Decelopmeo~tal Brain Research, 70 (1992) 223-229 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-3806/92/$05.00
223
BRESD 51546
Ontogeny of hippocampal afterdischarges in the urethane-anesthetized rat Janet L. Stringer
a
and Eric W. Lot hman
b
a Department of Pharmacology, Baylor College of Medicine, Ho~:siOn, TX 77030 (USA) and t, Department of Neurology, Unil'ersity of Virginia Medical Center, Chariottest'ille, VA 22908 (USA) (Accepted 21 July 1992)
Key words: Epilepsy; Afterdischarge; Dentate gyrus; Ontogeny; CAI; Extracellular potassium; EEG
Experimental studies have shown that seizure manifestations vary as the brain develops. This study investigated the characteristics of afterdischarges in the hippocampal circuits at various ages in the developing rat. Rats from the following post-natal periods were tested: PN 10-11, PN 14-15, PN 17-19, PN 21-23 and PN 25-27. Animals were anesthetized with urethane and recording electrodes placed in the hippocampus bilaterally. Stimulating electrodes were placed in the left CA3 region and in the angular bundle. Afterdischarges were produced in all animals using stimulus trains of 20 or 50 Hz. Rats in the PN 10-11 and 14-15 age groups had afterdischarges that consisted of population spikes in CAI and broad positive potentials in the dentate gyrus. Between PN 17 and 19, maximal dentate activation, which consists of bursts of large amplitude populatie,n spikes in the dentate gyrus, first appeared in response to 20 Hz stimulation to CA3 or either 20 or 50 Hz stimulation to the angular bundle. Rats older than 21 days had afterdischarge patterns like those recorded in the adult. These data indicate that, in the rat, the seizure capabilities of the limbic circuits 8o through a major transition period around PN 17-19. The appearance of maximal dentate activation marks the ability of the developing rat brain to produce and sustain reverberatory seizure discharges.
INTRODUCTION An understanding of epileptiform activity in young mammals, and development of techniques for its control, are important challenges to both clinical and laboratory researchers. Seizures occur with a relatively high frequency among young children, and our treatments for these forms of epilepsy are much less satisfactory than treatment for seizures in adults. Experimental studies in animals show that the behavioral and electrographic manifestations of seizures vary greatly as the brain develops t5'2~. An increased propensity for seizures has been described in the young animal in response to both chemical convulsants and electrical stimulation 1,3-7.9,"u7,19. It is difficult to make conclusions from these studies because of the different methods of seizure initiation and because the different brain regions being studied develop at different times and r a t e s 2'1°'13'14J6'26'27. In the urethane-anesthetized adult rat, two types of afterdischarges have been recorded in the dentate
gyrus2°. The first consisted of bursts of large amplitude population spikes associated with a negative shift of the DC potential and a secondary rise in the extracellular potassium. This triad of responses has been termed maximal dentate activation and has been shown to be generated by the granule cells 23'25. Maximal dentate activation appears to represent the synchronous activation of the granule cells in the dentate gyrus and to be a marker for reverberatory seizure activity in hippocampal-parahippocampal circuits 21'22. The second type of afterdischarge recorded in the dentate gyrus consisted of broad positive potentials and was termed a non-MDA (maximal dentate activation) afterdischarge. Non-MDA afterdischarges are elicited by stimuli to the CA3 region just shorter in duration than needed to produce maximal dentate activation; 50 Hz/3 s consistently elicited the non-MDA afterdischarge. These non-MDA afterdischarges could not be elicited by angular bundle stimulation. With repeated elicitation, the duration of maximal dentate activation readily lengthens in the adult, while the non-MDA afterdischarges
Correspondence: J.L. Stringer, Department of Pharmacology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. Fax: (1) (713) 798-3145.
224 do not. The non-MDA afterdischarge appears to be
predominantly mediated by the pyramidal cells of the hippocampus "~u and to represent an afterdischarge that is distinct from the afterdischarges that consist o f maximal dentate activation. This study examined the ability o f rats at different developmental stages to express afterdischarges in the C A I region and the dentate gyrus. O f particular interest was the age at which the two types o f afterdischarges could be distinguished and the age at which maximal dentate activation appeared. The results were then used to try to correlate some o f the findings from other laboratories that use other models of seizure generation in the developing rat.
repeated every 10 min while the right recording electrode was lowered through the CAI region, into, and through the dentate gyms. The electrode was lowered 50 pm each step through the cell layers and 100/zm each step between cell layers. For each stimulus train the duration of the afterdischarge was measured. After completion of this laminar map, the right electrode was raised to the CAI cell layer. Stimulus trains of 50 Hz/3 s were then given to the CA3 region to determine the type of afterdischarge that could be produced. Finally, stimulus trains of 20 and 50 Hz (maximum of 30 s, 3 mA) were given through the angular bundle stimulating e;ectrode. At the conclusion of all experiments, current was passed through the metal electrodes and the animals were perfused through the heart with 1% potassium ferrocyanide in 10% buffered formalin. The brains were subsequently equilibrated in 30% sucrose/|0% buffered formalin. Sections were cut on a sliding microtome and stained with Cresyl violet. The positions of the stimulating electrodes were indicared by blue spots and the tracks for the recording electrodes were identified.
RESULTS MATERIALS AND METHODS Three litters of Sprague-Dawley rats were used for these experiments. Pregnant rats were obtained at mid-gestation and after birth the litter~ were reduced to 10 pups. Two rats were used from each litter for each of 5 post-natal (PN) time points (PN 10-11 (21-24 g), PN 14-15 (26-36 g), PN 17-19 (38-42 g), PN 21-23 (52-62 g), PN 25-27 (62-90 g)). Both male and female pups were used. Five adult (275-350 g) male Sprague-Dawley rats were used as controls. The rats were anesthetized with urethane (!.2-1,5 g/kg i.p.) and placed in a ster,~otaxic frame. For the youngest animals (PN 10-15), the ear bars were pl:tced gently in the external auditory canal and the incisor bar (0.0) pieced under the tipper jaw. Body temperature was maintained tit 36-37°C with a heating pad. A concentric bipolar electrode (SNEX 100, Rhodes Medical Inst.) was placed in the CA3 region of the left hippocampus tit tin tingle of 10°. For the rats in the PN 10-11 group the electrode was placed 2.2 mm posterior to hregma and 3.1 mm lateral to the midline. For ,11 others, the anterior coordinate was increased 0.2 mm tor each IS g of body weight and the lateral dimension increased 0.1 mm until the adult coordinates were reached. In the ,dult these coordinates were 3.0 mm posterior and 3.S mm lateral. A second ,timulating electrode (twisted stainless steel) was placed in the angular bundle on the left side. A glass micro~:lectrode filled with I% Fast green in 2 M NaCI was pieced in the left dentate gyros in the same AP plane as the C A 3 stimulating electrode (lateral 1,2-1.8 mm). A double barrel potassium.sensitivemicroelectrode''~was placed in the CAI ceil layer on the right side, also lateral i.2-i,8 ram. The animals were grounded through a subcutaneous Ag/AgCI wire in the scapular region. Extracellular field potentialswere amplified and displayed on a digitaloscilloscopeand simultaneously displayed on a chart recorder for monitoring of the D C potentialchanges. The extracellulerpotassium was recorded by a differentialamplifier :,nd displayed on the chart recorder,The potassium electrodes were calibrated after each experiment. Stimulation was controlled by a digitaltimer led to a constant current isolatedstimulator. !n,lividualstimuli consisted of O ~ ms biphasic pulses. Stimulus tra.,~ were administered through the C A 3 or ;regularbundle stimulath~, ,.~:ctrodes,An afterdischarge was defined as any activity that occurred after the end of the stimulus and itsduration (in seconds)was measured from the end of the stimulus through the last burst of the afterdischarge.Maximal dentate a~t.ivationwas defined by the presence of bursts of large amplitude population spikes,a secondary increase in the extracellular potassium, and a negative shiftof the D C potential. With the left recording electrode in the dentate gyrus and the right recording electrode in the CAI cell layer, the efterdischarge threshold was determined with 20 Hz stimulus trains to the C A 3 region. The maximam stimulus duration was 30 s and the maximum stimulus intensitywas 3 mA. After determination of the afterdischarge threshold the right recording electrode was raised 200-2S0 # m above the CAI celllayer.Stimulus trainsto the CA3 region were
The initial preparation allowed examination of the evoked responses from left CA3 to right CAI and from the angular bundle to ipsilateral dentate gyms. I n agreement with previous reports in C A I s't3, the latency to the population spike decreased, the amplitude o f the population spike and the field EPSP increased and the width o f the population spike decreased as the animals aged (Fig. 1). In the dentate gyrus, a field EPSP did not appear until PN 14-15 and the latency changes were much smaller than in the C A I system. In both regions, the evoked responses were adult-like by
C A 3 .-~cCA1
A B -,~iDG
Fig, I, Evoked responses recorded in animals from different age groups, The left column of responseswere recorded in the right CAI cell layer (cCAI) after stimulation of the left CA3 region. The right column of responses were recorded in the left dentate wrus (iDG) after stimulation of the left angular bundle. The age of the rat from which the responses ~,ere obtained is given to the left in days (PH = post natal). Each response is preceded by a calibration pulse of $ mV by I ms and the time of stimulation is indicated by the arrow (positivity upwards). With increasing age, the latency to the responses get shorter and the EPSP and population spike increase in amplitude.
225 PN 21-23 (Fig. 1). The responses are presented for direct comparison to the epileptic discharges produced later in the same animals. The EEG that was recorded in the hippocampus also evolved over the same age range (Fig. 2). PN 10-11 the EEG was almost completely flat. The amplitude and frequency of activity began to pick up by PN 14-15. The EEG pattern was comparable to the adult pattern by PN 25. The afterdischarge threshold was determined in each animal using 20 Hz stimulation to the left CA3 region and then averaged within each age group. The PN 10-11 age group had the highest mean afterdischarge threshold (2,000+400), followed by the PN 14-15 (1,400 + 300). By PN 17-19 the afterdischarge thresholds (400 + 70) were not significantly different from the adult group (200 + 60, P > 0.05). The groups from PN 21-23 (360 + 100) and PN 25-27 (330 + 60) were also not different from the adult group. Both PN 10-11 and PN 14-15 were significantly different from the other age groups (P0.05, Newman-Keuls). Others have also shown that the afterdischarge threshold varies with age tt. Stimulus trains of 20 Hz to the left CA3 region readily produced afterdischarges in all animals. However, the characteristics of these afterdischarges varied across the age groups examined (Fig.s 3 and 4). At PN 10-11, the afterdischarges consisted of 5-10 mV broad population spikes in the right CAI (similar to the evoked responses in Fig. 1) and very small (1-2 mV) positive potentials in the left dentate 8yrus. There was no burst pattern to this afterdischarge. At PN 14-15, the positive potentials in the dentate 8yrus were more PN 10
-
--
PN 15
- - .........
PN 18
-~- -
'~. . . . . . .
_
. . . . . . -~.w
PN~
PN 25
Ad.,, i[ amv
Fig. 2. EEG recordings at different ages of the rat. A 40 s segment of the baseline EEG recorded in the hippocampub is shown for each age group studied. The ages are given to the left of the chart recordings. The vertical calibration is the same for all of the recordings and is indicated on the adult trace. For animals under urethane anesthesia, the amplitude of the background EEG increases with age.
R . C A I I_~:':.=__ ~ L - D~G
_-'-;~-:--.-:::-.-
I
RCA]
ri
I
,
II
R.CA1
I
I I
"
I
•
~
ItIBv~
I
-J"
Fig. 3. Afterdischarges recorded in young rats. Each panel p,:esents the afterdischarge recorded simultaneously in the left dentate gyrus (L-DG) and right CAI cell layer (R-CAI) of one animal. The age of the animal is indicated in the panel. The afterdischarges were produced by 20 Hz stimulation of the left CA3 region, except the bottom right adult example, which was produced by 50 Hz/3 s stimulation. The calibrations are indicated in each panel. Note that the time scale is constant, but that the vertical scale varies. Before PH 19 the discharges consisted of broad positive potentials in the dentate gyrus along with population spikes in CAl. Bursts of population spikes in the dentate wrus (maximal dentate activation) did not appear until after PN 19.
easily discerned and the population spikes in the CAI cell layer were sharper and larger in amplitude (similar to the change in the evoked response in Fig. 1). In the PN 17-19 group, there were two types of afterdischarges recorded in the dentate gyrus (Figs. 3 and 4). The first consisted of broad positive potentials with occasional small population spikes (see PN 17, Figs, 3 and 4A). As with the younger animals there was no burst pattern to this afterdischarge. The second type of afterdischarge consisted of larger population spikes in the dentate gyrus that occurred in bursts and was associated with a negative shift of the DC potential and a secondary rise in the extracellular potassium (see PN 19, Figs. 3 and 4B,C). Although the frequency of the population spikes within the bursts was less than in the adult (compare PN 19 to adult Fig. 3), this afterdischarge was considered to represent maximal dentate activation. Maximal dentate activation was recorded in
226
A
PN 14
PN I I
Io
")[ K+
mM
"~"
~!
i
,
,
.
,' .=
C
q
°:[ io
,my[
E
31. Fig. 4. Chart r;:cordings of the stimulus and afterdischarges from selected age groups. Each panel presents the reference (top) and extracellular potassium (bottom) recordings from the electrode in the right dentate gyrus during one stimulus train to the left CA3 region. The duration of the stimulus train is indicated by the bar under the reference trace on each panel. The abe of the animal is given for each set of recordings. The onset of maximal dentate activation is indicated by the arrow in B, C and D. A lind C are from the ,tame animal, The afterdischarge in A did not have population spikes in the dentate gyrus. In B, maximal dentate activation appeared during the stimulus train. Maximal dentate activation appeared during the afterdischarge in C. Note the change in the pattern of the aftcrdischarge lifter the appearance of maximal dentate activation. Adult examples of a maximal dentate activation afterdischarge (20 Hz stimulus, D) and an afterdischarge that does not consist of maximal dentate activation (50 tlz, 3 s stimulus, E) are shown for comparimm purposes.
three of the six animals in the PN 17-19 age group tone animal at each age). In two of these three animals, maximal dentate activation was only elicited intermittently. Two seizures from one of these 'intermittent' animals are illustrated in Fig. 4 (A,C). in this animal, maximal dentate activation appeared during the afterdischarge and the change in the burst pattern, as well as the secondary increase in the extracellular potassium, can be seen on the chart recording (Fig. 4C). By PN 21-23, the afterdischarges were indistinguishable from the adult in appearance, with bursts of population spikes in the dentate gyrus associated with a negative shift of the DC potential and a secondary rise in the extracellular potassium in response to 20 Hz stimulation of the contralateral CA3. When these 20 Hz stimulus trains were repeated every 10 rain the afterdischarges that consisted of maximal dentate activation lengthened (Figs. 5 and 6). The afterdischarges that consisted of population spikes in
223
BRESD 51546
Ontogeny of hippocampal afterdischarges in the urethane-anesthetized rat Janet L. Stringer
a
and Eric W. Lot hman
b
a Department of Pharmacology, Baylor College of Medicine, Ho~:siOn, TX 77030 (USA) and t, Department of Neurology, Unil'ersity of Virginia Medical Center, Chariottest'ille, VA 22908 (USA) (Accepted 21 July 1992)
Key words: Epilepsy; Afterdischarge; Dentate gyrus; Ontogeny; CAI; Extracellular potassium; EEG
Experimental studies have shown that seizure manifestations vary as the brain develops. This study investigated the characteristics of afterdischarges in the hippocampal circuits at various ages in the developing rat. Rats from the following post-natal periods were tested: PN 10-11, PN 14-15, PN 17-19, PN 21-23 and PN 25-27. Animals were anesthetized with urethane and recording electrodes placed in the hippocampus bilaterally. Stimulating electrodes were placed in the left CA3 region and in the angular bundle. Afterdischarges were produced in all animals using stimulus trains of 20 or 50 Hz. Rats in the PN 10-11 and 14-15 age groups had afterdischarges that consisted of population spikes in CAI and broad positive potentials in the dentate gyrus. Between PN 17 and 19, maximal dentate activation, which consists of bursts of large amplitude populatie,n spikes in the dentate gyrus, first appeared in response to 20 Hz stimulation to CA3 or either 20 or 50 Hz stimulation to the angular bundle. Rats older than 21 days had afterdischarge patterns like those recorded in the adult. These data indicate that, in the rat, the seizure capabilities of the limbic circuits 8o through a major transition period around PN 17-19. The appearance of maximal dentate activation marks the ability of the developing rat brain to produce and sustain reverberatory seizure discharges.
INTRODUCTION An understanding of epileptiform activity in young mammals, and development of techniques for its control, are important challenges to both clinical and laboratory researchers. Seizures occur with a relatively high frequency among young children, and our treatments for these forms of epilepsy are much less satisfactory than treatment for seizures in adults. Experimental studies in animals show that the behavioral and electrographic manifestations of seizures vary greatly as the brain develops t5'2~. An increased propensity for seizures has been described in the young animal in response to both chemical convulsants and electrical stimulation 1,3-7.9,"u7,19. It is difficult to make conclusions from these studies because of the different methods of seizure initiation and because the different brain regions being studied develop at different times and r a t e s 2'1°'13'14J6'26'27. In the urethane-anesthetized adult rat, two types of afterdischarges have been recorded in the dentate
gyrus2°. The first consisted of bursts of large amplitude population spikes associated with a negative shift of the DC potential and a secondary rise in the extracellular potassium. This triad of responses has been termed maximal dentate activation and has been shown to be generated by the granule cells 23'25. Maximal dentate activation appears to represent the synchronous activation of the granule cells in the dentate gyrus and to be a marker for reverberatory seizure activity in hippocampal-parahippocampal circuits 21'22. The second type of afterdischarge recorded in the dentate gyrus consisted of broad positive potentials and was termed a non-MDA (maximal dentate activation) afterdischarge. Non-MDA afterdischarges are elicited by stimuli to the CA3 region just shorter in duration than needed to produce maximal dentate activation; 50 Hz/3 s consistently elicited the non-MDA afterdischarge. These non-MDA afterdischarges could not be elicited by angular bundle stimulation. With repeated elicitation, the duration of maximal dentate activation readily lengthens in the adult, while the non-MDA afterdischarges
Correspondence: J.L. Stringer, Department of Pharmacology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. Fax: (1) (713) 798-3145.
224 do not. The non-MDA afterdischarge appears to be
predominantly mediated by the pyramidal cells of the hippocampus "~u and to represent an afterdischarge that is distinct from the afterdischarges that consist o f maximal dentate activation. This study examined the ability o f rats at different developmental stages to express afterdischarges in the C A I region and the dentate gyrus. O f particular interest was the age at which the two types o f afterdischarges could be distinguished and the age at which maximal dentate activation appeared. The results were then used to try to correlate some o f the findings from other laboratories that use other models of seizure generation in the developing rat.
repeated every 10 min while the right recording electrode was lowered through the CAI region, into, and through the dentate gyms. The electrode was lowered 50 pm each step through the cell layers and 100/zm each step between cell layers. For each stimulus train the duration of the afterdischarge was measured. After completion of this laminar map, the right electrode was raised to the CAI cell layer. Stimulus trains of 50 Hz/3 s were then given to the CA3 region to determine the type of afterdischarge that could be produced. Finally, stimulus trains of 20 and 50 Hz (maximum of 30 s, 3 mA) were given through the angular bundle stimulating e;ectrode. At the conclusion of all experiments, current was passed through the metal electrodes and the animals were perfused through the heart with 1% potassium ferrocyanide in 10% buffered formalin. The brains were subsequently equilibrated in 30% sucrose/|0% buffered formalin. Sections were cut on a sliding microtome and stained with Cresyl violet. The positions of the stimulating electrodes were indicared by blue spots and the tracks for the recording electrodes were identified.
RESULTS MATERIALS AND METHODS Three litters of Sprague-Dawley rats were used for these experiments. Pregnant rats were obtained at mid-gestation and after birth the litter~ were reduced to 10 pups. Two rats were used from each litter for each of 5 post-natal (PN) time points (PN 10-11 (21-24 g), PN 14-15 (26-36 g), PN 17-19 (38-42 g), PN 21-23 (52-62 g), PN 25-27 (62-90 g)). Both male and female pups were used. Five adult (275-350 g) male Sprague-Dawley rats were used as controls. The rats were anesthetized with urethane (!.2-1,5 g/kg i.p.) and placed in a ster,~otaxic frame. For the youngest animals (PN 10-15), the ear bars were pl:tced gently in the external auditory canal and the incisor bar (0.0) pieced under the tipper jaw. Body temperature was maintained tit 36-37°C with a heating pad. A concentric bipolar electrode (SNEX 100, Rhodes Medical Inst.) was placed in the CA3 region of the left hippocampus tit tin tingle of 10°. For the rats in the PN 10-11 group the electrode was placed 2.2 mm posterior to hregma and 3.1 mm lateral to the midline. For ,11 others, the anterior coordinate was increased 0.2 mm tor each IS g of body weight and the lateral dimension increased 0.1 mm until the adult coordinates were reached. In the ,dult these coordinates were 3.0 mm posterior and 3.S mm lateral. A second ,timulating electrode (twisted stainless steel) was placed in the angular bundle on the left side. A glass micro~:lectrode filled with I% Fast green in 2 M NaCI was pieced in the left dentate gyros in the same AP plane as the C A 3 stimulating electrode (lateral 1,2-1.8 mm). A double barrel potassium.sensitivemicroelectrode''~was placed in the CAI ceil layer on the right side, also lateral i.2-i,8 ram. The animals were grounded through a subcutaneous Ag/AgCI wire in the scapular region. Extracellular field potentialswere amplified and displayed on a digitaloscilloscopeand simultaneously displayed on a chart recorder for monitoring of the D C potentialchanges. The extracellulerpotassium was recorded by a differentialamplifier :,nd displayed on the chart recorder,The potassium electrodes were calibrated after each experiment. Stimulation was controlled by a digitaltimer led to a constant current isolatedstimulator. !n,lividualstimuli consisted of O ~ ms biphasic pulses. Stimulus tra.,~ were administered through the C A 3 or ;regularbundle stimulath~, ,.~:ctrodes,An afterdischarge was defined as any activity that occurred after the end of the stimulus and itsduration (in seconds)was measured from the end of the stimulus through the last burst of the afterdischarge.Maximal dentate a~t.ivationwas defined by the presence of bursts of large amplitude population spikes,a secondary increase in the extracellular potassium, and a negative shiftof the D C potential. With the left recording electrode in the dentate gyrus and the right recording electrode in the CAI cell layer, the efterdischarge threshold was determined with 20 Hz stimulus trains to the C A 3 region. The maximam stimulus duration was 30 s and the maximum stimulus intensitywas 3 mA. After determination of the afterdischarge threshold the right recording electrode was raised 200-2S0 # m above the CAI celllayer.Stimulus trainsto the CA3 region were
The initial preparation allowed examination of the evoked responses from left CA3 to right CAI and from the angular bundle to ipsilateral dentate gyms. I n agreement with previous reports in C A I s't3, the latency to the population spike decreased, the amplitude o f the population spike and the field EPSP increased and the width o f the population spike decreased as the animals aged (Fig. 1). In the dentate gyrus, a field EPSP did not appear until PN 14-15 and the latency changes were much smaller than in the C A I system. In both regions, the evoked responses were adult-like by
C A 3 .-~cCA1
A B -,~iDG
Fig, I, Evoked responses recorded in animals from different age groups, The left column of responseswere recorded in the right CAI cell layer (cCAI) after stimulation of the left CA3 region. The right column of responses were recorded in the left dentate wrus (iDG) after stimulation of the left angular bundle. The age of the rat from which the responses ~,ere obtained is given to the left in days (PH = post natal). Each response is preceded by a calibration pulse of $ mV by I ms and the time of stimulation is indicated by the arrow (positivity upwards). With increasing age, the latency to the responses get shorter and the EPSP and population spike increase in amplitude.
225 PN 21-23 (Fig. 1). The responses are presented for direct comparison to the epileptic discharges produced later in the same animals. The EEG that was recorded in the hippocampus also evolved over the same age range (Fig. 2). PN 10-11 the EEG was almost completely flat. The amplitude and frequency of activity began to pick up by PN 14-15. The EEG pattern was comparable to the adult pattern by PN 25. The afterdischarge threshold was determined in each animal using 20 Hz stimulation to the left CA3 region and then averaged within each age group. The PN 10-11 age group had the highest mean afterdischarge threshold (2,000+400), followed by the PN 14-15 (1,400 + 300). By PN 17-19 the afterdischarge thresholds (400 + 70) were not significantly different from the adult group (200 + 60, P > 0.05). The groups from PN 21-23 (360 + 100) and PN 25-27 (330 + 60) were also not different from the adult group. Both PN 10-11 and PN 14-15 were significantly different from the other age groups (P0.05, Newman-Keuls). Others have also shown that the afterdischarge threshold varies with age tt. Stimulus trains of 20 Hz to the left CA3 region readily produced afterdischarges in all animals. However, the characteristics of these afterdischarges varied across the age groups examined (Fig.s 3 and 4). At PN 10-11, the afterdischarges consisted of 5-10 mV broad population spikes in the right CAI (similar to the evoked responses in Fig. 1) and very small (1-2 mV) positive potentials in the left dentate 8yrus. There was no burst pattern to this afterdischarge. At PN 14-15, the positive potentials in the dentate 8yrus were more PN 10
-
--
PN 15
- - .........
PN 18
-~- -
'~. . . . . . .
_
. . . . . . -~.w
PN~
PN 25
Ad.,, i[ amv
Fig. 2. EEG recordings at different ages of the rat. A 40 s segment of the baseline EEG recorded in the hippocampub is shown for each age group studied. The ages are given to the left of the chart recordings. The vertical calibration is the same for all of the recordings and is indicated on the adult trace. For animals under urethane anesthesia, the amplitude of the background EEG increases with age.
R . C A I I_~:':.=__ ~ L - D~G
_-'-;~-:--.-:::-.-
I
RCA]
ri
I
,
II
R.CA1
I
I I
"
I
•
~
ItIBv~
I
-J"
Fig. 3. Afterdischarges recorded in young rats. Each panel p,:esents the afterdischarge recorded simultaneously in the left dentate gyrus (L-DG) and right CAI cell layer (R-CAI) of one animal. The age of the animal is indicated in the panel. The afterdischarges were produced by 20 Hz stimulation of the left CA3 region, except the bottom right adult example, which was produced by 50 Hz/3 s stimulation. The calibrations are indicated in each panel. Note that the time scale is constant, but that the vertical scale varies. Before PH 19 the discharges consisted of broad positive potentials in the dentate gyrus along with population spikes in CAl. Bursts of population spikes in the dentate wrus (maximal dentate activation) did not appear until after PN 19.
easily discerned and the population spikes in the CAI cell layer were sharper and larger in amplitude (similar to the change in the evoked response in Fig. 1). In the PN 17-19 group, there were two types of afterdischarges recorded in the dentate gyrus (Figs. 3 and 4). The first consisted of broad positive potentials with occasional small population spikes (see PN 17, Figs, 3 and 4A). As with the younger animals there was no burst pattern to this afterdischarge. The second type of afterdischarge consisted of larger population spikes in the dentate gyrus that occurred in bursts and was associated with a negative shift of the DC potential and a secondary rise in the extracellular potassium (see PN 19, Figs. 3 and 4B,C). Although the frequency of the population spikes within the bursts was less than in the adult (compare PN 19 to adult Fig. 3), this afterdischarge was considered to represent maximal dentate activation. Maximal dentate activation was recorded in
226
A
PN 14
PN I I
Io
")[ K+
mM
"~"
~!
i
,
,
.
,' .=
C
q
°:[ io
,my[
E
31. Fig. 4. Chart r;:cordings of the stimulus and afterdischarges from selected age groups. Each panel presents the reference (top) and extracellular potassium (bottom) recordings from the electrode in the right dentate gyrus during one stimulus train to the left CA3 region. The duration of the stimulus train is indicated by the bar under the reference trace on each panel. The abe of the animal is given for each set of recordings. The onset of maximal dentate activation is indicated by the arrow in B, C and D. A lind C are from the ,tame animal, The afterdischarge in A did not have population spikes in the dentate gyrus. In B, maximal dentate activation appeared during the stimulus train. Maximal dentate activation appeared during the afterdischarge in C. Note the change in the pattern of the aftcrdischarge lifter the appearance of maximal dentate activation. Adult examples of a maximal dentate activation afterdischarge (20 Hz stimulus, D) and an afterdischarge that does not consist of maximal dentate activation (50 tlz, 3 s stimulus, E) are shown for comparimm purposes.
three of the six animals in the PN 17-19 age group tone animal at each age). In two of these three animals, maximal dentate activation was only elicited intermittently. Two seizures from one of these 'intermittent' animals are illustrated in Fig. 4 (A,C). in this animal, maximal dentate activation appeared during the afterdischarge and the change in the burst pattern, as well as the secondary increase in the extracellular potassium, can be seen on the chart recording (Fig. 4C). By PN 21-23, the afterdischarges were indistinguishable from the adult in appearance, with bursts of population spikes in the dentate gyrus associated with a negative shift of the DC potential and a secondary rise in the extracellular potassium in response to 20 Hz stimulation of the contralateral CA3. When these 20 Hz stimulus trains were repeated every 10 rain the afterdischarges that consisted of maximal dentate activation lengthened (Figs. 5 and 6). The afterdischarges that consisted of population spikes in
