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Rate and extent of recovery from dark rearing in the visual cortex of the mouse
BRAIN RESEARCH
1
Research Reports
R A T E A N D E X T E N T OF RECOVERY F R O M D A R K R E A R I N G IN T H E VISUAL C O R T E X OF T H E MOUSE
F. VALVERDE Secci6n de Neuroanatomia Comparada, lnstituto Cajal, Madrid (Spain)
(Accepted April 7th, 1971)
INTRODUCTION Mice raised in complete darkness from birth show marked diminution of the number of dendritic spines in the apical shafts of layer V pyramidal cells of the visual cortex 15. The loss of dendritic spines is greatest during the first week after the spontaneous opening of the eyes20. Mice kept in complete darkness for longer periods of time also exhibit a statistically significant diminution of the number of spines with respect to normal mates of the same age 11. From these studies we have concluded that visual deprivation might have some effects on the spines, some of which would not grow in absence of normal visual inputs 11. The above results aroused our interest in whether or not these effects are permanent or to what extent the number of dendritic spines could reach normal values in mice raised in complete darkness but subsequently placed under normal conditions for various periods of time. MATERIALAND METHODS The present observations are based on the data obtained from 156 brains from a closed colony of black mice of an inbred strain (C57BL/6J). The mice were divided into the following 3 groups. (1) Eighty-four were raised under normal conditions exposed to ordinary day-night light variations. (2) Forty-seven were raised in complete darkness from birth to various ages and killed immediately for study after removal from the dark chamber. (3) Twenty-five were raised in darkness from birth to 20 days and then placed under normal conditions where they were allowed to live for additional periods of 1, 2, 4, 6, 10 or 30 days. The brains were stained by the rapid Golgi method as described elsewhere 17. Sections 150-200 # m in thickness were cut in the coronal plane and mounted serially. The number of dendritic spines was counted in one standard segment measuring 100 Brain Research, 33 (1971) 1-11
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Fig. 1. A, Mean number of dendritic spines per segment in apical dendrites of layer V pyramidal cells of the visual cortex as a function of age. The number of animals from which the mean values were obtained for each age group is indicated below the horizontal axis. B, First derivative function indicating the rate of increase of the number of spines in an arbitrary scale as a function of age. Geometrical reconstructions from fitted curves of A. C, The location of the standard segment in which the number of spines has been counted on each apical dendrite.
/ , m in each apical dendrite o f the layer V pyramidal cells o f the visual cortex in not less than 20 different dendrites for each brain. Part o f the present data includes values o f spine counts carried out previously in normal and dark-raised mice for the study o f the dendritic spine distribution11, 20. Brain Research, 33 (1971) 1-11
RECOVERY FROM DARK REARING
3
RESULTS
In previous studies we had found that the mean number of spines per consecutive segments of 50 #m in length along the apical dendrites increased exponentially with higher values in the third and fourth segmentsz0. In the present study these portions of the apical dendrite were considered as a single segment 100 #m in length beginning 100 #m apart from the origin of the apical dendrite in the pyramidal cell body (Fig. 1C). This segment, located in layer IV of the area striata, represents the standard segment of apical dendrites whose spines were counted to collect the data of the present analysis. Spine counts were carried out to obtain the mean values and standard deviation for each age group. In Fig. 1A the mean number of dendritic spines of the standard segment is plotted as a function of age in normal, dark-raised and darkness-light animals. The number of animals, usually one or two litters from which each mean value was obtained, is indicated in 3 horizontal rows of figures below the horizontal axis. The sequence of mean values for normal mice (Fig. 1A, open circles) followed a skew S-shaped curve. The number of spines increased slowly during the first postnatal week. There was a sharp increase around the time before and after the normal opening of the eyes (point indicated by an arrow between the 13th and 14th days) until the 20th postnatal day when the increase of spines continued steadily to maturity. The increase of the number of dendritic spines in the third postnatal week seemed most related to the opening of the eyes. For instance, two litters, consisting of 6 and 2 mice respectively that had not opened their eyes by the time they were killed (14 days and 12 h, and 16 days) gave lower mean values than other normal animals of similar age. The corresponding mean values of these 2 litters are indicated by 2 arrows in Fig. 1A. The mean values of animals that had been maintained in complete darkness since birth (Fig. 1A, solid circles) followed the same sequence as normal animals until approximately day 15 (just after normal opening of the eyes), where the mean values of spines in the standard segment stabilized until day 25. Thereafter the number of spines increased at a slow rate towards normal values which, however, they never reached. In fact the difference was still significant in animals that had been maintained in darkness for 6 months in comparison with normal mice of the same age 11,21 when tests were used to compare statistically the entire distribution of dendritic spines along the apical dendrites. The comparison of the sequence of mean values between normal and dark-raised mice shows that the difference was greater at the end of the third week. Therefore it was decided to raise the third group of animals in darkness until the 20th postnatal day and leave them to live under normal conditions for additional periods of time up to a total of 50 days. The sequence of mean values for this third group (half solid circles in Fig. 1A) shows that the mean number of dendritic spines reached nearly normal values in about one week after the animals were taken from the dark chamber. The comparison of means (t-test) shows statistically significant differences Brain Research, 33 (1971) 1-11
4
F. VALVI!RI)t~
between normal and dark-raised mice from day 19 to day 36. No significant differences were found before the 19th day and at 50 days. The difference was significant between the darkness-light group and the normal group at 24 days but not thereafter. The graph of Fig. IB shows, on an arbitrary scale, the increase of the number of spines in the 3 groups of normal, dark-raised and darkness-light animals as a function of age, calculated by using the first derivatives of fitted curves of Fig. IA. The rapid increase of the number of spines during the first two postnatal weeks in normal mice (Fig. 1B, continuous line) reached a peak at the ]3th-14th day, coinciding with the normal opening of the eyes. From this point the rate of increase fell off rapidly to become stabilized after the 30th day. The arrest of dendritic spine growth in dark-raised mice after the 14th postnatal day is reflected in the corresponding geometric first derivative (Fig. 1B, dashed line) by a vertical descent. A slight increase of the rate of spine growth is represented by a following wave with a very slow time course from 20 days onwards. The time course of the rate of increase of the number of spines in the darknesslight group after the 20th day, when the animals were placed under normal conditions (Fig. I B, dotted line), reached a peak 4-5 days later, with characteristics similar to those of the first peak for normal animals, but not so high. Thus, the entire time course of the rate of increase of the number of spines in the darkness-light group can be considered as follows: first it will coincide (Fig. 1B) with the continuous line from the origin of coordinates to reach the first peak at the 14th day; it will then fall off, as in the dashed line, to the base line between day 15 to day 20, finally to follow from day 20 the dotted line of the second peak. The preceding observations suggest the existence of two different populations of dendritic spines which might be related to two functionally different types of pyramidal ceils. The first would be represented by the spines that develop in absence of normal visual inputs (first peak in Fig. I B); the second would be those requiring the influence of light stimuli to grow normally (second peak in Fig. 1B). The slow increase of the number of spines in the dark-raised group after the 20th day, however, indicates that a third group of spines might grow in total darkness depending upon the existence of other unknown factors. Functionally different types of dendritic spines associated with the existence of different populations of pyramidal cells are further illustrated in the study of histograms showing the relative frequency of individual standard segments. From a large series of histograms made in each of the 35 age groups in the 3 conditions studied, we have selected the 8 histograms corresponding to 13, 24 and 50 days old as the most representative ones (Fig. 2). The histograms corresponding to mice 13 days old (Fig. 2A) show a normally distributed population of segments for both control and dark-raised groups with no significant difference between their corresponding mean values. The group of normal mice 24 days old (Fig. 2B) also shows a symmetrical frequency distribution, and so apparently does the 24 days dark-raised group. Here, however, the difference between both groups is most significant. Mice kept in darkness for 20 days and subsequently placed under normal condiBrain Research, 33 (1971) 1-11
RECOVERY FROM DARK REARING
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Fig. 2. Frequency histograms of dendritic spines per segment in 3 representative age groups of normal (line), dark-raised (solid black), and darkness-light (hatched) mice. A, 13 days old; B, 24 days old; C, 50 days old. The mean value (solid black triangles) and i S.D. (open triangles) are indicated. tions for 4 days (Fig. 2B, hatched histogram) showed a distribution that clearly indicates the existence of at least two populations of segments in different pyramidal cells: those that we assumed to have recovered a normal number of spines (having a mode at 80), and those that retained a low number o f spines (having a mode at 28). The third group of histograms, corresponding to animals 50 days old (Fig. 2C), shows that, while normal mice present a normally distributed population of segments, both dark-raised mice and the 20-30 days darkness-light group show an asymmetrical Brain Research, 33 (1971) 1-11
6
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Fig. 3. Mouse M429. Dark-raised for 20 days since birth and allowed to live for 2 days under normal conditions. Two categories of dendritic segments were projected on the surface of the right hemisphere. The area containing segments with less than 40 spines nearly coincides with the boundaries of the visual cortex. The asterisk indicates the region of defective impregnation. Arrow A points to the rostral limit of the staining brain block from which the 41 serial sections used in this reconstruction were obtained. Arrow B indicates the dorsal edge of the corpus call•sum.
distribution towards lower ranges of the number of spines. They suggest that most pyramidal cells might have recuperated to nearly normal spine numbers, but still others remained with low spine density. Those histograms that most clearly showed two different populations of segments from different pyramidal cells corresponded to animals of the third group that were allowed to live under normal conditions for I, 2 and 4 days. Yet we have no way of judging the limits of populations of segments in older animals of the darkness and darkness-light groups. The sampling of data is biased, apart from understandable subjective factors and the difficulties of collecting a large number of spine counts, by the presence of overlapping populations of segments with normal dendritic spines, those affected by light deprivation and those having recuperated. On account of these limitations it should be noted that the mean values of the darkness and darkness-light groups in Fig. 1A were obtained from the entire population of segments. For the above reasons, no attempt was made to differentiate any populations other than those clearly separated. Therefore the mean values in these groups should be interpreted only as estimates of the general trend followed by the spine outgrowth as a function of age. From the data obtained in some animals of the darkness-light group it has been possible to make reconstructions like that illustrated in Fig. 3. The brain of M429, a Brain Research, 33 (1971) 1-11
RECOVERY FROM DARK REARING
7
Fig. 4. Representative examples of apical dendrites of the visual cortex in mice raised in darkness for 20 days and allowed to live under normal conditions for several days. A, From a mouse allowed to live for 2 days after dark rearing showing 'normal' (left) and 'deprived' (right) pyramidal cells. B, From the same mouse. Pyramidal cell of layer V showing basal dendrites (arrows) devoid of spines. C, From a mouse allowed to live for 2 days after dark rearing showing a segment almost deprived of dendritic spines (left) and a segment with normal spine density (right). D, Segment of apical dendrite from a mouse allowed to live 4 days under normal conditions after dark rearing. E, Segment of apical dendrite from a mouse allowed to live for 10 days under normal conditions after dark rearing, Scale in C is valid for D and E.
Brain Research, 33 (1971) 1-11
t . VAI.VI RI)I
mouse that was allowed to live for two days under normal conditions after 20 days o1 dark rearing, showed a complete Golgi impregnation of two clearly different types of pyramidal cells (Fig. 4A, C). Pyramidal cells having apical shafts with less than 40 spines per standard segment were projected on the three-dimensional reconstruction oF the surface of the right hemisphere as solid circles (Fig. 3); apical shafts with more than 60 spines per standard segment were drawn as open circles. By this recording we found that pyramidal cells having less than 40 spines per segment occupied an area that nearly corresponded with the extension of the visual cortex according to the most usual maps of the visual area in lissencephalic mammals. The proportion of solid circles is about twice the number of open circles included within the striate cortex outline. This suggests that about one-third of the pyramidal ceils recuperated a normal number of spines after two days of exposure to light. Fig. 4D and E illustrate two segments of apical dendrites which we estimated to be representative of the spine density in mice killed 4 and 10 days, respectively, after being 20 days in darkness since birth. The absence of dendritic spines in mice raised in darkness is demonstrable not only along the entire apical shaft (Fig. 4A, pyramidal cell at right) but also in the basal dendrites which in many cases appear almost devoid of spines (Fig. 4B). DISCUSSION
Our present observations bring the following results for discussion. Firstly, the existence of a potential condition for a rapid recovery of the number of spines in pyramidal cells of the visual cortex in mice raised in darkness but subsequently placed under normal conditions. Second, the possible existence of two populations of pyramidal cells, one consisting of those able to react to light stimuli by virtue of the just-mentioned potential reserve, and the other including pyramidal cells that seem to remain permanently damaged or with little recuperation of spines. In relation to the first condition our results clearly indicate that the rapid growing of spines is triggered by the arrival of normal visual inputs. If light stimuli are not allowed to act, as occurs in mice reared in total darkness, the mean number of dendritic spines will never reach normal values, even though there is some recuperation of spines. The latter might depend upon spontaneous retinal activity existing in dark-raised mice reported to be capable of stimulating slow postnatal differentiation of the visual centers 7. We ignore the question whether or not this potential condition to recuperate dendritic spines is permanent or whether it decreases with longer periods of dark rearing. Additional series of experiments to check for spine recuperation, starting for instance 30 days after dark rearing, might lead to false interpretations since the spontaneous recuperation of spines, which seems to occur in dark-raised mice, will conceal the results. The existence of two groups of apical dendrites, possibly associated with different populations of pyramidal cells, containing few, and a normal number of spines, suggests a situation in which one finds pyramidal cells having different rates of spine Brain Research, 33 (1971) 1-11
RECOVERY FROM DARK REARING
9
recovery. This was particularly evident in the frst days of exposure of dark-raised animals to light. In 50-day-old mice of the darkness and darkness-light groups it was interesting to find the occurrence of a 'remaining' population of pyramidal cells with low spine density. This confirms the findings of Wiesel and Hubel ~3 that in certain circumstances some part of the deficiency produced by sensory deprivation becomes irreversible through the loss of potentiality to establish proper neuronal connections when the time to do it is past. Globus and Scheibel 6 and Jones and Powell 9 have postulated that specific thalamo-cortical afferents end on certain dendrites or dendritic segments. In animals enucleated at birth, loss of spines on apical dendrites is more pronounced at the level of layer IV and adjoining regions of layer 1116, 16 suggesting that dendritic spines at this level might be affected transneuronally. However, even though early studies of Walberg 2~ and Colonnied, 2 have emphasized the reaction of postsynaptic structures to degenerating terminals, Jones and Powell 8 reported that dendritic spines are apparently not affected after interruption of cortical afferents. We have discussed in previous publications the notion that sensory deprivation can lead to morphological alterations in related neural centers in terms of spine lOSS11,16,19 or of dendritic modifications12,16. It has recently been pointed out by Cowan 4 that it would be important to distinguish, whenever possible, strictly atrophic or transneuronal conditions from those leading to retardation or cessation of growth. Now it seems clear that in dark-raised mice spines are not removed secondarily to functional disuse and they will not grow in absence of normal visual inputs. This view has recently been discussed by Szentfigothai and HfimorP 3 who also showed that spine outgrowth in the lateral geniculate bodies of dark-raised dogs depends on specific function. According to our observations, cortical afferent fibers do not seem to select any specific dendrite or cell type12,18,20. Therefore we believe that the growing arrest of spines cannot be imposed by direct effects of particular domains of afferent fibers upon a given set of spines ~,10. We had previously found that the distribution of dendritic spines along the entire apical shaft is affected as a whole in dark-raised mice 20. Moreover, and as the present study indicates, the arrested growing of spines in young dark-raised mice is not only demonstrable along the entire apical dendrite, but also in the basal dendrites. Globus and Scheibel 6 and Colonnier and Rossignol ~ have suggested that different sets of dendritic spines could show discriminative functions in relation to different afferent inputs and therefore the spines could react in more or less perfect topographical image to the damage of their corresponding afferent terminals. In dark-raised mice the situation seems to be entirely different. It could be said that the pyramidal cell 'knows' somehow that it cannot, or does not, function properly, a circumstance that can lead to certain intrinsic metabolic alterations (of the proper cell) leading to arrested spine growth affecting all dendrites. The experiments of Talwar e t al. 14 and Cragg 5 on the dependence of the biosynthesis of proteins and of the structure of synapses in the visual cortex on first exposure of animals to light links extremely well with questions of this sort. Brain Research, 33 (1971) 1-11
l0
|:. VALVERD[
SUMMARY
(1) A n u m b e r o f dendritic spines (those found before opening o f eyes) in the apical dendrites o f pyramidal cells o f the visual cortex develop through the induction o f morphogenetic agencies. The growing o f these spines is not dependent on the presence or absence o f visual stimuli. (2) A second g r o u p o f dendritic spines depends on normal arrival o f visual impulses after the spontaneous opening o f the eyes. (3) If the animals are kept in darkness during the time o f eye-lid opening the second g r o u p o f spines does not develop, and/or possibly a third g r o u p o f spines grows in response to non-visual stimuli. The total n u m b e r o f spines will then never reach n o r m a l values. (4) I f the animals are removed f r o m darkness after lid opening and are allowed to live under normal conditions, a permanent numerical loss in spines persists in some o f the apical dendrites, while in others the n u m b e r o f spines reaches normal average values. (5) The recovery o f dendritic spines for some apical dendrites is noticeable two days after removal f r o m darkness. ACKNOWLEDGEMENTS This study was supported by a research grant f r o m Fundaci6n 'Eugenio Rodriguez Pascual'. This generous support is gratefully acknowledged. The a u t h o r is indebted to Miss M. L. Poves, Miss I. Alia and Mrs. Eva V. Valero for excellent technical assistance.
REFERENCES 1 COLONNIER, M., Experimental degeneration in the cerebral cortex, J. Anat. (Lond.), 98 (1964) 47-53. 2 COLONNIER,M., The structural design of the neocortex. In J. C. ECCLES(Ed.), Brain and Conscious Experience, Springer, Berlin, 1966, pp. 1-23. 3 COLONNIER, M., AND ROSSIGNOL, S., On the heterogeneity of the cerebral cortex. In H. JASPER, A. POPEANDA. WARD(Eds.), Basic Mechanisms of the Epilepsies, Little, Brown and Co., Boston, Mass., 1969, p. 29. 4 COWAN, W. M., Anterograde and retrograde transneuronal degeneration in the central and peripheral nervous system. In W. J. H. NAUTAAND S. O. E. EBBESSON(Eds.), Contemporary Research Methods in Neuroanatomy, Springer, Berlin, 1970, pp. 217-251. 5 CRAOG,B. G., Changes in visual cortex on first exposure of rats to light, Nature (Lond.), 215 (1967) 251-253. 6 GLOBUS,A., AND SCHEIBEL, A. B., Synaptic loci on visual cortical neurons of the rabbit: the specific afferent radiation, Exp. Neurol., 18 (1967) 116-13l. 7 GYLLENSTEN,L., MALFORMS,T., AND NORRLIN-GRETTVE, M. L., Visual and non-visual factors in the centripetal stimulation of postnatal growth of the visual centers in mice, J. comp. NeuroL, 131 (1967) 549-558. 8 JONES,E. G., AND POWELL, T. P. S., An electron microscopic study of terminal degeneration in the neocortex of the cat, Phil. Trans. B, 257 (1970) 29-43. 9 JONES,E. G., AND POWELL, T. P. S., An electron microscopic study of the laminar pattern and Brain Research, 33 (1971) 1-11
RECOVERY FROM DARK REARING
10 11 12 13
14
15 16 17
18 19
20 21
22 23
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mode of termination of afferent fibre pathways in the somatic sensory cortex of the cat, Phil. Trans. B, 257 (1970) 45-62. PETERS, A., AND KAISERMAN-ABRAMOF,I. R., The small pyramidal neuron of the rat cerebral cortex. The perikaryon, dendrites and spines, Amer. J. Anat., 127 (1970) 321-356. RUIZ-MARCOS, A., AND VALVERDE,F., The temporal evolution of the distribution of dendritic spines in the visual cortex of normal and dark raised mice, Exp. Brain Res., 8 (1969) 284-294. RUIZ-MARCOS, A., ANt) VALVERt)E,F., Dynamic architecture of the visual cortex, Brain Research, 19 (1970) 25-39. SZENTAGOTHAI,J., AND HAMORI, J., Growth and differentiation of synaptic structures under circumstances of deprivation of function and of distant connections. In S. H. BARONDES(Ed.), Syrup. Int. Soc. Cell Biology, Vol. 8, Cellular Dynamics of the Neuron, Academic Press, New York, 1969, pp. 301-320. TALWAR, (3. P., CrlOPRA, S. P., GOEL, B. K., AND D'MONTE, B., Correlation of the functional activity of the brain with metabolic parameters. III. Protein metabolism of the occipital cortex in relation to light stimulus, J. Neurochem., 13 (1966) 109-116. VALVERDE,F., Apical dendritic spines of the visual cortex and light deprivation in the mouse, Exp. Brain Res., 3 (1967) 337-352. VALVERt)E,F., Structural changes in the area striata of the mouse after enucleation, Exp. Brain Res., 5 (1968) 274-292. VALVERt)E,F., The (3olgi method. A tool for comparative structural analyses. In W. J. H. NAUTA ANt) S. O. E. EBBESSON(Eds.), Contemporary Research Methods in Neuroanatomy, Springer, Berlin, 1970, pp. 12-31. VAtWRt)E, F., Short axon neuronal subsystems in the visual cortex of the monkey, Int. J. Neurosci. 1 (1971) 181-197. VALVERDE,F., AND ESTEBAN, M. E., Peristriate cortex of mouse; location and the effects of enucleation on the number of dendritic spines, Brain Research, 9 (1968) 145-148. VALVERDE,F., AND RUIz-MARCOS, A., Dendritic spines in the visual cortex of the mouse. Introduction to a mathematical model, Exp. Brain Res., 8 (1969) 269-283. VALVERDE,F., AND RUIZ-MARCOS, A., The effects of sensory deprivation on dendritic spines; a mathematical model of spine distribution. In F. A. YOUNG AND D. B. LINDSLEY (Eds.), Early Experience and Visual Information Processing in Perceptual and Reading Disorders, National Academy of Sciences, Washington, 1970, pp. 261-290. WAtBERG, F., Role of normal dendrites in removal of degenerating terminal boutons, Exp. Neurol., 8 (1963) 112-124. WIESEL, T. N., AND HUBEL, D. H., Extent of recovery from the effects of visual deprivation in kittens, J. Neurophysiol., 28 (1965) 1060-1072.
Brain Research, 33 (1971) 1-11
1
Research Reports
R A T E A N D E X T E N T OF RECOVERY F R O M D A R K R E A R I N G IN T H E VISUAL C O R T E X OF T H E MOUSE
F. VALVERDE Secci6n de Neuroanatomia Comparada, lnstituto Cajal, Madrid (Spain)
(Accepted April 7th, 1971)
INTRODUCTION Mice raised in complete darkness from birth show marked diminution of the number of dendritic spines in the apical shafts of layer V pyramidal cells of the visual cortex 15. The loss of dendritic spines is greatest during the first week after the spontaneous opening of the eyes20. Mice kept in complete darkness for longer periods of time also exhibit a statistically significant diminution of the number of spines with respect to normal mates of the same age 11. From these studies we have concluded that visual deprivation might have some effects on the spines, some of which would not grow in absence of normal visual inputs 11. The above results aroused our interest in whether or not these effects are permanent or to what extent the number of dendritic spines could reach normal values in mice raised in complete darkness but subsequently placed under normal conditions for various periods of time. MATERIALAND METHODS The present observations are based on the data obtained from 156 brains from a closed colony of black mice of an inbred strain (C57BL/6J). The mice were divided into the following 3 groups. (1) Eighty-four were raised under normal conditions exposed to ordinary day-night light variations. (2) Forty-seven were raised in complete darkness from birth to various ages and killed immediately for study after removal from the dark chamber. (3) Twenty-five were raised in darkness from birth to 20 days and then placed under normal conditions where they were allowed to live for additional periods of 1, 2, 4, 6, 10 or 30 days. The brains were stained by the rapid Golgi method as described elsewhere 17. Sections 150-200 # m in thickness were cut in the coronal plane and mounted serially. The number of dendritic spines was counted in one standard segment measuring 100 Brain Research, 33 (1971) 1-11
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Fig. 1. A, Mean number of dendritic spines per segment in apical dendrites of layer V pyramidal cells of the visual cortex as a function of age. The number of animals from which the mean values were obtained for each age group is indicated below the horizontal axis. B, First derivative function indicating the rate of increase of the number of spines in an arbitrary scale as a function of age. Geometrical reconstructions from fitted curves of A. C, The location of the standard segment in which the number of spines has been counted on each apical dendrite.
/ , m in each apical dendrite o f the layer V pyramidal cells o f the visual cortex in not less than 20 different dendrites for each brain. Part o f the present data includes values o f spine counts carried out previously in normal and dark-raised mice for the study o f the dendritic spine distribution11, 20. Brain Research, 33 (1971) 1-11
RECOVERY FROM DARK REARING
3
RESULTS
In previous studies we had found that the mean number of spines per consecutive segments of 50 #m in length along the apical dendrites increased exponentially with higher values in the third and fourth segmentsz0. In the present study these portions of the apical dendrite were considered as a single segment 100 #m in length beginning 100 #m apart from the origin of the apical dendrite in the pyramidal cell body (Fig. 1C). This segment, located in layer IV of the area striata, represents the standard segment of apical dendrites whose spines were counted to collect the data of the present analysis. Spine counts were carried out to obtain the mean values and standard deviation for each age group. In Fig. 1A the mean number of dendritic spines of the standard segment is plotted as a function of age in normal, dark-raised and darkness-light animals. The number of animals, usually one or two litters from which each mean value was obtained, is indicated in 3 horizontal rows of figures below the horizontal axis. The sequence of mean values for normal mice (Fig. 1A, open circles) followed a skew S-shaped curve. The number of spines increased slowly during the first postnatal week. There was a sharp increase around the time before and after the normal opening of the eyes (point indicated by an arrow between the 13th and 14th days) until the 20th postnatal day when the increase of spines continued steadily to maturity. The increase of the number of dendritic spines in the third postnatal week seemed most related to the opening of the eyes. For instance, two litters, consisting of 6 and 2 mice respectively that had not opened their eyes by the time they were killed (14 days and 12 h, and 16 days) gave lower mean values than other normal animals of similar age. The corresponding mean values of these 2 litters are indicated by 2 arrows in Fig. 1A. The mean values of animals that had been maintained in complete darkness since birth (Fig. 1A, solid circles) followed the same sequence as normal animals until approximately day 15 (just after normal opening of the eyes), where the mean values of spines in the standard segment stabilized until day 25. Thereafter the number of spines increased at a slow rate towards normal values which, however, they never reached. In fact the difference was still significant in animals that had been maintained in darkness for 6 months in comparison with normal mice of the same age 11,21 when tests were used to compare statistically the entire distribution of dendritic spines along the apical dendrites. The comparison of the sequence of mean values between normal and dark-raised mice shows that the difference was greater at the end of the third week. Therefore it was decided to raise the third group of animals in darkness until the 20th postnatal day and leave them to live under normal conditions for additional periods of time up to a total of 50 days. The sequence of mean values for this third group (half solid circles in Fig. 1A) shows that the mean number of dendritic spines reached nearly normal values in about one week after the animals were taken from the dark chamber. The comparison of means (t-test) shows statistically significant differences Brain Research, 33 (1971) 1-11
4
F. VALVI!RI)t~
between normal and dark-raised mice from day 19 to day 36. No significant differences were found before the 19th day and at 50 days. The difference was significant between the darkness-light group and the normal group at 24 days but not thereafter. The graph of Fig. IB shows, on an arbitrary scale, the increase of the number of spines in the 3 groups of normal, dark-raised and darkness-light animals as a function of age, calculated by using the first derivatives of fitted curves of Fig. IA. The rapid increase of the number of spines during the first two postnatal weeks in normal mice (Fig. 1B, continuous line) reached a peak at the ]3th-14th day, coinciding with the normal opening of the eyes. From this point the rate of increase fell off rapidly to become stabilized after the 30th day. The arrest of dendritic spine growth in dark-raised mice after the 14th postnatal day is reflected in the corresponding geometric first derivative (Fig. 1B, dashed line) by a vertical descent. A slight increase of the rate of spine growth is represented by a following wave with a very slow time course from 20 days onwards. The time course of the rate of increase of the number of spines in the darknesslight group after the 20th day, when the animals were placed under normal conditions (Fig. I B, dotted line), reached a peak 4-5 days later, with characteristics similar to those of the first peak for normal animals, but not so high. Thus, the entire time course of the rate of increase of the number of spines in the darkness-light group can be considered as follows: first it will coincide (Fig. 1B) with the continuous line from the origin of coordinates to reach the first peak at the 14th day; it will then fall off, as in the dashed line, to the base line between day 15 to day 20, finally to follow from day 20 the dotted line of the second peak. The preceding observations suggest the existence of two different populations of dendritic spines which might be related to two functionally different types of pyramidal ceils. The first would be represented by the spines that develop in absence of normal visual inputs (first peak in Fig. I B); the second would be those requiring the influence of light stimuli to grow normally (second peak in Fig. 1B). The slow increase of the number of spines in the dark-raised group after the 20th day, however, indicates that a third group of spines might grow in total darkness depending upon the existence of other unknown factors. Functionally different types of dendritic spines associated with the existence of different populations of pyramidal cells are further illustrated in the study of histograms showing the relative frequency of individual standard segments. From a large series of histograms made in each of the 35 age groups in the 3 conditions studied, we have selected the 8 histograms corresponding to 13, 24 and 50 days old as the most representative ones (Fig. 2). The histograms corresponding to mice 13 days old (Fig. 2A) show a normally distributed population of segments for both control and dark-raised groups with no significant difference between their corresponding mean values. The group of normal mice 24 days old (Fig. 2B) also shows a symmetrical frequency distribution, and so apparently does the 24 days dark-raised group. Here, however, the difference between both groups is most significant. Mice kept in darkness for 20 days and subsequently placed under normal condiBrain Research, 33 (1971) 1-11
RECOVERY FROM DARK REARING
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Fig. 2. Frequency histograms of dendritic spines per segment in 3 representative age groups of normal (line), dark-raised (solid black), and darkness-light (hatched) mice. A, 13 days old; B, 24 days old; C, 50 days old. The mean value (solid black triangles) and i S.D. (open triangles) are indicated. tions for 4 days (Fig. 2B, hatched histogram) showed a distribution that clearly indicates the existence of at least two populations of segments in different pyramidal cells: those that we assumed to have recovered a normal number of spines (having a mode at 80), and those that retained a low number o f spines (having a mode at 28). The third group of histograms, corresponding to animals 50 days old (Fig. 2C), shows that, while normal mice present a normally distributed population of segments, both dark-raised mice and the 20-30 days darkness-light group show an asymmetrical Brain Research, 33 (1971) 1-11
6
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Fig. 3. Mouse M429. Dark-raised for 20 days since birth and allowed to live for 2 days under normal conditions. Two categories of dendritic segments were projected on the surface of the right hemisphere. The area containing segments with less than 40 spines nearly coincides with the boundaries of the visual cortex. The asterisk indicates the region of defective impregnation. Arrow A points to the rostral limit of the staining brain block from which the 41 serial sections used in this reconstruction were obtained. Arrow B indicates the dorsal edge of the corpus call•sum.
distribution towards lower ranges of the number of spines. They suggest that most pyramidal cells might have recuperated to nearly normal spine numbers, but still others remained with low spine density. Those histograms that most clearly showed two different populations of segments from different pyramidal cells corresponded to animals of the third group that were allowed to live under normal conditions for I, 2 and 4 days. Yet we have no way of judging the limits of populations of segments in older animals of the darkness and darkness-light groups. The sampling of data is biased, apart from understandable subjective factors and the difficulties of collecting a large number of spine counts, by the presence of overlapping populations of segments with normal dendritic spines, those affected by light deprivation and those having recuperated. On account of these limitations it should be noted that the mean values of the darkness and darkness-light groups in Fig. 1A were obtained from the entire population of segments. For the above reasons, no attempt was made to differentiate any populations other than those clearly separated. Therefore the mean values in these groups should be interpreted only as estimates of the general trend followed by the spine outgrowth as a function of age. From the data obtained in some animals of the darkness-light group it has been possible to make reconstructions like that illustrated in Fig. 3. The brain of M429, a Brain Research, 33 (1971) 1-11
RECOVERY FROM DARK REARING
7
Fig. 4. Representative examples of apical dendrites of the visual cortex in mice raised in darkness for 20 days and allowed to live under normal conditions for several days. A, From a mouse allowed to live for 2 days after dark rearing showing 'normal' (left) and 'deprived' (right) pyramidal cells. B, From the same mouse. Pyramidal cell of layer V showing basal dendrites (arrows) devoid of spines. C, From a mouse allowed to live for 2 days after dark rearing showing a segment almost deprived of dendritic spines (left) and a segment with normal spine density (right). D, Segment of apical dendrite from a mouse allowed to live 4 days under normal conditions after dark rearing. E, Segment of apical dendrite from a mouse allowed to live for 10 days under normal conditions after dark rearing, Scale in C is valid for D and E.
Brain Research, 33 (1971) 1-11
t . VAI.VI RI)I
mouse that was allowed to live for two days under normal conditions after 20 days o1 dark rearing, showed a complete Golgi impregnation of two clearly different types of pyramidal cells (Fig. 4A, C). Pyramidal cells having apical shafts with less than 40 spines per standard segment were projected on the three-dimensional reconstruction oF the surface of the right hemisphere as solid circles (Fig. 3); apical shafts with more than 60 spines per standard segment were drawn as open circles. By this recording we found that pyramidal cells having less than 40 spines per segment occupied an area that nearly corresponded with the extension of the visual cortex according to the most usual maps of the visual area in lissencephalic mammals. The proportion of solid circles is about twice the number of open circles included within the striate cortex outline. This suggests that about one-third of the pyramidal ceils recuperated a normal number of spines after two days of exposure to light. Fig. 4D and E illustrate two segments of apical dendrites which we estimated to be representative of the spine density in mice killed 4 and 10 days, respectively, after being 20 days in darkness since birth. The absence of dendritic spines in mice raised in darkness is demonstrable not only along the entire apical shaft (Fig. 4A, pyramidal cell at right) but also in the basal dendrites which in many cases appear almost devoid of spines (Fig. 4B). DISCUSSION
Our present observations bring the following results for discussion. Firstly, the existence of a potential condition for a rapid recovery of the number of spines in pyramidal cells of the visual cortex in mice raised in darkness but subsequently placed under normal conditions. Second, the possible existence of two populations of pyramidal cells, one consisting of those able to react to light stimuli by virtue of the just-mentioned potential reserve, and the other including pyramidal cells that seem to remain permanently damaged or with little recuperation of spines. In relation to the first condition our results clearly indicate that the rapid growing of spines is triggered by the arrival of normal visual inputs. If light stimuli are not allowed to act, as occurs in mice reared in total darkness, the mean number of dendritic spines will never reach normal values, even though there is some recuperation of spines. The latter might depend upon spontaneous retinal activity existing in dark-raised mice reported to be capable of stimulating slow postnatal differentiation of the visual centers 7. We ignore the question whether or not this potential condition to recuperate dendritic spines is permanent or whether it decreases with longer periods of dark rearing. Additional series of experiments to check for spine recuperation, starting for instance 30 days after dark rearing, might lead to false interpretations since the spontaneous recuperation of spines, which seems to occur in dark-raised mice, will conceal the results. The existence of two groups of apical dendrites, possibly associated with different populations of pyramidal cells, containing few, and a normal number of spines, suggests a situation in which one finds pyramidal cells having different rates of spine Brain Research, 33 (1971) 1-11
RECOVERY FROM DARK REARING
9
recovery. This was particularly evident in the frst days of exposure of dark-raised animals to light. In 50-day-old mice of the darkness and darkness-light groups it was interesting to find the occurrence of a 'remaining' population of pyramidal cells with low spine density. This confirms the findings of Wiesel and Hubel ~3 that in certain circumstances some part of the deficiency produced by sensory deprivation becomes irreversible through the loss of potentiality to establish proper neuronal connections when the time to do it is past. Globus and Scheibel 6 and Jones and Powell 9 have postulated that specific thalamo-cortical afferents end on certain dendrites or dendritic segments. In animals enucleated at birth, loss of spines on apical dendrites is more pronounced at the level of layer IV and adjoining regions of layer 1116, 16 suggesting that dendritic spines at this level might be affected transneuronally. However, even though early studies of Walberg 2~ and Colonnied, 2 have emphasized the reaction of postsynaptic structures to degenerating terminals, Jones and Powell 8 reported that dendritic spines are apparently not affected after interruption of cortical afferents. We have discussed in previous publications the notion that sensory deprivation can lead to morphological alterations in related neural centers in terms of spine lOSS11,16,19 or of dendritic modifications12,16. It has recently been pointed out by Cowan 4 that it would be important to distinguish, whenever possible, strictly atrophic or transneuronal conditions from those leading to retardation or cessation of growth. Now it seems clear that in dark-raised mice spines are not removed secondarily to functional disuse and they will not grow in absence of normal visual inputs. This view has recently been discussed by Szentfigothai and HfimorP 3 who also showed that spine outgrowth in the lateral geniculate bodies of dark-raised dogs depends on specific function. According to our observations, cortical afferent fibers do not seem to select any specific dendrite or cell type12,18,20. Therefore we believe that the growing arrest of spines cannot be imposed by direct effects of particular domains of afferent fibers upon a given set of spines ~,10. We had previously found that the distribution of dendritic spines along the entire apical shaft is affected as a whole in dark-raised mice 20. Moreover, and as the present study indicates, the arrested growing of spines in young dark-raised mice is not only demonstrable along the entire apical dendrite, but also in the basal dendrites. Globus and Scheibel 6 and Colonnier and Rossignol ~ have suggested that different sets of dendritic spines could show discriminative functions in relation to different afferent inputs and therefore the spines could react in more or less perfect topographical image to the damage of their corresponding afferent terminals. In dark-raised mice the situation seems to be entirely different. It could be said that the pyramidal cell 'knows' somehow that it cannot, or does not, function properly, a circumstance that can lead to certain intrinsic metabolic alterations (of the proper cell) leading to arrested spine growth affecting all dendrites. The experiments of Talwar e t al. 14 and Cragg 5 on the dependence of the biosynthesis of proteins and of the structure of synapses in the visual cortex on first exposure of animals to light links extremely well with questions of this sort. Brain Research, 33 (1971) 1-11
l0
|:. VALVERD[
SUMMARY
(1) A n u m b e r o f dendritic spines (those found before opening o f eyes) in the apical dendrites o f pyramidal cells o f the visual cortex develop through the induction o f morphogenetic agencies. The growing o f these spines is not dependent on the presence or absence o f visual stimuli. (2) A second g r o u p o f dendritic spines depends on normal arrival o f visual impulses after the spontaneous opening o f the eyes. (3) If the animals are kept in darkness during the time o f eye-lid opening the second g r o u p o f spines does not develop, and/or possibly a third g r o u p o f spines grows in response to non-visual stimuli. The total n u m b e r o f spines will then never reach n o r m a l values. (4) I f the animals are removed f r o m darkness after lid opening and are allowed to live under normal conditions, a permanent numerical loss in spines persists in some o f the apical dendrites, while in others the n u m b e r o f spines reaches normal average values. (5) The recovery o f dendritic spines for some apical dendrites is noticeable two days after removal f r o m darkness. ACKNOWLEDGEMENTS This study was supported by a research grant f r o m Fundaci6n 'Eugenio Rodriguez Pascual'. This generous support is gratefully acknowledged. The a u t h o r is indebted to Miss M. L. Poves, Miss I. Alia and Mrs. Eva V. Valero for excellent technical assistance.
REFERENCES 1 COLONNIER, M., Experimental degeneration in the cerebral cortex, J. Anat. (Lond.), 98 (1964) 47-53. 2 COLONNIER,M., The structural design of the neocortex. In J. C. ECCLES(Ed.), Brain and Conscious Experience, Springer, Berlin, 1966, pp. 1-23. 3 COLONNIER, M., AND ROSSIGNOL, S., On the heterogeneity of the cerebral cortex. In H. JASPER, A. POPEANDA. WARD(Eds.), Basic Mechanisms of the Epilepsies, Little, Brown and Co., Boston, Mass., 1969, p. 29. 4 COWAN, W. M., Anterograde and retrograde transneuronal degeneration in the central and peripheral nervous system. In W. J. H. NAUTAAND S. O. E. EBBESSON(Eds.), Contemporary Research Methods in Neuroanatomy, Springer, Berlin, 1970, pp. 217-251. 5 CRAOG,B. G., Changes in visual cortex on first exposure of rats to light, Nature (Lond.), 215 (1967) 251-253. 6 GLOBUS,A., AND SCHEIBEL, A. B., Synaptic loci on visual cortical neurons of the rabbit: the specific afferent radiation, Exp. Neurol., 18 (1967) 116-13l. 7 GYLLENSTEN,L., MALFORMS,T., AND NORRLIN-GRETTVE, M. L., Visual and non-visual factors in the centripetal stimulation of postnatal growth of the visual centers in mice, J. comp. NeuroL, 131 (1967) 549-558. 8 JONES,E. G., AND POWELL, T. P. S., An electron microscopic study of terminal degeneration in the neocortex of the cat, Phil. Trans. B, 257 (1970) 29-43. 9 JONES,E. G., AND POWELL, T. P. S., An electron microscopic study of the laminar pattern and Brain Research, 33 (1971) 1-11
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10 11 12 13
14
15 16 17
18 19
20 21
22 23
1l
mode of termination of afferent fibre pathways in the somatic sensory cortex of the cat, Phil. Trans. B, 257 (1970) 45-62. PETERS, A., AND KAISERMAN-ABRAMOF,I. R., The small pyramidal neuron of the rat cerebral cortex. The perikaryon, dendrites and spines, Amer. J. Anat., 127 (1970) 321-356. RUIZ-MARCOS, A., AND VALVERDE,F., The temporal evolution of the distribution of dendritic spines in the visual cortex of normal and dark raised mice, Exp. Brain Res., 8 (1969) 284-294. RUIZ-MARCOS, A., ANt) VALVERt)E,F., Dynamic architecture of the visual cortex, Brain Research, 19 (1970) 25-39. SZENTAGOTHAI,J., AND HAMORI, J., Growth and differentiation of synaptic structures under circumstances of deprivation of function and of distant connections. In S. H. BARONDES(Ed.), Syrup. Int. Soc. Cell Biology, Vol. 8, Cellular Dynamics of the Neuron, Academic Press, New York, 1969, pp. 301-320. TALWAR, (3. P., CrlOPRA, S. P., GOEL, B. K., AND D'MONTE, B., Correlation of the functional activity of the brain with metabolic parameters. III. Protein metabolism of the occipital cortex in relation to light stimulus, J. Neurochem., 13 (1966) 109-116. VALVERDE,F., Apical dendritic spines of the visual cortex and light deprivation in the mouse, Exp. Brain Res., 3 (1967) 337-352. VALVERt)E,F., Structural changes in the area striata of the mouse after enucleation, Exp. Brain Res., 5 (1968) 274-292. VALVERt)E,F., The (3olgi method. A tool for comparative structural analyses. In W. J. H. NAUTA ANt) S. O. E. EBBESSON(Eds.), Contemporary Research Methods in Neuroanatomy, Springer, Berlin, 1970, pp. 12-31. VAtWRt)E, F., Short axon neuronal subsystems in the visual cortex of the monkey, Int. J. Neurosci. 1 (1971) 181-197. VALVERDE,F., AND ESTEBAN, M. E., Peristriate cortex of mouse; location and the effects of enucleation on the number of dendritic spines, Brain Research, 9 (1968) 145-148. VALVERDE,F., AND RUIz-MARCOS, A., Dendritic spines in the visual cortex of the mouse. Introduction to a mathematical model, Exp. Brain Res., 8 (1969) 269-283. VALVERDE,F., AND RUIZ-MARCOS, A., The effects of sensory deprivation on dendritic spines; a mathematical model of spine distribution. In F. A. YOUNG AND D. B. LINDSLEY (Eds.), Early Experience and Visual Information Processing in Perceptual and Reading Disorders, National Academy of Sciences, Washington, 1970, pp. 261-290. WAtBERG, F., Role of normal dendrites in removal of degenerating terminal boutons, Exp. Neurol., 8 (1963) 112-124. WIESEL, T. N., AND HUBEL, D. H., Extent of recovery from the effects of visual deprivation in kittens, J. Neurophysiol., 28 (1965) 1060-1072.
Brain Research, 33 (1971) 1-11