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Substantia nigra single unit activity during penicillin-induced focal cortical epileptiform discharge in the rat
Brain
ResearchBulletin, Vol. 11, pp. 11-13, 1983.QAnkho International Inc. FVintal in the U.S.A.
Substantia Nigra Single Unit Activity During Penicillin-Induced Focal Cortical Epileptiform Discharge in the Rat T. E. KANIFF,
C. M. CHUMAN
AND
E. J. NEAFSEY’
Departments of Anatomy (EIN) and Surgery (TEK, CMC) Loyola University Medical Center, 2160 S. First Avenue, Maywood, IL 60163 Received
23 March
1983
KANIFF, T. E., C. M. CHUMAN AND E. J. NEAFSEY. Substantia nigra single unit activity during penicillin-induced cortical epiieptiform discharge in the rat. BRAIN RES BULL 11(l) 11-13, 1983.~In urethane anesthetized rats singIe unit activity was recorded in the substantia nigra (SN) during focal cortical epileptiform discharges induced by topical application of penicillin to the cortical surface. Eighty percent of SN units responded during the cortical interictal spike discharge, 50% with an initial burst or increase in tiring rate and 30% with an initial inhibition or decrease in tiring rate. In view of the SN’s widespread projections to thalamus and brainstem, these results suggest the SN may be a prominent element of the pathway involved in the spread and generalization of cortical epileptiforrn activity.
focal
SN single unit activity
Epileptiform discharge
Penicillin
lateral. The dura was then reflected from this area. A silver ball electrode was then placed on the surface of the sensorimotor cortex rostral to bregma to record the electrocorticographic activity (E&G) of the focus. A small pledget of gelfoam (2x2 mm) satmated with sodium penicillin (50,000 U/ml) was placed on the exposed sensorimotor cortex. Within two to three minutes spontaneous interictal spikes (IIS) began to occur at about once every five seconds. The pledget of geifoam was then removed. Unit recordings were made using a glass-insulated tungsten microelectrode [ 111 driven into the brain using a hydraulic microdrive. In each animal five to seven vertical electrode penetrations were made, using K6nig and Klippel’s stereotaxic coordinates for the SN (Al to A3, L.5 to L1.5). Unit activity and ECoG activity were simultaneously displayed on an oscilloscope, and it was noted if any unit recorded in the vicinity of the SN appeared to consistently change its firing pattern during or after each IIS that occurred while the unit was studied (at least 25 IIS’s for each unit). Responses in many cases were recorded on Polaroid pictures of storage scope displays. During each electrode track the depth of each unit from the cortical surface was noted, as was the depth at which electrical fiber activity correlated with the IIS was encountered in the crus cerebri. Following the completion of unit recordings from each electrode track, one or two marking lesions (10 Clamps for 10 set) were made at a known depth either dorsal to the recorded units or at the location of the electrophysiologically defined crus cerebri fiber activity. The animals were sacrificed by transcardial perfusion with 10%
LOCAL microinjections of GABA into the substantia nigra (SN) have recently been reported to exert a significant anticonvulsant effect on seizures produced by either electroshock or intravenous pentylenetetrazole [B]. Several other studies [4,13] have also suggested that .$he SN and other nuclei of the basal ganglia (caudate, putamen, globus pallidus) are important elements in the pathway of spread and generalization of focal cortical epileptiform discharge. Our own laboratory has previously shown that such cortical discharges produce dramatic changes in the activity of single units recorded in the caudate and entopeduncular nuclei [ 101. However, unit activity in the substantia nigra (which has widespread projections to the thalamus and brainstem [ll) has not yet been studied during cortical epileptiform activity. (This cortical activity does produce spiking in the SN EEG [13].) The present study was undertaken to investigate this question. METHOD
Unit recordings in the SN were done in five Long Evans hooded rats (250-350 g). The animals were anesthetized with urethane (1.3 g/kg, IP) and placed in a stereotaxic frame. A heating pad under the rat was electronically controlled to maintain the animal’s rectal temperature within one degree of 37 degrees C. In each experiment the cistema magna was opened to prevent cortical swelling. A craniotomy was then performed on one side exposing the brain from lambda to about 2 mm rostral to bregma and from the midline to 4 mm ‘Requests for reprints should be addressed to Dr. E. J. Neafsey.
11
12
KANIFF.
CHUMAN
AND NFAFSF.)
FIG. I. Photomicrograph of coronal section through SN with marking lesion (arrow) seen in pars compacta. Scale bar denotes I mm.
formalin. The brain was removed and postfixed in a 30% sucrose solution in 10% formalin. After the brain had sunk in this solution, it was frozen sectioned coronally; the sections were stained for both fibers and cells using eriochrome cyanine R as a fiber stain (31 and neutral red as a cell stain. Each electrode track was reconstructed on a drawing of the appropriate histological section; the location of the units was determined in relation to the marking lesions and the level of the fiber activity in the crus cerebri. Only units identified on these criteria as being in the SN were included in this study. Figure 1 illustrates a marking lesion just above the SN on one electrode track.
FIG. 2. A. SN unit with burst response to IIS seen in EEG tracing. B. SN unit with burst response followed bv inhibition. Time bar indicates 0.5 set in A and 0.2 set in B.
RESULTS
A total of 81 units were recorded within the SN, of which 6.5 (80%) were clearly and consistently responsive to the IIS. In 41 units (50% of all cells) the response consisted of an initial high frequency burst of spikes during the IIS. For 25 units this burst was the only component of the response; for the other 16 units the initial burst was followed by an apparent period of inhibition or decreased firing rate. Figures 2A and 2B illustrate examples of these initially excitatory responses. The second type of response was found in 24 units (30% of all cells) and consisted of an initial inhibition or decrease in activity during the IIS. In 17 units this initial decrease in firing was the only detectable response; the other 7 units showed a burst of higher frequency tiring following the initial decrease. Figures 3A and 3B depict examples of these initially inhibitory responses. The bar graph in Fig. 4 summarizes these results. The percentage of responsive units was the same (80%) in all parts of the SN. However, there appeared to be a progressive increase in the percentage of cells with initial inhibitory responses as the electrode penetrations moved from rostra1 to caudal in the SN. Thus, the ratio of excitatory to inhibitory responses (E/I) was 3/l in the rostral third of the SN, 2/l in the middle third, and l/l in the caudal third. Only a few units (~5) appeared to be recorded in the pars compacta of the SN; the responses of these cells were similar to those of the pars reticulata neurons (both initial excitations and inhibitions).
DISCUSSION
The response of SN neurons to interictal focal cortical epileptiform activity is either a burst of action potentials (50%) or decrease in firing rate below baseline (30%). This basic pattern is similar to that we have previously described in the entopeduncular nucleus [lo]. The entopeduncular nucleus and the SN receive a large projection from the caudate [l]. Since the entopeduncular responses depend on transmission through the caudate ]2], it would not be surprising if the same pathway also was responsible for the SN responses. The presence of both excitatory and inhibitory responses is not surprising since both substance P (a putative excitatory transmitter in the SN [6]) and GABA (a putative inhibitory transmitter in the SN [7]) have been localized to axon terminals in the SN [5,12]. Increasing the GABA levels in the SN appears to have an anticonvulsant effect (81. It is possible that this effect is due to shifting the balance of excitatory and inhibitory inputs to the SN in favor of inhibition. The finding that excitatory responses predominated in the rostra1 SN while inhibitory responses were more prevalent in the caudal SN was unexpected since no differential distribution of substance P and GABA within the SN has been reported. However, the source of substance P terminals in the SN is the rostra1 caudate-putamen while GABA terminals in the SN arise from the caudal caudate-putamen 191. Thus there may be a differential distribution of terminals of these two transmitters in the SN which remains to be discovered.
13
SUBSTANTIA NIGRA AND CORTICAL EPILEPSY
EEG Y
-of
80
8Ncah 04of80 20
B
M
I
II8
HI
FIG. 4. Bar graph illustrating percentage of various SN responses to cortical IIS, B. Burst; B/I, Burst followed by in~bi~n; I, Inhibition; I/B, In~bition followed by burst or increased firing; NR, No response.
FIG. 3. A. SN unit with inhibitor response to IIS. B. SN unit with inhibitory response followed by an increase in firing rate (10 superimposed traces). Time barindicates0.2 set in A and0.05 set in B.
In conclusion, the results of this study are consistent with the rest&s of similar studies of unit responses in other parts of the basaI ganglia during corticaJ ep~ep~~ activity f2,10] in that approximately 80% of the neurons in the SN responded in some way to the IIS. Since these neurons have prominent projections to the thalamus and brainstem [l] it is possible that they could he a significant part of the pathway
1. Carpenter, M. B. Anatomy of the corpus striatum and brain stem integration systems. In: Uundbook of Physiology. Section I: The Ndrvous System. Vol. II. Motor Cbntrbl. P&i 2, edited
by V. B. Bronks. Bethesda, MD: American Physiological Society, 1981, pp. 947-995. 2. Chuman, C. M and E. J. Neafsey. Propagation of focal cortical epileptiform discbarge to the entopeduncular nuckus: effect of caudate ksions. Extr Neurof 79: 152-159, 1983. 3. Clark, G. (Ed.)St&ing Procedures. Fourth Edition. Baltimore, MD: Williams and Wilkins, 1981, pp. l&110. 4. Collins, R. C., C. Kennedy, L. Sokoloff and F. Plum. Metabolic anatomy of focal motor seizures. Arch Neural 33: 536-542, 1978.
5, Cuello, A. C, and I. Kanaxawa. The dist~but~n of substance P immunoreactive fibers in the rat central nervous system. J Comp Neural 178: 129-156, 1978. 6. Davies, J. and A. Dray. Substance P in the substantia nigra. Bruin Res la7: 623-627, 1976. 7. Feltz, P. Gamma-aminobutyric acid and a caudate-nigral inhibition. Con J Physiol Pbarmacol49: 1113-1115, 1971.
responsihk epilepsy.
for the spread and generahxation
of cortical
ACKNOWLEDGEMENTS This study was supportedby grant No. S42-14(Potts Fund) from Loyola University to CMC and by NIH grant NS16146 to EJN.
8. Iadarola, M. J. and K. Gale. Substantia nigra: site of anticonvulsant activity mediated by gamma-aminobutyric acid. Science 218: 1237-1240, 1982. 9. Jessel, T. M., P. C. Emson, G. Paxinos and A. C. Cuello. Topographic projections of substance P and GABA pathways in the strlato and atria system: a bln&emkal and itemstochemical study. Brain Res 152: 487-499,1978. 10. Neafsey, E. J., C. M. Chuman and A. A. Ward, Jr. Propagation of focal cortical epilept&m discharge to the basal ganglia. Exp Neural 66: w-108, 1979. 11. Neafsey, E. J. A simple method for glass insulating tungsten microelectrodes. Brain Res BIlll6: 95-%, 1981. 12. RIM, C. E., J. E. Vaughn. K. Saito, R. Barber and E. Roberts. irnrn~~~~rn~ localixation of ghttamate decarboxylase in rat substantia nigra. Brain Res 116: 28X!98, 1976. 13. Udvarbelyi, G. 0. and A. E. Walker. Dissemination of acute focal seizures in the monkey. I. From cortical foci. Arch Neural 12: 33>3%, 1%5.
ResearchBulletin, Vol. 11, pp. 11-13, 1983.QAnkho International Inc. FVintal in the U.S.A.
Substantia Nigra Single Unit Activity During Penicillin-Induced Focal Cortical Epileptiform Discharge in the Rat T. E. KANIFF,
C. M. CHUMAN
AND
E. J. NEAFSEY’
Departments of Anatomy (EIN) and Surgery (TEK, CMC) Loyola University Medical Center, 2160 S. First Avenue, Maywood, IL 60163 Received
23 March
1983
KANIFF, T. E., C. M. CHUMAN AND E. J. NEAFSEY. Substantia nigra single unit activity during penicillin-induced cortical epiieptiform discharge in the rat. BRAIN RES BULL 11(l) 11-13, 1983.~In urethane anesthetized rats singIe unit activity was recorded in the substantia nigra (SN) during focal cortical epileptiform discharges induced by topical application of penicillin to the cortical surface. Eighty percent of SN units responded during the cortical interictal spike discharge, 50% with an initial burst or increase in tiring rate and 30% with an initial inhibition or decrease in tiring rate. In view of the SN’s widespread projections to thalamus and brainstem, these results suggest the SN may be a prominent element of the pathway involved in the spread and generalization of cortical epileptiforrn activity.
focal
SN single unit activity
Epileptiform discharge
Penicillin
lateral. The dura was then reflected from this area. A silver ball electrode was then placed on the surface of the sensorimotor cortex rostral to bregma to record the electrocorticographic activity (E&G) of the focus. A small pledget of gelfoam (2x2 mm) satmated with sodium penicillin (50,000 U/ml) was placed on the exposed sensorimotor cortex. Within two to three minutes spontaneous interictal spikes (IIS) began to occur at about once every five seconds. The pledget of geifoam was then removed. Unit recordings were made using a glass-insulated tungsten microelectrode [ 111 driven into the brain using a hydraulic microdrive. In each animal five to seven vertical electrode penetrations were made, using K6nig and Klippel’s stereotaxic coordinates for the SN (Al to A3, L.5 to L1.5). Unit activity and ECoG activity were simultaneously displayed on an oscilloscope, and it was noted if any unit recorded in the vicinity of the SN appeared to consistently change its firing pattern during or after each IIS that occurred while the unit was studied (at least 25 IIS’s for each unit). Responses in many cases were recorded on Polaroid pictures of storage scope displays. During each electrode track the depth of each unit from the cortical surface was noted, as was the depth at which electrical fiber activity correlated with the IIS was encountered in the crus cerebri. Following the completion of unit recordings from each electrode track, one or two marking lesions (10 Clamps for 10 set) were made at a known depth either dorsal to the recorded units or at the location of the electrophysiologically defined crus cerebri fiber activity. The animals were sacrificed by transcardial perfusion with 10%
LOCAL microinjections of GABA into the substantia nigra (SN) have recently been reported to exert a significant anticonvulsant effect on seizures produced by either electroshock or intravenous pentylenetetrazole [B]. Several other studies [4,13] have also suggested that .$he SN and other nuclei of the basal ganglia (caudate, putamen, globus pallidus) are important elements in the pathway of spread and generalization of focal cortical epileptiform discharge. Our own laboratory has previously shown that such cortical discharges produce dramatic changes in the activity of single units recorded in the caudate and entopeduncular nuclei [ 101. However, unit activity in the substantia nigra (which has widespread projections to the thalamus and brainstem [ll) has not yet been studied during cortical epileptiform activity. (This cortical activity does produce spiking in the SN EEG [13].) The present study was undertaken to investigate this question. METHOD
Unit recordings in the SN were done in five Long Evans hooded rats (250-350 g). The animals were anesthetized with urethane (1.3 g/kg, IP) and placed in a stereotaxic frame. A heating pad under the rat was electronically controlled to maintain the animal’s rectal temperature within one degree of 37 degrees C. In each experiment the cistema magna was opened to prevent cortical swelling. A craniotomy was then performed on one side exposing the brain from lambda to about 2 mm rostral to bregma and from the midline to 4 mm ‘Requests for reprints should be addressed to Dr. E. J. Neafsey.
11
12
KANIFF.
CHUMAN
AND NFAFSF.)
FIG. I. Photomicrograph of coronal section through SN with marking lesion (arrow) seen in pars compacta. Scale bar denotes I mm.
formalin. The brain was removed and postfixed in a 30% sucrose solution in 10% formalin. After the brain had sunk in this solution, it was frozen sectioned coronally; the sections were stained for both fibers and cells using eriochrome cyanine R as a fiber stain (31 and neutral red as a cell stain. Each electrode track was reconstructed on a drawing of the appropriate histological section; the location of the units was determined in relation to the marking lesions and the level of the fiber activity in the crus cerebri. Only units identified on these criteria as being in the SN were included in this study. Figure 1 illustrates a marking lesion just above the SN on one electrode track.
FIG. 2. A. SN unit with burst response to IIS seen in EEG tracing. B. SN unit with burst response followed bv inhibition. Time bar indicates 0.5 set in A and 0.2 set in B.
RESULTS
A total of 81 units were recorded within the SN, of which 6.5 (80%) were clearly and consistently responsive to the IIS. In 41 units (50% of all cells) the response consisted of an initial high frequency burst of spikes during the IIS. For 25 units this burst was the only component of the response; for the other 16 units the initial burst was followed by an apparent period of inhibition or decreased firing rate. Figures 2A and 2B illustrate examples of these initially excitatory responses. The second type of response was found in 24 units (30% of all cells) and consisted of an initial inhibition or decrease in activity during the IIS. In 17 units this initial decrease in firing was the only detectable response; the other 7 units showed a burst of higher frequency tiring following the initial decrease. Figures 3A and 3B depict examples of these initially inhibitory responses. The bar graph in Fig. 4 summarizes these results. The percentage of responsive units was the same (80%) in all parts of the SN. However, there appeared to be a progressive increase in the percentage of cells with initial inhibitory responses as the electrode penetrations moved from rostra1 to caudal in the SN. Thus, the ratio of excitatory to inhibitory responses (E/I) was 3/l in the rostral third of the SN, 2/l in the middle third, and l/l in the caudal third. Only a few units (~5) appeared to be recorded in the pars compacta of the SN; the responses of these cells were similar to those of the pars reticulata neurons (both initial excitations and inhibitions).
DISCUSSION
The response of SN neurons to interictal focal cortical epileptiform activity is either a burst of action potentials (50%) or decrease in firing rate below baseline (30%). This basic pattern is similar to that we have previously described in the entopeduncular nucleus [lo]. The entopeduncular nucleus and the SN receive a large projection from the caudate [l]. Since the entopeduncular responses depend on transmission through the caudate ]2], it would not be surprising if the same pathway also was responsible for the SN responses. The presence of both excitatory and inhibitory responses is not surprising since both substance P (a putative excitatory transmitter in the SN [6]) and GABA (a putative inhibitory transmitter in the SN [7]) have been localized to axon terminals in the SN [5,12]. Increasing the GABA levels in the SN appears to have an anticonvulsant effect (81. It is possible that this effect is due to shifting the balance of excitatory and inhibitory inputs to the SN in favor of inhibition. The finding that excitatory responses predominated in the rostra1 SN while inhibitory responses were more prevalent in the caudal SN was unexpected since no differential distribution of substance P and GABA within the SN has been reported. However, the source of substance P terminals in the SN is the rostra1 caudate-putamen while GABA terminals in the SN arise from the caudal caudate-putamen 191. Thus there may be a differential distribution of terminals of these two transmitters in the SN which remains to be discovered.
13
SUBSTANTIA NIGRA AND CORTICAL EPILEPSY
EEG Y
-of
80
8Ncah 04of80 20
B
M
I
II8
HI
FIG. 4. Bar graph illustrating percentage of various SN responses to cortical IIS, B. Burst; B/I, Burst followed by in~bi~n; I, Inhibition; I/B, In~bition followed by burst or increased firing; NR, No response.
FIG. 3. A. SN unit with inhibitor response to IIS. B. SN unit with inhibitory response followed by an increase in firing rate (10 superimposed traces). Time barindicates0.2 set in A and0.05 set in B.
In conclusion, the results of this study are consistent with the rest&s of similar studies of unit responses in other parts of the basaI ganglia during corticaJ ep~ep~~ activity f2,10] in that approximately 80% of the neurons in the SN responded in some way to the IIS. Since these neurons have prominent projections to the thalamus and brainstem [l] it is possible that they could he a significant part of the pathway
1. Carpenter, M. B. Anatomy of the corpus striatum and brain stem integration systems. In: Uundbook of Physiology. Section I: The Ndrvous System. Vol. II. Motor Cbntrbl. P&i 2, edited
by V. B. Bronks. Bethesda, MD: American Physiological Society, 1981, pp. 947-995. 2. Chuman, C. M and E. J. Neafsey. Propagation of focal cortical epileptiform discbarge to the entopeduncular nuckus: effect of caudate ksions. Extr Neurof 79: 152-159, 1983. 3. Clark, G. (Ed.)St&ing Procedures. Fourth Edition. Baltimore, MD: Williams and Wilkins, 1981, pp. l&110. 4. Collins, R. C., C. Kennedy, L. Sokoloff and F. Plum. Metabolic anatomy of focal motor seizures. Arch Neural 33: 536-542, 1978.
5, Cuello, A. C, and I. Kanaxawa. The dist~but~n of substance P immunoreactive fibers in the rat central nervous system. J Comp Neural 178: 129-156, 1978. 6. Davies, J. and A. Dray. Substance P in the substantia nigra. Bruin Res la7: 623-627, 1976. 7. Feltz, P. Gamma-aminobutyric acid and a caudate-nigral inhibition. Con J Physiol Pbarmacol49: 1113-1115, 1971.
responsihk epilepsy.
for the spread and generahxation
of cortical
ACKNOWLEDGEMENTS This study was supportedby grant No. S42-14(Potts Fund) from Loyola University to CMC and by NIH grant NS16146 to EJN.
8. Iadarola, M. J. and K. Gale. Substantia nigra: site of anticonvulsant activity mediated by gamma-aminobutyric acid. Science 218: 1237-1240, 1982. 9. Jessel, T. M., P. C. Emson, G. Paxinos and A. C. Cuello. Topographic projections of substance P and GABA pathways in the strlato and atria system: a bln&emkal and itemstochemical study. Brain Res 152: 487-499,1978. 10. Neafsey, E. J., C. M. Chuman and A. A. Ward, Jr. Propagation of focal cortical epilept&m discharge to the basal ganglia. Exp Neural 66: w-108, 1979. 11. Neafsey, E. J. A simple method for glass insulating tungsten microelectrodes. Brain Res BIlll6: 95-%, 1981. 12. RIM, C. E., J. E. Vaughn. K. Saito, R. Barber and E. Roberts. irnrn~~~~rn~ localixation of ghttamate decarboxylase in rat substantia nigra. Brain Res 116: 28X!98, 1976. 13. Udvarbelyi, G. 0. and A. E. Walker. Dissemination of acute focal seizures in the monkey. I. From cortical foci. Arch Neural 12: 33>3%, 1%5.