79
Developmental Brain Research, 61 (1991) 79-85 © 1991 Elsevier Science Publishers B.V. 0165-3806/91/$03.50 ADONIS 0165380691512886 BRESD 51288
An ontogenetic study of kindling using rapidly recurring hippocampal seizures Hillary B. Michelson and Eric W. Lothman Department of Neurology, University of Virginia Health Sciences Center, Charlottesville, VA 22908 (U.S.A.) (Accepted 19 March 1991) Key words: Kindling; Ontogeny; Hippocampus; Development
The present study investigated the acquisition and retention of kindling in immature rats. Postnatal (PN) 7-28-day-old rats were electrically kindled in the ventral hippocampus. Ten-second, 20-Hz stimulus trains were delivered every 5 min for 6 h on one day (short interval rapidly recurring hippocampal seizures, RRHS) or every 30 min for 9 h on each of two consecutive days (long interval RRHS). Afterdischarge durations (ADD) and behavioral seizure scores (BSS) were recorded following each stimulation. Animals of all ages kindled with both short and long interval RRHS, as manifested by lengthening of ADD and increasing BSS. With short interval RRHS, the course of kindling was erratic; with long interval RRHS, kindling proceeded smoothly over both test days. In PN 14-28 rats, the degree of kindling obtained on the first day of long interval RRHS was retained at the start of the second experimental day. In contrast, PN 7 rats showed a transient decrease in ADD and BSS from day 1 to day 2. Afterdischarge thresholds declined with maturation. Among the PN 14-28 animals, younger rats exhibited longer seizures at the outset of kindling and proceeded through kindling faster. Once established, kindled motor seizures also occurred with 2-s, 50-Hz stimulus trains. We conclude that rapid kindling occurs at all ages; however, PN 7 rats are less capable of retaining the kindling effect than are older rats.
INTRODUCTION The epilepsies are a group of neurological disorders characterized by paroxysmal synchronous discharges within a population of neurons. It is well known clinically that seizures occur more readily in the young brain 5'29 and that most cases of epilepsy begin in childhood 5'1°. Studies have shown that seizure susceptibility is higher in the developing nervous system 24'25'28"39, and that distinct types of seizures are associated with specific periods of development 5'6'1°'29. The need for correlating mechanisms of seizure activity with brain maturation is underscored by studies which indicate that seizure activity early in life may result in neurological damage or abnormal nervous system development 1'35. Studies of the hippocampus have demonstrated the marked susceptibility of this structure to the generation of synchronous activity 33'36'42. The hippocampus is a structure particularly suited for the study of seizure processes; its laminar structure and basic circuitry have been well described 4. The developing hippocampus is more seizure-prone than other structures 31. This increased susceptibility probably results from alterations in
the balance between excitatory and inhibitory synaptic interactions during development. Several studies suggest that the maturation of inhibitory processes lags behind that of excitation in the hippocampus 2t'34'38'39. Additional in vitro studies of the hippocampus have demonstrated the heightened ability of this region to generate prolonged ictal-like events during development 39. This enhancement appears to diminish by postnatal (PN) days 24-25. Increased seizure susceptibility in the developing rat has also been noted using several in vivo models of epilepsy 3"23'25. The kindling p h e n o m e n o n 8 is one such model which is well suited for the study of seizure generation and propagation, as the electrographic and behavioral stages through which kindling proceeds have been thoroughly characterized 8"32. It has been reported that younger animals have a lower seizure threshold and can be kindled at shorter interstimulus intervals than adult rats 24'25. However, the range of ages that has been studied is limited and little is known regarding kindling in animals younger than two weeks of age. Gilbert 7, using short duration, high frequency stimuli (60-Hz, 1-s trains) to the amygdala, found that rats younger than PN 14
Correspondence: H.B. Michelson. Present address: Department of Pharmacology, Box 29, SUNY/Health Science Center at Brooklyn, 450 Clarkson Avenue, Brooklyn, NY 11203, U.S.A. Fax: (1) (718) 270-2241.
80 pre-weanling pups were returned to their mothers. Older rats were housed in groups of 3-4 after surgery.
TABLE I
Coordinates for electrodeplacement Coordinates were measured from bregma. Silicone molds were used to secure PN 7 rats in the stereotaxic flame, as described in Materials and Methods. Skull position was flat in PN 7 rats, as measured between bregraa and lambda. For PN 14-21 rats, incisor bar was set at 0.0. For PN 28 rats, incisor bar was set at 5.0.
Age
AP
ML
DV
PN 7 PN 14 PN21 PN 28
-3.3 -3.0 -2.9 -3.6
+4.3 +3.9 +3.7 +4.9
+4.4 +4.2 +3.8 +5.0
cannot support the kindling p h e n o m e n o n . O n e particular difficulty in extending 'traditional' kindling paradigms to the i m m a t u r e rat is that the duration of the experimental protocol (e.g. 10-14 days with daily amygdala kindling) becomes impractical at a time when development is occurring at such a rapid rate. We have previously described the p h e n o m e n o n of rapidly recurring hippocampal seizures ( R R H S ) in which rats can be kindled during a single day using long d u r a t i o n stimuli delivered every 5 rain 14'1s'19. The short timespan of this experimental protocol readily lends itself to the examination of seizure generation in the developing animal. Holmes and T h o m p s o n 11 have described the rapid kindling p h e n o m e n o n in rats 30 days of age, and have f o u n d that these rats behave as adults in the R R H S protocol. More recently, we have found that increasing the stimulus interval in the R R H S protocol to 30 rain results in smoother kindling acquisition in adult rats, and that 20-Hz stimuli are more effective than 50-Hz stimuli for achieving the rapid kindling effect 17. The present
Afierdischarge thresholds The day following surgery, thresholds to produce an afterdischarge using a 10-s train of 20-1-Iz, 1-ms biphasic square wave pulses were determined using methods previously described 19. These stimulation parameters were chosen as they provided optimal electrographic responses in preliminary studies across the various age groups. All subsequent kindling stimulations used the same 10-s pulse train at a current intensity of at least twice that of the afterdischarge threshold.
Kindling paradigms Kindling was initiated 30 rain following threshold determination. Afterdischarge durations (ADD) and behavioral seizure scores (BSS) were assessed following each stimulation. Motor seizures for PN 14 and older rats were scored as follows: Stage 1, facial or head movements; Stage 2, pronounced head nodding or chewing; Stage 3, unilateral forelimb clonus; Stage 4, bilateral forelimb clonus; Stage 5, bilateral forelimb clonus with loss of postural control. PN 7 rats showed distinctly different motor seizure patterns. The scale used to score behavioral seizures in PN 7 rats was as follows: Stage 1, head movements; Stage 2, pedalling of forelimbs without locomotion; Stage 3, pedalling with steady locomotion or tottering; Stage 4, tottering with accelerated locomotion; Stage 5, running fit with forelimb clonus. Other investigators7'23 have described comparable motor seizure scoring systems for infant rats. Two separate RRHS kindling paradigms were used in this study. Both paradigms used the 20-Hz, 10-s stimulus described above for determination of afterdischarge threshold. In one group (short interval RRHS), stimuli were delivered every 5 min for a total of 72 stimulations (6 h, completed in one day). In the second group (long interval RRHS), rats were stimulated every 30 rain for a total of 36 stimulations (18 stimulations on each of two consecutive days). Thirty minutes following completion of long interval RRHS, a 'crossover' afterdischarge threshold to a 2-s, 50-Hz train of 1-ms duration pulses was determined, using the approach specified above. Comparable electrode placement in the ventral hippocampus across age was verified h i s t o l o # l y by tract or tip lesion observation at the completion of the experiment.
Analysis
ered every 5 min) and long interval (every 30 min) R R H S paradigms.
Pooled results are presented as mean + S.E.M. As specified below, results were analyzed for statistical significance using Student's t-tests for single pair analysis, Mann-Whitney U-tests or one-way analysis of variance (ANOVA) to compare data obtained from the different age groups studied. Newman-Keuls tests were used after ANOVAs where appropriate to determine which pairs of all possible pairs were significantly different. For all analyses, significance was set at P < 0.05.
MATERIALS AND METHODS
RESULTS
study extends these findings to rats as young as 7 days of age. The ability of different ages to support and maintain kindling was examined with short interval (stimuli deliv-
Surgery Four age groups of Sprague-Dawley rats (n = 8-12 per group) were studied: PN 7 (rats 7-10 days of age), PN 14 (14-16 days old), PN 21 (20-22 days old) and PN 28 (27-30 days old). All rats were anesthetized with ketamine (50 mg/kg, i.p.) and xylazine (12.5 mg/kg, i.p.). A twisted, insulated bipolar stainless steel electrode is' 19 was lowered into the ventral hiplx~ampus. Eleetrnde placement coordinates for the various groups of rats are listed in Table I. Preliminary studies histologically confirmed comparable electrode placement across the various age groups. It should be noted that the coordinates used for electrode placement were in part determined by the angle of the head during surgery, a factor which differed across age groups (see Table I). The electrodes were placed into Amphenol connectors and secured to the skull with jeweller's screws and acrylic cement. Following recovery from the anesthesia, the
Pre-kindling afterdischarge thresholds for the various age groups studied are listed in the first row of Table II. Within each age group, thresholds b e t w e e n the short and long interval R R H S paradigms were not statistically different (t-tests) and were therefore grouped together for analysis. Afterdiseharge thresholds were different across the age groups studies ( A N O V A , F = 10.81; P < 0.01). A m o n g group comparisons revealed that the afterdischarge threshold in PN 7 rats was significantly greater than all other age groups. T h e r e were no other significant differences in threshold across age, although
81 TABLE II Characteristics of rapid kindling at various ages
Data presented as mean ± S.E.M. N/A indicates not applicable because behavioral seizures in PN 7 rats were scored on a different scale.
Afterdischarge threshold ~uA)a Stimulations to first Stage 5 seizure First ADD c (s)
PN 7
PN 14
PN 21
PN 28
354.5 + 57.6* (n = 11) N/A
157.5 ± 40.3 (n = 8) 20.8 i 1.30 (n = 5) 44.8 ± 8.9 (n = 5) 63.4 ± 4.1 (n = 5)
198.2 ± 26.6 (n = 8) 26.7 ± 1.6b (n = 6) 28.1 ± 2.5 (n = 7) 52.3 ± 5.0 (n = 7)
110.0 ± 11.3 (n = 10) 33.0 ± 1.7 (n = 5) 47.6 ± 9.4 (n = 5) 74.7 ± 7.5 (n = 5)
30.6 ± 1.6 (n = 6) 126.2 ± 23.7 (n = 6)
Last ADD (s) d'e
* indicates significantly different from all other age groups of rats (ANOVA, Newman-Keuls). ~ Data for afterdischarge thresholds pooled from both short and long interval RRHS protocols. Data in other rows from long interval RRHS paradigm only; for corresponding analyses see Table III. b Significantly different from all older age groups (Newman-Keuls tests), c Significant differences across age groups (F = 4.28, P < 0.05). d Significant differences across age groups (F = 3.40, P < 0.05). e For all age groups except PN 14, last ADDs were longer than first ADDs (Student's t-test).
there was a trend towards decreasing afterdischarge threshold as the animal m a t u r e d . A l l ages of rats showed kindling acquisition with short interval R R H S , as evidenced by lengthening of afterdischarges and progressive worsening of behavioral seizures. E a r l y in the course of short interval R R H S , A D D s were consistently shorter. H o w e v e r , as kindling progressed and behavioral seizures intensified, longer afterdischarges associated with m o r e severe m o t o r responses a l t e r n a t e d with shorter afterdischarges and minimal m o t o r seizures. To m o r e accurately d e t e r m i n e afterdischarge lengthening following kindling, the A D D of the first 6 stimulations (stimulations 1-6) were averaged and c o m p a r e d to the average A D D of the last 6 stimulations (stimulations 67-72). These d a t a are summarized in Table III. PN 14 rats showed significantly longer seizures at the outset of short interval R R H S c o m p a r e d to all o t h e r ages of rats ( A N O V A , F = 9.38, P < 0.05). N o o t h e r
TABLE III Comparison of afterdischarge lengthening following short interval RRHS paradigm
All animals were kindled every 5 rain with the short interval RRHS protocol. Because the short interval RRHS paradigm results in cycling of short and long afterdischarges, comparison of initial and final ADD does not accurately reflect afterdischarge lengthening. Averaged (mean ± S.E.M.) responses for first 6 stimulus trains (Initial block ADD) and for last 6 stimulus trains (Final block ADD) of the 72 stimulation protocol are given for purposes of comparison. Age
~alb~ckADD ~
~nalb~ckADD~)
PN7 PN14 PN21 PN28
~.9±3.0 45.3±4.7 30.1±4.2 25.3±1.9
44.2±3.0 62.2±4.0 51.1±2.8 39.9±7.7
statistically significant differences in initial A D D were detected. N o differences in the m e a n final A D D across age groups were d e t e c t e d ( A N O V A , F = 1.56, n.s.). Paired analyses of A D D b e f o r e and after kindling (t-tests) within individual age groups indicated that A D D s in PN 14 rats l e n g t h e n e d during kindling, but the difference in the initial and final A D D m e a s u r e m e n t was not significant. PN 7 and PN 21 rats showed significant A D D lengthening during kindling, and PN 28 rats showed a trend t o w a r d lengthening which did not reach significance. Fully k i n d l e d m o t o r responses (Stages 4 and 5) associated with longer afterdischarges were o b s e r v e d in all age groups by the end of the short interval R R H S paradigms. A s a result of the difficulties of analysis associated with the cycling of responses with short interval R R H S , we focused a m o r e detailed analysis on the results of the long interval R R H S kindling protocol. Using the long interval R R H S p a r a d i g m , all ages of rats kindled as d e t e r m i n e d by lengthening of A D D and increasing severity of BSS. These d a t a are s u m m a r i z e d in Table II and illustrated in Figs. 1 and 2. Following kindling, seizure duration had l e n g t h e n e d by 414% in PN 7 rats, 145% in PN 14 rats, 179% in PN 21 rats, and 163% in PN 28 rats. A D D lengthening r e a c h e d statistical significance in all but the PN 14 age groups. The age-related susceptibility to the acquisition of kindling was further assessed by c o m p a r i n g the n u m b e r of stimulations required to reach each behavioral stage of kindling. Because the PN 7 rats were scored on a different scale than the o t h e r ages, this age group was not included in the comparison. Pertinent d a t a are summarized in Table IV. Overall, PN 14 rats proceeded through the kindling process quickest, reaching Stages 3-5 earlier than PN 21 and 28 animals. PN 28 rats were consistently slower to reach each stage of kindling than the younger rats.
82 TABLE IV
ONTOGENY OF RAPID KINDLING ELECTROGRAPHIC SEIZURES
Number of stimulations to reach each stage of kindling Behavioral stage
~o I PN 14
l
2
3
4
5
1.0±0 1.0±0 1.0±0 2.2±0.2 F=~.5 a P
Developmental Brain Research, 61 (1991) 79-85 © 1991 Elsevier Science Publishers B.V. 0165-3806/91/$03.50 ADONIS 0165380691512886 BRESD 51288
An ontogenetic study of kindling using rapidly recurring hippocampal seizures Hillary B. Michelson and Eric W. Lothman Department of Neurology, University of Virginia Health Sciences Center, Charlottesville, VA 22908 (U.S.A.) (Accepted 19 March 1991) Key words: Kindling; Ontogeny; Hippocampus; Development
The present study investigated the acquisition and retention of kindling in immature rats. Postnatal (PN) 7-28-day-old rats were electrically kindled in the ventral hippocampus. Ten-second, 20-Hz stimulus trains were delivered every 5 min for 6 h on one day (short interval rapidly recurring hippocampal seizures, RRHS) or every 30 min for 9 h on each of two consecutive days (long interval RRHS). Afterdischarge durations (ADD) and behavioral seizure scores (BSS) were recorded following each stimulation. Animals of all ages kindled with both short and long interval RRHS, as manifested by lengthening of ADD and increasing BSS. With short interval RRHS, the course of kindling was erratic; with long interval RRHS, kindling proceeded smoothly over both test days. In PN 14-28 rats, the degree of kindling obtained on the first day of long interval RRHS was retained at the start of the second experimental day. In contrast, PN 7 rats showed a transient decrease in ADD and BSS from day 1 to day 2. Afterdischarge thresholds declined with maturation. Among the PN 14-28 animals, younger rats exhibited longer seizures at the outset of kindling and proceeded through kindling faster. Once established, kindled motor seizures also occurred with 2-s, 50-Hz stimulus trains. We conclude that rapid kindling occurs at all ages; however, PN 7 rats are less capable of retaining the kindling effect than are older rats.
INTRODUCTION The epilepsies are a group of neurological disorders characterized by paroxysmal synchronous discharges within a population of neurons. It is well known clinically that seizures occur more readily in the young brain 5'29 and that most cases of epilepsy begin in childhood 5'1°. Studies have shown that seizure susceptibility is higher in the developing nervous system 24'25'28"39, and that distinct types of seizures are associated with specific periods of development 5'6'1°'29. The need for correlating mechanisms of seizure activity with brain maturation is underscored by studies which indicate that seizure activity early in life may result in neurological damage or abnormal nervous system development 1'35. Studies of the hippocampus have demonstrated the marked susceptibility of this structure to the generation of synchronous activity 33'36'42. The hippocampus is a structure particularly suited for the study of seizure processes; its laminar structure and basic circuitry have been well described 4. The developing hippocampus is more seizure-prone than other structures 31. This increased susceptibility probably results from alterations in
the balance between excitatory and inhibitory synaptic interactions during development. Several studies suggest that the maturation of inhibitory processes lags behind that of excitation in the hippocampus 2t'34'38'39. Additional in vitro studies of the hippocampus have demonstrated the heightened ability of this region to generate prolonged ictal-like events during development 39. This enhancement appears to diminish by postnatal (PN) days 24-25. Increased seizure susceptibility in the developing rat has also been noted using several in vivo models of epilepsy 3"23'25. The kindling p h e n o m e n o n 8 is one such model which is well suited for the study of seizure generation and propagation, as the electrographic and behavioral stages through which kindling proceeds have been thoroughly characterized 8"32. It has been reported that younger animals have a lower seizure threshold and can be kindled at shorter interstimulus intervals than adult rats 24'25. However, the range of ages that has been studied is limited and little is known regarding kindling in animals younger than two weeks of age. Gilbert 7, using short duration, high frequency stimuli (60-Hz, 1-s trains) to the amygdala, found that rats younger than PN 14
Correspondence: H.B. Michelson. Present address: Department of Pharmacology, Box 29, SUNY/Health Science Center at Brooklyn, 450 Clarkson Avenue, Brooklyn, NY 11203, U.S.A. Fax: (1) (718) 270-2241.
80 pre-weanling pups were returned to their mothers. Older rats were housed in groups of 3-4 after surgery.
TABLE I
Coordinates for electrodeplacement Coordinates were measured from bregma. Silicone molds were used to secure PN 7 rats in the stereotaxic flame, as described in Materials and Methods. Skull position was flat in PN 7 rats, as measured between bregraa and lambda. For PN 14-21 rats, incisor bar was set at 0.0. For PN 28 rats, incisor bar was set at 5.0.
Age
AP
ML
DV
PN 7 PN 14 PN21 PN 28
-3.3 -3.0 -2.9 -3.6
+4.3 +3.9 +3.7 +4.9
+4.4 +4.2 +3.8 +5.0
cannot support the kindling p h e n o m e n o n . O n e particular difficulty in extending 'traditional' kindling paradigms to the i m m a t u r e rat is that the duration of the experimental protocol (e.g. 10-14 days with daily amygdala kindling) becomes impractical at a time when development is occurring at such a rapid rate. We have previously described the p h e n o m e n o n of rapidly recurring hippocampal seizures ( R R H S ) in which rats can be kindled during a single day using long d u r a t i o n stimuli delivered every 5 rain 14'1s'19. The short timespan of this experimental protocol readily lends itself to the examination of seizure generation in the developing animal. Holmes and T h o m p s o n 11 have described the rapid kindling p h e n o m e n o n in rats 30 days of age, and have f o u n d that these rats behave as adults in the R R H S protocol. More recently, we have found that increasing the stimulus interval in the R R H S protocol to 30 rain results in smoother kindling acquisition in adult rats, and that 20-Hz stimuli are more effective than 50-Hz stimuli for achieving the rapid kindling effect 17. The present
Afierdischarge thresholds The day following surgery, thresholds to produce an afterdischarge using a 10-s train of 20-1-Iz, 1-ms biphasic square wave pulses were determined using methods previously described 19. These stimulation parameters were chosen as they provided optimal electrographic responses in preliminary studies across the various age groups. All subsequent kindling stimulations used the same 10-s pulse train at a current intensity of at least twice that of the afterdischarge threshold.
Kindling paradigms Kindling was initiated 30 rain following threshold determination. Afterdischarge durations (ADD) and behavioral seizure scores (BSS) were assessed following each stimulation. Motor seizures for PN 14 and older rats were scored as follows: Stage 1, facial or head movements; Stage 2, pronounced head nodding or chewing; Stage 3, unilateral forelimb clonus; Stage 4, bilateral forelimb clonus; Stage 5, bilateral forelimb clonus with loss of postural control. PN 7 rats showed distinctly different motor seizure patterns. The scale used to score behavioral seizures in PN 7 rats was as follows: Stage 1, head movements; Stage 2, pedalling of forelimbs without locomotion; Stage 3, pedalling with steady locomotion or tottering; Stage 4, tottering with accelerated locomotion; Stage 5, running fit with forelimb clonus. Other investigators7'23 have described comparable motor seizure scoring systems for infant rats. Two separate RRHS kindling paradigms were used in this study. Both paradigms used the 20-Hz, 10-s stimulus described above for determination of afterdischarge threshold. In one group (short interval RRHS), stimuli were delivered every 5 min for a total of 72 stimulations (6 h, completed in one day). In the second group (long interval RRHS), rats were stimulated every 30 rain for a total of 36 stimulations (18 stimulations on each of two consecutive days). Thirty minutes following completion of long interval RRHS, a 'crossover' afterdischarge threshold to a 2-s, 50-Hz train of 1-ms duration pulses was determined, using the approach specified above. Comparable electrode placement in the ventral hippocampus across age was verified h i s t o l o # l y by tract or tip lesion observation at the completion of the experiment.
Analysis
ered every 5 min) and long interval (every 30 min) R R H S paradigms.
Pooled results are presented as mean + S.E.M. As specified below, results were analyzed for statistical significance using Student's t-tests for single pair analysis, Mann-Whitney U-tests or one-way analysis of variance (ANOVA) to compare data obtained from the different age groups studied. Newman-Keuls tests were used after ANOVAs where appropriate to determine which pairs of all possible pairs were significantly different. For all analyses, significance was set at P < 0.05.
MATERIALS AND METHODS
RESULTS
study extends these findings to rats as young as 7 days of age. The ability of different ages to support and maintain kindling was examined with short interval (stimuli deliv-
Surgery Four age groups of Sprague-Dawley rats (n = 8-12 per group) were studied: PN 7 (rats 7-10 days of age), PN 14 (14-16 days old), PN 21 (20-22 days old) and PN 28 (27-30 days old). All rats were anesthetized with ketamine (50 mg/kg, i.p.) and xylazine (12.5 mg/kg, i.p.). A twisted, insulated bipolar stainless steel electrode is' 19 was lowered into the ventral hiplx~ampus. Eleetrnde placement coordinates for the various groups of rats are listed in Table I. Preliminary studies histologically confirmed comparable electrode placement across the various age groups. It should be noted that the coordinates used for electrode placement were in part determined by the angle of the head during surgery, a factor which differed across age groups (see Table I). The electrodes were placed into Amphenol connectors and secured to the skull with jeweller's screws and acrylic cement. Following recovery from the anesthesia, the
Pre-kindling afterdischarge thresholds for the various age groups studied are listed in the first row of Table II. Within each age group, thresholds b e t w e e n the short and long interval R R H S paradigms were not statistically different (t-tests) and were therefore grouped together for analysis. Afterdiseharge thresholds were different across the age groups studies ( A N O V A , F = 10.81; P < 0.01). A m o n g group comparisons revealed that the afterdischarge threshold in PN 7 rats was significantly greater than all other age groups. T h e r e were no other significant differences in threshold across age, although
81 TABLE II Characteristics of rapid kindling at various ages
Data presented as mean ± S.E.M. N/A indicates not applicable because behavioral seizures in PN 7 rats were scored on a different scale.
Afterdischarge threshold ~uA)a Stimulations to first Stage 5 seizure First ADD c (s)
PN 7
PN 14
PN 21
PN 28
354.5 + 57.6* (n = 11) N/A
157.5 ± 40.3 (n = 8) 20.8 i 1.30 (n = 5) 44.8 ± 8.9 (n = 5) 63.4 ± 4.1 (n = 5)
198.2 ± 26.6 (n = 8) 26.7 ± 1.6b (n = 6) 28.1 ± 2.5 (n = 7) 52.3 ± 5.0 (n = 7)
110.0 ± 11.3 (n = 10) 33.0 ± 1.7 (n = 5) 47.6 ± 9.4 (n = 5) 74.7 ± 7.5 (n = 5)
30.6 ± 1.6 (n = 6) 126.2 ± 23.7 (n = 6)
Last ADD (s) d'e
* indicates significantly different from all other age groups of rats (ANOVA, Newman-Keuls). ~ Data for afterdischarge thresholds pooled from both short and long interval RRHS protocols. Data in other rows from long interval RRHS paradigm only; for corresponding analyses see Table III. b Significantly different from all older age groups (Newman-Keuls tests), c Significant differences across age groups (F = 4.28, P < 0.05). d Significant differences across age groups (F = 3.40, P < 0.05). e For all age groups except PN 14, last ADDs were longer than first ADDs (Student's t-test).
there was a trend towards decreasing afterdischarge threshold as the animal m a t u r e d . A l l ages of rats showed kindling acquisition with short interval R R H S , as evidenced by lengthening of afterdischarges and progressive worsening of behavioral seizures. E a r l y in the course of short interval R R H S , A D D s were consistently shorter. H o w e v e r , as kindling progressed and behavioral seizures intensified, longer afterdischarges associated with m o r e severe m o t o r responses a l t e r n a t e d with shorter afterdischarges and minimal m o t o r seizures. To m o r e accurately d e t e r m i n e afterdischarge lengthening following kindling, the A D D of the first 6 stimulations (stimulations 1-6) were averaged and c o m p a r e d to the average A D D of the last 6 stimulations (stimulations 67-72). These d a t a are summarized in Table III. PN 14 rats showed significantly longer seizures at the outset of short interval R R H S c o m p a r e d to all o t h e r ages of rats ( A N O V A , F = 9.38, P < 0.05). N o o t h e r
TABLE III Comparison of afterdischarge lengthening following short interval RRHS paradigm
All animals were kindled every 5 rain with the short interval RRHS protocol. Because the short interval RRHS paradigm results in cycling of short and long afterdischarges, comparison of initial and final ADD does not accurately reflect afterdischarge lengthening. Averaged (mean ± S.E.M.) responses for first 6 stimulus trains (Initial block ADD) and for last 6 stimulus trains (Final block ADD) of the 72 stimulation protocol are given for purposes of comparison. Age
~alb~ckADD ~
~nalb~ckADD~)
PN7 PN14 PN21 PN28
~.9±3.0 45.3±4.7 30.1±4.2 25.3±1.9
44.2±3.0 62.2±4.0 51.1±2.8 39.9±7.7
statistically significant differences in initial A D D were detected. N o differences in the m e a n final A D D across age groups were d e t e c t e d ( A N O V A , F = 1.56, n.s.). Paired analyses of A D D b e f o r e and after kindling (t-tests) within individual age groups indicated that A D D s in PN 14 rats l e n g t h e n e d during kindling, but the difference in the initial and final A D D m e a s u r e m e n t was not significant. PN 7 and PN 21 rats showed significant A D D lengthening during kindling, and PN 28 rats showed a trend t o w a r d lengthening which did not reach significance. Fully k i n d l e d m o t o r responses (Stages 4 and 5) associated with longer afterdischarges were o b s e r v e d in all age groups by the end of the short interval R R H S paradigms. A s a result of the difficulties of analysis associated with the cycling of responses with short interval R R H S , we focused a m o r e detailed analysis on the results of the long interval R R H S kindling protocol. Using the long interval R R H S p a r a d i g m , all ages of rats kindled as d e t e r m i n e d by lengthening of A D D and increasing severity of BSS. These d a t a are s u m m a r i z e d in Table II and illustrated in Figs. 1 and 2. Following kindling, seizure duration had l e n g t h e n e d by 414% in PN 7 rats, 145% in PN 14 rats, 179% in PN 21 rats, and 163% in PN 28 rats. A D D lengthening r e a c h e d statistical significance in all but the PN 14 age groups. The age-related susceptibility to the acquisition of kindling was further assessed by c o m p a r i n g the n u m b e r of stimulations required to reach each behavioral stage of kindling. Because the PN 7 rats were scored on a different scale than the o t h e r ages, this age group was not included in the comparison. Pertinent d a t a are summarized in Table IV. Overall, PN 14 rats proceeded through the kindling process quickest, reaching Stages 3-5 earlier than PN 21 and 28 animals. PN 28 rats were consistently slower to reach each stage of kindling than the younger rats.
82 TABLE IV
ONTOGENY OF RAPID KINDLING ELECTROGRAPHIC SEIZURES
Number of stimulations to reach each stage of kindling Behavioral stage
~o I PN 14
l
2
3
4
5
1.0±0 1.0±0 1.0±0 2.2±0.2 F=~.5 a P
