r e a t m e n t with the opiate antagonist naloxone 9`t°, suggesting that electrical stimulation of the mossy fibers can result in the release of opioid peptides. Because r e p e a t e d infusions of low concentrations of opioid p e p t i d e s into the D G and h i p p o c a m p u s can kindle seizures 5"6, it would be of interest to assess the susceptibility of the D G to kindling with electrical stimulation. Several previous studies have described kindling in different regions of the h i p p o c a m p a l formation. Differences have been noted in the rate and pattern of kindling in the p y r a m i d a l cell fields of the dorsal and ventral h i p p o c a m p u s lx'26. Kindling with stimulation of the perforant path (PP), the m a j o r projection from the entorhinal cortex to the granule cells of the D G , has been described 12"19"2°'2x~31"32. H o w e v e r , only limited information is available ~2'14 concerning kindling of the hilus of the D G , the region between the u p p e r and lower blades
of granule cells. We therefore characterized hilar kindling in terms of rate and p a t t e r n of d e v e l o p m e n t of clinical seizures; threshold of afterdischarge ( A D ) ; frequency, amplitude, and duration of A D ; n u m b e r of days off (i.e. days in which stimulation failed to e v o k e A D after it had previously been elicited); and behavioral effects. We also m o n i t o r e d field potentials (population spikes and population excitatory postsynaptic potentials (EPSPs)) in the hilus e v o k e d by stimulation of the PP during kindling. These electrophysiological correlates of hilar kindling are r e p o r t e d in the accompanying p a p e r ~5'1. MATERIALS AND METHODS
Animals Male hooded rats of the Long-Evans strain were used, weighing between 300 and 375 g at the time of surgery. The rats were housed in individual stainless steel cages with food and water available ad libitum. A 12 h light-dark cycle was in effect throughout the study, and all testing was conducted during the light portion of the cycle. Surgery Using pentobarbital anesthesia (60 mg/kg), chronic electrodes were implanted in the hilus of the DG and in the PP. The hilar electrode was used for delivery of kindling stimulation and recording of EEG activity. The PP electrode was used for recording EEG during kindling sessions and for evoking field potentials in the DG, as described separately~5~'. Each electrode consisted of a single strand of stainless-steel wire, 761~m in diameter, coated with Teflon to a total diameter of 114 urn. Cemented to each electrode, approximately 5 mm above the tip, was a small plastic bead that served to anchor the electrode in the dental acrylic. Between 0.25 and (I.5 mm of insulation on the tips of the electrodes was removed. Two skull screws, connected to uninsulated stainless-steel wire,
Correspondence. R.W. Skelton, Department of Psychology, University of Victoria, P.O. Box 1700, Victoria, B.C., Canada V8W 2Y2, 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
25(I served as current-return and ground-reference electrodes. Goldplated pins (Amphenol 220-S02) were soldered to the ends of the 4 wires, inserted into a small connector, and affixed to the skull using dental acrylic and two additional anchoring skull screws. Electrodes were implanted in the left side of the brain under electrophysiological guidance. With the skull level between bregma and lambda, coordinates for the hilus were 4.0 mm posterior to bregma, 2.0 mm lateral to the midline, and 3.3 mm ventral to the surface of the cortex; coordinates for the PP were 8.1 mm posterior to bregma, 4.3 mm lateral to the midline, and 2.4 mm ventral to the surface of the cortex. As both electrodes were lowered, a storage oscilloscope displayed field potentials recorded from the electrode in the hilus, evoked by single square-wave pulses delivered to the PP electrode (0.1 ms, 0.2 Hz, 400/xA). The positions and depths of the electrodes were adjusted to produce a hilar evoked potential with a population spike of maximum amplitude and minimum threshold. After a 12 day recovery period, population spike amplitudes were measured in response to a stimulus intensity of 400 flA. Rats with amplitudes of less than 6 mV were excluded from the study. Field potentials were monitored on a regular basis throughout kindling, and rats that exhibited unstable field potentials were also excluded.
on each day, and spike frequency was measured from the 4 s block containing the highest frequency spikes recorded from that electrode for each rat on each day.
Histology At the end of the experiment, rats were given an overdose of pentobarbital, and were perfused with normal saline followed by 10% formalin. Brains were sectioned into 40 um slices, mounted on slides, and stained with thionin.
Statistical analysis The data were analyzed with independent t-tests, with two-way analysis of variance (ANOVA; group x kindling period) with repeated measures, and, where appropriate, with multivariate analysis of variance. A rejection criterion of P < 0.05 was used.
RESULTS A f t e r r e c o v e r y f r o m s u r g e r y , 20 r a t s d i s p l a y e d field potentials with spike amplitudes above 6 mV (range 9-20
Determination of A D threshold
m V ) in r e s p o n s e to a s i n g l e 4 0 0 / ~ A p u l s e . D u r i n g t h e
Electrical stimulation for kindling consisted of a 1 s train of constant current balanced biphasic square-wave pulses, with a pulse width of 1.0 ms and a frequency of 60/s. On the first day, a series of stimulations was delivered, once every 3 min, starting at 30 ~tA (base-to-peak) and increasing in 20/~A steps until AD was elicited. On the following day, the stimulation intensity was decreased by steps of 10/xA from this first 'threshold' until the AD was no longer evoked. Threshold was defined as the minimum intensity sufficient to evoke AD on this second day.
study, 4 rats were rejected due to problems with their e l e c t r o d e s so t h a t u l t i m a t e l y a t o t a l o f 16 r a t s u n d e r w e n t the entire kindling procedure.
Kindling rates Ten
of the
16 r a t s
developed
generalized
s e i z u r e s , w i t h i n a m e a n o f 18.2 ( + 1 2 . 3
stage-5
S.D.) stimula-
tions. T h e r e m a i n i n g 6 r a t s p r o g r e s s e d o n l y t o s t a g e 1
Kindling
(n = 1), s t a g e 2 (n = 4), o r s t a g e 3 ( n = 1) s e i z u r e s ,
Rats were stimulated once daily at their threshold intensities, and the intensity and duration of the seizures were noted (see ref. 25 for behavioral classification system used). When stage-5 generalized seizures were noted on 3 consecutive stimulation days, stimulation was discontinued for 3 weeks. After this rest period, rats were rekindled by administering stimulation once daily until a stage-5 seizure was displayed, in order to test the permanency of the kindling. Rats that did not exhibit any stage-5 seizures were stimulated for a maximum of 60 days. Before and after each stimulation, referential EEG was recorded from both the hilus and PP, and evoked AD was subsequently analysed for total duration of all episodes, and for spike frequency and spike amplitude. Mean amplitude of AD was measured from the 4 s block containing the largest amplitude spikes recorded from that electrode for each rat
a l t h o u g h t h e y w e r e s t i m u l a t e d f o r 60 d a y s (i.e. 3.4 S . D . l o n g e r ) . B e c a u s e o f t h i s s t r i k i n g d i f f e r e n c e in k i n d l i n g , we
considered
Generalized
the
former
10 r a t s
kindling group
and
as c o n s t i t u t i n g
the
l a t t e r 6 r a t s as
c o n s t i t u t i n g a P a r t i a l k i n d l i n g g r o u p . T a b l e I lists t h e number of ADs to each stage of seizure and number of regressions for both groups. between
the
Partial
and
There
was no difference
Generalized
groups
in t h e
n u m b e r o f A D s t o t h e first stage-1 s e i z u r e (/14 = 0~t0, P = 0 . 9 2 ) , b u t r a t s in t h e G e n e r a l i z e d g r o u p r e q u i r e d f e w e r
TABLE I
Number of A D s to each stage of seizure and number of regressions Criterion: 3 consecutive stage-5 seizures. Regression: prior to criterion, n = number of rats displaying this seizure stage.
Criterion
Regressions
18.2 3.9 10
28.8 3.7 10
6.0 1.3 10
-
-
-
-
Stageofseizure
Generalized group Mean S.E.M. n Partial group Mean S.E.M. n
a
0
1
2
3
4
10
4.4 0.7 10
13.8 1.8 8
22.0 3.0 2
16.8 2.6 4
6
4.5 0.6 6
38.8 5.4 5
46.0 . l
.
. -
.
8.8 1.7 6
251 become prostrate and exhibited generalized clonic jerking; they did not display the rearing behavior commonly seen in an amygdaloid stage-5 seizure, or the usual progression through the seizure stages (1-5). As is commonly observed with limbic kindling, the rats would typically remain still after the clonic component of the convulsion and appeared to be unresponsive to the environment. This was followed by several minutes of automatisms, during which the rats would appear to be trying to climb the walls of the recording box. For several minutes after the automatisms, the rats showed signs of hyperreactivity to sensory stimuli.
ADs to develop stage-2 seizures than rats in the Partial group (t H = 5.24, P < 0.01). Development of stage-3 and stage-4 seizures was rare, and occurred only once or twice in a few rats. Regression to less severe behavioral seizure stages was equally common in both groups (t14 = 1.31, P = 0.21). The Generalized group displayed large variability in the number of ADs to the first stage-5 seizure (range = 5-41; mean --- 18.2) and in the number of ADs to 3 consecutive stage-5 seizures (range = 14-47; mean = 28.8). Most rats in this group developed seizures abruptly and progressed from stage-1 seizures directly to stage-5 seizures, without developing the intermediate stages of seizure characteristic of limbic kindling s'15"25. After the 3-week rest period, all rats in the Generalized group displayed a stage-5 seizure after only one or two ADs. However, one rat demonstrated 4 days off (i.e. no AD in response to stimulation) in the process. In addition to the unusual pattern of seizure development, the Generalized group also displayed unusual stage-5 seizure topography, During AD, the rats often
A
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A D threshold The mean threshold for A D in all 16 rats was 42/~A (S.E.M. = 8, range -- 15-130). Rats in the Generalized group had slightly lower thresholds (mean = 37.5, S.E.M. -- 7.27, range = 15-80) than rats in the Partial group (mean = 49, S.D. = 18.4, range -- 15-130), but the difference was not significant (t14 -0.70, P = 0.50).
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Fig. 1. Sample electroencephalogram (EEG) illustrating the changes in afterdischarge (AD) that occurred during kindling. The lower trace is a record from the stimulation/recording site in the hilus of the dentate gyrus (DG), and the upper trace is a record from the recording electrode in the ipsilateral perforant path (PP). A: the 1st A D induced by hilar stimulation; there was no behavioral seizure. B: the 8th A D from the same rat, during a partial seizure. Note the increase in A D frequency from the hilus relative to the first A D . C - E : continuous record of the 26th A D from the same rat, during a generalized seizure. Note the increased duration and complexity of the A D from both the DG and PP electrodes. Calibrations: 5 s, upper trace, 600 ~V; lower trace, 3.0 mV.
i
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A
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b 12
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o
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KINDLING PERIOD
Fig. 3. Changes in the duration of afterdischarge (AD) recorded from the dentate gyrus (DG) and perforant path (PP) in the Generalized (Gen) and Partial (Part) groups. Duration of AD (s) was the sum of primary and secondary ADs for each rat on each day, averaged over each of three equal phases of kindling (1/3 total number of kindling stimulations).
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Mid(lie AD E P I S O D E
Final
Fig. 2. Changes in afterdischarge (AD) recorded from the dentate gyrus (DG) and perforant path (PP) electrodes over kindling in the Generalized (Gen) and Partial (Part) groups. A: spike frequency (spikes/s), measured in the 4 s block of AD containing the highest frequency spikes of the first, middle, and final AD of kindling. B: spike amplitude (uV peak-to-peak), measured in the 4 s block of AD containing the largest spikes of the first, middle, and final AD of kindling.
General morphology of AD The A D recorded from both PP and hilar electrodes typically began 2-4 s after the kindling stimulation ended. Hilar A D usually subsided first, and the AD recorded from the PP electrode continued for several more seconds (see Fig. 1). The AD in the hilus was almost always followed by a period of flat E E G and then gradual recovery of E E G amplitude to pre-stimulation levels. Although the amplitude of E E G recorded from the PP was lower than baseline after the AD, it was never as flat as that recorded from the hilus. There was usually a secondary episode of AD, briefer and less intense than the first in terms of both frequency and intensity, which was generally recorded from both electrode locations. Occasionally, the secondary episodes were limited to the PP region. The waveform of the A D changed over the course of kindling. A D was initially composed of regularly spaced simple biphasic spikes. However, with further stimulation
the waveform became more complex and variable, with periods of notched or double spike configurations or very high-frequency bursts. This progression of waveform complexity has also been noted in amygdaloid A D 25. The frequency of the epileptiform spikes in the A D increased significantly over the course of kindling, in both the hilar and the PP regions (hilus: F2,13 = 31.0, P < 0.01; PP: F2,13 = 12.62, P < 0.01) (see Fig. 2A). The AD frequency in the Generalized group was greater than that in the Partial group, but the difference was significant only in the PP region (F1:4 = 10.12, P < 0.01) and not in the hilus (F~: 4 = 3.59, P = 0.08). There was no significant group by phase interaction for either electrode location. The amplitude of A D was much higher in the hilus than in the PP region (see Fig. 2B). Marginal increases in amplitude were observed in both locations, a n d the increase was significant only in the PP region (F2,13 = 6.17, P < 0.02) and not in the hilus (F2,]3 = 0.30, P = 0.74). Like A D frequency, A D amplitude was greater in the Generalized group than in the Partial group, and this difference was again significant only in the PP region (Fl.14 = 9.09, P < 0.01) and not in the hilus (F1.14 = 0.73, P = 0.41). Similarly, there was no significant group by phase interaction for either electrode location. Low-frequency postictal spiking was observed in both the Generalized and Partial groups, although the proportion of rats displaying postictal spikes was higher in the Generalized group (8 of 10 rats) than in the Partial group (3 of 6 rats).
Growth of AD In order to assess changes in A D duration over kindling, and to compare rats that received different
253
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Fig. 4. Mean number of wet dog shakes per day for each rat in the Generalized (Gen) and Partial (Part) groups, within each of 3 equal phases of kindling (1/3 total number of kindling stimulations).
numbers of stimulation days, each rat's stimulation series was divided into 3 equal phases (early, middle and late). The total A D duration for each day (i.e. the sum of primary and secondary episodes) was then averaged for each rat over each phase. This analysis revealed that AD duration was greater in the PP region than in the hilus throughout the experiment (see Fig. 3). AD duration was greater in the Generalized group than in the Partial group, in both electrode locations (hilus: Fl,14 --- 20.48, P < 0.01; PP: Fl,14 = 25.26, P < 0.01). Taking both groups together, there was no main effect of phase in either electrode location (hilus: F2.~3 = 1.84, P = 0.20; PP: F2,~3 = 0.91, P = 0.43). In fact, A D duration in the first two phases was very stable in both electrode locations for both Generalized and Partial groups (see Fig. 3). However, in the third phase, AD duration in both the hilus and PP region increased in the Generalized group but decreased in the Partial group. This interaction between group and phase was significant in the hilus (F2,13 = 6.62, P = 0.01) but not in the PP (F2A 3 = 2.26, P = 0.14).
Days off Days off are defined as days when rats did not exhibit any A D in response to kindling stimulation after A D had previously been evoked. Rats in the Partial group tended to display more days off than rats in the Generalized group (mean = 3.0 + 1.3 vs mean = 2,2 +_ 0.6), but this difference was not significant (q4 = 0.63, P = 0.54).
Ictal and postictal behaviors A number of behaviors were observed during kindling, some during AD but before the behavioral seizure (ictal behaviors) and others after termination of the AD and seizure (postictal behaviors). The ictal behaviors noted most frequently in both groups of rats were grooming, stretching, and wet dog shakes (WDS). Yawns and
sneezes were also observed postictally, but much less frequently than the ictal behaviors. Grooming and stretching were observed as often in the Generalized group as in the Partial group and did not increase or decrease over the course of the experiment. In contrast, WDS were more prevalent in the Generalized group, particularly during the first third of kindling, but the incidence of WDS declined as kindling progressed (see Fig. 4). A multivariate repeated measures A N O V A revealed a significant effect of kindling phase (F2,13 = 12.20, P < 0.001) but no overall difference between groups (F1,14 = 0.99) and a nearly significant interaction between group and phase (F2,13 = 3.54, P < 0.059). An exploratory post hoc analysis of the linear trends in the two groups showed that WDS in the Generalized group declined significantly over the three phases (FI.9 = 13.63, P < 0.005) and that there was no significant change in the Partial group (F1.,s = 0.98).
Histology All electrodes but one were found to be localized to their appropriate target, either the PP or the hilus of the DG. The location of the hilar electrode of one rat in the Generalized group could not be determined. There were no obvious differences in electrode placement between the two groups. In particular, hilar electrodes from rats in the Generalized group were not located nearer to the convergence of the mossy fibers than electrodes from rats in the Partial group. DISCUSSION The present experiment demonstrated that the characteristics of kindling with stimulation of the hilus of the DG are different from those of kindling at most other limbic sites. For example, we noted that rats kindled with hilar stimulation seemed to constitute two distinct subgroups, those rats that developed stage-5 seizures (Generalized group) and those that developed only stage-1 or stage-2 seizures (Partial group) during the 60 day period of stimulation. Compared to the Partial group, rats in the Generalized group required significantly fewer ADs to reach the first stage-2 seizure. They had lower thresholds for A D and higher frequency, amplitude, and overall duration of AD during kindling. They showed an increase in duration of AD during the course of kindling. Finally, they tended to have fewer days off and were more likely to show postictai spiking. In other words, they simply were more susceptible to developing generalized seizures than were rats in the Partial group. The reasons for their greater susceptibility to kindling are not immediately apparent, particularly since there were no obvious histological differences between the groups in
254 location of their hilar electrodes. Although kindling with stimulation of the hilus was typical of limbic kindling in terms of the threshold for AD and the relatively late development of motor seizures, it differed in important ways from the prototypical form of limbic kindling, amygdaloid kindling. One of the most obvious differences was in rate of kindling. With stimulation of the amygdala in rats, stage-5 seizures typically develop after about 12 ADs and are reliably evoked by all subsequent A D s 2"7A5"25'36. In contrast, the rats in the Generalized group in the present study required about 18 ADs to develop their first stage-5 seizure and an additional 11 ADs to develop 3 consecutive stage-5 seizures. This rate of seizure development resembles kindling observed previously in other hippocampal locations 1s'26. A second important difference was that there was a subgroup of rats (the Partial group) with comparable electrode placements in the hilus that failed to develop seizures more intense than stage 1 or stage 2 during the 60 days of stimulation. Other investigators have made similar observations of these odd characteristics of hilar kindling but have not studied them systematically nor described them in detail 12~4. In the present study we found hilar kindling to be highly unstable, with rats in both Generalized and Partial groups displaying many regressions and days off (i.e. sessions in which AD and seizures were not evoked). Even after a stage-5 seizure had been kindled, regressions back to stage-1 seizures were common. Instability of kindled seizures has previously been described only with anterior neocortical stimulation 3'29"3° and with stimulation of the pp19,20,21, and has not been reported with kindling at sites in the hippocampal formation itself. The electrophysiological characteristics of hilar kindling are also somewhat unusual and, not unexpectedly, resemble those of hippocampal kindling (e.g. CA1, CA3, subiculum) more than kindling at other limbic sites (e.g. amygdala). For example, during the course of kindling the duration of AD in both the hilus and the PP increased only slightly (in the Generalized group) or not at all (in the Partial group). This is similar to the pattern sometimes reported with stimulation of the hippocampus 228. but contrasts with the rapid growth of AD typically oberved with amygdaloid kindling 2's'25. Also, the occurrence of flat E E G or a 'silent period' in the hilus after the primary A D is characteristic of hippocampal kindling 2' 18,24,26 but not of kindling at other limbic sites. Finally, we noted that hilar kindling is associated with a variety of ictal and postictal behaviors, including grooming, stretching, yawning and sneezing, and WDS. The other behaviors we observed have not previously been reported, but WDS are commonly observed during kindling of other limbic sites such as the amygdala ~7,
septum 17, and hippocampus ~2~'. In agreement with most previous reports investigating the frequency of WDS during limbic kindling ~7"Js, we found that WDS occur with greatest frequency during the early stages and decrease as kindling progresses, reaching a minimum by the time generalized seizures have developed (phase 3 in our analysis). It may be significant that, throughout the experiment, the occurrence of WDS was infrequent in the rats that failed to develop generalized seizures (i.e. the Partial group). Thus both the appearance and subsequent decline in WDS were apparently related to the development of generalized seizures. It is not surprising that epileptogenic stimulation of the hilus can evoke WDS, given that the DG appears to be critical for the occurrence of WDS after stimulation of the PP. Damiano and Connor l~ found that stimulation of the DG at nonepileptogenic levels would induce WDS but that stimulation of the PP would trigger WDS only at epileptogenic levels and only when population spikes were induced in the DG. However, the DG does not appear to be required for kindling of hippocampal afferent structures. Sutula et al. 33 found that extensive destruction of dentate granule cells by intra-dentate infusion of colchicine raised the threshold for AD but did not affect the rate of kindling in the entorhinal cortex. The causes of the peculiar characteristics of hilar kindling described here, particularly the instability and regressions in kindled seizures, can only be speculated on. Perhaps they are due to strong local inhibitory processes evoked by direct stimulation of the mossy fibers in the hilus. The existence of powerful recurrent (feedback) inhibition in the DG is well documented 16'34, and there is compelling evidence for feedforward inhibition as well 4, Activation of inhibitory interneurons directly by electrical stimulation or synaptically by granule cell discharge could presumably have contributed to the marked instability of hilar kindling and kindled seizures observed here, and to the resistance of the dentate to electrically induced kindling observed here and previously ~2. Kindling in the hippocampal formation seems to be accompanied by increased or potentiated inhibitory effects, both in the hippocampus proper ~6'23 and in the D G 12'16"21'22"34. This effect is correlated with increased densities of benzodiazepine receptors 35 and involves both early and late components of inhibition 12' 23. It seems possible that some of the effects we observed were due to a progressive potentiation of inhibition in local feedforward and feedback circuits in the DG, produced by the kindling stimulation. Although we recognize that there may be no simple relation between the resistance of the DG to kindling and the efficacy of inhibitory conduction in the hilus, we offer this idea as a speculative but plausible explanation for the present results.
255 The slow and unstable kindling p r o d u c e d by electrical stimulation of the hilus contrasts sharply with the rapid and reliable kindling induced by r e p e a t e d infusions of o p i o i d p e p t i d e s into the D G 5'6. High-frequency stimulation like that used here produces naloxone-reversible behavioral effects, p r e s u m a b l y by causing the release of e n d o g e n o u s opioid peptides 9"m, and it thus seems likely that the stimulation we used for kindling was also effective in producing the release of e n d o g e n o u s opioids. If seizure d e v e l o p m e n t in the D G involves opioid peptides, as suggested by the opioid kindling data, electrical stimulation of the hilus and intra-hilar infusions of o p i o i d p e p t i d e s should have similar epileptogenic effects. The present results indicate that they have differing epileptogenic effects. T h e r e are several plausible explanations for the app a r e n t discrepancy in the susceptibility of the D G to o p i o i d kindling and electrical kindling. O n e is based on the idea that the ability of d e n t a t e granule cells to p r o p a g a t e epileptiform activity is severely restricted by the powerful inhibitory influences discussed earlier. O p i o i d infusions and electrical stimulation would probably have different effects on such inhibition. Infusions would be e x p e c t e d to have long-lasting effects that could lead to failure of inhibition l, resulting in a progressive disinhibition of dentate granule cells. In contrast, brief REFERENCES 1 Ben-Ari, Y., Krnjevic, K. and Reinhardt, W., Hippocampal seizures and failure of inhibition, Can. J. Physiol. Pharmacol., 57 (1979) 1462-1466. 2 Burnham, W.M., Primary and 'transfer' seizure development in the kindled rat, In J.A. Wada lEd.), Kindling, Raven, New York, 1976, pp. 61-83. 3 Burnham, W.M., Cortical and limbic kindling: similarities and differences. In K.E. Livingston and O. Hornykiewicz (Eds.), Lirnbic Mechanisms, Plenum, New York, 1978, pp. 507-519. 4 Buzsaki, G., Feed-forward inhibition in the hippocampal formation, Prog. Neurobiol., 22 (1984) 131-153. 5 Cain, D.P. and Corcoran, M.E., Intracerebral fl-endorphin, met-enkephalin and morphine: kindling of seizures and handling-induced potentiation of epileptiform effects, Life Sci., 34 (1984) 2535-2542. 6 Cain, D.P. and Corcoran, M.E., Epileptiform effects of metenkephalin, fl-endorphin and morphine: kindling of generalized seizures and potentiation of epileptiform effects by handling, Brain Research, 338 (1985) 327-336. 7 Corcoran, M.E, and Mason, S.T,, Role of forebrain catecholamines in amygdaloid kindling, Brain Research, 190 (1980) 473-484. 8 Corcoran, M.E., Urstad, H., McCaughran, J.A. and Wada, J.A., Frontal lobe and kindling in the rat, Can. J. Neurol. Sci., 2 (1975) 501-508. 9 Collier, T.J. and Rounenberg, A., Electrical self-stimulation of dentate gyrus granule cells, Behav. Neurol. Biol,, 42 (1984) 85-89. 10 Collier, T.J. and Routtenberg, A., Selective impairment of declarative memory following stimulation of dentate gyrus granule cells: a naloxone-sensitive effect, Brain Research, 310 (1984) 384-387.
trains of high-frequency electrical stimulation might produce only transient activation of inhibitory mechanisms, and this would not only not lead to a failure of inhibition 34 but also might actually retard the epileptogenic effects of granule cell activation. A n alternative explanation is that the epileptiform effects of infusions of opioid p e p t i d e s are not due to direct activation of granule cells, but rather are due to inhibition of hilar inhibitory interneurons, similar to the disinhibitory effect of opioids that has been described in field CA137. Electrical stimulation of the hilus would not p r o d u c e such a specific disinhibition. A final explanation is based on evidence suggesting that opioid peptides released by the mossy fibers p r o d u c e excitatory effects on p y r a m i d a l cells in field C A 3 of the h i p p o c a m p u s 27. This finding raises the possibility that diffusion to C A 3 is responsible for the epileptiform effects of i n t r a - d e n t a t e infusions of opioid peptides. W h a t e v e r the correct explanation for the discrepancies b e t w e e n electrical and chemical kindling of the D G turns out to be, we conclude that the present results are not necessarily inconsistent with a role for opioid peptides in the seizure susceptibility of this region. Acknowledgements. This research was supported by operating grants from the Natural Sciences and Engineering Research Council of Canada awarded to M.E.C. and to R.W.S.G.M.G. was supported by a Postgraduate Scholarship from NSERC.
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