Brain Research, 97 (1975) 215-233 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
215
T I M E - D E P E N D E N T C H A N G E S IN C O M M I S S U R A L FIELD POTENTIALS IN T H E D E N T A T E G Y R U S F O L L O W I N G LESIONS OF T H E E N T O R H I N A L CORTEX IN A D U L T RATS
JAMES R. WEST, SAM DEADWYLER, CARL W. COTMAN AND GARY LYNCH
Department of Psychobiology, University of California, Irvine, Calif. 92664 (U.S.A.) (Accepted April 21st, 1975)
SUMMARY
Previous neuroanatomical work has shown that lesions of the entorhinal cortex in adult rats cause the commissural projections to spread from their normally restricted locus in the inner molecular layer approximately 40-50 #m into the outer molecular layer (that is, into the zone deafferented by the lesion). In the present study we measured the effects of the entorhinal lesion on the distribution of shortlatency potentials elicited by commissural stimulation in the molecular layer. Studies with animals tested at various times after the lesion and with a preparation that permitted recording from the same rat at several post-lesion intervals both indicated that the commissural response spread 100-150 # m towards the deafferented outer molecular layer, while the maximum response spread 50-100 #m. These effects were first detectable by 9 days after the lesion and were fully developed by 15 days post-lesion. These findings suggest that the growth of the commissural system seen after entorhinal lesions results in the rapid formation of functional terminals and are discussed in relationship to the behavioral consequences of brain lesions.
INTRODUCTION
In recent years, neuroanatomical studies have provided convincing evidence that if one afferent to a given brain region is removed, the remaining axonal projections to that region will undergo morphological changes which result in reoccupancy of vacated synaptic space2,7,11,12,14,16,1s,19,23,z4,28,z9,31,33-36. This phenomenon (now generally referred to as 'sprouting') while not universally obtained7,8, 2° has nonetheless been observed at several levels of the CNS. Several investigators have suggested that sprouting could play a central role in the behavioral sequelae to brain lesions (e.g. ref. 25). For example it might be responsible for some aspects of the
216 recovery or compensation which is commonly observed after certain types of brain damage in adult mammalsa,4, 32. But there are many questions about the sprouting effect which must be resolved before it will be possible to evaluate these hypotheses. For one it will be necessary to establish whether the new connections function (transmit) or not. There is good evidence from studies in the peripheral nervous system that certain types of aberrant connections formed by post-lesion axonal growth are non-functionalS, 2~-~4. It is possible then that the new connections which form in brain after lesions are inoperative, a conclusion which would certainly alter current thinking on the possible behavioral significance of sprouting. If it is assumed that the new connections formed after lesions are functional then we need to consider the question of how much time is required for the completion of these new synapses. Recovery from brain lesions in mammals occurs at varying rates but in many cases it is completed within 30-40 days. The problem arises then whether post-lesion axonal growth and terminal formation could occur with sufficient speed to be involved in this process (e.g., refs. 3 and 15). Histochemical studies have reached the exciting conclusion that sprouting is evident within 5-10 days after the lesion2,~6, 34. Recently, Raisman and Field 31 have reported electron microscopic data showing that the process is well-advanced in the first post-lesion week and is essentially completed within 30 days. These data suggest that sprouting occurs with sufficient speed to play a major role in the brain's efforts to correct the behavioral deficits created by lesions. However, while these elegant studies provide us with a time course for the formation of new synapses they give no hint about the time required for these connections to begin to operate (assuming of course that they ever do operate). In the experiments reported in the present paper we attempted to investigate these two problems. In earlier paperslS, 19 we demonstrated that the deafferentation of the outer molecular layer of the dentate gyrus produced by lesions of the entorhinal cortex1, 30 causes the commissural projections to the dentate gyrus to sprout and invade the outer molecular layer and that this effect is more dramatic in neonatal than in mature rats 19. Furthermore, the expansion of the commissural system into the outer molecular layer in immature rats is accompanied by an outward shift in the locus of the maximum short-latency potential recorded in the molecular layer to commissural stimulationlL These latter studies suggested that the commissural fibers which invaded the outer molecular layer following neonatal entorhinal lesions established functional connections in that layer. In the experiments described in the present paper we studied the effects of entorhinal lesions made in mature rats on the response of the dentate gyrus to commissural stimulation as a function of time after the lesion. METHODS
Adult Sprague-Dawley rats (300-350 g) were used for all experiments. Unilateral electrolytic lesions of the entorhinal cortex were performed under sodium pentobarbital anesthesia as previously described 16. Following various time periods of
217 post-lesion recovery, the lesioned rats were again anesthetized with either sodium pentobarbital (50 mg/kg) or urethane (1.0 g/kg). The skull was opened bilaterally above the hippocampus and a monopolar or twisted wire (30-gauge stainless steel) bipolar stimulating electrode was placed stereotaxically into the hippocampal field CA3 contralateral to the entorhinal lesion. A low resistance (1~4 Mr2) glass microelectrode, having a 1-5 # m tip and filled with 2 M NaC1 inserted in the hippocampus ipsilateral to the entorhinal lesion, was used to record unit activity and field potentials. This experimental arrangement is illustrated in Fig. 1. Field potentials and unit recordings were obtained from the same electrode and the field potentials were separated using high-pass filters, averaged with a Nuclear Chicago data retrieval computer, and written out on an X - Y plotter. Laminar profiles of field potentials were constructed in the following manner. The recording electrode was lowered through the cortex until it encountered the stratum pyramidale of CA1 (which was readily identified by unitary injury discharges). The stimulating electrode was then lowered so that low-voltage stimulation produced a short-latency (4-7 msec) potential which was positive in the contralateral pyramidal cell layer, and turned abruptly negative as the electrode was advanced into the apical dendritic zone of these cells. The recording electrode was lowered in 50 #m intervals through the apical dendritic fields of the regio superior pyramidal cells into molecular and granular cell layers of the dentate gyrus. If the granule cell layer of the dentate gyrus (also identified by injury discharges) was not encountered 800-900 # m below the pyramidal cell layer, the recording electrode was raised and another track initiated. This procedure provided assurance that the portion of the dentate gyrus under study was that row of granule cells which runs parallel to the obliterated fissure and the CA1 stratum pyramidale of the rostral (dorsal) hippocampus. With the recording electrode positioned in this manner, the field potential profile of the dentate gyrus to low-voltage stimulation of the contralateral field CA3 was constructed. In some
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218 cases it was necessary to raise or lower the stimulating electrode slightly in order to maximize the field potentials in the dentate gyrus. The recording electrode was then raised in 50 # m intervals and 8 potentials were averaged and recorded at each position. The process was then repeated as the recording electrode was lowered back through the molecular layer (50 increments) into the granule cell layer. At the completion of each recording sequence, a small amount of Fast Green dye or horseradish peroxidase 3v was ejected from the electrode tip at various levels within the microelectrode tract. Three parallel penetrations through this region of the dentate gyrus were made for each rat. Five normal unlesioned adult rats were tested in this way and 29 adult rats with unilateral entorhinal lesions were tested at the following times after the lesion: 3 days, N = 2; 4 days, N = 2; 5 days, N = 1 ; 6 days, N = 1 ; 7 days, N = 1 ; 8 days, N = 5; 9 days, N = 4; 10 days, N = 1; 11 days, N ~ 2; 12 days, N = 3; 13 days, N = 1; 15 days, N = 3; 88 days, N = 1; 90 days, N = 1; and 250 days, N=I. At the completion of testing, all animals were sacrificed with an overdose of Nembutal and perfused through the heart with 1 0 ~ formalin. The brains were removed and post-fixed in 1 0 ~ formalin for several days after which they were divided in half by a coronal cut through the descending arch of the hippocampus. The caudal half of the forebrain was sliced horizontally and the entorhinal lesion reconstructed. The rostral half of the brain was then sectioned coronally on a freezing microtome and stained in order to verify the location of the marks left by the recording microelectrode and the electrolytic lesion made by the stimulating electrode. A second group of rats (N = 5) was prepared so that the same animal could be tested for the commissural response at several different time intervals following a unilateral entorhinal lesion. Animals were anesthetized with sodium pentobarbital and tested as described above with the following exception. After the electrodes were correctly positioned, the stimulating electrode was permanently affixed to the skull with acrylic cement. This procedure did not disturb the response of the contralateral dentate gyrus to CA3 stimulation. Next, the microelectrode was withdrawn and an internally threaded 2.5 m m diameter metal well was affixed to the skull over the exposed cortex. A small amount of sterile mineral oil was then placed in the well and a metal screw with an Epoxylite tip threaded into the well so that the tip was flush with the skull. The mineral oil and screw cap acted as a seal for the cortex which prevented swelling and distortion of the underlying structures. At the beginning of each subsequent recording session, animals were anesthetized in the manner described above, the protective cap was removed from the well and the recording microelectrode was lowered through the overlying cortex into the hippocampus. Following the conclusion of the last recording session the sites of the stimulation and recording electrodes were verified as described above. RESULTS
In an earlier paper 5 we described the characteristics of the field responses of the
219
dentate gyrus to stimulation of the contralateral hippocampal subfield CA3c. The field potential was shown to be the extracellular reflection of the dendritically located EPSP by the following criteria: (1) in the inner molecular layer where the commissural terminals are located the field potential was a short latency (2-4 msec) negativity which was preceded in some cases by smaller triphasic 'ripples' representing presynaptic fiber potentialsS,13; (2) in the outer dendrites and granule cell layers the negative potential reversed to a positivity suggesting that the 'sink' of the synaptic current was fed from two passive 'sources', indicating an electrotonic conduction of the synaptic currents away from the point of initial dendritic depolarization; (3) in the cell layer the positive reflection of the dendritically negative potential was associated with unit discharges of small amplitude and short duration; (4) high-frequency stimulation (200 Hz) did not eliminate the early components of the negative potential; and
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Fig. 2. Field potentials and laminar profile resulting from stimulation of the contralateral CA3 region, recorded from the dentate gyrus and CA1 fields of the hippocampus. A: stylized representation of a CA1 pyramidal cell (pyr) and dentate granule cell (Gr) with juxtaposed apical dendrites (F = obliterated hippocampal fissure). B: field potential elicited by stimulation of the contralateral CA3 region in tract running parallel to the cell configuration shown in A. Arrows indicate loci of recordings (50/zm intervals). C: laminar profile (voltage v s . depth analysis) of field potentials illustrated in B. From the prestimulus baseline (see reference lines in B), measurements were taken at a latency corresponding to the peak negativity recorded within the dentate molecular layer. Distance from the granule cell layer is indicated at the left (note the reversal point at 50/~m from the top of the granule cell bodies).
220
(5) the negative potential was eliminated by severing the commissural fibers within the ventral psalterium. The amplitude and polarity of the commissural field potentials in the dentate gyrus were measured and plotted against the depth of penetration in order to yield laminar profiles which could be compared across animals. The profiles were constructed by measuring the amplitude of the field potential at a constant latency from onset. The latency always corresponded to the peak o f the largest negative field potential (approximately 2.0 msec). The potentials were measured at this latency in order to more clearly define the extent to which they could be detected in the outer molecular layer in lesioned animals. This latency measure also prevented the contamination from other later potentials which might have been volume conducted from the overlying CA1 region (Fig. 2). In most cases the amplitude of the field potentials was determined by measuring the voltage difference between the prestimulus baseline and 2.0 msec after onset.
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221 However, in a few cases this was not possible since the start of the computer sweep coincided with the onset of the stimulus. In those cases the poststimulus baseline prior to the onset of the negative field potential was used as the reference point. This was necessary in some cases employing monopolar stimulation since the field potentials were superimposed upon a longer duration capacitive component of the stimulus artifact which elevated them above the prestimulus baseline. Fig. 3 shows a comparison of the two types of measurements and the disparities between monopolar and bipolar stimulation. The measures agree to the extent that the largest difference (using the prestimulus vs. poststimulus baseline) in amplitude of the field potentials was less than 0.5 mV; and in the placement of the potential within the molecular layer less than 50 mV. In the following results most of the potentials were measured from the prestimulus baseline. In those cases where measurements from this reference point could not be obtained then measurements were made from the poststimulus baseline prior to the onset of the negative field potential (see Fig. 4). Fig. 2 shows the results of a typical experiment in which the field potentials were recorded from various depths in the dentate gyrus (dorsal leaf) of a normal rat. The point of negative to positive reversal of the field potential was approximately 50 #m above the granule cell layer. In the outer two-thirds of the molecular layer the potential also reverses to a positive potential which is somwehat delayed in time from the negative peak. The positivity in both the granule cell and outer molecular regions presumably reflects the passive conduction of hyperpolarizing currents generated within the granule cells following their activation by commissural fibersS, x3. It can be seen that the maximal negative response to CA3 stimulation in the dentate gyrus occurred between 100 and 150 #m above the layer of granule cells, which correlates well with the circumscribed dendritic zone occupied by the commissttral terminals 19. The close correlation between the known anatomical location of the commissural terminals and maximal negative response were verified by noting the location of the Fast Green dye or horseradish peroxidase ejected from the recording electrodes. The farthest point from the granule cell layer at which the negativity could be recorded in the normal rat was marked and found to be approximately 150-200 #m above the granule cells (see below, Fig. 7). The negative potential was consistently observed to reverse to positive approximately 50 #m above the regions where granule cell unit discharges could be clearly distinguished (Figs. 2 and 4). This reversal point was therefore a more reliable physiological indicator of depth than granule cell activity and hence was utilized as the 'anchor' point from which to compare any changes in either the location, or amplitude of the negative potential. The distance between the pyramidal cell and the granule cell layers was always 850-900 #m which corresponds to the measured distance between these layers in shrinkage adjusted histological reconstructions. A second longer latency negative potential (20-25 msec) was often recorded from within the molecular layer of the dentate to commissural stimulation in the normal rat (Fig. 5). This 'late' negative potential was maximal in the outer twothirds of the molecular layer, and reversed positive in the cell layer. It was eliminated by ipsilateral, entorhinal lesions. In subsequent experiments we have determined that
222 the late potential originates in the ipsilateral entorhinal cortex. Hence its disappearance provided independent evidence for the effectiveness of the lesion 37. Characteristics o f the commissural response following a unilateral lesion o f the entorhinal cortex in adult rats Stimulation of the contralateral CA3 field in adult rats allowed to recover at least 15 days following an ipsilateral entorhinal lesion elicited the normal short-
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215
T I M E - D E P E N D E N T C H A N G E S IN C O M M I S S U R A L FIELD POTENTIALS IN T H E D E N T A T E G Y R U S F O L L O W I N G LESIONS OF T H E E N T O R H I N A L CORTEX IN A D U L T RATS
JAMES R. WEST, SAM DEADWYLER, CARL W. COTMAN AND GARY LYNCH
Department of Psychobiology, University of California, Irvine, Calif. 92664 (U.S.A.) (Accepted April 21st, 1975)
SUMMARY
Previous neuroanatomical work has shown that lesions of the entorhinal cortex in adult rats cause the commissural projections to spread from their normally restricted locus in the inner molecular layer approximately 40-50 #m into the outer molecular layer (that is, into the zone deafferented by the lesion). In the present study we measured the effects of the entorhinal lesion on the distribution of shortlatency potentials elicited by commissural stimulation in the molecular layer. Studies with animals tested at various times after the lesion and with a preparation that permitted recording from the same rat at several post-lesion intervals both indicated that the commissural response spread 100-150 # m towards the deafferented outer molecular layer, while the maximum response spread 50-100 #m. These effects were first detectable by 9 days after the lesion and were fully developed by 15 days post-lesion. These findings suggest that the growth of the commissural system seen after entorhinal lesions results in the rapid formation of functional terminals and are discussed in relationship to the behavioral consequences of brain lesions.
INTRODUCTION
In recent years, neuroanatomical studies have provided convincing evidence that if one afferent to a given brain region is removed, the remaining axonal projections to that region will undergo morphological changes which result in reoccupancy of vacated synaptic space2,7,11,12,14,16,1s,19,23,z4,28,z9,31,33-36. This phenomenon (now generally referred to as 'sprouting') while not universally obtained7,8, 2° has nonetheless been observed at several levels of the CNS. Several investigators have suggested that sprouting could play a central role in the behavioral sequelae to brain lesions (e.g. ref. 25). For example it might be responsible for some aspects of the
216 recovery or compensation which is commonly observed after certain types of brain damage in adult mammalsa,4, 32. But there are many questions about the sprouting effect which must be resolved before it will be possible to evaluate these hypotheses. For one it will be necessary to establish whether the new connections function (transmit) or not. There is good evidence from studies in the peripheral nervous system that certain types of aberrant connections formed by post-lesion axonal growth are non-functionalS, 2~-~4. It is possible then that the new connections which form in brain after lesions are inoperative, a conclusion which would certainly alter current thinking on the possible behavioral significance of sprouting. If it is assumed that the new connections formed after lesions are functional then we need to consider the question of how much time is required for the completion of these new synapses. Recovery from brain lesions in mammals occurs at varying rates but in many cases it is completed within 30-40 days. The problem arises then whether post-lesion axonal growth and terminal formation could occur with sufficient speed to be involved in this process (e.g., refs. 3 and 15). Histochemical studies have reached the exciting conclusion that sprouting is evident within 5-10 days after the lesion2,~6, 34. Recently, Raisman and Field 31 have reported electron microscopic data showing that the process is well-advanced in the first post-lesion week and is essentially completed within 30 days. These data suggest that sprouting occurs with sufficient speed to play a major role in the brain's efforts to correct the behavioral deficits created by lesions. However, while these elegant studies provide us with a time course for the formation of new synapses they give no hint about the time required for these connections to begin to operate (assuming of course that they ever do operate). In the experiments reported in the present paper we attempted to investigate these two problems. In earlier paperslS, 19 we demonstrated that the deafferentation of the outer molecular layer of the dentate gyrus produced by lesions of the entorhinal cortex1, 30 causes the commissural projections to the dentate gyrus to sprout and invade the outer molecular layer and that this effect is more dramatic in neonatal than in mature rats 19. Furthermore, the expansion of the commissural system into the outer molecular layer in immature rats is accompanied by an outward shift in the locus of the maximum short-latency potential recorded in the molecular layer to commissural stimulationlL These latter studies suggested that the commissural fibers which invaded the outer molecular layer following neonatal entorhinal lesions established functional connections in that layer. In the experiments described in the present paper we studied the effects of entorhinal lesions made in mature rats on the response of the dentate gyrus to commissural stimulation as a function of time after the lesion. METHODS
Adult Sprague-Dawley rats (300-350 g) were used for all experiments. Unilateral electrolytic lesions of the entorhinal cortex were performed under sodium pentobarbital anesthesia as previously described 16. Following various time periods of
217 post-lesion recovery, the lesioned rats were again anesthetized with either sodium pentobarbital (50 mg/kg) or urethane (1.0 g/kg). The skull was opened bilaterally above the hippocampus and a monopolar or twisted wire (30-gauge stainless steel) bipolar stimulating electrode was placed stereotaxically into the hippocampal field CA3 contralateral to the entorhinal lesion. A low resistance (1~4 Mr2) glass microelectrode, having a 1-5 # m tip and filled with 2 M NaC1 inserted in the hippocampus ipsilateral to the entorhinal lesion, was used to record unit activity and field potentials. This experimental arrangement is illustrated in Fig. 1. Field potentials and unit recordings were obtained from the same electrode and the field potentials were separated using high-pass filters, averaged with a Nuclear Chicago data retrieval computer, and written out on an X - Y plotter. Laminar profiles of field potentials were constructed in the following manner. The recording electrode was lowered through the cortex until it encountered the stratum pyramidale of CA1 (which was readily identified by unitary injury discharges). The stimulating electrode was then lowered so that low-voltage stimulation produced a short-latency (4-7 msec) potential which was positive in the contralateral pyramidal cell layer, and turned abruptly negative as the electrode was advanced into the apical dendritic zone of these cells. The recording electrode was lowered in 50 #m intervals through the apical dendritic fields of the regio superior pyramidal cells into molecular and granular cell layers of the dentate gyrus. If the granule cell layer of the dentate gyrus (also identified by injury discharges) was not encountered 800-900 # m below the pyramidal cell layer, the recording electrode was raised and another track initiated. This procedure provided assurance that the portion of the dentate gyrus under study was that row of granule cells which runs parallel to the obliterated fissure and the CA1 stratum pyramidale of the rostral (dorsal) hippocampus. With the recording electrode positioned in this manner, the field potential profile of the dentate gyrus to low-voltage stimulation of the contralateral field CA3 was constructed. In some
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218 cases it was necessary to raise or lower the stimulating electrode slightly in order to maximize the field potentials in the dentate gyrus. The recording electrode was then raised in 50 # m intervals and 8 potentials were averaged and recorded at each position. The process was then repeated as the recording electrode was lowered back through the molecular layer (50 increments) into the granule cell layer. At the completion of each recording sequence, a small amount of Fast Green dye or horseradish peroxidase 3v was ejected from the electrode tip at various levels within the microelectrode tract. Three parallel penetrations through this region of the dentate gyrus were made for each rat. Five normal unlesioned adult rats were tested in this way and 29 adult rats with unilateral entorhinal lesions were tested at the following times after the lesion: 3 days, N = 2; 4 days, N = 2; 5 days, N = 1 ; 6 days, N = 1 ; 7 days, N = 1 ; 8 days, N = 5; 9 days, N = 4; 10 days, N = 1; 11 days, N ~ 2; 12 days, N = 3; 13 days, N = 1; 15 days, N = 3; 88 days, N = 1; 90 days, N = 1; and 250 days, N=I. At the completion of testing, all animals were sacrificed with an overdose of Nembutal and perfused through the heart with 1 0 ~ formalin. The brains were removed and post-fixed in 1 0 ~ formalin for several days after which they were divided in half by a coronal cut through the descending arch of the hippocampus. The caudal half of the forebrain was sliced horizontally and the entorhinal lesion reconstructed. The rostral half of the brain was then sectioned coronally on a freezing microtome and stained in order to verify the location of the marks left by the recording microelectrode and the electrolytic lesion made by the stimulating electrode. A second group of rats (N = 5) was prepared so that the same animal could be tested for the commissural response at several different time intervals following a unilateral entorhinal lesion. Animals were anesthetized with sodium pentobarbital and tested as described above with the following exception. After the electrodes were correctly positioned, the stimulating electrode was permanently affixed to the skull with acrylic cement. This procedure did not disturb the response of the contralateral dentate gyrus to CA3 stimulation. Next, the microelectrode was withdrawn and an internally threaded 2.5 m m diameter metal well was affixed to the skull over the exposed cortex. A small amount of sterile mineral oil was then placed in the well and a metal screw with an Epoxylite tip threaded into the well so that the tip was flush with the skull. The mineral oil and screw cap acted as a seal for the cortex which prevented swelling and distortion of the underlying structures. At the beginning of each subsequent recording session, animals were anesthetized in the manner described above, the protective cap was removed from the well and the recording microelectrode was lowered through the overlying cortex into the hippocampus. Following the conclusion of the last recording session the sites of the stimulation and recording electrodes were verified as described above. RESULTS
In an earlier paper 5 we described the characteristics of the field responses of the
219
dentate gyrus to stimulation of the contralateral hippocampal subfield CA3c. The field potential was shown to be the extracellular reflection of the dendritically located EPSP by the following criteria: (1) in the inner molecular layer where the commissural terminals are located the field potential was a short latency (2-4 msec) negativity which was preceded in some cases by smaller triphasic 'ripples' representing presynaptic fiber potentialsS,13; (2) in the outer dendrites and granule cell layers the negative potential reversed to a positivity suggesting that the 'sink' of the synaptic current was fed from two passive 'sources', indicating an electrotonic conduction of the synaptic currents away from the point of initial dendritic depolarization; (3) in the cell layer the positive reflection of the dendritically negative potential was associated with unit discharges of small amplitude and short duration; (4) high-frequency stimulation (200 Hz) did not eliminate the early components of the negative potential; and
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Fig. 2. Field potentials and laminar profile resulting from stimulation of the contralateral CA3 region, recorded from the dentate gyrus and CA1 fields of the hippocampus. A: stylized representation of a CA1 pyramidal cell (pyr) and dentate granule cell (Gr) with juxtaposed apical dendrites (F = obliterated hippocampal fissure). B: field potential elicited by stimulation of the contralateral CA3 region in tract running parallel to the cell configuration shown in A. Arrows indicate loci of recordings (50/zm intervals). C: laminar profile (voltage v s . depth analysis) of field potentials illustrated in B. From the prestimulus baseline (see reference lines in B), measurements were taken at a latency corresponding to the peak negativity recorded within the dentate molecular layer. Distance from the granule cell layer is indicated at the left (note the reversal point at 50/~m from the top of the granule cell bodies).
220
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221 However, in a few cases this was not possible since the start of the computer sweep coincided with the onset of the stimulus. In those cases the poststimulus baseline prior to the onset of the negative field potential was used as the reference point. This was necessary in some cases employing monopolar stimulation since the field potentials were superimposed upon a longer duration capacitive component of the stimulus artifact which elevated them above the prestimulus baseline. Fig. 3 shows a comparison of the two types of measurements and the disparities between monopolar and bipolar stimulation. The measures agree to the extent that the largest difference (using the prestimulus vs. poststimulus baseline) in amplitude of the field potentials was less than 0.5 mV; and in the placement of the potential within the molecular layer less than 50 mV. In the following results most of the potentials were measured from the prestimulus baseline. In those cases where measurements from this reference point could not be obtained then measurements were made from the poststimulus baseline prior to the onset of the negative field potential (see Fig. 4). Fig. 2 shows the results of a typical experiment in which the field potentials were recorded from various depths in the dentate gyrus (dorsal leaf) of a normal rat. The point of negative to positive reversal of the field potential was approximately 50 #m above the granule cell layer. In the outer two-thirds of the molecular layer the potential also reverses to a positive potential which is somwehat delayed in time from the negative peak. The positivity in both the granule cell and outer molecular regions presumably reflects the passive conduction of hyperpolarizing currents generated within the granule cells following their activation by commissural fibersS, x3. It can be seen that the maximal negative response to CA3 stimulation in the dentate gyrus occurred between 100 and 150 #m above the layer of granule cells, which correlates well with the circumscribed dendritic zone occupied by the commissttral terminals 19. The close correlation between the known anatomical location of the commissural terminals and maximal negative response were verified by noting the location of the Fast Green dye or horseradish peroxidase ejected from the recording electrodes. The farthest point from the granule cell layer at which the negativity could be recorded in the normal rat was marked and found to be approximately 150-200 #m above the granule cells (see below, Fig. 7). The negative potential was consistently observed to reverse to positive approximately 50 #m above the regions where granule cell unit discharges could be clearly distinguished (Figs. 2 and 4). This reversal point was therefore a more reliable physiological indicator of depth than granule cell activity and hence was utilized as the 'anchor' point from which to compare any changes in either the location, or amplitude of the negative potential. The distance between the pyramidal cell and the granule cell layers was always 850-900 #m which corresponds to the measured distance between these layers in shrinkage adjusted histological reconstructions. A second longer latency negative potential (20-25 msec) was often recorded from within the molecular layer of the dentate to commissural stimulation in the normal rat (Fig. 5). This 'late' negative potential was maximal in the outer twothirds of the molecular layer, and reversed positive in the cell layer. It was eliminated by ipsilateral, entorhinal lesions. In subsequent experiments we have determined that
222 the late potential originates in the ipsilateral entorhinal cortex. Hence its disappearance provided independent evidence for the effectiveness of the lesion 37. Characteristics o f the commissural response following a unilateral lesion o f the entorhinal cortex in adult rats Stimulation of the contralateral CA3 field in adult rats allowed to recover at least 15 days following an ipsilateral entorhinal lesion elicited the normal short-
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