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Retention disruption following post-trial picrotoxin injection into the substantia nigra
620
Brain Research. 113 (1976)621} 625 :c Elsevier Scientific Publishing Company, Amsterdam Printed in The Netherh~ds
Retention disruption following post-trial picrotoxin injection into the substantia nigra
H A I N G - J A KIM and A R Y E H R O U T T E N B E R G
Cresap Neuroscience Laboratory, Northwestern University, Evanston, IlL 60201 (U.S.A.) (Accepted May 18th, 1976)
Memory for simple tasks can be disrupted by treatments that alter the physiological activity of the mammalian brain shortly after learning 23. Localization of these disruptive treatment effects to particular brain regions has been achieved ~ 3 . For example, Routtenberg and Holzman 35 found that electrical stimulation of the pars compacta of substantia nigra (SN), either during learning or immediately following learning, disrupted retention performance 24 h later. The problem with ascribing the observed retention deficit to disruption of nigral neuronal activity is that stimulation of peri-nigral fibers of passage ls-~0 may also have played a role. In the present study, therefore, an intracranial chemical injection technique 32 was used, since neurons originating in SN, but not fibers of passage, could be modified by chemical agents which alter the action of a putative neurotransmitter naturally affecting these neurons. The SN receives fiber projections from neostriatum and globus paltidus 12. Some of these fibers are gamma-aminobutyric acid (GABA)-containing 16,24. These GABA-containing striatonigral fibers inhibit SN neurons4,9,ag: iontophoretic application of GABA in SN mimics the inhibition produced by electrical caudatal stimulation 9 and this inhibition is blocked by intravenous 29 or iontophoretic 4 administration ofpicrotoxim a proposed GABA-specific antagonist 8,26. It would be expected, then, that blockade of the GABA-mediated inhibitory action on SN neurons by injection of picrotoxin into SN would cause a temporary 17 alteration in the nigral neuronal activity. If such an alteration were responsible for the memory disruptive effect of electrical stimulation observed previously35, then injection of picrotoxin into SN should also produce a similar amnesic effect. The present results confirm this expectation. Male albino rats weighing 200-300 g at the time of surgery received unilateral cannula implantations aimed at the rostral SN. Tubing of 27-gauge and M-gauge was employed for the guide and injection cannula, respectively. Following a 4-day recovery period, animals were placed on the platform of a step-down passive avoidance learning apparatus, described previously aS. Animals usually descended from the platform within 15 sec. When they did so, they received 0.5 mA foot shock through the electrified grid floor until they ascended onto the platform to escape the shock. As soon as the subject
621 reached the learning criterion by remaining on the platform for 2 min without a descent, the rat was removed from the learning chamber. Approximately 5 min after learning, the animal was subjected to intranigral injection. Drugs were dissolved in 0.5 #1 of sterile saline (pH -- 5.7) and injected at the rate of 0.5/zl/2 min using a Sage syringe pump (Model 352). Retention was studied 24 h later. The criterion for perfect retention was achieved if the subject stayed on the platform for 5 rain without a descent. Any subject which descended from the platform during the 5-min retention test period was considered to have shown a retention deficit. The latency to first-descent was recorded for later statistical analysis. The preparation of histological material was the same as described previously zS. An injection site was considered a SN placement when the tip of the cannula was found either in contact with the cells in the pars compacta or in the pars reticularis of SN without contacting or piercing the cerebral peduncle situated beneath SN. Picrotoxin injected into SN at dosages of 0.01,0.05, 0.1 and 0.2/~g (pH -- 6.1-6.3) led to a decrement in the mean latencies to descent in the retention test compared to the mean descent latency for the saline control group (Table I; Newman-Keuls test, P < 0.05). The pooled frequency of picrotoxin-treated rats which showed retention disruption was higher than the combined frequency of disrupted control subjects (Z 2, P < 0.01). Of 68 picrotoxin-treated animals, 31 subjects were found to have their cannula tips either in the cerebral peduncle or penetrating its lower boundary. Only 8 of these 31 animals showed retention deficits. This ratio of disruption was not different from that obtained for the pooled control groups in Table I (Z 2, P > 0.05)" nor was the mean latency to descent for these animals (243.08 ± 27.38 sec) different from that for the implanted control group (t, P > 0.05). Injection ofpicrotoxin through a cannula ventral to SN, therefore, had no significant effect on retention performance. Intranigral injection of picrotoxin at the highest dosage (0.2/tg) occasionally produced a mild behavioral convulsion, but was less effective in inducing a retention deficit than the lower doses of picrotoxin which produced no signs of behavioral conTABLE ! E[lect o f 5 mhl post-learning injection o f pierotoxin into the substantia nigra on retention studied 24 h later Group
% disruption (number o f subjects)
Mean latency o f first descent in sec ± S.E.M.
Unoperated Implanted Saline Strychnine Picrotoxin 0.01 /~g 0.05 t~g 0.1 /tg 0.2 ttg
0 20 10 25
(10) (10) (10) (8)
300.0 -3- 0.0 259.5 ± 28.1 298.0 ± 2.1) 270.0 ~ 20.0
63 90* 89* 67
(8) (10) (9) (9)
161.9 ± 47.2 126.8 ± 38.5* 117.3 _+ 42.8* 154.0 _+_28.0
(1) All picrotoxin groups were significantly different (P < 0.05) from the saline control group. (2) *Indicates a significant difference (P < 0.05) from the strychnine-treated group.
622 vulsion. It seems unlikely, therefore, that the observed amnesic effect in picrotoxintreated animals was due to the same neurophysiological factor associated with the convulsion. Additionally, if picrotoxin injected into SN produced the observed retention deficit by inducing l o c a l electrophysiological seizures, it would be predicted that within limits the resulting amnesic effect would increase rather than decrease as the dose of picrotoxin is increased. The present results indicate, however, that this is not the case (see Table I). Circling behavior was also observed in the present experiments following intranigral injection of picrotoxin at dosages higher than 0.05/~g. It is important to note that, at the 0.01 #g dosage, picrotoxin injection did not produce any sign of circling behavior, though it produced a retention deficit. Therefore, it is unlikely that the observed retention deficit is related to circling behavior induced by picrotoxin injected into SN. When strychnine (0.125 #g/0.5 #1, pH .... 8.0), a convulsant drug 5 which is not a GABA antagonist ~;, was injected into SN, the retention performance of these animals did not differ from the other 3 control groups (Table I) in both frequency of disruption (Fisher's exact test, P :~ 0.05) and in mean descent latency (Newman-Keuls, P :,- 0.05). In addition, the performance of strychnine-treated animals during the retention test was superior to that of picrotoxin animals (see Table l). Both picrotoxin and strychnine are known to be central nervous system (CNS) stimulants 5. Therefore, the present results strongly suggest that the retention deficit observed following picrotoxin injection is due to an interaction of this drug with the GABA-mediated inputs to SN, as opposed to some non-specific stimulation of the CNS. We also examined the effect of intranigral injection of picrotoxin given 22 h after learning, i.e., 2 h before the retention test. As shown in Table 11, we found that this group did not differ from the 2-h preretention control groups. For all these 2-h preretention injection groups, the percentage of rats descending on a retention trial was no more than 30 °,~i, which is similar to the percentage disruption for those 5-rain post-learning control injection groups. The differences in the descent latencies between any two groups in Table II were not significant (Newman-Keuls, P • 0.05). These results lend support to the hypothesis that memory consolidation is a time-dependent TABLE
I1
Effect o f 22 h post-learning #~iection o f picrotoxin into the substantia nigra on retention studied 24 h after learnblg Group
% disruption
Mean first descent latency in sec ~ S.E.M.
10 20 10 30
285.8 -j 14.2 273.2 ~- 19.2 276.3 ::k 23.7 268.5 :[ 17.0
Picrotoxin
(0.05 tzg) Unoperated Implanted Saline
(1) Ten subjects were used for each group. (2) All groups were significantly different (P <.- 0.01) from the picrotoxin (0.05/~g)-treated group shown in Table I, which was injected 5 min post-learning.
623 process 22, and suggest that picrotoxin injected 5 min after learning acted selectively on memory consolidation processes rather than retrieval processes. In addition, the absence of an amnesic effect following picrotoxin injection 2 h before retention testing mitigates the possibility that picrotoxin injected into SN 5 min post-learning might have produced the observed retention deficit by causing some proactive changes in performance variables during the retention test. The present data lend support to the earlier report of Routtenberg and H olzman 35 which implicated SN in retention of learned passive avoidance behavior. Evidence in support of this possibility is found in an electrophysiological study 27 showing that these neurons exhibit short-latency learned unit responses with conditioning. A lesion study z5 has also implicated SN as one of the neural substrates concerned with avoidance learning. Since picrotoxin injected into SN blocks the GABA-mediated inputs from neostriatum 16 and globus pallidus12, 24 to SN, it is possible that this blockade interfered with the physiological activity in those pathways originating in1°,13,z°,21 or terminating onlZ, 3° SN which are involved in the mediation of the avoidance retention. It is also possible that activation of SN neurons as a result of the blockade of inhibitory GABA action, might have affected, transsynaptically, the memory consolidation process occurring following learning in certain nigral projection areas. Of particular interest in this regard is to note that several studies have demonstrated retention disruption by either electricalal,2s, 3s or chemical v,14 manipulation of neostriatum shortly following learning. Furthermore, it has been reported that, during or following learning, alterations of electrophysiologica137 and chemical34, 36 events occur in caudate nucleus. Whichever mechanism underlies the observed retention deficit, the abundance ofdopaminergic cells in SN 2 suggests that the dopaminergic projection pathways originating in SNI,13, z°, particularly the nigroneostriatal bundle, may have played a role in the observed disruptive effects of picrotoxin injected into this region. It should be pointed out, however, that presumed GABA-containing fibers terminate on both dopaminergic and non-dopaminergic cells in SN v~,16,24. Accordingly, non-dopaminergic systems, such as the nigrothalamic projection21, 31, which could have been influenced by picrotoxin injected into SN, should also be given consideration. In contrast to the present finding, it has been reported that post-trial peripheral injection of picrotoxin facilitates avoidance learning 3. This finding is not necessarily inconsistent with the results obtained in the present study, since in the case of peripheral injection other brain structures as well as SN, which are innervated by GABA-mediated fibers, would be affected by injected picrotoxin. This research was supported by N I M H Grant MH 25281, N1H Grant NS HL 10768, NSF Grant BMS74-19481, and by The Alfred P. Sloan Foundation to A. Routtenberg. Gratitude is expressed to David Miskit for assistance and to Betty Wells for preparation of the manuscript.
624 1 Anden, N. E., Carlsson, A., Dahlstr6m, A., Fuxe, K., Hillarp, N. A. and Larsson, K., l)emolastration and mapping out of nigro-neostriatal dopamine neurons, Life Sci., 3 (1964) 523-~53~. 2 BjiSrklund, A. and Lindvall, O., Dopamine in dendrites of substantia nigra neurons: suggestions for a role in dendritic terminals, Bra#~ Research, 83 (1975) 531-537. 3 Breen, R. A. and McGaugh, J. L., Facilitation of maze learning with post-trial injections of picrotoxin, J. comp. physiol. Psychol., 54 (1961) 498-501. 4 Crossman, A. R., Walker, R. J. and Woodruff, G. N., Picrotoxin antagonism of ~,-aminobutyric acid inhibitory responses and synaptic inhibition in the rat substantia nigra, Brit. J. Pharmacot.. 49 (1973) 696 698. 5 Curtis, D. R., The pharmacology of central and peripheral inhibition, Pharmacol. Rev., 15 (1963~ 333--364. 6 Curtis, D. R., H6sli, L., Johnston, G. A. R., and Johnston, 1. H., The hyperpolarization of spinal motoneurons by glycine and related amino acids, Exp. Brain Res., 5 (1968) 235- 258. 7 Deadwyler, S. A., Montgomery, D. and Wyers, E. J., Passive avoidance and carbachol excitation of the caudate nucleus, Physiol. Behav., 8 (1972) 631-635. 8 Engberg, I. and Thaller, S., On the interaction of picrotoxin with GABA and glycine in the spinal cord, Brain Research, 19 (1970) 151-154. 9 Feltz, P., 7-Aminobutyric acid and a caudato-nigral inhibition, Canad. J. Physiol. Pharmacol.. 49 (1971) 1113-1115. 10 Graybiel, A. M. and Sciascia, T. R., Origin and distribution of nigrotectal fibers in the cat. Neuroscience Abstr., 1 (1975) 174. 11 Grinberg-Zylberbaum, J., Carranza, M. B., Celzeda, G. V., Vale, T. C. and Steinberg, N. N., Caudate nucleus stimulation impairs the processes of perceptual integration, Physiol. Behav., 12 (1973) 913--918. 12 Hattori, T., Fibiger, H. C. and McGeer, P. L., Demonstration of a pallido-nigral projection innervating dopaminergic neurons, J. comp. Neurol., 162 (1975) 487-504. 13 Hattori, T., Fibiger, H. C., McGeer, P. L. and Maler, L., Analysis of the fine structure of the dopaminergic nigrostriatal projection by electron microscopic autoradiography, Exp. Neurol., 41 (1973) 599-611. 14 Haycock, J. W., Deadwyler, S. A., Sideroff, S. I. and McGaugh J. L., Retrograde amnesia and cholinergic systems in the caudate-putamen complex and dorsal hippocampus of the rat, Exp. Neurol., 41 (1973) 201-213. 15 Kesner, R. P. and Wilburn, M. W., A review of electrical stimulation of the brain in context of learning and retention, Behav. BioL, 10 (1974) 259-294. 16 Kim, J. S., Bak, I. J., Hassler, R. and Okada, Y., Role of T-aminobutyric acid (GABA) in the extrapyramidal motor system. 2. Some evidence for the existence of a type of GABA-rich strio-nigrat neurons, Exp. Brain Res., 14 (1971) 95-104. 17 Klivington, K. A., Effects of pharmacological agents on subcortical resistance shifts, Exp. Neurol., 46 (1975) 78-86. 18 Leonard, C. M., The prefrontal cortex of the rat. 1. Cortical projection of the mediodorsal nucleus. 1I. Efferent connections, Brain Research, 12 (1969) 321-343. 19 Lindvall, O. and Bj6rklund, A., The organization of the ascending catecholamine neuron system in the rat brain as revealed by the glyoxylic acid fluorescence method, Acta physiol, stand., Suppl. 412 (1974) I 45. 20 Lindvall, O., Bj6rklund, A., Moore, R. Y., and Stenevi, U., Mesencephalic dopamine neurons projecting to neocortex, Brain Research, 81 (1974) 325-331. 21 Maler, L., Fibiger, H. C. and McGeer, P. L., Demonstration of the nigrostriatal projection by silver staining after nigral injections of 6-OHDA, Exp. NeuroL, 40 (1973) 505-515. 22 McGaugh, J. L., Time-dependent processes in memory storage, Science, 153 (1966) 1351-1358. 23 McGaugh, J. L. and Herz, M. J., Memory Consolidation, Albion Company, San Francisco, Calif., 1972. 24 McGeer, P. L. and McGeer, E. G., Evidence for glutamic acid decarboxylase-containing interneurons in the neostriatum, Brain Research, 91 (1975) 331-335. 25 Mitcham, J. C. and Thomas, R. K., Effects of substantia nigra and caudate nucleus lesions on avoidance learning in rats, J. comp. physiol. Psychol., 81 (1972) 101-107. 26 Obata, K. and Highstein, S. M., Blocking by picrotoxin of both vestibular inhibition and GABA action on rabbit oculomotor neurons, Brain Research, 18 (1970) 538-541. 27 Olds, J., Disterhoft, J. F., Segal, M., Kornblith, C. L. and Hirsh, R., Learning centers of rat brain mapped by measuring latencies of conditioned unit responses, J. Neurophysiol., 35 (1972) 202. 219,
625 28 Peeke, H. V. S. and Herz, M. J., Caudate stimulation retroactively impairs complex maze learning in the rat, Science, 173 (1971) 80-82. 29 Precht, W. and Yoshida, M., Blockage of caudate evoked inhibition of neurons in the substantia nigra by picrotoxin, Brain Research, 32 (1971) 229-233. 30 Rinvik, E., The cortico-nigral projection in the cat. An experimental study with silver impregnation methods, J. comp. Neurol., 126 (1966) 241-254. 31 Rinvik, E., Demonstration of nigrothalamic connections in the cat by retrograde axonal transport of horseradish peroxidase, Brain Research, 90 (1975) 313-318. 32 Routtenberg. A., Intracranial chemical injection and behavior: a critical review, Behav. Biol~ 7 (1972) 601-641. 33 Routtenberg, A., Significance ofintracranial self-stimulation pathways for memory consolidation. In P. B. Bradley (Ed.), Methods in Brain Research, Wiley, New York, 1975. 34 Routtenberg, A., Ehrlich, Y. H. and Rabjohns, R., Effect of a training experience on phosphorylation of a specific protein in neocortical and subcortical membrane preparations, Fed. Proc., 34 (1975) 293. 35 Routtenberg, A. and Holzman, N., Memory disruption by electrical stimulation of substantia nigra, pars compacta, Science, 181 (1973) 83 86. 36 Routtenberg, A., George, D. R., Davis, L G. and Brunngraber, E., Memory consolidation and fucosylation of crude synaptosomal glycoproteins resolved by gel electrophoresis: a regional study, Behav. Biol., 12 (1974) 461475. 37 Soltysik, S., Hull, C. D., Buchwald, N. A. and Fekete, T., Single unit activity in basal ganglia of monkeys during performance of a delayed response task, Electroenceph. clin. Neurophysiol., 39 (1975) 65-78. 38 Wyers, E. J., Peeke, H. V. S., Williston, J. S. and Herz, M. J., Retroactive impairment of passive avoidance learning by stimulation of the caudate nucleus Exp. Neurol., 22 (1968) 350-366. 39 Yoshida, M. and Precht, W., Monosynaptic inhibition of neurons of the substantia nigra by caudatonigral fibers, Brain Research, 32 (1971) 225-228.
Brain Research. 113 (1976)621} 625 :c Elsevier Scientific Publishing Company, Amsterdam Printed in The Netherh~ds
Retention disruption following post-trial picrotoxin injection into the substantia nigra
H A I N G - J A KIM and A R Y E H R O U T T E N B E R G
Cresap Neuroscience Laboratory, Northwestern University, Evanston, IlL 60201 (U.S.A.) (Accepted May 18th, 1976)
Memory for simple tasks can be disrupted by treatments that alter the physiological activity of the mammalian brain shortly after learning 23. Localization of these disruptive treatment effects to particular brain regions has been achieved ~ 3 . For example, Routtenberg and Holzman 35 found that electrical stimulation of the pars compacta of substantia nigra (SN), either during learning or immediately following learning, disrupted retention performance 24 h later. The problem with ascribing the observed retention deficit to disruption of nigral neuronal activity is that stimulation of peri-nigral fibers of passage ls-~0 may also have played a role. In the present study, therefore, an intracranial chemical injection technique 32 was used, since neurons originating in SN, but not fibers of passage, could be modified by chemical agents which alter the action of a putative neurotransmitter naturally affecting these neurons. The SN receives fiber projections from neostriatum and globus paltidus 12. Some of these fibers are gamma-aminobutyric acid (GABA)-containing 16,24. These GABA-containing striatonigral fibers inhibit SN neurons4,9,ag: iontophoretic application of GABA in SN mimics the inhibition produced by electrical caudatal stimulation 9 and this inhibition is blocked by intravenous 29 or iontophoretic 4 administration ofpicrotoxim a proposed GABA-specific antagonist 8,26. It would be expected, then, that blockade of the GABA-mediated inhibitory action on SN neurons by injection of picrotoxin into SN would cause a temporary 17 alteration in the nigral neuronal activity. If such an alteration were responsible for the memory disruptive effect of electrical stimulation observed previously35, then injection of picrotoxin into SN should also produce a similar amnesic effect. The present results confirm this expectation. Male albino rats weighing 200-300 g at the time of surgery received unilateral cannula implantations aimed at the rostral SN. Tubing of 27-gauge and M-gauge was employed for the guide and injection cannula, respectively. Following a 4-day recovery period, animals were placed on the platform of a step-down passive avoidance learning apparatus, described previously aS. Animals usually descended from the platform within 15 sec. When they did so, they received 0.5 mA foot shock through the electrified grid floor until they ascended onto the platform to escape the shock. As soon as the subject
621 reached the learning criterion by remaining on the platform for 2 min without a descent, the rat was removed from the learning chamber. Approximately 5 min after learning, the animal was subjected to intranigral injection. Drugs were dissolved in 0.5 #1 of sterile saline (pH -- 5.7) and injected at the rate of 0.5/zl/2 min using a Sage syringe pump (Model 352). Retention was studied 24 h later. The criterion for perfect retention was achieved if the subject stayed on the platform for 5 rain without a descent. Any subject which descended from the platform during the 5-min retention test period was considered to have shown a retention deficit. The latency to first-descent was recorded for later statistical analysis. The preparation of histological material was the same as described previously zS. An injection site was considered a SN placement when the tip of the cannula was found either in contact with the cells in the pars compacta or in the pars reticularis of SN without contacting or piercing the cerebral peduncle situated beneath SN. Picrotoxin injected into SN at dosages of 0.01,0.05, 0.1 and 0.2/~g (pH -- 6.1-6.3) led to a decrement in the mean latencies to descent in the retention test compared to the mean descent latency for the saline control group (Table I; Newman-Keuls test, P < 0.05). The pooled frequency of picrotoxin-treated rats which showed retention disruption was higher than the combined frequency of disrupted control subjects (Z 2, P < 0.01). Of 68 picrotoxin-treated animals, 31 subjects were found to have their cannula tips either in the cerebral peduncle or penetrating its lower boundary. Only 8 of these 31 animals showed retention deficits. This ratio of disruption was not different from that obtained for the pooled control groups in Table I (Z 2, P > 0.05)" nor was the mean latency to descent for these animals (243.08 ± 27.38 sec) different from that for the implanted control group (t, P > 0.05). Injection ofpicrotoxin through a cannula ventral to SN, therefore, had no significant effect on retention performance. Intranigral injection of picrotoxin at the highest dosage (0.2/tg) occasionally produced a mild behavioral convulsion, but was less effective in inducing a retention deficit than the lower doses of picrotoxin which produced no signs of behavioral conTABLE ! E[lect o f 5 mhl post-learning injection o f pierotoxin into the substantia nigra on retention studied 24 h later Group
% disruption (number o f subjects)
Mean latency o f first descent in sec ± S.E.M.
Unoperated Implanted Saline Strychnine Picrotoxin 0.01 /~g 0.05 t~g 0.1 /tg 0.2 ttg
0 20 10 25
(10) (10) (10) (8)
300.0 -3- 0.0 259.5 ± 28.1 298.0 ± 2.1) 270.0 ~ 20.0
63 90* 89* 67
(8) (10) (9) (9)
161.9 ± 47.2 126.8 ± 38.5* 117.3 _+ 42.8* 154.0 _+_28.0
(1) All picrotoxin groups were significantly different (P < 0.05) from the saline control group. (2) *Indicates a significant difference (P < 0.05) from the strychnine-treated group.
622 vulsion. It seems unlikely, therefore, that the observed amnesic effect in picrotoxintreated animals was due to the same neurophysiological factor associated with the convulsion. Additionally, if picrotoxin injected into SN produced the observed retention deficit by inducing l o c a l electrophysiological seizures, it would be predicted that within limits the resulting amnesic effect would increase rather than decrease as the dose of picrotoxin is increased. The present results indicate, however, that this is not the case (see Table I). Circling behavior was also observed in the present experiments following intranigral injection of picrotoxin at dosages higher than 0.05/~g. It is important to note that, at the 0.01 #g dosage, picrotoxin injection did not produce any sign of circling behavior, though it produced a retention deficit. Therefore, it is unlikely that the observed retention deficit is related to circling behavior induced by picrotoxin injected into SN. When strychnine (0.125 #g/0.5 #1, pH .... 8.0), a convulsant drug 5 which is not a GABA antagonist ~;, was injected into SN, the retention performance of these animals did not differ from the other 3 control groups (Table I) in both frequency of disruption (Fisher's exact test, P :~ 0.05) and in mean descent latency (Newman-Keuls, P :,- 0.05). In addition, the performance of strychnine-treated animals during the retention test was superior to that of picrotoxin animals (see Table l). Both picrotoxin and strychnine are known to be central nervous system (CNS) stimulants 5. Therefore, the present results strongly suggest that the retention deficit observed following picrotoxin injection is due to an interaction of this drug with the GABA-mediated inputs to SN, as opposed to some non-specific stimulation of the CNS. We also examined the effect of intranigral injection of picrotoxin given 22 h after learning, i.e., 2 h before the retention test. As shown in Table 11, we found that this group did not differ from the 2-h preretention control groups. For all these 2-h preretention injection groups, the percentage of rats descending on a retention trial was no more than 30 °,~i, which is similar to the percentage disruption for those 5-rain post-learning control injection groups. The differences in the descent latencies between any two groups in Table II were not significant (Newman-Keuls, P • 0.05). These results lend support to the hypothesis that memory consolidation is a time-dependent TABLE
I1
Effect o f 22 h post-learning #~iection o f picrotoxin into the substantia nigra on retention studied 24 h after learnblg Group
% disruption
Mean first descent latency in sec ~ S.E.M.
10 20 10 30
285.8 -j 14.2 273.2 ~- 19.2 276.3 ::k 23.7 268.5 :[ 17.0
Picrotoxin
(0.05 tzg) Unoperated Implanted Saline
(1) Ten subjects were used for each group. (2) All groups were significantly different (P <.- 0.01) from the picrotoxin (0.05/~g)-treated group shown in Table I, which was injected 5 min post-learning.
623 process 22, and suggest that picrotoxin injected 5 min after learning acted selectively on memory consolidation processes rather than retrieval processes. In addition, the absence of an amnesic effect following picrotoxin injection 2 h before retention testing mitigates the possibility that picrotoxin injected into SN 5 min post-learning might have produced the observed retention deficit by causing some proactive changes in performance variables during the retention test. The present data lend support to the earlier report of Routtenberg and H olzman 35 which implicated SN in retention of learned passive avoidance behavior. Evidence in support of this possibility is found in an electrophysiological study 27 showing that these neurons exhibit short-latency learned unit responses with conditioning. A lesion study z5 has also implicated SN as one of the neural substrates concerned with avoidance learning. Since picrotoxin injected into SN blocks the GABA-mediated inputs from neostriatum 16 and globus pallidus12, 24 to SN, it is possible that this blockade interfered with the physiological activity in those pathways originating in1°,13,z°,21 or terminating onlZ, 3° SN which are involved in the mediation of the avoidance retention. It is also possible that activation of SN neurons as a result of the blockade of inhibitory GABA action, might have affected, transsynaptically, the memory consolidation process occurring following learning in certain nigral projection areas. Of particular interest in this regard is to note that several studies have demonstrated retention disruption by either electricalal,2s, 3s or chemical v,14 manipulation of neostriatum shortly following learning. Furthermore, it has been reported that, during or following learning, alterations of electrophysiologica137 and chemical34, 36 events occur in caudate nucleus. Whichever mechanism underlies the observed retention deficit, the abundance ofdopaminergic cells in SN 2 suggests that the dopaminergic projection pathways originating in SNI,13, z°, particularly the nigroneostriatal bundle, may have played a role in the observed disruptive effects of picrotoxin injected into this region. It should be pointed out, however, that presumed GABA-containing fibers terminate on both dopaminergic and non-dopaminergic cells in SN v~,16,24. Accordingly, non-dopaminergic systems, such as the nigrothalamic projection21, 31, which could have been influenced by picrotoxin injected into SN, should also be given consideration. In contrast to the present finding, it has been reported that post-trial peripheral injection of picrotoxin facilitates avoidance learning 3. This finding is not necessarily inconsistent with the results obtained in the present study, since in the case of peripheral injection other brain structures as well as SN, which are innervated by GABA-mediated fibers, would be affected by injected picrotoxin. This research was supported by N I M H Grant MH 25281, N1H Grant NS HL 10768, NSF Grant BMS74-19481, and by The Alfred P. Sloan Foundation to A. Routtenberg. Gratitude is expressed to David Miskit for assistance and to Betty Wells for preparation of the manuscript.
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