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Dendritic branching: some preliminary results of training and complexity in rat visual cortex
SHORT COMMUNICATIONS
393
Dendritic branching: some preliminary results of training and complexity in rat visual cortex It has not been until recent years that direct evidence has been produced showing that environmental complexity of stimuli can positively effect the neurohistology of the cerebral cortex. Bennett et al. 1 and Rosenzweig et al. 7 reported that an increase of environmental complexity and training (ECT condition) produced significant changes in brain weight and brain biochemistry in rats. Diamond et al. 2 have replicated these studies and have given a more detailed analysis of the histological changes in the visual and somatosensory cortex. It was suggested that these changes might have been accompanied by increased dendritic branching, although this aspect was not studied by the authors. This present communication presents evidence which clearly supports the suggestion that dendritic branching is increased in the visual cortex of ECT animals, i.e. that environmental complexity does effect the finer structure of the cerebral cortex. It must be emphasized at the onset that while the results obtained to date are clearly positive, larger samples must be tested to corroborate the results reported herein. Using Ramon-Moliner's ~ modification of the Golgi-Cox method for staining nerve processes, two separate groups of rats of the $1 strain were processed after training under ECT and isolated condition (IC) conditions. These conditions have been described in detail elsewhereS, 7. Briefly, male litter mates are separated after weaning and placed in one of two conditions: ECT and IC. ECT conditions provide communal living, daily exposure to different 'toys' or stimulus objects, exploration, and handling by the experimenter. Under 1C conditions, each animal lives in a separate cage, is not provided with stimulus variety, and receives no handling by the experimenter. Each of the two groups studied had been put through exactly the same environmental schedule, and were processed histologically in the same way. At the end of the test conditions, after about 85 days, the animals were sacrificed, each being processed individually. Following ether narcosis, each animal was decapitated, the brain carefully removed to avoid any pressure or deformation of the surface. The posterior half of the brain was separated from the remaining brain, and placed in the impregnating solution described by Ramon-Moliner~. Following his procedure, the brains were imbedded in celloidin, sectioned at 100/z perpendicular to the cortical surface, mounted, and stored. Individual stellate neurons from the second layer of the visual cortex within a 60 ° angle from the corpus callosum were selected for drawing. Using high intensity illumination and a prism mounted in a binocular microscope, each stellate cell was projected through the prism onto an 8½" x 1 I" piece of white drawing paper in a dark room. While varying the depth of focus, each neuron selected was traced onto the paper until all processes, including the axon were traced. Thus, a three-dimensional configuration was reduced to two dimensions; no correction being made to convert projected lengths to actual lengths. A stage micrometer was placed under the same lens as used for tracing, and the grid lines transferred to the paper by tracing. Using ShoWs method s of concentric shells or circles, each neuron traced was surrounded with circles until all of the dendritic Brain Research, 2 (1966) 393-396
394
SHORT COMMUNICATIONS
processes were enclosed. The circles were drawn with a compass with increasing radii of 20 # around the center of the cell body, using the traced micrometer grid lines to measure radii. Since each cell body traced measured no more than about ~" in diameter, the center could easily be chosen by inspection. Each of these shells or circles was numbered, and the number of times each shell was crossed by dendrites was counted. Table 1 summarizes the results from the first, or pilot study. The table gives the E C T - I C litter mate pairs, the number of neurons traced, mean total shell perforation, standard deviation, and test of significant differences between means, using Fisher's t-test for uncorrelated, small samples. The results illustrate that variation in litter mate pairs is high. In this first group, 4 out of 6 pairs show greater dendritic branching for the ECT member, but none of the differences are significant. In the second study, 12 pairs were processed, 3 of which were lost during era-
TABLE I PILOT STUDY SHELL PERFORATIONS FOR
Litter pair I II III IV V VI
ECT IC ECT IC ECT IC ECT 1C ECT IC ECT IC
ECT-IC
LITTER PAIRS
No. neurons Total perforations (mean)
Stand. dev.
P.
8 7 7 4 5 7 II 8 8 7 5 8
11.2 16.6 11.7 11.2 19.9 12.6 17.7 8.8 28.3 9.4 12.5 9.5
n.s.
62.0 68.8 71.6 70.0 58.6 75.5 73.3 72.3 68.6 64.3 66.8 63.0
n.s. 0.1 n.s. n.s. n.s.
bedding. Of the remaining 9 pairs, only 6 were suitable for quantification using shell perforation as the parameter. Of the other 3, however, mean values of shell perforation were available for one of the litter mates, and are included in Table II. The differences between Table I and Table lI are very striking. Four out of 5 of the full pairs show differences that are significant. The pooled ECT average is distinct from the pooled average for IC members, and this difference is significant at better than the 0.05 level. Special comment is deserved for litter pairs l, II, III, and VIII. In pairs l, I1, and VIII, the shell perforation values are high for the ECT animals. Indeed, 3 are above the pooled ECT average. In pair I1, the IC value is very low, and appraisal of the branching of the ECT member indicated that it was heavier than any of the other ECT members in this second study. It seems highly probable to the experimenter that the difference between the ECT and IC litter mates would have been highly significant had it been possible to draw unambiguously the neurons for the ECT Brain Research, 2 (1966) 393-396
395
SHORT COMMUNICATIONS
T A B L E II SECOND STUDY WITH LARGER SAMPLE~ SHOWING SHELL PERFORATIONS FOR
Litter pair I 1I 111 IV V VI VII VIII IX
ECT IC ECT IC ECT IC ECT IC ECT IC ECT IC ECT IC ECT IC ECT IC
E C T - I C LITTER PAIRS
No. neurons Total perforations (mean)
Stand. dev.
P.
8 . -8 9 -7 8 6 4 8 8 I0 7 9 -11 8
12.3
--
-13.0 14.8
? <0.05
92.5 .
. . (very h e a v y branching) 70.0 99.4 -87.5 73.7 100.0 72.0 74.1 89.0 93.8 94.8 89.4 -99.2 85.1
9.0 14.7 16.0 16.1 12.2 9.2 9.4 21.3 11.3
-> 0 . 0 5 <0.1 >0.02 <0.05 > 0.1 < 0 . 2 n.s. --
14.4 11.9
>0.02 <0.05
12.4 16.0
>0.02 <0.05
Pooled averages: ECT IC
8* 6
92.0 80.7
* N o . o f animals.
member of this pair. Since there is an element of subjective judgement in this case, a question mark has been placed along pair 1I in Table 1I. It might be mentioned, however, that this judgement was made p r i o r to knowing the identity of the members of pair 11. When pairs I1, I11, IV, V, VI, VI1, and 1X are considered, ECT's have greater values than their 1C litter pairs in 5 out of 7 cases. If pairs 1 and VIII are included on the basis of their high ECT values, dendritic branching is increased in 7 out of 9 litter pairs. The results clearly favor this inclusion as reasonable. Thus the results of the second experiment are clearly favorable to the hypothesis under test, but cannot be regarded as conclusive. Combining first and second studies, ECT's have more dendritic branching than IC litter mates in 11 out of 15 cases. Elsewhere3, 4, I have detailed experimental, ontogenetic, and comparative studies and their possible implications for relating complexity of behavior to molecular neuroanatomical configurations within an evolutionary framework. It should be added that increased dendritic branching in the ECT animals is also concordant with the biochemical changes reported by Rosenzweig et al. 7 with respect to increased acetylcholine activity (measured by cholinesterase hydrolization). Increased dendritic branching suggests more synapses, thus more acetylcholine and cholinesterase activity. In sum, the histological picture provided by Diamond et al. 2 is in agreement with those changes found from other lines of evidence. This communication provides the Brain Research, 2 (1966) 393-396
396
SHORT COMMUNI(;A]"IONS
first direct positive evidence c o n c e r n i n g dendritic branching, a finding also in agreem e n t with these other lines of evidence. The behavioral p r e p a r a t i o n of the animals and first part of the a n a t o m i c a l procedures were carried out at the University of California; this was done with s u p p o r t from U S P H S G r a n t MH 07903 to M. R. Rosenzweig, D. Krech, E. L. Bennett and M. C. D i a m o n d and from A r m y Medical Research C o n t r a c t DA 49-193MD-2329 to D. Krech, M. R. Rosenzweig, E. L. B e n n e t t and M. C. D i a m o n d . The early part of this investigation was s u p p o r t e d in part by an N . I . M . H . Postdoctoral Fellowship, I - F 2 - M H- 17,171-01. Department of Anthropology, Columbia University, New York 27, N.Y., (U.S.A.)
RALPH L. HOLLOWAY JR,
1 BENNETT,E. L., DIAMOND,M. C., KRECH,D., AND ROSENZWEIG,M. R., Chemical and anatomical plasticity of brain, Science, 146 (1964) 610-619. 2 DIAMOND, M. C., KRECH, D., AND ROSENZWEIG, M. R., The effects of an enriched environment on the histology of the rat cerebral cortex, J. eomp. Neurol., 132 (1964) 111-119. 3 HOLLOWAY,R. L., JR., Some Aspects of Quantitative Relations in the Primate Brain, Doct. Diss., University of California, Berkeley, 1964, Unpublished. 4 HOLLOWAY, R. L., JR., Cranial capacity, neural reorganization, and hominid evolution: a search for more suitable parameters, Amer. Anthropol., 68 (1966) 103-121. 5 KRECH,D., ROSENZWEIG,M. R., AND BENNETT,E. L., Effects of complex environment and blindness on rat brain, Arch. Neurol., 8 (1963) 403-412. 6 RAMON-MOUNER,E., The histology of the postcruciate gyrus in the cat. I. Quantitative studies, J. comp. Neurol., 117 (1961)43-62. 7 ROSENZWEIG,M. R., KRECH, D., BENNETT,E. L., AND DIAMOND, M. C., Effects of environmental complexity and training on brain chemistry and anatomy: a replication and extension, J. comp. physiol. Psychol., 55 0962) 429-437. 8 SHOLL, D. A., The Organization of the Cerebral Cortex, London, Methuen, 1956.
(Received July 18th, 1966)
Brain Research, 2 (1966) 393-396
393
Dendritic branching: some preliminary results of training and complexity in rat visual cortex It has not been until recent years that direct evidence has been produced showing that environmental complexity of stimuli can positively effect the neurohistology of the cerebral cortex. Bennett et al. 1 and Rosenzweig et al. 7 reported that an increase of environmental complexity and training (ECT condition) produced significant changes in brain weight and brain biochemistry in rats. Diamond et al. 2 have replicated these studies and have given a more detailed analysis of the histological changes in the visual and somatosensory cortex. It was suggested that these changes might have been accompanied by increased dendritic branching, although this aspect was not studied by the authors. This present communication presents evidence which clearly supports the suggestion that dendritic branching is increased in the visual cortex of ECT animals, i.e. that environmental complexity does effect the finer structure of the cerebral cortex. It must be emphasized at the onset that while the results obtained to date are clearly positive, larger samples must be tested to corroborate the results reported herein. Using Ramon-Moliner's ~ modification of the Golgi-Cox method for staining nerve processes, two separate groups of rats of the $1 strain were processed after training under ECT and isolated condition (IC) conditions. These conditions have been described in detail elsewhereS, 7. Briefly, male litter mates are separated after weaning and placed in one of two conditions: ECT and IC. ECT conditions provide communal living, daily exposure to different 'toys' or stimulus objects, exploration, and handling by the experimenter. Under 1C conditions, each animal lives in a separate cage, is not provided with stimulus variety, and receives no handling by the experimenter. Each of the two groups studied had been put through exactly the same environmental schedule, and were processed histologically in the same way. At the end of the test conditions, after about 85 days, the animals were sacrificed, each being processed individually. Following ether narcosis, each animal was decapitated, the brain carefully removed to avoid any pressure or deformation of the surface. The posterior half of the brain was separated from the remaining brain, and placed in the impregnating solution described by Ramon-Moliner~. Following his procedure, the brains were imbedded in celloidin, sectioned at 100/z perpendicular to the cortical surface, mounted, and stored. Individual stellate neurons from the second layer of the visual cortex within a 60 ° angle from the corpus callosum were selected for drawing. Using high intensity illumination and a prism mounted in a binocular microscope, each stellate cell was projected through the prism onto an 8½" x 1 I" piece of white drawing paper in a dark room. While varying the depth of focus, each neuron selected was traced onto the paper until all processes, including the axon were traced. Thus, a three-dimensional configuration was reduced to two dimensions; no correction being made to convert projected lengths to actual lengths. A stage micrometer was placed under the same lens as used for tracing, and the grid lines transferred to the paper by tracing. Using ShoWs method s of concentric shells or circles, each neuron traced was surrounded with circles until all of the dendritic Brain Research, 2 (1966) 393-396
394
SHORT COMMUNICATIONS
processes were enclosed. The circles were drawn with a compass with increasing radii of 20 # around the center of the cell body, using the traced micrometer grid lines to measure radii. Since each cell body traced measured no more than about ~" in diameter, the center could easily be chosen by inspection. Each of these shells or circles was numbered, and the number of times each shell was crossed by dendrites was counted. Table 1 summarizes the results from the first, or pilot study. The table gives the E C T - I C litter mate pairs, the number of neurons traced, mean total shell perforation, standard deviation, and test of significant differences between means, using Fisher's t-test for uncorrelated, small samples. The results illustrate that variation in litter mate pairs is high. In this first group, 4 out of 6 pairs show greater dendritic branching for the ECT member, but none of the differences are significant. In the second study, 12 pairs were processed, 3 of which were lost during era-
TABLE I PILOT STUDY SHELL PERFORATIONS FOR
Litter pair I II III IV V VI
ECT IC ECT IC ECT IC ECT 1C ECT IC ECT IC
ECT-IC
LITTER PAIRS
No. neurons Total perforations (mean)
Stand. dev.
P.
8 7 7 4 5 7 II 8 8 7 5 8
11.2 16.6 11.7 11.2 19.9 12.6 17.7 8.8 28.3 9.4 12.5 9.5
n.s.
62.0 68.8 71.6 70.0 58.6 75.5 73.3 72.3 68.6 64.3 66.8 63.0
n.s. 0.1 n.s. n.s. n.s.
bedding. Of the remaining 9 pairs, only 6 were suitable for quantification using shell perforation as the parameter. Of the other 3, however, mean values of shell perforation were available for one of the litter mates, and are included in Table II. The differences between Table I and Table lI are very striking. Four out of 5 of the full pairs show differences that are significant. The pooled ECT average is distinct from the pooled average for IC members, and this difference is significant at better than the 0.05 level. Special comment is deserved for litter pairs l, II, III, and VIII. In pairs l, I1, and VIII, the shell perforation values are high for the ECT animals. Indeed, 3 are above the pooled ECT average. In pair I1, the IC value is very low, and appraisal of the branching of the ECT member indicated that it was heavier than any of the other ECT members in this second study. It seems highly probable to the experimenter that the difference between the ECT and IC litter mates would have been highly significant had it been possible to draw unambiguously the neurons for the ECT Brain Research, 2 (1966) 393-396
395
SHORT COMMUNICATIONS
T A B L E II SECOND STUDY WITH LARGER SAMPLE~ SHOWING SHELL PERFORATIONS FOR
Litter pair I 1I 111 IV V VI VII VIII IX
ECT IC ECT IC ECT IC ECT IC ECT IC ECT IC ECT IC ECT IC ECT IC
E C T - I C LITTER PAIRS
No. neurons Total perforations (mean)
Stand. dev.
P.
8 . -8 9 -7 8 6 4 8 8 I0 7 9 -11 8
12.3
--
-13.0 14.8
? <0.05
92.5 .
. . (very h e a v y branching) 70.0 99.4 -87.5 73.7 100.0 72.0 74.1 89.0 93.8 94.8 89.4 -99.2 85.1
9.0 14.7 16.0 16.1 12.2 9.2 9.4 21.3 11.3
-> 0 . 0 5 <0.1 >0.02 <0.05 > 0.1 < 0 . 2 n.s. --
14.4 11.9
>0.02 <0.05
12.4 16.0
>0.02 <0.05
Pooled averages: ECT IC
8* 6
92.0 80.7
* N o . o f animals.
member of this pair. Since there is an element of subjective judgement in this case, a question mark has been placed along pair 1I in Table 1I. It might be mentioned, however, that this judgement was made p r i o r to knowing the identity of the members of pair 11. When pairs I1, I11, IV, V, VI, VI1, and 1X are considered, ECT's have greater values than their 1C litter pairs in 5 out of 7 cases. If pairs 1 and VIII are included on the basis of their high ECT values, dendritic branching is increased in 7 out of 9 litter pairs. The results clearly favor this inclusion as reasonable. Thus the results of the second experiment are clearly favorable to the hypothesis under test, but cannot be regarded as conclusive. Combining first and second studies, ECT's have more dendritic branching than IC litter mates in 11 out of 15 cases. Elsewhere3, 4, I have detailed experimental, ontogenetic, and comparative studies and their possible implications for relating complexity of behavior to molecular neuroanatomical configurations within an evolutionary framework. It should be added that increased dendritic branching in the ECT animals is also concordant with the biochemical changes reported by Rosenzweig et al. 7 with respect to increased acetylcholine activity (measured by cholinesterase hydrolization). Increased dendritic branching suggests more synapses, thus more acetylcholine and cholinesterase activity. In sum, the histological picture provided by Diamond et al. 2 is in agreement with those changes found from other lines of evidence. This communication provides the Brain Research, 2 (1966) 393-396
396
SHORT COMMUNI(;A]"IONS
first direct positive evidence c o n c e r n i n g dendritic branching, a finding also in agreem e n t with these other lines of evidence. The behavioral p r e p a r a t i o n of the animals and first part of the a n a t o m i c a l procedures were carried out at the University of California; this was done with s u p p o r t from U S P H S G r a n t MH 07903 to M. R. Rosenzweig, D. Krech, E. L. Bennett and M. C. D i a m o n d and from A r m y Medical Research C o n t r a c t DA 49-193MD-2329 to D. Krech, M. R. Rosenzweig, E. L. B e n n e t t and M. C. D i a m o n d . The early part of this investigation was s u p p o r t e d in part by an N . I . M . H . Postdoctoral Fellowship, I - F 2 - M H- 17,171-01. Department of Anthropology, Columbia University, New York 27, N.Y., (U.S.A.)
RALPH L. HOLLOWAY JR,
1 BENNETT,E. L., DIAMOND,M. C., KRECH,D., AND ROSENZWEIG,M. R., Chemical and anatomical plasticity of brain, Science, 146 (1964) 610-619. 2 DIAMOND, M. C., KRECH, D., AND ROSENZWEIG, M. R., The effects of an enriched environment on the histology of the rat cerebral cortex, J. eomp. Neurol., 132 (1964) 111-119. 3 HOLLOWAY,R. L., JR., Some Aspects of Quantitative Relations in the Primate Brain, Doct. Diss., University of California, Berkeley, 1964, Unpublished. 4 HOLLOWAY, R. L., JR., Cranial capacity, neural reorganization, and hominid evolution: a search for more suitable parameters, Amer. Anthropol., 68 (1966) 103-121. 5 KRECH,D., ROSENZWEIG,M. R., AND BENNETT,E. L., Effects of complex environment and blindness on rat brain, Arch. Neurol., 8 (1963) 403-412. 6 RAMON-MOUNER,E., The histology of the postcruciate gyrus in the cat. I. Quantitative studies, J. comp. Neurol., 117 (1961)43-62. 7 ROSENZWEIG,M. R., KRECH, D., BENNETT,E. L., AND DIAMOND, M. C., Effects of environmental complexity and training on brain chemistry and anatomy: a replication and extension, J. comp. physiol. Psychol., 55 0962) 429-437. 8 SHOLL, D. A., The Organization of the Cerebral Cortex, London, Methuen, 1956.
(Received July 18th, 1966)
Brain Research, 2 (1966) 393-396