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Ontogeny of epileptogenesis in the rat hippocampus: a study of the influence of GABAergic inhibition
Developmental Brain Research, 66 (1991) 237-243 Elsevier
237
BRESD 51438
Ontogeny of epileptogenesis in the rat hippocampus: a study of the influence of GABAergic inhibition Hillary B. Michelson*
a n d E r i c W. L o t h m a n
Department of Neurology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908 (USA)
(Accepted 24 December 1991) Key words: Epilepsy; Ontogeny; Hippocampus; Development; 7-Aminobutyric acid-ergic inhibition; Seizure; Afterdischarge; Kindling
In vivo experiments were carried out to examine whether the period during which y-aminobutyric acid (GABA)ergic inhibition in the hippocampus matures is associated with a decrease in epileptogenesis. Seizures were elicited with bipolar electrodes stcreotactically positioned in the hippocampus of urethane-anesthetized rat pups from postnatal (PN) 7 through 28 days of age. No clinical seizure activity was detected but electrographic seizures (afterdischarges) were induced at all ages. Afterdischarge thresholds (ADT) varied inverse!y with age. However, the durations of initial afterdischarges and the degree of lengthening of afterdischarges with the rapidly recurring hippocampal seizure (RRHS) protocol were not different for the various age animals studied. Paired pulse inhibition was assessed with a twin pulse paradigm that has been shown to monitor GABAergic inhibition. Measurements were made before and 60 min after a single seizure and again 60 rain after the RRHS protocol. At no age was there a significant change'in paired pulse inhibition after a single seizure. After RRHS there was a significant reduction ot paired pulse inhibition only in the groups that had manifested adult levels of paired pulse inhibition in preseizure measurements (->PN 21). These studies indicate that heightened epileptogenesis in the young hippocampus cannot simply be explained on the basis of an immaturity of GABA-mediated inhibition. INTRODUCTION Based on animal and clinical observations~ the idea has been advanced that the occurrence of seizures leads to a chronic disturbance of brain function which, in turn, promotes further seizures s't°'~s'tg'29. Thus, a thorough examination of the pathophysiology of epilepsy involves more than just the factors that can induce seizures. Certain phenomena also emphasize the age-dependent aspects in epilepsy. The younger brain is more prone to seizures, and the clinical expression of seizures can vary tre¢~e,adously as a function of age 7'1s. It also seems that the sensitivity of the young brain to the deleteriou~ effects of seizures may change with age 19. Therefore, it is important to determine mechanisms of epileptogenesis at various ages and to determine the consequences of repeated seizures when they occur at different stages of development of the brain. Attention has been drawn to the hippocampus in human epilepsy because of striking morphological and functional abnormalities detected in this structure in certain patients with intractable complex partial seizures. In the literature on experimental epilepsy in animals, there has been a corresponding interest in the hippocampus
because of the well-known ease with which seizures can be elicited in this region and the utility of bippocampal slices for detailed electrophysiological studie~. Kindling ~°'2s is widely accepted as a powerful animal model of complex partial seizures with secondary generalization. The procedure traditionally used for kindling relies on stimuli given once daily. This approach impacts on experimental design in several ways. For instance, the traditional protocol is not practical for studying kindling in immature rats since the time needed to kindle spans a major portion of development of the brain. In addition, it may be technically difficult to measure electrophysiological responses before and after kindling. To deal with these issues, we have developed and characterized 'rapid kindling' protocols that elicit focal seizures every few minutes through hippocampal electrodes ~3'2t. Using this approach we have examined excitatory and inhibitou; neurotransmission before and after rapid kindling in adult rats and found evidence for substvntial decreases in 7-aminobutyric acid (GABA)ergic inhibition in the CA1 region lt't4. In other recent work 24 we have developed techniques for assessing excitatory and inhibitory neurotransmission in the hippocampal formation of rats down to postnatal
* Present address: Department of Pharmacology, Box 29, SUNY/Health Science Center at Brooklyn, 450 Clarkson Avenue, Brooklyn, N.Y. 11203, USA. Correspondence: H. Michelson, Department of Pharmacology, Box 29, SUNY/Health Science Center at Brooklyn, 450 Ciarkson Avenue, Brooklyn, N.Y. 11203, USA. Fax: (1) (718) 270-2241.
238 7 days of age (PN 7). We found that there was a delay in the maturation of paired pulse inhibition in the C A I region of the ~ippocampus for interpulse intervals that reflect G A B A e r g i c inhibition t2aS. These findings are in agreement with dual intracellular m e a s u r e m e n t s of G A B A e r g i c inhibition in CA1 pyramidal cells 3°'33. We also reported 25 that when the rapidly recurring hippocampal stimulus pretocol is applied to unanesthetized young rats (postnatal (PN) 7-28), both motor and electrographic kindling take place. The studies reported below were u n d e r t a k e n to examine the influence of the maturation of G A B A e r g i c inhibition on epileptogenesis in the rat hi ppoc a m pa l formation. I m m a t u r e rats from PN 7 to PN 28 were studied, ages which span a period from the absence to the mature expression of G A B A - m e d i a t e d inhibition in C A I , as revealed by intracellulat and extracellular studies 25'33. Both epileptogenesis in the 'baseline' conditior~ (as defined by afterdischarges thresholds and initial afterdischarges durations) and seizure-induced alterations (lengthening of afterdischarges with rapid kindling and changes in paired-pulse responses at interpulse intervals that reflect G A B A e r g i c inhibition) were assessed. MATERIALS AND METHODS Sprague-Dawley rats, PN ages 7 to 28 days, were anesthetized with urethane (1.2 g/kg, i.p,). A concentric bipolar insulated stainless steel stimulating electrode (SNEX 200, Rhodes Instruments, Tujunga, CA) was lowered into the CA3 region of the right hippocampus. A glass recording microeleetrode filled with 2% Fast green in 2 M NaCI (2-4 MfJ) was lowered into the contralateral CAI region. The potential from the tip of the recording electrode was antplified and referred to an Ag/AgCI electrode placed subcutaneously in the scapular area. Five age groups were studied: PN 7 (7-10 days of age: n -- 5), PN 14 (rats 13-15 days: n - 4)~ PN 18 (17-19 days: n -- 7): PN 21 (20-22 days: n = 4), and PN 28 (27-29 days: n = 5). Stereotaxic coordinates and surgical procedures are described elsewhere (cf. Table II in ref. 25), Core body temperaturc was maintained at 37°C throughout the course of the experiment using either a heating pad or a heat lamp. Following completion of the experiments, locations of the stimulating and recording electrodes were marked by iron deposition and microiontophoresis, respectively t3. Histologic analysis verified stimulating and recording electrode placements at the targeted sites for all the age groups. Determination of input-output curves and paired pulse inhibition Methods for determination of input-output curves and pairedpulse inhibition are described in detail elsewhere t4'24. Briefly, extracellular field potentials were recorded in the stratum pyramidale of the CAI hippocampal region in response to stimulation (0.4 ms, 0.1 Hz, 1-80 V) of the contralateral CA3 region. Final positions of the electrodes were adjusted with micromanipulators to maximize population spike (PS) amplitudes in response to CA3 stimulation. Once the final position of the electrode was determined, an additional 45-60 rain were allowed to elapse prior to data collection. Data analysis was performed with computer assistance according to the methods of Aitken ~. As previously reported 24. considerable variation in population excitatory postsynaptic potentials slopes was observed within each age group: therefore, analyses focused on the population spike. For
each animal, a minimum of 6 responses to single test pulses at specific stimulus intensities were averaged as the stimulus strength was gradually increased frem subthreshold to supramaximal values for PS amplitudes. These values were used to generate 'input-output' (stimulus intensity vs. PS ami~!izude) curves. Paired stimuli were used to assess the potency of paired pulse inhibition14"24. Inhibition was quantified by comparing the ratio of the test (second) ~,iJulation spike amplitude [PS(T)] to that of the conditionin~ (fi~) population spike [PS(C)]. With this procedure, the degree of paired pulse inhibition varies in relation to the amplitude of PS(C) (and thus the stimulus intensity used) and the interpulse interval. However, above a certain stimulus intensity, the degree of inhibition at a specific interpulse interval becomes independent of PS(C) and remains constant 14"24. In the present studies, the determination of paired pulse inhibition was conducted using stimulus intensities high enough to exceed this critical threshold and elicit a population spike of maximal size. Data was collected and averaged for an interpulse interval of 20 ms, which represents the point at which inhibition was maximized in the region of the PS(T)/PS(C) vs. interpulse interval cu.Tye that monitors inhibition mediated by the GABAA receptor 12't4ab'. Therefore, as quantified in our prior studies, data will be presented in terms of an index of maximum inhibition (the PS(T)/PS(C) rates at 20 ms IPI). By subtracting the indices of maximal inhibition obtained after seizures from indices of maximal inhibition obtained before seizures, another parameter, amount of inhibition lost, was derived TM. Afterdischarge threshold determination Afterdischarge thresholds (ADT) were determined using a 10 s train of 20 Hz, 1 ms biphasic square wave pulses according to methods described before 21, increasing current intensity up to a maximum of 3000 pA peak-to-peak. In some younger rats (PN 7), no afterdischarge was obtained with 3000/~A. These animals were excluded from the study. Alteratiops in input-output relations and paired pulse inhibition 30 and 60 min following a single seizure were assessed by measurements made following ADT determination for the separate age groups examined. Rats were then used in subsequent kindling studies.
Kindling protocol Following the ADT determinations and electrophysiological measurements just described, rapid kindling with a rapidly recurring hippocampal seizure (RRHS) protocol was initiated. The kindling stimulus was a 20 Hz, 10 s train of I ms biphasic square wave pulses delivered once every 5 rain at a current intensity set at twice ADT. These stimulus parameters were chosen after preliminary studies determined that the younger animals could follow a RRHS protocol when the stim,:!ation was delivered at a 20 Hz, but not a 50 Hz frequency. Twenty Hz, 10 s trains were also used in a prior study of RRHS in unanesthetized rats ~. Rats were stimulated every 5 min for 6 h, for a total of 72 stimulations. The afterdischarge durations associated with each stimulation were measured. With the urethane anesthesia used, no motor seizures occurred and, therefore, behavioral scores were not quantified. Because of variability of the lengths of individual serial afterdischarges (see ref. 25), for purposes of analysis averages of the first 6 afterdischarge durations and of the final 6 afterdischarge durations were also calculated. These determinations were tabulated and ane~lyzedas initial block afterdischarge durations and final block afterdischarge durations, respectively. Calculations were done on afterdischarges recorded both from the stimulating and the contralateral electrodes. One hour following completion of RRHS, input-output curves of stimulus intensity vs. PS amplitude were again determined and paired pulse inhibition again quantified. Statistical analysis for significant differences between groups was performed on pooled data using a one-way analysis of variance (ANOVA). When appropriate Newman-Keuls tests were subsequently performed to determine which pairs were different, t-Tests were used for pairwise comparisons as indicated below. For all tests
239 A
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POSTNATAL AGE IN DAYS
Fig, 1. Afterdischarge thresholds (ADT) for various ages rats. ADT in/~A (mean + S.E.M,, ordinate) are given for rats of various postnatal ages (abscissa). There was a significant difference of ADT across the 5 age groups studied (ANOVA F = 11.2, P < 0.01). Asterisk indicates significant difference from all other ages (P < 0.05, Newman-Keuis tests). For all ages -> PN 14, afterdischarges were obtained in all animals. In 3 of the 8 animals tested at PN 7, there were no afterdischarges (with stimulus intensities up to 3000 /~A). These animals were excluded from the study; the data presented reflects only animals exhibiting afterdischarges.
a P < 0.05 level was set for statistically significant differences. RESULTS Findings with the initial seizure As the first assessment of epileptogenicity in different ages of rats, we determined ADT. To allow comparisons with our previous studies on awake animals, stimuli trains were 20 Hz, 10 s trains of I ms biphasic pulses. A D T were found to vary inversely with age: 1025.4- 225 (mean :l: S . E . M . ) , 488 -4- 43, 328 :l: 50, 318 :l= 110, and 251 + 33/~A for PN 7, PN 14, PN 18, PN 21, PN 28 groups, respectively. While there was a trend for A D T to decrease throughout the ages studied (Fig. 1), only the
ADTs for the youngest group (PN 7) were statistically different from all older other ages. A similar pattern of ontogeny of A D T was found in awake animals not anesthetized with urethane 2s. In the urethane-anesthetized rats in this study, ADTs were consistently higher than those in unanesthetized animals. In our prior study on the unanesthetized animals, the thresholds (means _+ S.E.M.'s) were 355 + 58, 158 + 40, 198 + 27, and 110 _+ 1 ! / ~ A for ages PN 7, PN 14, PN 21, and PN 28, respectively. Except for the PN 21 groups, the ADTs in the urethane-anesthetized animals were higher than the corresponding thresholds in the awake animals (P < 0.05, grouped t-tests). In our previous studies on the influences of seizures on paired pulse inhibition in the hippocampus of adult rats 14, we found no difference in the potency of inhibition measured before and 60 min after an isolated seizure. Therefore, in the present study, we examined the immature animals in the same fashion. The potency of paired pulse inhibition was determined before and 1 h after a single seizure for an interpulse interval of 20 ms. Prior work (see refs. 12 and 14 for details) has shown that the measurements at this particular interpulse interval reliably assess changes in the potency of GABAergic inhibition. Baseline (preseizure) indices o f maximal inhibition for the different aged rats in this study are given in the top row of Table I. For PN 7, PS(T)/PS(C) measurements showed no evidence of inhibition, but rather reflected paired pulse facilitation. For all other ages, inhibition was detected which varied with age. These data for individual ages are comparable to those reported previously (Table III, ref. 24) and showed statistically significant differences across the study groups ( A N O V A F = 25.2, P < 0.001 for 5 sets of data, top row in Table I). Indices of maximal inhibition for the PN 7 and PN 14 groups were significantly different from all older age groups (P < 0.05, Newman-Keuls tests).
TABLE I Changes in indices of maximal inhibition after single seizure and after recurrent seizures at different postnatal ages
Test population spike amplitude/conditioning population spike amplitude (PS(T)/PS(C)) rates at 20 ms interpulse intervals; (cf. Materials and Methods). For values less than 1.0, paired pulse inhibition was present; for values greater than 1.0, responses represented paired pulse facilitation, aNote that baseline values show significant age-dependence, bAmount of inhibition lost under specified condition [Index of maximal inhibition after seizure(s) subtracted for index of maximal inhibition before seizure(s); negative numbers denote a decrease in potency of paired pulse inhibition while positive numbers denote an increased potency].
Baseline a 60 lPin b after single seizure 60 rainb after RRHS
PN 7
PN 14
PN 18
PN 21
PN 28
1.51 + 0.20 +0.10 __. 0.15 -0.21 +_,0.19
0.93 + 0.30 -0.20 ± 0.19 -0.07 + 0.13
0.21 + 0.04 -0.13 + 0.06 -0.14 + 0.19
0.06 + 0.04 -0.14 _ 0.06 -0.28 + 0.12"
0.07 -4- 0.04 -0.06 + 0.02 -0.24 + 0.06*
* Difference between baseline and after seizure(s) measurement for specified conditions (age; single vs. recurrent seizures); P < 0.05 paired t-tests. Data presented as mean + S.E.M.
240
The amount of inhibition lost 60 min after a single seizure for the various ages studied are given in the second row of Table I. There was a tendency for larger loss of inhibition in the animals of intermediate ages (PN 14, PN 18, and PN 21), the period when GABA-mediated inhibition was rapidly n~aturing. However, none of the determinations achieved statistical significance (paired t-tests comparing indices of maximal inhibition before and after single seizure). The amount of inhibition lost at the various ages examined was not significantly different (ANOVA F = 1.21, P > 0.05 for row 2 of Table I). Although we did not carry out a detailed study, it was nc.ted that there was occasionally a small rightward shift of input-output curves for PS after the single seizures.
Findings with repetitive seizures Our next step was to determine the consequences of RRHS on the electrophysiological parameters introduced above. As in prior studies 14'21'25, we found in the current experiments that seizures were reliably elicited by each stimulus. Furthermore, when tile seizures were repeated, they lengthened.. Individual recordings showed that seizures recorded on the two sides were well syn-
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chronized and terminated within a few seconds of each other (Fig.2). Measurements on afterdischarges for all ages studied are summarized in Table II. For each age group the initial block afterdischarge durations were not different between ipsilateral (to stimulation) and contralateral sides (grouped t-tests). For each age group studied, the afterdischarges on the two side.~ showed similar lengthening (no significant difference in ipsilateral or contralatera! final block afterdischarge durations, grouped t-tests). For each age group studied, there was substantial lengthening (statistically significant difference between final block afterdischarge durations and initial block afterdischarge durations -- P < 0.05 in all cases, paired t-tests), with a nearly two- to three-fold increment. There was no significant difference among the 5 age groups studied with respect to initial block afterdischarge durations ( A N O V A F = 2.40 ipsilateral and F -- 1.29 contralateral), with respect to final block afterdischarge durations, (F = 2.57 ipsilateral, F = 2.69 contralateral), or to degree of lengthening ( F = 2.53 ipsilateral, F = 2.49 contralateral). There was no discernable behavioral accompaniment to the electrographic seizures at any time throughout the recurrent hippocampal seizure protocol in any of the age groups examined. The amount of inhibition lost after the R R H S protocol for the various ages is given in row 3 of Table I. For the animals in which there was some paired pulse inhibition present before seizures (ages -- PN t4), there was
a trend for the amount of inhibition lost to increase in parallel with age, even though this did not achieve statistical significance ( A N O V A F = 1.88, P > 0.05 for row
!
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3 data in Table I). There was a statistically significant
TABLE II
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Electrographic responses of developing rats to RRHS
2. Electrographic
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the bippocampi of young rats. In each panel the top two traces give electroencephalographic tracings recorded from stimulation site (1) and from recording site in contralaterai (2) hippocampus (see Materials and Methods), Bottom tracings mark I s between short lines and 5 s between long lines (note change in paper speed in B). During interval indicated by dotted line, bipolar electrode at stimulus site was switched from recording to stimulus mode to give a 20 Hz, I0 s train of 1 ms pulses; at other times this electrode was in record mode (for details see ref. 21). Note synchronicity and similarity of waveforms of afterdischarges at the two recording sites. Calibration bar at left betwee, traces 1 and 2 in each panel: 3 mV for 1 and 2 mV for 2. A: frot3 72nd stimulus in rapidly recurring hippocampal seizure (RRHS) protocol in PN 14 rat; B: from 72nd stimulus in RRHS protocol in PN 28 rat.
Ipsilateral side PN 7 PN 14 PN 18 PN 21 PN 28
10,6 + 1.7 15.4 ± 3.2 11.0 :!: 2,0 10,3 + 2,1 9.9 ± 1.4
18,6 ± 33.7 ± 21.6 ± 33.2 + 27.8 ~
2.8 5.1 3,5 3.6 3.5
1.9 2,4 2.2 3.4 3.0
± ± ± ± ±
0.3 0.4 0.4 0.4 0.2
Contralateral side PN 7 10.5 ± 1.7 PN 14 15.1 + 3.1 PN 18 12.3 ± 2.2 PN 21 10.1 ± 2.3 PN 28 9.0 ± 1.5
18.8 + 33.6 + 21,8 + 32.l ± 27.7 ±
3.0 5.1 3.6 2.9 2.9
1.9 2.4 2.2 3.6 3.4
+ + ± ± ±
0.4 0.5 0.5 0.5 0.4
Data presented as mean + S.E.M. For all ages, there was a significant lengthening of afterdischarges on both the ipsilateral and contralateral sides.
241 loss of inhibition after repeated seizures only for the two older groups for which the indices of maximal inhibition had reached adult values (grouped t-tests for PN 21 and PN 28 P < 0.05; P > 0.05 in all other cases). As was the case after single seizures (see above) and as has been observed in adult animals ~4, there was a tendency for the input-output curves for PS to show a rightward shift. However, this was not systematically analyzed. DISCUSSION Two main conclusions were drawn from the results presented above. First, epileptogenic properties in the rat hippocampus are well-developed as early as PN 7. Second, in our studies, which were performed throughout the period when GABAergic inhibition in the hippocampus was increasing, there was no age-related decrease in hippocampai epileptogenicity. The findings indicate a complex interplay between GABAergic inhibition and epileptogenesis as the rat hippocampal formation matures. Two observations from the foregoing experiments support the contention that epileptogenesis in the hippocampus is well-developed early in life. Robust afterdischarges could be elicited in all age groups examined. Furthermore, all ages showed the capability of substantial increases in afterdischarge duration during the RRHS protocol. Thus, the present findings, in conjunction with our previous studies 2s, demonstrate that, as with adult rats 14'2~'2~'2s,rapid kindling of afterdischarges takes place in rat pups both under urethane anesthesia, or in awake animals. Findings of the current study are in agreement with prior in vivo work 2'24 and in vitro 3°'33 work that demonstrate that the potency of paired pulse inhibition varied greatly across the various age groups examined, from none detectable (at PN 7) to fully developed, levels. GABA-mediated inhibition has received considerable attention as a major factor in epileptogenesis4'5'u-~3'~6'23' 27.3~,32. In several of these studies, investigators have noted a deterioration of GABA.mediated inhibition during a seizure or after a series of seizures. However, all these studies were done in adult animals. Fewer workers have investigated the role of GABAergic inhibition in modulating seizure activity in the immature brain, although deFeo et al. ~ have demonstrated that young rats are sensitive to seizures via blockade of GABAergic inhibition. Since a diminution of GABA-mediated inhibition is considered as a means to promote epileptogenesis, one would expect that with less potent inhibition, seizure activity would be more severe. However, this was not
found in the above experiments. In fact, for the youngest age group studied, in which paired pulse inhibition was not detected (see also ref. 24) and intraceUular recordings have documented the absence of GABAergic inhibition, the ADTs were highest; whereas the lowest ADTs were found in the animals with the most potent paired pulse inhibition, an age at which GABAA-mediated IPSPs are known to be present 3°'33. Thus, in the present experiments, ADTs were found to be highly age-dependent, and inversely related to the strength of GABAA-mediated inhibition. Moshe et al. 26 have obtained the same results with different methods. Gilbert also reported a resistance of the very young brain to afterdischarges induced by electrical stimulation 9. In addition, initial block afterdischarge durations did not decrease with age as would be predicted if epileptogenesis were merely a function of the strength of inhibition. One might argue that it is not the baseline potency of inhibition before seizures that matters, but rather how inhibition can withstand seizure discharges, especially when they recur. Nonetheless, certain findings presented above speak against this last idea. For instance, the deterioration of paired pulse inhibition with recurrent seizures was largest for the two oldest groups, whereas the younger animals, with less baseline GABAergic inhibition, showed less deterioration of inhibition following kindling. However, the older groups did not show any greater electrographic kindling. It should also be noted at this point that our results indicate that seizure-induced plasticity of inhibition, as measured by changes in indices of maximal inhibition, does not occur only in the adult hippocampus, but also at younger ages. Interestingly, only small changes are s e e n at earlier ages, when paired pulse inhibition is not as potent as at the later ages. Thus, it seems that seizure-induced 'disinhibition' requires a certain level, or certain degree of maturity, before it can develop. In contrast to the age-dependent changes of ADTs, we found no differences in the age groups examined with respect to either the initial block of afterdischarge durations or to the propensity of afterdischarges to lengthen when repeated. For technical reasons, the stimulus trains use,J in this study and in our prior studies on adult rats under urethane anesthesia n'14 were quite similar but not identical. Thus, rigorous comparisons will not be made between the res,~!ts of the current work and previous findings. However, the initial afterdischarge durations in young rats and the durations in adults are comparable and the degrees of lengthening in the various age groups with repeated seizures are also similar 12'~4. It is of interest that the electrographic seizures recorded at the site of hippocampal stimulation and in the
242 contralateral hippocampus were synchronized during the discharges and terminated at virtually the same time. This finding and the fact that the afterdischarges on the two sides showed identical lengthening during the RRHS protocol support the concept of a tight linkage between the two hippocampi in terms of seizures in our studies. Previous studies indicate that, in adult rats, seizures elicited with hippocampal stimulation spread from the site of stimulation to the contralateral hippocampus via the hippocampal commissure 34'35. Based on the electroencephalic recordings in all groups in the current work, it seems that a likely pathway by which the two hippocampi in young rats become synchronized during seizures is the hippocampal commissures. How can one then account for the seemingly paradoxical findings that, with very poorly developed inhibition in the young hippocampus there is not a greater epileptogenicity, revealed by longer afterdischarges, more robust electrographic kindling, or lower ADT. It may be that there is some system in the young brain that retards or opposes seizures, thereby counteracting the immaturity of GABA-mediated inhibition, that later recedes as REFERENCES 1 Aitken, P.J., Kainic acid and penicillin: differential effects of excitatory and inhibitory interactions in the CAt region of the hippocampal slice, Brain Res., 325 (1985) 261-269. 2 Bekenstein, J.W. and Lothman, E.W., A comparison of excitatory and inhibitory neurotransmission in the CA1 region and dentate gyrus of the rat hippocampal formation, Dev. Brain Res., 63 (1991) 237-224. 3 Bekenstein, J.W. and Lothman, E.W., An in vivo study of the ontogeny of long-term potentiation (LTP) in the CAI region and in the dentate gyrus of the rat hippocampal formation, Dev. Brain Res., 63 (1991) 245-252. 4 Ben.Ari, Y., Krnjevic, K., Reiffenstein, R.J. and Reinhardt, W., Inhibitory condt,ctance changes and action of gamma-ami. nobutyrate in rat hippocampus, Neuroscience, 6 (1981) 24452463. 5 Ben-Ari, Y., Krnjevic, K. and Reinhardt, W., Hippocampal sei. zures and failure of inhibition, Can. J. Physiol. Pharmacol., 57 (1979) 1462-1466. 6 de Feo, M.R.. MecareUi, O. and Ricci, G.F., Bicuculline. att0 allyglycine-induced epilepsy in developing rats, Exp. Neurol., 90 (1985) 411-421. 7 Dreifuss, F.E., Pediatric Epileptoiogy, John Wright, London, 1983, 302 pp. 8 Engel Jr., J., Epilepsy and Seizures, EA. Davis, Philadelphia, 1989, 536 pp. 9 Gilbert, M.E. and Cain, D.P., A developmental study of kindling in the rat, Dev. Brain Res., 2 (1982) 321-328. 10 Goddard, G.V., Mclntyre, D.C. and Leech, C.K., A permanent change in brain functioning resulting from daily electrical stimulation, Exp. Neurol., 25 (1969) 295-330. 11 Kapur, J., Bennett Jr., J.R, Wooten, G.E and Lothman, E.W., Evidence for a chronic loss of inhibition in the hippocampus after kindling: biochemical studies, Epilepsy Res., 4 (1989) 100.108. 12 Kapur, J. and Lothman, E.W., NMDA receptor activation mediates the loss of GABAergic inhibition induced by recurrent seizures, Epilepsy Res., 5 (1990) 103-111.
the animal ages. Alternatively it may be that, in the young brain, systems important for supporting seizures are not yet present or fully developed. This may relate to molecular aspects of excitatory neurotransmission like long-term potentiation 2'3 or aspects of circuit development and the ability to propagate seizures ~s'24. As ontogeny progresses, these systems would mature in parallel with the maturation of inhibition, so that the two would balance each other. These ideas are at present speculations raised to deal with the findings encountered in the present studies. A resolution of these issues, as well as the puzzle of age-dependence of seizure-associated changes of inhibition, awaits further studies. Presently we can only conclude that the ontogeny of epileptogenesis in the hippocampus is complex and that GABA-mediated inhibition is but one contributing factor.
Acknowledgements. Supported in part by USPHS Grsnts NS 21671, NS 25605 and HD 07323. We thank Rose Powelt, Marie Noel, and Patricia Caines for assistance in preparation of the manuscript.
13 Kapur, J., Michelson, H.B., Buterl~augh, G.G. and Lothman, E.W., Evidence for a chronic loss of inhibition in the hippocampus after kindling: electrophysiological studies, Epilepsy Res., 4 (1989) 90-99. 14 Kapur, J., Stringer, J.L. and Lothman, E.W., Evidence that repetitive seizures in the hippocampus cause a lasting reduction of GABAergic inhibition, J. Neurophysiol., 61 (1989) 417-426. 15 Kehi, S.J. and McLennen, H., Evidence for a bicuculline.in. sensitive long-lasting inhibition in the CA3 region of the rat hippocampal slice, Brain Res., 299 (1983) 278-281. 16 Krnjevic, K., GABA-mediated inhibitory mechanisms in rela. tion to epileptic discharges. In H.H. Jasper and N.M. van Gelder (Eds.), Basic Mechanisms of Neuronal Hyperexcltabll. it),, A.R. List, New York, 1983, pp. 249-280. 17 Lee, S..S., Murata, R. and Matsura, S., Developmental study of hippocampal kindling, Epilepsia, 30 (1989) 266-270. 18 Lothman, E.W., Pathophysiology of seizure and epilepsy in the mature and immature brain: cells, synapses and circuits. In J.W. Pellock and E. Dodson (Eds.), Epilepsy in Childhood, Demos, New York, in press. 19 Lothman, E.W. and Bertram III, E.H., Epileptogenic effects of status epilepticus, Epilepsia, in press. 20 Lothman, E.W., Bertram III, E.H. and Stringer, J.L., Functional anatomy of hippocampal seizures, Prog. Ne~robiol., 37 (1991) 1-82. 21 Lothman, E.W., Hatlelid, J.M., Zorumski, C.F., Conry, J.A., Moon, P.E and Perlin, J.B., Kindling with rapidly recurring hippocampai seizures, Brain Res., 360 (1985) 83-91. 22 Lothman, E.W., Salerno, R.A., Perlin, J.B. and Kaiser, D.L., Screening and characterization of antiepileptic drugs with rapidly recurring hippocampal seizures, Epilepsy Res., 2 (1988) 367-379. 23 McCarren, M. and Alger, B.E., Use-dependent depression of lPSPs in rat hippocampai pyramidal cells in vitro, J. Neuro. physiol., 53 (1985) 557-571. 24 Micheison, H.B. and Lothman, E.W., An in vivo electrophysiological study of the ontogeny of excitatory and inhibitory processes in the rat hippocampus, Dev. Brain Res., 47 (1989) 113122.
243 25 Michelson, H.B. and Lothman, E.W., An ontogenetic study of kindling using rapidly recurring hippocampal seizures, Dev. Brain Res., 61 (1991) 79-85. 26 Moshe, S.L., Sharpless, N.S. and Kaplan, J., Kindling in developing rats: variability of afterdischarge thresholds with age, Brain Res., 211 (1981) 190-195. 27 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. 28 Racine, R.J., Modification of seizure activity by electrical stimulation. I. After-discharge threshold, EEG Clin. NeuropiiysioL, 32 (1972) 269-279. 29 Rodin, E., The Prognosis in Patients with Epilepsy, Charles C. Thomas, Springfield, Illinois, 1968, 455 pp. 30 Schwartzkroin, P.A., Development of rabbit hippocampus: physiology, Dev. Brain Res., 2 (1982) 469-486.
31 Sloviter, R.S., Decreased hippocampal inhibition and a selective loss of interneurons in experimental epilepsy, Science, 235 (1987) 73-76. 32 Stelzer, A., Siater, N.T. and ten Bruggencate, G., Activation of NMDA receptors blocks GABAergic inhibition in an in vitro model of epilepsy, Nature, 326 (1987) 698-701. 33 Swann, J.W., Brady, R.J. and Martin, D.L., Postsynaptic development of GABA-me,~iated synaptic inhibition in rat hippocampus, Neuroscience, 28 (1988) 551-561. 34 VanLandingham, K.E. and Lothman, E.W., Self-sustaining limbic status epilepticus. I. Acute and chronic cerebral metabolic studies -- limbic hyperm~:tabolism and neocortical hypometabolism, Neurology, in press. 35 VanLandingham, K.E. arid Lothman, E.W., Self-sustaining limbic status epilepticus. I1. Role of hippocampal commissures in metabolic responses, Neurology, in press.
237
BRESD 51438
Ontogeny of epileptogenesis in the rat hippocampus: a study of the influence of GABAergic inhibition Hillary B. Michelson*
a n d E r i c W. L o t h m a n
Department of Neurology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908 (USA)
(Accepted 24 December 1991) Key words: Epilepsy; Ontogeny; Hippocampus; Development; 7-Aminobutyric acid-ergic inhibition; Seizure; Afterdischarge; Kindling
In vivo experiments were carried out to examine whether the period during which y-aminobutyric acid (GABA)ergic inhibition in the hippocampus matures is associated with a decrease in epileptogenesis. Seizures were elicited with bipolar electrodes stcreotactically positioned in the hippocampus of urethane-anesthetized rat pups from postnatal (PN) 7 through 28 days of age. No clinical seizure activity was detected but electrographic seizures (afterdischarges) were induced at all ages. Afterdischarge thresholds (ADT) varied inverse!y with age. However, the durations of initial afterdischarges and the degree of lengthening of afterdischarges with the rapidly recurring hippocampal seizure (RRHS) protocol were not different for the various age animals studied. Paired pulse inhibition was assessed with a twin pulse paradigm that has been shown to monitor GABAergic inhibition. Measurements were made before and 60 min after a single seizure and again 60 rain after the RRHS protocol. At no age was there a significant change'in paired pulse inhibition after a single seizure. After RRHS there was a significant reduction ot paired pulse inhibition only in the groups that had manifested adult levels of paired pulse inhibition in preseizure measurements (->PN 21). These studies indicate that heightened epileptogenesis in the young hippocampus cannot simply be explained on the basis of an immaturity of GABA-mediated inhibition. INTRODUCTION Based on animal and clinical observations~ the idea has been advanced that the occurrence of seizures leads to a chronic disturbance of brain function which, in turn, promotes further seizures s't°'~s'tg'29. Thus, a thorough examination of the pathophysiology of epilepsy involves more than just the factors that can induce seizures. Certain phenomena also emphasize the age-dependent aspects in epilepsy. The younger brain is more prone to seizures, and the clinical expression of seizures can vary tre¢~e,adously as a function of age 7'1s. It also seems that the sensitivity of the young brain to the deleteriou~ effects of seizures may change with age 19. Therefore, it is important to determine mechanisms of epileptogenesis at various ages and to determine the consequences of repeated seizures when they occur at different stages of development of the brain. Attention has been drawn to the hippocampus in human epilepsy because of striking morphological and functional abnormalities detected in this structure in certain patients with intractable complex partial seizures. In the literature on experimental epilepsy in animals, there has been a corresponding interest in the hippocampus
because of the well-known ease with which seizures can be elicited in this region and the utility of bippocampal slices for detailed electrophysiological studie~. Kindling ~°'2s is widely accepted as a powerful animal model of complex partial seizures with secondary generalization. The procedure traditionally used for kindling relies on stimuli given once daily. This approach impacts on experimental design in several ways. For instance, the traditional protocol is not practical for studying kindling in immature rats since the time needed to kindle spans a major portion of development of the brain. In addition, it may be technically difficult to measure electrophysiological responses before and after kindling. To deal with these issues, we have developed and characterized 'rapid kindling' protocols that elicit focal seizures every few minutes through hippocampal electrodes ~3'2t. Using this approach we have examined excitatory and inhibitou; neurotransmission before and after rapid kindling in adult rats and found evidence for substvntial decreases in 7-aminobutyric acid (GABA)ergic inhibition in the CA1 region lt't4. In other recent work 24 we have developed techniques for assessing excitatory and inhibitory neurotransmission in the hippocampal formation of rats down to postnatal
* Present address: Department of Pharmacology, Box 29, SUNY/Health Science Center at Brooklyn, 450 Clarkson Avenue, Brooklyn, N.Y. 11203, USA. Correspondence: H. Michelson, Department of Pharmacology, Box 29, SUNY/Health Science Center at Brooklyn, 450 Ciarkson Avenue, Brooklyn, N.Y. 11203, USA. Fax: (1) (718) 270-2241.
238 7 days of age (PN 7). We found that there was a delay in the maturation of paired pulse inhibition in the C A I region of the ~ippocampus for interpulse intervals that reflect G A B A e r g i c inhibition t2aS. These findings are in agreement with dual intracellular m e a s u r e m e n t s of G A B A e r g i c inhibition in CA1 pyramidal cells 3°'33. We also reported 25 that when the rapidly recurring hippocampal stimulus pretocol is applied to unanesthetized young rats (postnatal (PN) 7-28), both motor and electrographic kindling take place. The studies reported below were u n d e r t a k e n to examine the influence of the maturation of G A B A e r g i c inhibition on epileptogenesis in the rat hi ppoc a m pa l formation. I m m a t u r e rats from PN 7 to PN 28 were studied, ages which span a period from the absence to the mature expression of G A B A - m e d i a t e d inhibition in C A I , as revealed by intracellulat and extracellular studies 25'33. Both epileptogenesis in the 'baseline' conditior~ (as defined by afterdischarges thresholds and initial afterdischarges durations) and seizure-induced alterations (lengthening of afterdischarges with rapid kindling and changes in paired-pulse responses at interpulse intervals that reflect G A B A e r g i c inhibition) were assessed. MATERIALS AND METHODS Sprague-Dawley rats, PN ages 7 to 28 days, were anesthetized with urethane (1.2 g/kg, i.p,). A concentric bipolar insulated stainless steel stimulating electrode (SNEX 200, Rhodes Instruments, Tujunga, CA) was lowered into the CA3 region of the right hippocampus. A glass recording microeleetrode filled with 2% Fast green in 2 M NaCI (2-4 MfJ) was lowered into the contralateral CAI region. The potential from the tip of the recording electrode was antplified and referred to an Ag/AgCI electrode placed subcutaneously in the scapular area. Five age groups were studied: PN 7 (7-10 days of age: n -- 5), PN 14 (rats 13-15 days: n - 4)~ PN 18 (17-19 days: n -- 7): PN 21 (20-22 days: n = 4), and PN 28 (27-29 days: n = 5). Stereotaxic coordinates and surgical procedures are described elsewhere (cf. Table II in ref. 25), Core body temperaturc was maintained at 37°C throughout the course of the experiment using either a heating pad or a heat lamp. Following completion of the experiments, locations of the stimulating and recording electrodes were marked by iron deposition and microiontophoresis, respectively t3. Histologic analysis verified stimulating and recording electrode placements at the targeted sites for all the age groups. Determination of input-output curves and paired pulse inhibition Methods for determination of input-output curves and pairedpulse inhibition are described in detail elsewhere t4'24. Briefly, extracellular field potentials were recorded in the stratum pyramidale of the CAI hippocampal region in response to stimulation (0.4 ms, 0.1 Hz, 1-80 V) of the contralateral CA3 region. Final positions of the electrodes were adjusted with micromanipulators to maximize population spike (PS) amplitudes in response to CA3 stimulation. Once the final position of the electrode was determined, an additional 45-60 rain were allowed to elapse prior to data collection. Data analysis was performed with computer assistance according to the methods of Aitken ~. As previously reported 24. considerable variation in population excitatory postsynaptic potentials slopes was observed within each age group: therefore, analyses focused on the population spike. For
each animal, a minimum of 6 responses to single test pulses at specific stimulus intensities were averaged as the stimulus strength was gradually increased frem subthreshold to supramaximal values for PS amplitudes. These values were used to generate 'input-output' (stimulus intensity vs. PS ami~!izude) curves. Paired stimuli were used to assess the potency of paired pulse inhibition14"24. Inhibition was quantified by comparing the ratio of the test (second) ~,iJulation spike amplitude [PS(T)] to that of the conditionin~ (fi~) population spike [PS(C)]. With this procedure, the degree of paired pulse inhibition varies in relation to the amplitude of PS(C) (and thus the stimulus intensity used) and the interpulse interval. However, above a certain stimulus intensity, the degree of inhibition at a specific interpulse interval becomes independent of PS(C) and remains constant 14"24. In the present studies, the determination of paired pulse inhibition was conducted using stimulus intensities high enough to exceed this critical threshold and elicit a population spike of maximal size. Data was collected and averaged for an interpulse interval of 20 ms, which represents the point at which inhibition was maximized in the region of the PS(T)/PS(C) vs. interpulse interval cu.Tye that monitors inhibition mediated by the GABAA receptor 12't4ab'. Therefore, as quantified in our prior studies, data will be presented in terms of an index of maximum inhibition (the PS(T)/PS(C) rates at 20 ms IPI). By subtracting the indices of maximal inhibition obtained after seizures from indices of maximal inhibition obtained before seizures, another parameter, amount of inhibition lost, was derived TM. Afterdischarge threshold determination Afterdischarge thresholds (ADT) were determined using a 10 s train of 20 Hz, 1 ms biphasic square wave pulses according to methods described before 21, increasing current intensity up to a maximum of 3000 pA peak-to-peak. In some younger rats (PN 7), no afterdischarge was obtained with 3000/~A. These animals were excluded from the study. Alteratiops in input-output relations and paired pulse inhibition 30 and 60 min following a single seizure were assessed by measurements made following ADT determination for the separate age groups examined. Rats were then used in subsequent kindling studies.
Kindling protocol Following the ADT determinations and electrophysiological measurements just described, rapid kindling with a rapidly recurring hippocampal seizure (RRHS) protocol was initiated. The kindling stimulus was a 20 Hz, 10 s train of I ms biphasic square wave pulses delivered once every 5 rain at a current intensity set at twice ADT. These stimulus parameters were chosen after preliminary studies determined that the younger animals could follow a RRHS protocol when the stim,:!ation was delivered at a 20 Hz, but not a 50 Hz frequency. Twenty Hz, 10 s trains were also used in a prior study of RRHS in unanesthetized rats ~. Rats were stimulated every 5 min for 6 h, for a total of 72 stimulations. The afterdischarge durations associated with each stimulation were measured. With the urethane anesthesia used, no motor seizures occurred and, therefore, behavioral scores were not quantified. Because of variability of the lengths of individual serial afterdischarges (see ref. 25), for purposes of analysis averages of the first 6 afterdischarge durations and of the final 6 afterdischarge durations were also calculated. These determinations were tabulated and ane~lyzedas initial block afterdischarge durations and final block afterdischarge durations, respectively. Calculations were done on afterdischarges recorded both from the stimulating and the contralateral electrodes. One hour following completion of RRHS, input-output curves of stimulus intensity vs. PS amplitude were again determined and paired pulse inhibition again quantified. Statistical analysis for significant differences between groups was performed on pooled data using a one-way analysis of variance (ANOVA). When appropriate Newman-Keuls tests were subsequently performed to determine which pairs were different, t-Tests were used for pairwise comparisons as indicated below. For all tests
239 A
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POSTNATAL AGE IN DAYS
Fig, 1. Afterdischarge thresholds (ADT) for various ages rats. ADT in/~A (mean + S.E.M,, ordinate) are given for rats of various postnatal ages (abscissa). There was a significant difference of ADT across the 5 age groups studied (ANOVA F = 11.2, P < 0.01). Asterisk indicates significant difference from all other ages (P < 0.05, Newman-Keuis tests). For all ages -> PN 14, afterdischarges were obtained in all animals. In 3 of the 8 animals tested at PN 7, there were no afterdischarges (with stimulus intensities up to 3000 /~A). These animals were excluded from the study; the data presented reflects only animals exhibiting afterdischarges.
a P < 0.05 level was set for statistically significant differences. RESULTS Findings with the initial seizure As the first assessment of epileptogenicity in different ages of rats, we determined ADT. To allow comparisons with our previous studies on awake animals, stimuli trains were 20 Hz, 10 s trains of I ms biphasic pulses. A D T were found to vary inversely with age: 1025.4- 225 (mean :l: S . E . M . ) , 488 -4- 43, 328 :l: 50, 318 :l= 110, and 251 + 33/~A for PN 7, PN 14, PN 18, PN 21, PN 28 groups, respectively. While there was a trend for A D T to decrease throughout the ages studied (Fig. 1), only the
ADTs for the youngest group (PN 7) were statistically different from all older other ages. A similar pattern of ontogeny of A D T was found in awake animals not anesthetized with urethane 2s. In the urethane-anesthetized rats in this study, ADTs were consistently higher than those in unanesthetized animals. In our prior study on the unanesthetized animals, the thresholds (means _+ S.E.M.'s) were 355 + 58, 158 + 40, 198 + 27, and 110 _+ 1 ! / ~ A for ages PN 7, PN 14, PN 21, and PN 28, respectively. Except for the PN 21 groups, the ADTs in the urethane-anesthetized animals were higher than the corresponding thresholds in the awake animals (P < 0.05, grouped t-tests). In our previous studies on the influences of seizures on paired pulse inhibition in the hippocampus of adult rats 14, we found no difference in the potency of inhibition measured before and 60 min after an isolated seizure. Therefore, in the present study, we examined the immature animals in the same fashion. The potency of paired pulse inhibition was determined before and 1 h after a single seizure for an interpulse interval of 20 ms. Prior work (see refs. 12 and 14 for details) has shown that the measurements at this particular interpulse interval reliably assess changes in the potency of GABAergic inhibition. Baseline (preseizure) indices o f maximal inhibition for the different aged rats in this study are given in the top row of Table I. For PN 7, PS(T)/PS(C) measurements showed no evidence of inhibition, but rather reflected paired pulse facilitation. For all other ages, inhibition was detected which varied with age. These data for individual ages are comparable to those reported previously (Table III, ref. 24) and showed statistically significant differences across the study groups ( A N O V A F = 25.2, P < 0.001 for 5 sets of data, top row in Table I). Indices of maximal inhibition for the PN 7 and PN 14 groups were significantly different from all older age groups (P < 0.05, Newman-Keuls tests).
TABLE I Changes in indices of maximal inhibition after single seizure and after recurrent seizures at different postnatal ages
Test population spike amplitude/conditioning population spike amplitude (PS(T)/PS(C)) rates at 20 ms interpulse intervals; (cf. Materials and Methods). For values less than 1.0, paired pulse inhibition was present; for values greater than 1.0, responses represented paired pulse facilitation, aNote that baseline values show significant age-dependence, bAmount of inhibition lost under specified condition [Index of maximal inhibition after seizure(s) subtracted for index of maximal inhibition before seizure(s); negative numbers denote a decrease in potency of paired pulse inhibition while positive numbers denote an increased potency].
Baseline a 60 lPin b after single seizure 60 rainb after RRHS
PN 7
PN 14
PN 18
PN 21
PN 28
1.51 + 0.20 +0.10 __. 0.15 -0.21 +_,0.19
0.93 + 0.30 -0.20 ± 0.19 -0.07 + 0.13
0.21 + 0.04 -0.13 + 0.06 -0.14 + 0.19
0.06 + 0.04 -0.14 _ 0.06 -0.28 + 0.12"
0.07 -4- 0.04 -0.06 + 0.02 -0.24 + 0.06*
* Difference between baseline and after seizure(s) measurement for specified conditions (age; single vs. recurrent seizures); P < 0.05 paired t-tests. Data presented as mean + S.E.M.
240
The amount of inhibition lost 60 min after a single seizure for the various ages studied are given in the second row of Table I. There was a tendency for larger loss of inhibition in the animals of intermediate ages (PN 14, PN 18, and PN 21), the period when GABA-mediated inhibition was rapidly n~aturing. However, none of the determinations achieved statistical significance (paired t-tests comparing indices of maximal inhibition before and after single seizure). The amount of inhibition lost at the various ages examined was not significantly different (ANOVA F = 1.21, P > 0.05 for row 2 of Table I). Although we did not carry out a detailed study, it was nc.ted that there was occasionally a small rightward shift of input-output curves for PS after the single seizures.
Findings with repetitive seizures Our next step was to determine the consequences of RRHS on the electrophysiological parameters introduced above. As in prior studies 14'21'25, we found in the current experiments that seizures were reliably elicited by each stimulus. Furthermore, when tile seizures were repeated, they lengthened.. Individual recordings showed that seizures recorded on the two sides were well syn-
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chronized and terminated within a few seconds of each other (Fig.2). Measurements on afterdischarges for all ages studied are summarized in Table II. For each age group the initial block afterdischarge durations were not different between ipsilateral (to stimulation) and contralateral sides (grouped t-tests). For each age group studied, the afterdischarges on the two side.~ showed similar lengthening (no significant difference in ipsilateral or contralatera! final block afterdischarge durations, grouped t-tests). For each age group studied, there was substantial lengthening (statistically significant difference between final block afterdischarge durations and initial block afterdischarge durations -- P < 0.05 in all cases, paired t-tests), with a nearly two- to three-fold increment. There was no significant difference among the 5 age groups studied with respect to initial block afterdischarge durations ( A N O V A F = 2.40 ipsilateral and F -- 1.29 contralateral), with respect to final block afterdischarge durations, (F = 2.57 ipsilateral, F = 2.69 contralateral), or to degree of lengthening ( F = 2.53 ipsilateral, F = 2.49 contralateral). There was no discernable behavioral accompaniment to the electrographic seizures at any time throughout the recurrent hippocampal seizure protocol in any of the age groups examined. The amount of inhibition lost after the R R H S protocol for the various ages is given in row 3 of Table I. For the animals in which there was some paired pulse inhibition present before seizures (ages -- PN t4), there was
a trend for the amount of inhibition lost to increase in parallel with age, even though this did not achieve statistical significance ( A N O V A F = 1.88, P > 0.05 for row
!
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3 data in Table I). There was a statistically significant
TABLE II
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Electrographic responses of developing rats to RRHS
2. Electrographic
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induced
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the bippocampi of young rats. In each panel the top two traces give electroencephalographic tracings recorded from stimulation site (1) and from recording site in contralaterai (2) hippocampus (see Materials and Methods), Bottom tracings mark I s between short lines and 5 s between long lines (note change in paper speed in B). During interval indicated by dotted line, bipolar electrode at stimulus site was switched from recording to stimulus mode to give a 20 Hz, I0 s train of 1 ms pulses; at other times this electrode was in record mode (for details see ref. 21). Note synchronicity and similarity of waveforms of afterdischarges at the two recording sites. Calibration bar at left betwee, traces 1 and 2 in each panel: 3 mV for 1 and 2 mV for 2. A: frot3 72nd stimulus in rapidly recurring hippocampal seizure (RRHS) protocol in PN 14 rat; B: from 72nd stimulus in RRHS protocol in PN 28 rat.
Ipsilateral side PN 7 PN 14 PN 18 PN 21 PN 28
10,6 + 1.7 15.4 ± 3.2 11.0 :!: 2,0 10,3 + 2,1 9.9 ± 1.4
18,6 ± 33.7 ± 21.6 ± 33.2 + 27.8 ~
2.8 5.1 3,5 3.6 3.5
1.9 2,4 2.2 3.4 3.0
± ± ± ± ±
0.3 0.4 0.4 0.4 0.2
Contralateral side PN 7 10.5 ± 1.7 PN 14 15.1 + 3.1 PN 18 12.3 ± 2.2 PN 21 10.1 ± 2.3 PN 28 9.0 ± 1.5
18.8 + 33.6 + 21,8 + 32.l ± 27.7 ±
3.0 5.1 3.6 2.9 2.9
1.9 2.4 2.2 3.6 3.4
+ + ± ± ±
0.4 0.5 0.5 0.5 0.4
Data presented as mean + S.E.M. For all ages, there was a significant lengthening of afterdischarges on both the ipsilateral and contralateral sides.
241 loss of inhibition after repeated seizures only for the two older groups for which the indices of maximal inhibition had reached adult values (grouped t-tests for PN 21 and PN 28 P < 0.05; P > 0.05 in all other cases). As was the case after single seizures (see above) and as has been observed in adult animals ~4, there was a tendency for the input-output curves for PS to show a rightward shift. However, this was not systematically analyzed. DISCUSSION Two main conclusions were drawn from the results presented above. First, epileptogenic properties in the rat hippocampus are well-developed as early as PN 7. Second, in our studies, which were performed throughout the period when GABAergic inhibition in the hippocampus was increasing, there was no age-related decrease in hippocampai epileptogenicity. The findings indicate a complex interplay between GABAergic inhibition and epileptogenesis as the rat hippocampal formation matures. Two observations from the foregoing experiments support the contention that epileptogenesis in the hippocampus is well-developed early in life. Robust afterdischarges could be elicited in all age groups examined. Furthermore, all ages showed the capability of substantial increases in afterdischarge duration during the RRHS protocol. Thus, the present findings, in conjunction with our previous studies 2s, demonstrate that, as with adult rats 14'2~'2~'2s,rapid kindling of afterdischarges takes place in rat pups both under urethane anesthesia, or in awake animals. Findings of the current study are in agreement with prior in vivo work 2'24 and in vitro 3°'33 work that demonstrate that the potency of paired pulse inhibition varied greatly across the various age groups examined, from none detectable (at PN 7) to fully developed, levels. GABA-mediated inhibition has received considerable attention as a major factor in epileptogenesis4'5'u-~3'~6'23' 27.3~,32. In several of these studies, investigators have noted a deterioration of GABA.mediated inhibition during a seizure or after a series of seizures. However, all these studies were done in adult animals. Fewer workers have investigated the role of GABAergic inhibition in modulating seizure activity in the immature brain, although deFeo et al. ~ have demonstrated that young rats are sensitive to seizures via blockade of GABAergic inhibition. Since a diminution of GABA-mediated inhibition is considered as a means to promote epileptogenesis, one would expect that with less potent inhibition, seizure activity would be more severe. However, this was not
found in the above experiments. In fact, for the youngest age group studied, in which paired pulse inhibition was not detected (see also ref. 24) and intraceUular recordings have documented the absence of GABAergic inhibition, the ADTs were highest; whereas the lowest ADTs were found in the animals with the most potent paired pulse inhibition, an age at which GABAA-mediated IPSPs are known to be present 3°'33. Thus, in the present experiments, ADTs were found to be highly age-dependent, and inversely related to the strength of GABAA-mediated inhibition. Moshe et al. 26 have obtained the same results with different methods. Gilbert also reported a resistance of the very young brain to afterdischarges induced by electrical stimulation 9. In addition, initial block afterdischarge durations did not decrease with age as would be predicted if epileptogenesis were merely a function of the strength of inhibition. One might argue that it is not the baseline potency of inhibition before seizures that matters, but rather how inhibition can withstand seizure discharges, especially when they recur. Nonetheless, certain findings presented above speak against this last idea. For instance, the deterioration of paired pulse inhibition with recurrent seizures was largest for the two oldest groups, whereas the younger animals, with less baseline GABAergic inhibition, showed less deterioration of inhibition following kindling. However, the older groups did not show any greater electrographic kindling. It should also be noted at this point that our results indicate that seizure-induced plasticity of inhibition, as measured by changes in indices of maximal inhibition, does not occur only in the adult hippocampus, but also at younger ages. Interestingly, only small changes are s e e n at earlier ages, when paired pulse inhibition is not as potent as at the later ages. Thus, it seems that seizure-induced 'disinhibition' requires a certain level, or certain degree of maturity, before it can develop. In contrast to the age-dependent changes of ADTs, we found no differences in the age groups examined with respect to either the initial block of afterdischarge durations or to the propensity of afterdischarges to lengthen when repeated. For technical reasons, the stimulus trains use,J in this study and in our prior studies on adult rats under urethane anesthesia n'14 were quite similar but not identical. Thus, rigorous comparisons will not be made between the res,~!ts of the current work and previous findings. However, the initial afterdischarge durations in young rats and the durations in adults are comparable and the degrees of lengthening in the various age groups with repeated seizures are also similar 12'~4. It is of interest that the electrographic seizures recorded at the site of hippocampal stimulation and in the
242 contralateral hippocampus were synchronized during the discharges and terminated at virtually the same time. This finding and the fact that the afterdischarges on the two sides showed identical lengthening during the RRHS protocol support the concept of a tight linkage between the two hippocampi in terms of seizures in our studies. Previous studies indicate that, in adult rats, seizures elicited with hippocampal stimulation spread from the site of stimulation to the contralateral hippocampus via the hippocampal commissure 34'35. Based on the electroencephalic recordings in all groups in the current work, it seems that a likely pathway by which the two hippocampi in young rats become synchronized during seizures is the hippocampal commissures. How can one then account for the seemingly paradoxical findings that, with very poorly developed inhibition in the young hippocampus there is not a greater epileptogenicity, revealed by longer afterdischarges, more robust electrographic kindling, or lower ADT. It may be that there is some system in the young brain that retards or opposes seizures, thereby counteracting the immaturity of GABA-mediated inhibition, that later recedes as REFERENCES 1 Aitken, P.J., Kainic acid and penicillin: differential effects of excitatory and inhibitory interactions in the CAt region of the hippocampal slice, Brain Res., 325 (1985) 261-269. 2 Bekenstein, J.W. and Lothman, E.W., A comparison of excitatory and inhibitory neurotransmission in the CA1 region and dentate gyrus of the rat hippocampal formation, Dev. Brain Res., 63 (1991) 237-224. 3 Bekenstein, J.W. and Lothman, E.W., An in vivo study of the ontogeny of long-term potentiation (LTP) in the CAI region and in the dentate gyrus of the rat hippocampal formation, Dev. Brain Res., 63 (1991) 245-252. 4 Ben.Ari, Y., Krnjevic, K., Reiffenstein, R.J. and Reinhardt, W., Inhibitory condt,ctance changes and action of gamma-ami. nobutyrate in rat hippocampus, Neuroscience, 6 (1981) 24452463. 5 Ben-Ari, Y., Krnjevic, K. and Reinhardt, W., Hippocampal sei. zures and failure of inhibition, Can. J. Physiol. Pharmacol., 57 (1979) 1462-1466. 6 de Feo, M.R.. MecareUi, O. and Ricci, G.F., Bicuculline. att0 allyglycine-induced epilepsy in developing rats, Exp. Neurol., 90 (1985) 411-421. 7 Dreifuss, F.E., Pediatric Epileptoiogy, John Wright, London, 1983, 302 pp. 8 Engel Jr., J., Epilepsy and Seizures, EA. Davis, Philadelphia, 1989, 536 pp. 9 Gilbert, M.E. and Cain, D.P., A developmental study of kindling in the rat, Dev. Brain Res., 2 (1982) 321-328. 10 Goddard, G.V., Mclntyre, D.C. and Leech, C.K., A permanent change in brain functioning resulting from daily electrical stimulation, Exp. Neurol., 25 (1969) 295-330. 11 Kapur, J., Bennett Jr., J.R, Wooten, G.E and Lothman, E.W., Evidence for a chronic loss of inhibition in the hippocampus after kindling: biochemical studies, Epilepsy Res., 4 (1989) 100.108. 12 Kapur, J. and Lothman, E.W., NMDA receptor activation mediates the loss of GABAergic inhibition induced by recurrent seizures, Epilepsy Res., 5 (1990) 103-111.
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Acknowledgements. Supported in part by USPHS Grsnts NS 21671, NS 25605 and HD 07323. We thank Rose Powelt, Marie Noel, and Patricia Caines for assistance in preparation of the manuscript.
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