- Email: [email protected]
Hippocampal stimulation and memory: Effects of stimulation parameters and reinforcement magnitude
Physiology and Behavior, Vol. 13, pp. 47-55. Brain Research Publications Inc., 1974. Printed in the U.S.A.
Hippocampal Stimulation and Memory: Effects of Stimulation Parameters and Reinforcement Magnitude' BRUCE S. KAPP, J E F F R E Y D. K A U F M A N AND DENNIS A. REPOLE
Department o f Psychology, University o f Vermont, Burlington, Vermont 05401
(Received 14 December. 1973)
KAPP, B. S., J. D. KAUFMAN AND D. A. REPOLE. Hippocampal stimulation and memory: effects of stimulation parameters and reinforcement magnitude. PHYSIOL. BEHAV. 13(1) 47-55, 1974. - The present study investigated the extent to which post-conditioning stimulation-induced hippocampal seizure activity is a necessary condition for the production of retrograde amnesia in rats. Groups of animals received dorsal hippocampal stimulation at several different current intensities immediately following one-trial passive avoidance conditioning. The results suggest that current intensifies far in excess of those which produce hippocampal seizure activity are a necesssry condition for the production of retrograde amnesia. Reinforcement magnitude is not a significant variable at hippocampal seizure threshold current intensities. Current intensities insufficient for the production of retrograde amnesia, but sufficient for the production of donml hippocampal seizure activity, produced a spread of seizure activity into the ventral hippocampal formation. The results suggest that caution be exercised in attributing a role to the hippocampus in memory consolidation processes in the rat. Memory consolidation
Passive avoidance conditioning
Hippocampal stimulation
THE CONTRIBUTION of specific neuroanatomical structures to memory consolidation processes has recently been investigated by using post-conditioning, low-level, intracranial electrical stimulation. The results have indicated that several subcortical structures including the caudate nucleus [9, 19, 20, 22], the amygdaloid complex [2, 3, 4, 7, 8], and particularly the hippocampal formation [ I , 6, 7, 8, 14, 15, 18, 21] may be involved in the consolidation o f newly acquired information. Stimulation of any one of the three structures immediately after conditioning has been reported to result in retention deficits 24 hr following stimulation in rats and mice. In any a t t e m p t to demonstrate the involvement of specific neuroanatomical sites in consolidation processes by using stimulation techniques i t becomes critical to define the extent to which the applied current produces abnormal high amplitude seizure activity which may propagate to structures other than the one in question. As a first step, therefore, it would seem desirable to use current levels which produce reliable retention deficits but which produce a minimal amount of abnormal neuroelectrical seizure activity in an a t t e m p t to confine this activity to the structure under investigation. Whether or not the production of seizure activity in the hippocampus is a necessary condition for the production of
Hippocampal seizure activity
retrograde amnesia is still uncertain. Wyers, Peeke, Wiili~ton and Herz [ 19] have reported that current levels subthreshold for the production o f hippocampal seizure activity were effective in producing retrograde amnesia in rats for onetrial l~assive avoidance conditioning. McDonough and Kesner [8] have reported similar results for the cat. On the other hand, Shinkman and Kaufman [14] have reported that current levels just exceeding hippocampal seizure threshold administered to rats after each of four daily trials in a CER paradigm have no effect on retention. Their results, however, differ from those o f Vardaris and Schwartz [18] who report retention deficits for one-trial passive avoidance using current levels just suprathreshold for the production of hippocampal seizure activity. Several variables may contribute to the inconsistent results, including species differences and differences in conditioning parameters (reinforcement magnitude, onetrial vs. multiple-trial paradigms). It has been reported, for example, that the retention deficits produced by another treatment, electroconvulsive shock (ECS), vary directly with species [13] and reinforcement magnitude [12]. It becomes essential, therefore, in attempting to define current levels which produce reliable retention deficits while producing minimal amounts of abnormal hippocampal neuroelectrical activity to (a) manipulate current
J This research was supported by research Grants MH-22404-01 from the National Institute of Mental Health and NSF-1972-128 from the Institutional Grants Committee of the University of Vermont. 47
48
KAPP, KAUFMAN AND REPOLE
intensity while maintaining constant conditioning parameters and (b) manipulate reinforcement magnitude while maintaining constant current stimulation parameters within a single species. The purpose of the present investigation was (a) to determine the minimal current intensity that, when applied to the dorsal hippocampal formation of rats immediately following one-trial passive avoidance conditioning, will produce reliable retention deficits and (b) to determine if reinforcement magnitude is a significant variable in defining the minimal current intensity needed to produce retention deficits.
EXPERIMENT 1 Experiment 1 was designed to determine if hippocampal seizure afterdischarge activity is a necessary condition for producing retention deficits for one-trial passive avoidance in rats. Current levels just suprathreshold for the production of hippocampal seizure activity were used. METHOD
A nimals Experimentally naive Sprague-Dawley male rats weighing 2 7 5 - 3 2 5 g at the time of surgery were used. The rats were maintained on a 12 hr light-dark cycle, given ad lib food and water and were group housed until surgery at which time they were placed in separate cages.
Surgical Procedures All animals with the exception of an unoperated control group were surgically prepared under Nembutal anesthesia (50 mg/ml [P) with bipolar electrodes constructed from 0.25 mm in diameter stainless steel wire, soldered to A m p h e n o l Subminax connectors, and insulated with Formvar except at the cross-sectional tip. Tip separation was 0.5 mm and the electrodes were implanted bilaterally into the dorsal hippocampal formation using the coordinates of Pellegrino and Cushman [ 10] : 2.5 mm posterior to bregrna, 2.5 mm lateral to the midline and 3.5 mm below dura. Immediately following surgery intramuscular injections of Wyeillin (30,000 units, Wyeth) were given.
A ppara tus The training apparatus was similar to the one used by Schneider, Kapp, Aron and Jarvik [13], constructed of Plexigias and enlarged to accomodate rats (64.0 x 28.0 x 6.0 cm). It consisted of a small compartment constructed of clear Plexigias connected to a larger black compartment by a 28.0 x 6.0 cm opening. The floor of each compartment consisted of pairs of parallel stainless-steel plates, one pair (16.0 x 2.5 cm) in the smaller and two pairs (23.0 x 2.5 cm) in the larger compartment. Adjacent plates were spaced 1.25 cm apart. The apparatus was trough-shaped with the width of each compartment increasing from a 6.0 cm base to a 21.0 cm top. The top of each compartment was covered by a hinged lid. The floor plates of the large black compartment were connected to a Grason-Stadler shock scrambler set to deliver a 2.0 sec footshock (FS). The stimulation and recording apparatus consisted of a second box (23.0 x 19.0 x 29.0 cm) constructed of Plexigias with a grid floor of stainless steel rods 0.25 cm in
diameter, spaced 1.0 cm apart. Attached to the top was a Scientific Prototype mercury commutator from which extended the leads used for stimulation and recording.
Procedure Hippocampal seizure threshold determination. In order to reliably produce post-conditioning hippocampal seizure activity in each animal, individual current thresholds suffid e n t to produce hippocampal seizure activity were determined. Following 7 - 1 0 days of post-operative recovery, each animal was placed in the recording apparatus and a 5 sec train of biphasic square waves of 100 pps with a pulse duration of 0.3 msec was applied to the dorsal hippocampal formation. Stimulation was given bilaterally using two constant current stimulators isolated from ground. Current intensity was increased in 3 uA increments every 3 min until hippocampal seizure activity was recorded on a 6-channel E&M Physiograph Six. Current intensity was monitored using a dual-beam oscilloscope by monitoring the voltage drop across a 10 Kohm resistor placed in the stimulation circuit in series with the animals. The intensity used for post-conditioning stimulation was 120% of the seizure threshold current intensity. Since hippocampal seizure threshold current levels differ from animal to animal, the above method was chosen to assure that all animals received equal amounts of post-conditioning current relative to their individual threshold current levels. Conditioning procedure. The conditioning procedure was a one-trial passive avoidance task commonly used to study both the effects of electroconvulsive shock [ 11,13 ] and hippocampal stimulation [7, 18, 21] on memory consolidation. One week following hippocampal seizure threshold testing all animals were given an habituation session in the training apparatus. After connecting the electrode leads, the animal was placed into the small compartment facing away from the large compartment and allowed to step into the large compartment for 10 sec of exploration. Twentyfour hr later the animal, with electrode leads attached, was again placed into the small compartment and a timer was started. When all four paws entered the large compartment, a guillotine door between the two compartments was closed, the timer stopped and a 1.6 mA, 2.0 sec FS was given. During FS the animal was disconnected from the stimulation and recording circuitry in order to prevent artifactual brain stimulation by crosstalk between peripheral FS and stimulation electrode leads. All animals were then immediately removed from the apparatus and placed in the stimulation and recording chamber. Electrode leads were connected to the stimulation-recording circuitry and the designated post-training manipulation was given. The circuitry was programmed so that hippocampal electrical activity was recorded from the site of stimulation 0.1 sec following the termination of the stimulus train. The FSstimulation interval ranged from 5 to 10 sec. Following stimulation and recording all animals were returned to their home cages. Post-training manipulation. Animals were assigned to one of four groups. (I) FS-C. Animals in this group were unoperated controls and received FS upon entering the large compartment. They were subject to the same handling procedure and length of time in the stimulation-recording apparatus as the other operated groups. This group was included to determine whether the lesions produced by hippocampal electrode implantation had any effect on re-
HIPPOCAMPAL STIMULATION PARAMETERS AND MEMORY tention. (2) FS-NESB. Animals received FS upon entering the large compartment followed by sham hippocampal stimulation. (3) FS-ESB. Animals received FS followed within 5 - 1 0 sec by hippocampal stimulation at 120% o f hippocampal seizure threshold current for 5.0sec. (4) NFS-ESB. Animals did not receive FS and were stimulated at 120% of hippocampal seizure threshold within 5 - 1 0 sec of entering the large compartment. This group was included to assess the aversive properties of the stimulation. Retention test procedure. Twenty-four hr following training all animals, with electrode leads attached (Group FS=C with no leads), were returned to the small compartment and step-through latencies (STL's) were taken. An increased STL compared with training STL indicated retention for the training experience. If the animal did not enter the large compartment within 300 sec, the test was terminated. Histology. After behavioral testing all animals were perfused with physiological saline followed by 10% formalsaline. The brains were removed, sliced into 40 u sections with a freezing rnlcrotome and examined for verification of implantation sites using the method of Thompson [ 17] for unstained sections with the aid of the Pellegrino and Cushman [ 10] brain atlas. RESULTS
Histological Only those animals with confirmed bilateral electrode placements within the dorsal hippocampai formation were
49
included in the data analysis. This resulted in a total of 9 animals in Group FS-NESB, 17 in Group FS-ESB and 10 in Group NFS-ESB. Group FS-C consisted of 9 animals. Electrode placements for Group FS-ESB are reconstructed in Fig. 1. No relationship was found between electrode placement within the dorsal hippocampal formation and the magnitude of retention test step-through latency.
Electrophysiological Threshold current levels sufficient to produce hippocampal seizure afterdischarge (AD) activity ranged from 3 2 - 1 6 0 u A with a mean of 76t~A. Animals received a mean of 25 stimulus presentations before reaching seizure AD threshold. Typical post-conditioning hippocampal seizure AD activity is shown in Fig. 2. F o u r animals in the FS-ESB group and one animal in the NFS-ESB group showed no signs of post-conditioning hippocampal seizure AD activity and were excluded from the behavioral data analysis, reducing the number of animals in Groups FS-ESB and NFS-ESB to 13 and 9, respectively. All animals showing signs of hippocampal AD activity with the exception of one animal in Group FS-ESB and one in Group NFS-ESB showed both primary afterdischarge (PAD) and secondary afterdischarge (SAD) activity with a period of post-ictal depression (PID) following the primary and preceding the s e c o n d a r y afterdischarge period. The two exceptions showed PAD activity with little or no post-ictal depression and no SAD activity. The mean duration o f the PAD was 15 sec, that of the post-ictal depression period 60 sec,
FIG. 1. Bilateral electrode placements for Group FS-ESB of Experiment 1. Each identical pair of letters represents bilateral placements in one animal.
50
KAPP, KAUFMAN AND REPOLE
2
'l,~,
'a'Iv~fi~~~''4/v~'vvwe'~''~'~N'~'~/~'~'~x'~'`''
,
'~"
'''
3
.
.
.
.
.
.
.
.
5
8
!
9 10 •- - J 100 uV 1 SEC FIG. 2. Typical bilateral dorsal hippocampal seizure activity recorded immediately following stimulus termination. Current level was 120% (73/~A) of hippocampal seizure threshold. Traces I and 2; base level hippocampal activity recorded one week prior to stimulation. Traces 3 and 4; primary afterdischarge activity (PAD) recorded immediately following stimulus termination. Traces 5 and 6; secondary afterdischarge (SAD) recorded approximately 45 sec following PAD. Traces 7 and 8; continuation of Traces 5 and 6. Traces 9 and 10; activity recorded 5 rain following stimulus termination. and that of the SAD activity 19 sec. No differences in posttraining AD characteristics were observed between Groups FS-ESB and NFS-ESB. Normal hippocampal activity returned from 5 to lO rain following the SAD period. Behavioral observations indicated that animals showing signs of hippocampal seizure activity generally demonstrated simultaneous headshaking and exploratory activity although none showed signs of behavioral clonus of convulsive activity. Behavioral
The retention test results are presented in Fig. 3. The 5 animals that showed no AD activity are excluded from the data analysis. A Kruskal-WaUis [16) analysis of variance revealed no significant differences in training day STL's between groups (H -- 2.5; d r = 3; p>0.1). Analysis of variance of retention test latencies revealed an overall main treatment effect (H = 55.6; d f = 3; p
that there were no significant differences between retention test latencies of Groups FS-ESB, FS-NESB and FS-C (p's>0.1). This indicated that neither post-training hippocampal stimulation at 120% of afterdischarge threshold, implant-produced lesions, nor pretraining afterdischarge determination had any significant effect on retention. Retention test STL's for Group NFS-ESB, however, did significantly differ from those of Groups FS-ESB, FS-NESB and FS-C (p's<0.001) but they did not significantly differ from their respective training day STL's (p> 0.1; Wilcoxon matched-pairs signed-ranks test). This indicated that the long retention test STL's in Group FS-ESB were not a result o f an aversive property of immediate post-conditioning stimulation. The four animals of Group FS-ESB in which post-conditioning stimulation produced no signs of h~ppocampal AD activity had a median retention test score of 300 sec and did not differ significantly from Groups FS-ESB, FS-NESB, or FS-C. The data from these 4 animals suggested that current .levels subthreshold for the produ¢-
HIPPOCAMPAL STIMULATION PARAMETERS AND MEMORY C ~ TRAINING DAY $ T L lib RETENTION D A Y STL
U g
z
lu
I_J .r
o o
I[ .r I-" 40 7
51
Electrophysiological Threshold current levels sufficient to produce hippocampal AD activity ranged from 2 3 - 9 3 ~A with a mean of 64 uA. Animals received a mean of 21 stimulus presentations before reaching seizure AD threshold. All animals in Group FS-5 showed PAD activity, post-ictal depression and SAD activity. Since recordings could not be taken for the first 90 sec in Group FS-90, no comparisons of the AD activity between this group and Group FS-5 were made. Seizure AD activity was occasionally recorded following the 90 sec o f stimulation and was followed by periods of postictal depression and subsequent recovery. Headshaking and hyperactivity were again observed during the abnormal EEG activity, but no signs of behavioral seizures were noticeable.
Behavioral It'll-C
PII-.Nt|D
PS.4ESD HI~EIO
FIG. 3. Median training day and retention test latencies for groups in Experiment 1 receiving post-trial stimulation at 120% hippocampal seizure threshold current levels. tion of hippocampal AD activity produced no effect on retention. EXPERIMENT 2 The results of Experiment 1 indicated that dorsal hippocampal stimulation just suprathreshold for the production of hippocampal seizure AD activity did not produce retention deficits at the conditioning and current parameters used. Experiment 2 was designed, therefore, in an a t t e m p t to produce retention deficits by increasing the current intensity and duration while maintaining constant conditioning parameters.
The behavioral results are presented in Fig. 4. A KruskalWallls analysis o f variance of retention test STL's revealed no main treatment effects (H = 2.1;dfffi 2; p>0.1), indicating that neither 5 nor 90 sec o f post-conditioning stimulation at 300% o f seizure AD threshold produced retention deficits. Although no control group was included in Experiment 2 to assess the aversive properties of 300% stimulation, a control group included in the following experiment (Experiment 3) appears to exclude the possibility that the lack of a retention deficit is due to the aversive properties of more intense current levels.
]
r-~ TRAMINO DAY STL ga RETENTION DAY STL
v
METHOD
Animals Experimentally naive Spragne-Dawley male rats weighing 2 7 5 - 3 2 5 g at the time of surgery were used.
Procedure Surgical, hippocampal seizure threshold determination, conditioning, histological and retention test procedures were the same as those described in Experiment I. The animals were divided into 3 groups, one group given 5 sec of immediate post-conditioning hippocampal stimulation (FS-5) at current levels 300% of seizure AD threshold, a second group given 9 0 s e c of post-conditionin~g hippocampal stimulation (FS-90) at current levels 300% of seizure AD threshold, and a third group which was trained but received sham ESB (FS-NESB). Following histological verification there were a total of 9 animals in Group FS-5, 9 animals in Group FS-90 and 6 animals in Group FS-NESB. RESULTS
Histological Electrode placements were confined to the dorsal hippocampal formation and were similar to those presented in Fig. 1. No relationship was found between electrode placement and the magnitude o f the retention test latency.
Fs-mKn
ms
Fs4o
FIG. 4. Median Ualning day and retention test latencies for groups in Experiment 2 receiving post-U-ial stimulation at 300~ hippocampal seizure threshold current levels. EXPERIMENT 3 The results of Experiment 2 indicated that current levels considerably suprathreshold for the production of hippocampal seizure AD activity were not sufficient to produce retention deficits for the one-trial passive avoidance task used. Experiment 3, therefore, was designed to determine whether or not very intense stimulation would produce a retention deficit at the conditioning parameters used in Experiments 1 and 2.
52
KAPP, KAUFMAN AND REPOLE METHOD
r-~ 1
Animals
Experimentally naive Sprague-Dawley male rats weighing 2 7 5 - 3 2 5 g at the time of surgery were used.
T R ~ ~ ~
DAy S T L DAY STL
300. z
2SO-
Procedure
,.J
Surgical, conditioning, histological and retention test procedures were the same as those described for Experiment 1. Hippoeampal seizure threshold determination, however, differed from that used in the previous experiments. In the present experiment, current intensity was increased in 100 u A increments every 3 rain until signs of an overt behavioral seizure were observed. These seizure responses were characterized by withdrawal responses and wild running followed by clonus and salivation. The current intensity used for post-conditioning stimulation was onehalf that required to produce an overt seizure response. One week following seizure threshold determination the animals were assigned to one of 4 groups. (1) FS-5. Animals were given 5 see of stimulation following training. (2) FS-90. AnimaLs were given 90 sec of stimulation following training. (3) F$-NESB. Animals were given sham stimulation following training. (4) NFS-90. Animals did not receive footshock but received 90 sec of stimulation within 5 I 0 sec of entering the large compartment. Following histological verification there were a total of 9 animals in Group FS-5, 10 in Group FS-90, I 0 in Group FS-NESB, and 9 in Group NFS-90. RESULTS Histological
Electrode placements were confined to the dorsal hippocampal formation and were similar to those presented in Fig. 1. As in Experiments 1 and 2 no relationship could be found between electrode tip localization within the dorsal hippocampal formation and the magnitude of the retention test score.
200180i-.
IA/ I-U) z
I0050.
"
FS-NEgll
Behavioral
Three animals showed signs of behavioral seizures to post-conditioning stimulation and were not given retention tests. All other animals showed typical head-shaking and hyperactivity to stimulation. The retention test results are presented in Fig. 5. Analysis of variance of retention test STL's revealed an overall main treatment effect (H = 47.6; d f = 3; p<0.001). Between group analyses by a two-tailed Mann-Whitney U test revealed no significant difference in retention test STL's between Groups FS-5 and FS-NESB (p>0.1). This indicated that 5 see of post-training hippo-
1:1-6
F|-O0
NPS-90
FIG. 5. Median training day and retention test lateneies for groups in Experiment 3 receiving post-trial stimulation at one-haif behavioral seizure current levels. campal stimulation at current levels one-half overt seizure threshold had no effect on retention. Group FS-90 differed significantly from both Group FS-5 (p0.1; Wilcoxon matchedpairs signed-ranks test). These results indicated that the intense stimulating current levels were not aversive.
Electrophysiological
Threshold current levels necessary to produce behavioral seizure responses ranged from 4 0 0 - 4 2 0 0 uA with a mean of 2 4 1 0 u A . Animals received a mean of 24 stimulus presentations before behavioral seizure responses were observed. All animals in Group FS-5 demonstrated the typical PAD, PID and SAD activity observed in Experiments 1 and 2. As in Experiment 2, since recordings were not taken for the first 90 see in Groups FS-90 and NFS-90, no comparisons o f hippocampal seizure activity between these groups and Group FS-5 were made.
i
EXPERIMENT 4 Taken together, the results of Experiments 1 - 3 indicate that a current level far suprathreshold for the production Of hippocampal seizure AD activity is a necessary condition for the production of retention deficits. The question arises, however, as to whether or not retention deficits could have been obtained with lower stimulation current levels if a lower reinforcement magnitude had been used. It has been reported [ 12] that the retention deficits produced by electroconvulsive shock vary directly as a function of reinforcement magnitude; the weaker the aversive footshock, the greater the retention deficit. Experiment 4, therefore, was designed to determine whether retention deficits could be produced by stimulation current levels similar to those used in Experiment 1 when using a footshock intensity (0.8 mA) considerably weaker than that used in Experiment 1. METHOD Animals
Experimentally naive Spragne-Dawley male rats weighing 2 7 5 - 3 2 5 g at the time of surgery were used.
HIPPOCAMPAL STIMULATION PARAMETERS AND MEMORY
53 EXPERIMENT 5
Procedure Surgical, hippocampal seizure threshold determination, conditioning, histological and retention test procedures were the same as those described for Experiment I. One group of animals (FS-5) was given 5 sec of hippocampal stimulation at 120% of afterdischarge threshold within 5 - 1 0 sec of conditioning, and a second group (FS-NESB) given sham stimulation following training. A third group (FS-C) served as an unoperated, conditioning control group. Following histological verification there were 8 animals in Group FS-5 and 9 in Group FS-NESB. Group FS-C consisted o f 9 animals. RESULTS
Histological Electrode placements were confined to the dorsal hippocampal formation. Again, no relationship was found between tip localization and the magnitude of the retention test score.
One possible explanation for the lack of retention deficits observed with lower intensity seizure-producing current levels applied to the dorsal hippocampus is that the seizure activity remained relatively localized to the dorsal area. The possibility exists that a necessary condition for the production of retention deficits is the spread of seizure activity throughout the entire hippocampal formation, including both dorsal and ventral areas. In Experiments 1 - 4 recordings were taken only from the dorsal hippocampal areas following stimulation and the extent of spread into the ventral areas was not determined. Experiment 5 was designed to determine the extent to which a current level insufficient for the production of retention deficits, but sufficient to produce seizure activity when applied to the dorsal hippocampus, produced a spread of seizures to the ventral area of the hippocampus. METHOD
Animals Six experimentally naive Sprague-Dawley male rats weighing 2 5 0 - 3 2 5 g at the time of surgery were used.
Electrophysiological Current levels sufficient to produce hippocampal afterdischarges ranged from 2 0 - 1 I 0 g A with a mean of 59 #A. Animals received a mean of 20 stimulus presentations before reaching seizure AD threshold. All animals included in Experiment 4 which received hippocampal stimulation demonstrated the typical seizure activity described in Experiment 1 following post-training stimulation.
Behavioral The behavioral results are presented in Fig. 6. Analyses of retention test scores by two-tailed Matin-Whitney U tests revealed no significant differences in retention test scores among the 3 groups. This result indicates that the lack of a retention deficit observed in Experiment 1 with current levels just suprathreshold for the production of hippocampal seizure AD activity does n o t appear to be a function o f reinforcement magnitude, at least for the FS levels used in the present series o f experiments.
Ill
TIUUNINGDAY SK III[TEIITNNI MY STL
Procedure All animals were surgically prepared with bipolar electrodes implanted into the dorsal hippocampal formation using the same coordinates and methods described in Experiment 1. Two additional bipolar electrodes were implanted into the ventral area of the hippocampus at the following coordinates of Pellegrino and Cushman [ 1 0 ] : 4.8 turn posterior to bregma, 5.0 mm lateral to the midline and 5.0 mm below dura. Approximately two weeks following surgery, the animals were placed in the recording and stimulation chamber and were stimulated in the dorsal hippocampus with a 100 pps, 0.3 msec, 5 sec train of biphasic square waves. Following stimulus termination bilateral recordings were taken from both dorsal and ventral areas. A constant intensity of 200 ~A was administered to all animals. F r o m the results of Experiments 1 - 4 , this intensity was greater than that required to produce dorsal hippocampal seizure activity but insufficient to produce retention deficits. Following the experiment all animals were perfused and verification o f electrode implantation sites made according to the method described in Experiment 1. RESULTS
|
Hippocampal seizure AD activity was recorded bilaterally from all animals in both dorsal and ventral hippocampal leads. A typical record is shown in Fig. 7. Seizure patterns were similar to those observed in the previous experiments. Both PAD and SAD activity were observed in all leads. These results suggest that seizure activity produced by current levels insufficient to produce retention deficits when applied to the dorsal hippocampus spreads into the ventral areas o f the hippocampus.
7 L I m
50-
IrS-C
FS-li[Sll
F$-S
FIG. 6. Median Uaining day and retention test latencies for groups in Experiment 4. Footshock intensity was 0.8 mA. Brain stimulation intensity was 120% hippocampal seizure threshold current levels.
DISCUSSION The results o f the present series of experiments, in which current intensity and reinforcement magnitude were varied, suggest that current levels just exceeding the threshold for dorsal hippocampal primary and secondary seizure after-
54
KAPP, KAUFMAN AND REPOLE ,,
lull
!
; ~':',CFr'r-'-'J
'!
' ':';.)!~-
O-
'1
till
~-r-'r-
f.f
),
I
,-~ ~
w~--- ~",--~-~,,~,~,~,~&..'X
. - ~ ' ~,- - . . . . . . .
tIHI , )~
i
)
~ ' '
[
IItl
Im
FIG. 7. Primary afterdischarge activity recorded bilaterally from dorsal and ventral hippocampal areas. RDH and LDH = right and left dorsal hippocampal recordings: RVH and LVH = right and left ventral hippocampal recordings. discharge activity do not produce retention deficits for one trial passive avoidance learning. Furthermore, current levels far in excess of those necessary for the production of seizure activity were required to produce retention deficits at the reinforcement magnitudes used. It is of interest that the results of Experiment 3 suggest that the duration of applied current was a critical factor in producing a retention deficit. Whether or not the longer duration of applied current produced seizure patterns different from the shorter duration current was not determined due to the inability to record during the former. The results of the present series of experiments appear to be at variance with several previous reports suggesting that retention deficits for passive avoidance conditioning are produced by current levels either subthreshold [ 19] or just suprathreshold [18] for the production of hippocampal seizure AD activity. Wyers e t al. [ 19] have reported that single pulse bilateral stimulation of the ventral hippocampus, at a current level subthreshold for seizure AD activity, produces substantial retention deficits for one-trial passive avoidance conditioning in which a very intense footshock was used. Since our electrodes were located in the dorsal hippocampus, we implanted electrodes into the ventral hippocampus in one additional group of animals (n = 8). No retention deficits were found with current levels of 300% afterdischarge threshold administered for 90 sec. The disparity between our results and those of Wyers e t al. [ 19] is u n k n o w n and may well be due to differential task parameters. Our results are also at variance with those of Vardaris and Schwartz [ 18] who reported that dorsal hippocampal stimulation at 120% of seizure threshold current levels
produced retention deficits. They used a maximum 3.0 sec training-stimulation interval compared with a maximum I0.0 sec interval used in the present experiments. Additional studies in our laboratory (manuscript in preparation), however, have demonstrated that stimulation within 1.0 sec of training does not result in retention deficits with suprathreshold seizure-producing current levels. It is difficult to explain the difference in results between the Vardaris and Schwartz [18] study and those of the present study since electrode placement, behavioral procedures and reinforcement magnitudes were similar. Our results are in essential agreement with those reported by Shinkman and Kaufman [14] in which hippocampal seizure threshold current levels administered to the dorsal hippocampus immediately after each trial during the acquisition phase of a four trial CER task failed to produce a subsequent retention deficit. The lack of a retention deficit observed by Shinkman and Kaufman [ 14], however, may have been due to their use of a 4 trial paradigm. Kesner and McDonough [5] have observed that an ECS treatment which is effective in causing retrograde amnesia after one-trial conditioning loses its effect on retention when given after each trial of a multiple trial conditioning paradigm. They propose that some information of training may survive each ECS treatment and summate to produce a functionally adequate memory of the training experience. The same interpretation may apply for hippocampal stimulation as an amnesic agent and, in the case of the Shinkman and Kaufman [ 14] study, may explain the lack of a retention deficit with hippocampal seizure threshold current levels. The results of the present series of experiments are
HIPPOCAMPAL STIMULATION PARAMETERS AND MEMORY not subject to this interpretation since a one trial, one stimulation paradigm was used. The results of the present study suggest that current levels just suprathreshold for the production of dorsal hippocampal seizure activity are not a sufficient condition for the production of a retention deficit. Zornetzer et al. [21] have recently reported the effects of both subthreshold (80%) and suprathreshold (125%) seizure current levels applied to the dorsal hippocampus of mice on the retention of one-trial passive avoidance conditioning. They found that mice in which both electrode tips were located in the dentate area demonstrated retention deficits at both current levels, whereas those mice with bilaterally asymmetrical placements within the extradentate hippocampal formation did not show retention deficits. Histological examination of the electrode placements in the present series of experiments revealed no relationship between placement and magnitude of the retention test score. Furthermore, animals in which both electrode tips were located in the dentate area demonstrated no consistent deficits when compared to animals with bilaterally asymmetrical placements within the extradentate regions of the dorsal hippocampal formation. In addition, the results of Experiment 4 suggest that current levels which produce seizure activity, but not retention deficits, produce a spread of seizure activity throughout the hippocampal formation
55
which would more than likely spread into the dentate area from extradentate hippocampal placements, thus producing disruption of normal electrical activity in the dentate area. It must be noted however, as Zornetzer et al. [21 ] have suggested, that the susceptibility of memory to disruption may be a function of the distinction between abnormal neuroelectrical activity initiated in rather than propagating to a particular structure. Whether or not this distinction may account for the inability to produce retention deficits with seizure-producing current levels in the rat hippocampus will depend upon further experimentation. In conclusion, the results of the present series of experiments suggest that current levels far in excess of hippocampal seizure threshold current levels are a necessary condition for the production of retention deficits. Furthermore, current levels insufficient for the production of retention deficits produce seizure activity which spreads throughout the hippocampus. This seizure activity has been observed to propagate to extrahippocampal structures (Kapp, unpublished observations). It is therefore difficult at the present time to conclude that abnormal electrical activity in the hippocampal formation is responsible for the retention deficits for one-trial passive avoidance conditioning since other structures may be equally affected by the extrahippocampal propagation of seizure activity.
REFERENCES 1. Barcik, J. D. Hippocampal afterdischarges and conditioned emotional wspmue. Psychon. Sc/. 20: 297-299, 1970. 2. Goddard, G. V. Amygdaloid stimulation and learning in the rat. J. comp. physiol. PSycho£ 58: 23-30, 1964. 3. Gold, P. E. and J. L. McGaugh. Retrograde amnesia produced by unilateral and bilateral subseizure stimulation of the amygdala. Paper presented at Society for Neuroscience, San Diego, Cal., November, 1973. 4. Kemer, R. P. and R. W. Doty. Amnesia produced in cats by local seizure activity initiated from the amygdala. Expl Neurol. 21: 58-68, 1968. 5. Kesner, R. P. and J. H. McDonough, Jr. Diminished amnesic effect of a second electroconvulsive seizure. Expl Neurol. 27: 527-533, 1970. 6. Kemer, R. P. and H. S. Conner. Independence of short and long term memory: A neural system analysis. Science 176: 432-434, 1972. 7. Lidsky, A. and B. M. Slotnick. Effects of posttrial limbic stimulation on retention of a one-trial passive avoidance response. J. comp. physioL Psychol. 76: 337-348, 1971. 8. McDonough, J. H. and R. P. Kesner. Amnesia produced by brief electrical stimulation of amygdala or dorsal hippocampus in cats..I, comp. physiol. PsychoL 77: 171-178, 1971. 9. Peeke, H. V. S. and M. J. Herz. Caudate nucleus stimulation retroactively impairs complex maze learning in the rat. Science 173: 80-82, 1971. 10. Pellergrino, L. J. and A. J. Cushman. A Stereotaxie Atlas of the Rat Brain. New York: Appleton-Century-Crofts, 1967. 11. Quartermain, D., R. M. Paolino and N. E. Miller. A brief temporal gradient of retrograde amnesia independent of situational change. Science 149: 1116-1118, 1965. 12. Ray, O. and L. Bivens. Reinforcement magnitude as a determinant of performance decrement after electroconvulsive shock. Science 160: 330-332, 1968.
13. Schneider, A. M., B. S. Kapp, C. Aron and M. E. Jarvik. Retroactive effects of transcomeai and transpinnate ECS on stepthrough latencies of mice and rats. J. comp. physiol. Psychol. 69: 506-509, 1969. 14. Shinkman, P. G. and K. P. Kaufman. Tune course of retroactive effects of hippocampal stimulation on learning. Expl Neurol. 34: 476-483, 1972. 15. Shinkman, P. G. and K. P. Kaufman. Posttrial hippocampal stimulation and CER acqui~tion nq the rat. J. comp. physiol. Psycho/. 80: 283-292, 1972. 16. Siegal, S. Nonl~arametric Statistics for the Behavioral Sciences. New York: McGraw-Hill, 1956. 17. Thompson, R. Introducing subcortical lesions by electrolytic methods. In: Methods in Psychobiology, edited by R. D. Meyers. New York: Academic Press, 1971, pp. 131-154. 18. Vardaris, R. M. and K. E. Schwartz. Retrograde amnesia for passive avoidance produced by stimulation of dorsal hippocampus.Physio/. Behav. 6: 131-135, 1971. 19. Wyers,E. J., H. V. S. Peeke, J. S. WiIlistonand M. J. Herz. Retroactive impairment of pasmve avoidance learning by stimulation of the caudate nuclenLExpi Neurol. 22: 350-366, 1968. 20. Wyers, E. J. and S. A. Deadwyler. Duration and nature of retrograde amnesia produced by stimulation of the caudate nucleus. Physiol. Behav. 6: 97-103, 1971. 21. Zometzer, S. F., R. B. Chronister and B. Ross. The hippocampus and retrograde amnesia: Localization of some positive and negative memory disruptive sites. Behav. Biol. 8: 507-518, 1973. 22. Zometzer, S. F. and R. B. Chtonister. Neuroanatomical localization of memory disruption: Relationship between brain structure and learning task. Physiol. Behav. 10: 747-750, 1973.
Hippocampal Stimulation and Memory: Effects of Stimulation Parameters and Reinforcement Magnitude' BRUCE S. KAPP, J E F F R E Y D. K A U F M A N AND DENNIS A. REPOLE
Department o f Psychology, University o f Vermont, Burlington, Vermont 05401
(Received 14 December. 1973)
KAPP, B. S., J. D. KAUFMAN AND D. A. REPOLE. Hippocampal stimulation and memory: effects of stimulation parameters and reinforcement magnitude. PHYSIOL. BEHAV. 13(1) 47-55, 1974. - The present study investigated the extent to which post-conditioning stimulation-induced hippocampal seizure activity is a necessary condition for the production of retrograde amnesia in rats. Groups of animals received dorsal hippocampal stimulation at several different current intensities immediately following one-trial passive avoidance conditioning. The results suggest that current intensifies far in excess of those which produce hippocampal seizure activity are a necesssry condition for the production of retrograde amnesia. Reinforcement magnitude is not a significant variable at hippocampal seizure threshold current intensities. Current intensities insufficient for the production of retrograde amnesia, but sufficient for the production of donml hippocampal seizure activity, produced a spread of seizure activity into the ventral hippocampal formation. The results suggest that caution be exercised in attributing a role to the hippocampus in memory consolidation processes in the rat. Memory consolidation
Passive avoidance conditioning
Hippocampal stimulation
THE CONTRIBUTION of specific neuroanatomical structures to memory consolidation processes has recently been investigated by using post-conditioning, low-level, intracranial electrical stimulation. The results have indicated that several subcortical structures including the caudate nucleus [9, 19, 20, 22], the amygdaloid complex [2, 3, 4, 7, 8], and particularly the hippocampal formation [ I , 6, 7, 8, 14, 15, 18, 21] may be involved in the consolidation o f newly acquired information. Stimulation of any one of the three structures immediately after conditioning has been reported to result in retention deficits 24 hr following stimulation in rats and mice. In any a t t e m p t to demonstrate the involvement of specific neuroanatomical sites in consolidation processes by using stimulation techniques i t becomes critical to define the extent to which the applied current produces abnormal high amplitude seizure activity which may propagate to structures other than the one in question. As a first step, therefore, it would seem desirable to use current levels which produce reliable retention deficits but which produce a minimal amount of abnormal neuroelectrical seizure activity in an a t t e m p t to confine this activity to the structure under investigation. Whether or not the production of seizure activity in the hippocampus is a necessary condition for the production of
Hippocampal seizure activity
retrograde amnesia is still uncertain. Wyers, Peeke, Wiili~ton and Herz [ 19] have reported that current levels subthreshold for the production o f hippocampal seizure activity were effective in producing retrograde amnesia in rats for onetrial l~assive avoidance conditioning. McDonough and Kesner [8] have reported similar results for the cat. On the other hand, Shinkman and Kaufman [14] have reported that current levels just exceeding hippocampal seizure threshold administered to rats after each of four daily trials in a CER paradigm have no effect on retention. Their results, however, differ from those o f Vardaris and Schwartz [18] who report retention deficits for one-trial passive avoidance using current levels just suprathreshold for the production of hippocampal seizure activity. Several variables may contribute to the inconsistent results, including species differences and differences in conditioning parameters (reinforcement magnitude, onetrial vs. multiple-trial paradigms). It has been reported, for example, that the retention deficits produced by another treatment, electroconvulsive shock (ECS), vary directly with species [13] and reinforcement magnitude [12]. It becomes essential, therefore, in attempting to define current levels which produce reliable retention deficits while producing minimal amounts of abnormal hippocampal neuroelectrical activity to (a) manipulate current
J This research was supported by research Grants MH-22404-01 from the National Institute of Mental Health and NSF-1972-128 from the Institutional Grants Committee of the University of Vermont. 47
48
KAPP, KAUFMAN AND REPOLE
intensity while maintaining constant conditioning parameters and (b) manipulate reinforcement magnitude while maintaining constant current stimulation parameters within a single species. The purpose of the present investigation was (a) to determine the minimal current intensity that, when applied to the dorsal hippocampal formation of rats immediately following one-trial passive avoidance conditioning, will produce reliable retention deficits and (b) to determine if reinforcement magnitude is a significant variable in defining the minimal current intensity needed to produce retention deficits.
EXPERIMENT 1 Experiment 1 was designed to determine if hippocampal seizure afterdischarge activity is a necessary condition for producing retention deficits for one-trial passive avoidance in rats. Current levels just suprathreshold for the production of hippocampal seizure activity were used. METHOD
A nimals Experimentally naive Sprague-Dawley male rats weighing 2 7 5 - 3 2 5 g at the time of surgery were used. The rats were maintained on a 12 hr light-dark cycle, given ad lib food and water and were group housed until surgery at which time they were placed in separate cages.
Surgical Procedures All animals with the exception of an unoperated control group were surgically prepared under Nembutal anesthesia (50 mg/ml [P) with bipolar electrodes constructed from 0.25 mm in diameter stainless steel wire, soldered to A m p h e n o l Subminax connectors, and insulated with Formvar except at the cross-sectional tip. Tip separation was 0.5 mm and the electrodes were implanted bilaterally into the dorsal hippocampal formation using the coordinates of Pellegrino and Cushman [ 10] : 2.5 mm posterior to bregrna, 2.5 mm lateral to the midline and 3.5 mm below dura. Immediately following surgery intramuscular injections of Wyeillin (30,000 units, Wyeth) were given.
A ppara tus The training apparatus was similar to the one used by Schneider, Kapp, Aron and Jarvik [13], constructed of Plexigias and enlarged to accomodate rats (64.0 x 28.0 x 6.0 cm). It consisted of a small compartment constructed of clear Plexigias connected to a larger black compartment by a 28.0 x 6.0 cm opening. The floor of each compartment consisted of pairs of parallel stainless-steel plates, one pair (16.0 x 2.5 cm) in the smaller and two pairs (23.0 x 2.5 cm) in the larger compartment. Adjacent plates were spaced 1.25 cm apart. The apparatus was trough-shaped with the width of each compartment increasing from a 6.0 cm base to a 21.0 cm top. The top of each compartment was covered by a hinged lid. The floor plates of the large black compartment were connected to a Grason-Stadler shock scrambler set to deliver a 2.0 sec footshock (FS). The stimulation and recording apparatus consisted of a second box (23.0 x 19.0 x 29.0 cm) constructed of Plexigias with a grid floor of stainless steel rods 0.25 cm in
diameter, spaced 1.0 cm apart. Attached to the top was a Scientific Prototype mercury commutator from which extended the leads used for stimulation and recording.
Procedure Hippocampal seizure threshold determination. In order to reliably produce post-conditioning hippocampal seizure activity in each animal, individual current thresholds suffid e n t to produce hippocampal seizure activity were determined. Following 7 - 1 0 days of post-operative recovery, each animal was placed in the recording apparatus and a 5 sec train of biphasic square waves of 100 pps with a pulse duration of 0.3 msec was applied to the dorsal hippocampal formation. Stimulation was given bilaterally using two constant current stimulators isolated from ground. Current intensity was increased in 3 uA increments every 3 min until hippocampal seizure activity was recorded on a 6-channel E&M Physiograph Six. Current intensity was monitored using a dual-beam oscilloscope by monitoring the voltage drop across a 10 Kohm resistor placed in the stimulation circuit in series with the animals. The intensity used for post-conditioning stimulation was 120% of the seizure threshold current intensity. Since hippocampal seizure threshold current levels differ from animal to animal, the above method was chosen to assure that all animals received equal amounts of post-conditioning current relative to their individual threshold current levels. Conditioning procedure. The conditioning procedure was a one-trial passive avoidance task commonly used to study both the effects of electroconvulsive shock [ 11,13 ] and hippocampal stimulation [7, 18, 21] on memory consolidation. One week following hippocampal seizure threshold testing all animals were given an habituation session in the training apparatus. After connecting the electrode leads, the animal was placed into the small compartment facing away from the large compartment and allowed to step into the large compartment for 10 sec of exploration. Twentyfour hr later the animal, with electrode leads attached, was again placed into the small compartment and a timer was started. When all four paws entered the large compartment, a guillotine door between the two compartments was closed, the timer stopped and a 1.6 mA, 2.0 sec FS was given. During FS the animal was disconnected from the stimulation and recording circuitry in order to prevent artifactual brain stimulation by crosstalk between peripheral FS and stimulation electrode leads. All animals were then immediately removed from the apparatus and placed in the stimulation and recording chamber. Electrode leads were connected to the stimulation-recording circuitry and the designated post-training manipulation was given. The circuitry was programmed so that hippocampal electrical activity was recorded from the site of stimulation 0.1 sec following the termination of the stimulus train. The FSstimulation interval ranged from 5 to 10 sec. Following stimulation and recording all animals were returned to their home cages. Post-training manipulation. Animals were assigned to one of four groups. (I) FS-C. Animals in this group were unoperated controls and received FS upon entering the large compartment. They were subject to the same handling procedure and length of time in the stimulation-recording apparatus as the other operated groups. This group was included to determine whether the lesions produced by hippocampal electrode implantation had any effect on re-
HIPPOCAMPAL STIMULATION PARAMETERS AND MEMORY tention. (2) FS-NESB. Animals received FS upon entering the large compartment followed by sham hippocampal stimulation. (3) FS-ESB. Animals received FS followed within 5 - 1 0 sec by hippocampal stimulation at 120% o f hippocampal seizure threshold current for 5.0sec. (4) NFS-ESB. Animals did not receive FS and were stimulated at 120% of hippocampal seizure threshold within 5 - 1 0 sec of entering the large compartment. This group was included to assess the aversive properties of the stimulation. Retention test procedure. Twenty-four hr following training all animals, with electrode leads attached (Group FS=C with no leads), were returned to the small compartment and step-through latencies (STL's) were taken. An increased STL compared with training STL indicated retention for the training experience. If the animal did not enter the large compartment within 300 sec, the test was terminated. Histology. After behavioral testing all animals were perfused with physiological saline followed by 10% formalsaline. The brains were removed, sliced into 40 u sections with a freezing rnlcrotome and examined for verification of implantation sites using the method of Thompson [ 17] for unstained sections with the aid of the Pellegrino and Cushman [ 10] brain atlas. RESULTS
Histological Only those animals with confirmed bilateral electrode placements within the dorsal hippocampai formation were
49
included in the data analysis. This resulted in a total of 9 animals in Group FS-NESB, 17 in Group FS-ESB and 10 in Group NFS-ESB. Group FS-C consisted of 9 animals. Electrode placements for Group FS-ESB are reconstructed in Fig. 1. No relationship was found between electrode placement within the dorsal hippocampal formation and the magnitude of retention test step-through latency.
Electrophysiological Threshold current levels sufficient to produce hippocampal seizure afterdischarge (AD) activity ranged from 3 2 - 1 6 0 u A with a mean of 76t~A. Animals received a mean of 25 stimulus presentations before reaching seizure AD threshold. Typical post-conditioning hippocampal seizure AD activity is shown in Fig. 2. F o u r animals in the FS-ESB group and one animal in the NFS-ESB group showed no signs of post-conditioning hippocampal seizure AD activity and were excluded from the behavioral data analysis, reducing the number of animals in Groups FS-ESB and NFS-ESB to 13 and 9, respectively. All animals showing signs of hippocampal AD activity with the exception of one animal in Group FS-ESB and one in Group NFS-ESB showed both primary afterdischarge (PAD) and secondary afterdischarge (SAD) activity with a period of post-ictal depression (PID) following the primary and preceding the s e c o n d a r y afterdischarge period. The two exceptions showed PAD activity with little or no post-ictal depression and no SAD activity. The mean duration o f the PAD was 15 sec, that of the post-ictal depression period 60 sec,
FIG. 1. Bilateral electrode placements for Group FS-ESB of Experiment 1. Each identical pair of letters represents bilateral placements in one animal.
50
KAPP, KAUFMAN AND REPOLE
2
'l,~,
'a'Iv~fi~~~''4/v~'vvwe'~''~'~N'~'~/~'~'~x'~'`''
,
'~"
'''
3
.
.
.
.
.
.
.
.
5
8
!
9 10 •- - J 100 uV 1 SEC FIG. 2. Typical bilateral dorsal hippocampal seizure activity recorded immediately following stimulus termination. Current level was 120% (73/~A) of hippocampal seizure threshold. Traces I and 2; base level hippocampal activity recorded one week prior to stimulation. Traces 3 and 4; primary afterdischarge activity (PAD) recorded immediately following stimulus termination. Traces 5 and 6; secondary afterdischarge (SAD) recorded approximately 45 sec following PAD. Traces 7 and 8; continuation of Traces 5 and 6. Traces 9 and 10; activity recorded 5 rain following stimulus termination. and that of the SAD activity 19 sec. No differences in posttraining AD characteristics were observed between Groups FS-ESB and NFS-ESB. Normal hippocampal activity returned from 5 to lO rain following the SAD period. Behavioral observations indicated that animals showing signs of hippocampal seizure activity generally demonstrated simultaneous headshaking and exploratory activity although none showed signs of behavioral clonus of convulsive activity. Behavioral
The retention test results are presented in Fig. 3. The 5 animals that showed no AD activity are excluded from the data analysis. A Kruskal-WaUis [16) analysis of variance revealed no significant differences in training day STL's between groups (H -- 2.5; d r = 3; p>0.1). Analysis of variance of retention test latencies revealed an overall main treatment effect (H = 55.6; d f = 3; p
that there were no significant differences between retention test latencies of Groups FS-ESB, FS-NESB and FS-C (p's>0.1). This indicated that neither post-training hippocampal stimulation at 120% of afterdischarge threshold, implant-produced lesions, nor pretraining afterdischarge determination had any significant effect on retention. Retention test STL's for Group NFS-ESB, however, did significantly differ from those of Groups FS-ESB, FS-NESB and FS-C (p's<0.001) but they did not significantly differ from their respective training day STL's (p> 0.1; Wilcoxon matched-pairs signed-ranks test). This indicated that the long retention test STL's in Group FS-ESB were not a result o f an aversive property of immediate post-conditioning stimulation. The four animals of Group FS-ESB in which post-conditioning stimulation produced no signs of h~ppocampal AD activity had a median retention test score of 300 sec and did not differ significantly from Groups FS-ESB, FS-NESB, or FS-C. The data from these 4 animals suggested that current .levels subthreshold for the produ¢-
HIPPOCAMPAL STIMULATION PARAMETERS AND MEMORY C ~ TRAINING DAY $ T L lib RETENTION D A Y STL
U g
z
lu
I_J .r
o o
I[ .r I-" 40 7
51
Electrophysiological Threshold current levels sufficient to produce hippocampal AD activity ranged from 2 3 - 9 3 ~A with a mean of 64 uA. Animals received a mean of 21 stimulus presentations before reaching seizure AD threshold. All animals in Group FS-5 showed PAD activity, post-ictal depression and SAD activity. Since recordings could not be taken for the first 90 sec in Group FS-90, no comparisons of the AD activity between this group and Group FS-5 were made. Seizure AD activity was occasionally recorded following the 90 sec o f stimulation and was followed by periods of postictal depression and subsequent recovery. Headshaking and hyperactivity were again observed during the abnormal EEG activity, but no signs of behavioral seizures were noticeable.
Behavioral It'll-C
PII-.Nt|D
PS.4ESD HI~EIO
FIG. 3. Median training day and retention test latencies for groups in Experiment 1 receiving post-trial stimulation at 120% hippocampal seizure threshold current levels. tion of hippocampal AD activity produced no effect on retention. EXPERIMENT 2 The results of Experiment 1 indicated that dorsal hippocampal stimulation just suprathreshold for the production of hippocampal seizure AD activity did not produce retention deficits at the conditioning and current parameters used. Experiment 2 was designed, therefore, in an a t t e m p t to produce retention deficits by increasing the current intensity and duration while maintaining constant conditioning parameters.
The behavioral results are presented in Fig. 4. A KruskalWallls analysis o f variance of retention test STL's revealed no main treatment effects (H = 2.1;dfffi 2; p>0.1), indicating that neither 5 nor 90 sec o f post-conditioning stimulation at 300% o f seizure AD threshold produced retention deficits. Although no control group was included in Experiment 2 to assess the aversive properties of 300% stimulation, a control group included in the following experiment (Experiment 3) appears to exclude the possibility that the lack of a retention deficit is due to the aversive properties of more intense current levels.
]
r-~ TRAMINO DAY STL ga RETENTION DAY STL
v
METHOD
Animals Experimentally naive Spragne-Dawley male rats weighing 2 7 5 - 3 2 5 g at the time of surgery were used.
Procedure Surgical, hippocampal seizure threshold determination, conditioning, histological and retention test procedures were the same as those described in Experiment I. The animals were divided into 3 groups, one group given 5 sec of immediate post-conditioning hippocampal stimulation (FS-5) at current levels 300% of seizure AD threshold, a second group given 9 0 s e c of post-conditionin~g hippocampal stimulation (FS-90) at current levels 300% of seizure AD threshold, and a third group which was trained but received sham ESB (FS-NESB). Following histological verification there were a total of 9 animals in Group FS-5, 9 animals in Group FS-90 and 6 animals in Group FS-NESB. RESULTS
Histological Electrode placements were confined to the dorsal hippocampal formation and were similar to those presented in Fig. 1. No relationship was found between electrode placement and the magnitude o f the retention test latency.
Fs-mKn
ms
Fs4o
FIG. 4. Median Ualning day and retention test latencies for groups in Experiment 2 receiving post-U-ial stimulation at 300~ hippocampal seizure threshold current levels. EXPERIMENT 3 The results of Experiment 2 indicated that current levels considerably suprathreshold for the production of hippocampal seizure AD activity were not sufficient to produce retention deficits for the one-trial passive avoidance task used. Experiment 3, therefore, was designed to determine whether or not very intense stimulation would produce a retention deficit at the conditioning parameters used in Experiments 1 and 2.
52
KAPP, KAUFMAN AND REPOLE METHOD
r-~ 1
Animals
Experimentally naive Sprague-Dawley male rats weighing 2 7 5 - 3 2 5 g at the time of surgery were used.
T R ~ ~ ~
DAy S T L DAY STL
300. z
2SO-
Procedure
,.J
Surgical, conditioning, histological and retention test procedures were the same as those described for Experiment 1. Hippoeampal seizure threshold determination, however, differed from that used in the previous experiments. In the present experiment, current intensity was increased in 100 u A increments every 3 rain until signs of an overt behavioral seizure were observed. These seizure responses were characterized by withdrawal responses and wild running followed by clonus and salivation. The current intensity used for post-conditioning stimulation was onehalf that required to produce an overt seizure response. One week following seizure threshold determination the animals were assigned to one of 4 groups. (1) FS-5. Animals were given 5 see of stimulation following training. (2) FS-90. AnimaLs were given 90 sec of stimulation following training. (3) F$-NESB. Animals were given sham stimulation following training. (4) NFS-90. Animals did not receive footshock but received 90 sec of stimulation within 5 I 0 sec of entering the large compartment. Following histological verification there were a total of 9 animals in Group FS-5, 10 in Group FS-90, I 0 in Group FS-NESB, and 9 in Group NFS-90. RESULTS Histological
Electrode placements were confined to the dorsal hippocampal formation and were similar to those presented in Fig. 1. As in Experiments 1 and 2 no relationship could be found between electrode tip localization within the dorsal hippocampal formation and the magnitude of the retention test score.
200180i-.
IA/ I-U) z
I0050.
"
FS-NEgll
Behavioral
Three animals showed signs of behavioral seizures to post-conditioning stimulation and were not given retention tests. All other animals showed typical head-shaking and hyperactivity to stimulation. The retention test results are presented in Fig. 5. Analysis of variance of retention test STL's revealed an overall main treatment effect (H = 47.6; d f = 3; p<0.001). Between group analyses by a two-tailed Mann-Whitney U test revealed no significant difference in retention test STL's between Groups FS-5 and FS-NESB (p>0.1). This indicated that 5 see of post-training hippo-
1:1-6
F|-O0
NPS-90
FIG. 5. Median training day and retention test lateneies for groups in Experiment 3 receiving post-trial stimulation at one-haif behavioral seizure current levels. campal stimulation at current levels one-half overt seizure threshold had no effect on retention. Group FS-90 differed significantly from both Group FS-5 (p
Electrophysiological
Threshold current levels necessary to produce behavioral seizure responses ranged from 4 0 0 - 4 2 0 0 uA with a mean of 2 4 1 0 u A . Animals received a mean of 24 stimulus presentations before behavioral seizure responses were observed. All animals in Group FS-5 demonstrated the typical PAD, PID and SAD activity observed in Experiments 1 and 2. As in Experiment 2, since recordings were not taken for the first 90 see in Groups FS-90 and NFS-90, no comparisons o f hippocampal seizure activity between these groups and Group FS-5 were made.
i
EXPERIMENT 4 Taken together, the results of Experiments 1 - 3 indicate that a current level far suprathreshold for the production Of hippocampal seizure AD activity is a necessary condition for the production of retention deficits. The question arises, however, as to whether or not retention deficits could have been obtained with lower stimulation current levels if a lower reinforcement magnitude had been used. It has been reported [ 12] that the retention deficits produced by electroconvulsive shock vary directly as a function of reinforcement magnitude; the weaker the aversive footshock, the greater the retention deficit. Experiment 4, therefore, was designed to determine whether retention deficits could be produced by stimulation current levels similar to those used in Experiment 1 when using a footshock intensity (0.8 mA) considerably weaker than that used in Experiment 1. METHOD Animals
Experimentally naive Spragne-Dawley male rats weighing 2 7 5 - 3 2 5 g at the time of surgery were used.
HIPPOCAMPAL STIMULATION PARAMETERS AND MEMORY
53 EXPERIMENT 5
Procedure Surgical, hippocampal seizure threshold determination, conditioning, histological and retention test procedures were the same as those described for Experiment I. One group of animals (FS-5) was given 5 sec of hippocampal stimulation at 120% of afterdischarge threshold within 5 - 1 0 sec of conditioning, and a second group (FS-NESB) given sham stimulation following training. A third group (FS-C) served as an unoperated, conditioning control group. Following histological verification there were 8 animals in Group FS-5 and 9 in Group FS-NESB. Group FS-C consisted o f 9 animals. RESULTS
Histological Electrode placements were confined to the dorsal hippocampal formation. Again, no relationship was found between tip localization and the magnitude of the retention test score.
One possible explanation for the lack of retention deficits observed with lower intensity seizure-producing current levels applied to the dorsal hippocampus is that the seizure activity remained relatively localized to the dorsal area. The possibility exists that a necessary condition for the production of retention deficits is the spread of seizure activity throughout the entire hippocampal formation, including both dorsal and ventral areas. In Experiments 1 - 4 recordings were taken only from the dorsal hippocampal areas following stimulation and the extent of spread into the ventral areas was not determined. Experiment 5 was designed to determine the extent to which a current level insufficient for the production of retention deficits, but sufficient to produce seizure activity when applied to the dorsal hippocampus, produced a spread of seizures to the ventral area of the hippocampus. METHOD
Animals Six experimentally naive Sprague-Dawley male rats weighing 2 5 0 - 3 2 5 g at the time of surgery were used.
Electrophysiological Current levels sufficient to produce hippocampal afterdischarges ranged from 2 0 - 1 I 0 g A with a mean of 59 #A. Animals received a mean of 20 stimulus presentations before reaching seizure AD threshold. All animals included in Experiment 4 which received hippocampal stimulation demonstrated the typical seizure activity described in Experiment 1 following post-training stimulation.
Behavioral The behavioral results are presented in Fig. 6. Analyses of retention test scores by two-tailed Matin-Whitney U tests revealed no significant differences in retention test scores among the 3 groups. This result indicates that the lack of a retention deficit observed in Experiment 1 with current levels just suprathreshold for the production of hippocampal seizure AD activity does n o t appear to be a function o f reinforcement magnitude, at least for the FS levels used in the present series o f experiments.
Ill
TIUUNINGDAY SK III[TEIITNNI MY STL
Procedure All animals were surgically prepared with bipolar electrodes implanted into the dorsal hippocampal formation using the same coordinates and methods described in Experiment 1. Two additional bipolar electrodes were implanted into the ventral area of the hippocampus at the following coordinates of Pellegrino and Cushman [ 1 0 ] : 4.8 turn posterior to bregma, 5.0 mm lateral to the midline and 5.0 mm below dura. Approximately two weeks following surgery, the animals were placed in the recording and stimulation chamber and were stimulated in the dorsal hippocampus with a 100 pps, 0.3 msec, 5 sec train of biphasic square waves. Following stimulus termination bilateral recordings were taken from both dorsal and ventral areas. A constant intensity of 200 ~A was administered to all animals. F r o m the results of Experiments 1 - 4 , this intensity was greater than that required to produce dorsal hippocampal seizure activity but insufficient to produce retention deficits. Following the experiment all animals were perfused and verification o f electrode implantation sites made according to the method described in Experiment 1. RESULTS
|
Hippocampal seizure AD activity was recorded bilaterally from all animals in both dorsal and ventral hippocampal leads. A typical record is shown in Fig. 7. Seizure patterns were similar to those observed in the previous experiments. Both PAD and SAD activity were observed in all leads. These results suggest that seizure activity produced by current levels insufficient to produce retention deficits when applied to the dorsal hippocampus spreads into the ventral areas o f the hippocampus.
7 L I m
50-
IrS-C
FS-li[Sll
F$-S
FIG. 6. Median Uaining day and retention test latencies for groups in Experiment 4. Footshock intensity was 0.8 mA. Brain stimulation intensity was 120% hippocampal seizure threshold current levels.
DISCUSSION The results o f the present series of experiments, in which current intensity and reinforcement magnitude were varied, suggest that current levels just exceeding the threshold for dorsal hippocampal primary and secondary seizure after-
54
KAPP, KAUFMAN AND REPOLE ,,
lull
!
; ~':',CFr'r-'-'J
'!
' ':';.)!~-
O-
'1
till
~-r-'r-
f.f
),
I
,-~ ~
w~--- ~",--~-~,,~,~,~,~&..'X
. - ~ ' ~,- - . . . . . . .
tIHI , )~
i
)
~ ' '
[
IItl
Im
FIG. 7. Primary afterdischarge activity recorded bilaterally from dorsal and ventral hippocampal areas. RDH and LDH = right and left dorsal hippocampal recordings: RVH and LVH = right and left ventral hippocampal recordings. discharge activity do not produce retention deficits for one trial passive avoidance learning. Furthermore, current levels far in excess of those necessary for the production of seizure activity were required to produce retention deficits at the reinforcement magnitudes used. It is of interest that the results of Experiment 3 suggest that the duration of applied current was a critical factor in producing a retention deficit. Whether or not the longer duration of applied current produced seizure patterns different from the shorter duration current was not determined due to the inability to record during the former. The results of the present series of experiments appear to be at variance with several previous reports suggesting that retention deficits for passive avoidance conditioning are produced by current levels either subthreshold [ 19] or just suprathreshold [18] for the production of hippocampal seizure AD activity. Wyers e t al. [ 19] have reported that single pulse bilateral stimulation of the ventral hippocampus, at a current level subthreshold for seizure AD activity, produces substantial retention deficits for one-trial passive avoidance conditioning in which a very intense footshock was used. Since our electrodes were located in the dorsal hippocampus, we implanted electrodes into the ventral hippocampus in one additional group of animals (n = 8). No retention deficits were found with current levels of 300% afterdischarge threshold administered for 90 sec. The disparity between our results and those of Wyers e t al. [ 19] is u n k n o w n and may well be due to differential task parameters. Our results are also at variance with those of Vardaris and Schwartz [ 18] who reported that dorsal hippocampal stimulation at 120% of seizure threshold current levels
produced retention deficits. They used a maximum 3.0 sec training-stimulation interval compared with a maximum I0.0 sec interval used in the present experiments. Additional studies in our laboratory (manuscript in preparation), however, have demonstrated that stimulation within 1.0 sec of training does not result in retention deficits with suprathreshold seizure-producing current levels. It is difficult to explain the difference in results between the Vardaris and Schwartz [18] study and those of the present study since electrode placement, behavioral procedures and reinforcement magnitudes were similar. Our results are in essential agreement with those reported by Shinkman and Kaufman [14] in which hippocampal seizure threshold current levels administered to the dorsal hippocampus immediately after each trial during the acquisition phase of a four trial CER task failed to produce a subsequent retention deficit. The lack of a retention deficit observed by Shinkman and Kaufman [ 14], however, may have been due to their use of a 4 trial paradigm. Kesner and McDonough [5] have observed that an ECS treatment which is effective in causing retrograde amnesia after one-trial conditioning loses its effect on retention when given after each trial of a multiple trial conditioning paradigm. They propose that some information of training may survive each ECS treatment and summate to produce a functionally adequate memory of the training experience. The same interpretation may apply for hippocampal stimulation as an amnesic agent and, in the case of the Shinkman and Kaufman [ 14] study, may explain the lack of a retention deficit with hippocampal seizure threshold current levels. The results of the present series of experiments are
HIPPOCAMPAL STIMULATION PARAMETERS AND MEMORY not subject to this interpretation since a one trial, one stimulation paradigm was used. The results of the present study suggest that current levels just suprathreshold for the production of dorsal hippocampal seizure activity are not a sufficient condition for the production of a retention deficit. Zornetzer et al. [21] have recently reported the effects of both subthreshold (80%) and suprathreshold (125%) seizure current levels applied to the dorsal hippocampus of mice on the retention of one-trial passive avoidance conditioning. They found that mice in which both electrode tips were located in the dentate area demonstrated retention deficits at both current levels, whereas those mice with bilaterally asymmetrical placements within the extradentate hippocampal formation did not show retention deficits. Histological examination of the electrode placements in the present series of experiments revealed no relationship between placement and magnitude of the retention test score. Furthermore, animals in which both electrode tips were located in the dentate area demonstrated no consistent deficits when compared to animals with bilaterally asymmetrical placements within the extradentate regions of the dorsal hippocampal formation. In addition, the results of Experiment 4 suggest that current levels which produce seizure activity, but not retention deficits, produce a spread of seizure activity throughout the hippocampal formation
55
which would more than likely spread into the dentate area from extradentate hippocampal placements, thus producing disruption of normal electrical activity in the dentate area. It must be noted however, as Zornetzer et al. [21 ] have suggested, that the susceptibility of memory to disruption may be a function of the distinction between abnormal neuroelectrical activity initiated in rather than propagating to a particular structure. Whether or not this distinction may account for the inability to produce retention deficits with seizure-producing current levels in the rat hippocampus will depend upon further experimentation. In conclusion, the results of the present series of experiments suggest that current levels far in excess of hippocampal seizure threshold current levels are a necessary condition for the production of retention deficits. Furthermore, current levels insufficient for the production of retention deficits produce seizure activity which spreads throughout the hippocampus. This seizure activity has been observed to propagate to extrahippocampal structures (Kapp, unpublished observations). It is therefore difficult at the present time to conclude that abnormal electrical activity in the hippocampal formation is responsible for the retention deficits for one-trial passive avoidance conditioning since other structures may be equally affected by the extrahippocampal propagation of seizure activity.
REFERENCES 1. Barcik, J. D. Hippocampal afterdischarges and conditioned emotional wspmue. Psychon. Sc/. 20: 297-299, 1970. 2. Goddard, G. V. Amygdaloid stimulation and learning in the rat. J. comp. physiol. PSycho£ 58: 23-30, 1964. 3. Gold, P. E. and J. L. McGaugh. Retrograde amnesia produced by unilateral and bilateral subseizure stimulation of the amygdala. Paper presented at Society for Neuroscience, San Diego, Cal., November, 1973. 4. Kemer, R. P. and R. W. Doty. Amnesia produced in cats by local seizure activity initiated from the amygdala. Expl Neurol. 21: 58-68, 1968. 5. Kesner, R. P. and J. H. McDonough, Jr. Diminished amnesic effect of a second electroconvulsive seizure. Expl Neurol. 27: 527-533, 1970. 6. Kemer, R. P. and H. S. Conner. Independence of short and long term memory: A neural system analysis. Science 176: 432-434, 1972. 7. Lidsky, A. and B. M. Slotnick. Effects of posttrial limbic stimulation on retention of a one-trial passive avoidance response. J. comp. physioL Psychol. 76: 337-348, 1971. 8. McDonough, J. H. and R. P. Kesner. Amnesia produced by brief electrical stimulation of amygdala or dorsal hippocampus in cats..I, comp. physiol. PsychoL 77: 171-178, 1971. 9. Peeke, H. V. S. and M. J. Herz. Caudate nucleus stimulation retroactively impairs complex maze learning in the rat. Science 173: 80-82, 1971. 10. Pellergrino, L. J. and A. J. Cushman. A Stereotaxie Atlas of the Rat Brain. New York: Appleton-Century-Crofts, 1967. 11. Quartermain, D., R. M. Paolino and N. E. Miller. A brief temporal gradient of retrograde amnesia independent of situational change. Science 149: 1116-1118, 1965. 12. Ray, O. and L. Bivens. Reinforcement magnitude as a determinant of performance decrement after electroconvulsive shock. Science 160: 330-332, 1968.
13. Schneider, A. M., B. S. Kapp, C. Aron and M. E. Jarvik. Retroactive effects of transcomeai and transpinnate ECS on stepthrough latencies of mice and rats. J. comp. physiol. Psychol. 69: 506-509, 1969. 14. Shinkman, P. G. and K. P. Kaufman. Tune course of retroactive effects of hippocampal stimulation on learning. Expl Neurol. 34: 476-483, 1972. 15. Shinkman, P. G. and K. P. Kaufman. Posttrial hippocampal stimulation and CER acqui~tion nq the rat. J. comp. physiol. Psycho/. 80: 283-292, 1972. 16. Siegal, S. Nonl~arametric Statistics for the Behavioral Sciences. New York: McGraw-Hill, 1956. 17. Thompson, R. Introducing subcortical lesions by electrolytic methods. In: Methods in Psychobiology, edited by R. D. Meyers. New York: Academic Press, 1971, pp. 131-154. 18. Vardaris, R. M. and K. E. Schwartz. Retrograde amnesia for passive avoidance produced by stimulation of dorsal hippocampus.Physio/. Behav. 6: 131-135, 1971. 19. Wyers,E. J., H. V. S. Peeke, J. S. WiIlistonand M. J. Herz. Retroactive impairment of pasmve avoidance learning by stimulation of the caudate nuclenLExpi Neurol. 22: 350-366, 1968. 20. Wyers, E. J. and S. A. Deadwyler. Duration and nature of retrograde amnesia produced by stimulation of the caudate nucleus. Physiol. Behav. 6: 97-103, 1971. 21. Zometzer, S. F., R. B. Chronister and B. Ross. The hippocampus and retrograde amnesia: Localization of some positive and negative memory disruptive sites. Behav. Biol. 8: 507-518, 1973. 22. Zometzer, S. F. and R. B. Chtonister. Neuroanatomical localization of memory disruption: Relationship between brain structure and learning task. Physiol. Behav. 10: 747-750, 1973.