Relationship of afferentation with soma size of nucleus gracilis neurons after bilateral dorsal column lesion in the rat

EXPERIMENTAL

NEUROLOGY

Relationship Gracilis

71,452-463

(1%:)

of Afferentation with Soma Size of Nucleus Neurons after Bilateral Dorsal Column Lesion in the Rat

JERALD

J. BERNSTEIN

AND DONALD

GANCHROW’

Laboratory of Central Nervous System Injury and Regeneration, V. A. Medical 50 Irving Street, Washington, D.C. 20422, and Department of Neurosurgery Physiology, George Washington School of Medicine, Washington, D.C. 20006; and the Department of Anatomy and Embryology, Hebrew University-Hadassah Medical School, P.O. Box 1172, Jerusalem, Israel Received

November

28, 1979; revision

received

July

Center, and

7. 1980

The effect of bilateral dorsal column lesion on the afferentation and diameter of neurons in the nucleus gracilis (NG) ofthe rat was studied during 120 postoperative days. After bilateral, low thoracic lesion of the dorsal column, the animals were killed 1.2,3,7,14,30,45,60,90, or 120dayslater, andcells in the rostral, middle, or caudal NG were selected and measured for soma diameter and number of boutons on these same somas. The resutts showed significant reduction in soma diameter during the first 2 weeks postoperatively compared with normals, and also significant changes (increases or decreases in diameter) in survival times during 120 postoperative days. Soma diameter and number of boutons on the soma were positively correlated at all NG levels and survival times. Extrapolations of those values to total surface area and total surface area of soma covered by boutons, show that (i) neither normal nor deafferented soma are totally innervated, and (ii) normal innervation values are maintained in parallel with fluctuations in the total surface area of the soma. These results emphasize the dynamic changes occurring upon and in the postsynaptic cell after deafferentation, and imply a mechanism which regulates the interaction of quantity of presynaptic afferent fibers upon, and size of, the postsynaptic cell body. Abbreviations: DPO-days postoperative; NG-nucleus gracilis; SD, BD-somal, bouton diameter; TSA, total surface area of the soma; TBA, total bouton area on surface of soma. ’ This research was supported by a grant from the United States-Israel Binational Science Foundation (BSF), Jerusalem, Israel, and the National Institutes of Health (NINCDS, NS-16979). The authors wish to thank Dr. Judith Ganchrow for her support and critical comments during all phases of the research. 452

0014-4886/81/030452-12$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved

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INTRODUCTION The morphology of a neuron in the central nervous system may be modified by deefferentation, or deafferentation. For example, axotomy may result in somal hypertrophy [see review in (21)J as well as changes in dendritic length of the parent cell (4, 30). Eye enucleation affects cell diameter in the lateral geniculate nucleus (7, 16) and removal of mystacial vibrassal follicles modifies the morphology and organization of recipient cortical “barrel” tissue (19, 29, 33). Central deafferentation either by hemisection (1,2) or dorsal column lesion (22) was shown to affect several parameters of neuronal measurement in the motor horn and nucleus gracilis (NG), respectively; moreover, deafferented postsynaptic cells underwent fluctuations in morphology during extended postoperative periods (3). These postsynaptic neuronal changes may be a reaction to signal parallel changes in their presynaptic innervation. On the other hand, the amount of total surface membrane of neuronal soma available as synaptic sites may, in part, regulate the degree of afferentation, either in the normal or lesion condition (5). The following studies in the rat were undertaken to determine the effect of dorsal column lesions on NG soma diameter (SD) for 120 postoperative days. In addition, the degree of afferentation of the soma was correlated with the soma diameter of these same NG neurons, for this same postoperative period. Finally, extrapolations were made from the data on soma diameter and the neuron’s innervation to determine the expected relation between total surface area of a neuron and total innervation areas of the soma. These results emphasize the fact that the degree of innervation on the soma fluctuates around normal values even when the amount of afferentation parallels changes in soma diameter, and suggest a regulatory mechanism linking the quantity of presynaptic afferent fibers to the postsynaptic neuron soma. MATERIALS

AND METHODS

Injections of atropine sulfate (2 mg/kg), followed by pentobarbital sodium (78 mg/kg) were administered to each of 44 adult, male Sabra strain rats. With an operation microscope, bilateral dorsal column lesions were made with a jeweler’s forceps in the spinal cord after removing the T12 vertebral arch and incising the meninges. A single dose of penicillin G (100,000 Units) was administered postsurgery. The animals were killed 1 (N = 3), 2 (3), 3 (3), 7 (3), 14 (6), 30 (5), 45 (5), 60 (5), 90 (5), or 120 (6) days postlesion. Normal rats (N = 13) of the same age were yoked to survival times of the animals with lesions. These 57 rats were also used in a previous study (15). Anesthetized rats were killed by intracardial flushing with 0.9%

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saline followed by perfusion with 10% neutral buffered Formalin. Medullospinal tissue containing the NG was processed for the light microscopic demonstration of silver-impregnated boutons (3,23,26). In rat spinal cord, a correspondence has been shown between normalized absolute bouton counts seen by light microscopy and the frequency of presynaptic boutons seen by electron microscopy (2, 3). Tissues were sectioned at 6 pm, and slides were chosen from the rostral, middle, and caudal portions of the NG [for rationale, see (15)], and coded. Counts were made of boutons on the soma of those neurons exhibiting a prominent nucleolus and nuclear and somal membranes in the medial two-thirds of the NG, previously determined in Nauta preparations as the terminal projection field of hind limb and tail afferent fibers traveling in the dorsal columns (13). At least eight cells per NG level per animal (i.e., four cells from each side) were selected for measuring somal diameter; counts of boutons on the soma were made on those same cells. The total sample from all 57 rats included 1546 neurons. The results for boutons on the soma are detailed elsewhere (15). Diameter of the soma (SD) was defined as one-half the sum of the measured minimum and maximum diameters. The circumference (= nSD) and total surface area [TSA [=rr(SD)2]] of the soma were derived from the neuron diameter. The average diameter of a normal rat bouton (BD) was determined from measurements of boutons in published electron micrographs in rat dorsal column nuclei (31), and was calculated to be 1.25 pm. The total area of somal membrane occupied by boutons (TBA) was determined from the following relationship: Nb x BD TBA =-3 zSD Z-(SD)* and therefore, TBA = Nb x BD x SD, where Nb = number of boutons on the circumference of the soma, measured in cross section. Measures of the SD and innervation of the soma (i.e., TBA/TSA ratios), at each NG level, were statistically tested by analysis of variance (37) and Duncan’s multiple range test (10, 37). If the analysis of variance was significant, individual t tests were applied, using the within-groups mean square error term from the analysis of variance (9). Changes in SD between any two levels of the NG, and the relation of diameter and boutons on the soma, at each NG level and at various intervals during 120 postoperative days were tested by Pearson product-moment correlations (12). Previous statistical analysis showed no right- versus left-side differences with respect to the SD or number of boutons on the soma. All statistical tests are reported at one-tailed significance values.

AFFERENTATION

1

2

3

455

AND SOMA SIZE

7 14 DAYS POSTOPERATIVE

30

45

60

90120

FIG. 1. Average soma diameter of neurons in rostra1 (R), middle (M), and caudal (C) portions of nucleus gracilis for 120 days after dorsal column lesion. Normal values at each level of the nucleus gracilis are represented by horizontal lines at the right side of the figure.

RESULTS Figure 1 shows the average somal diameter (SD) by survival time of experimental and control groups determined for the three levels studied in the nucleus gracilis (NG). Tables 1 and 2 summarize the comparative analyses for those data. In the rowal NG, it is seen that already at 1 day postoperative (DPO), the SD was significantly smaller than in the normal rats, the 3 DPO rats, and all groups between 14 and 120 DPO (Fig. 1; see Table 1 for significance values). Moreover, the SD of 2- and 7-DPO animals remained significantly smaller than that of 45 and 90-DPO rats; 7-DPO animals also had a significantly smaller SD compared with 60-DPO rats (Table 1). Conversely, it was apparent that at 90 DPO, the SD was significantly larger than in the l-, 2-, 7-, and 120-DPO groups (Fig. 1 and Table 1). These results in the rostra1 NG were reflected in a significant change in the SD for all experimental and normal groups [F(10,490) = 2.32, P < 0.0251. In the middle NG, the SD was significantly

rats compared with other survival-time

smaller in 2-, 7-, and 14-DPO groups and normal controls (Fig. 1;

456

BERNSTEIN

AND GANCHROW TABLE

1

Significance Values of t-Test Comparisons of Soma Diameter for Postoperative and Normal Groups, in the Rostra/ Nucleus Gracilis DAYS POSTOPERATIVE

2

3

7

14 30 45 60 90120 1430456090120N

N

1

Nofe. Blank boxes represent comparisons which were not statistically significant.

see Table 2 for significance values). In addition, and as reported for the rostra1 NG above, the SD at 90 DPO was significantly larger than at 2,7, and 14 DPO, though not different from normals (Table 2). These results in middle NG were reflected in a significant change in the SD for all experimental and normal groups [F(10,510) = 1.85, P < 0.051. TABLE

2

Significance Values of r-Test Comparisons of Soma Diameter for Postoperative and Normal Groups, in the Middle nucleus gracilis DAYS POSTOPERATIVE

Nofe. Blank boxes represent comparisons which were not statistically significant.

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AND SOMA SIZE

700

680

620 9-N I &80

540

500

ROSTAAL

7

14

30

45 DAYS

6’0 POSTOPERATIVE

90

11

FIG. 2. Averaged total surface areas of the soma (TSA, solid line) and percentage of the total soma surface occupied by boutons (TBA/TSA, dotted line; TBA-total bouton area on the soma) for 120 days after dorsal column lesion, of neurons in the rostra1 portion of the nucleus gracilis (see Materials and Methods). Normal(N) values are designated by horizontal solid or dotted lines. Note that the normal value for TSA has been placed at the ordinate.

In the caudal NG, the SD at 7 DPO was significantly smaller than at 2 DPO (P < 0.025) and 90 DPO (P < 0.025) (Fig. 1). These relatively few changes were reflected in a nonsignificant difference for the SD for all experimental and normal groups [F(10,513) = 1.18, P > 0.251. To test whether or not changes in the SD varied similarly during 120 postoperative days, Pearson product-moment correlations were carried out between any two of the three levels of the nucleus. The results showed that the caudal-middle NG [r(8) = 0.467, P > 0.051 and caudal-rostral NG [r(8) = 0.358, P > 0.051 were not, whereas the rostral-middle NG [r(8) = 0.752, P > 0.011 were significantly correlated. Did those differences in the postsynaptic SD reflect the degree of presynaptic innervation upon these soma cells (15), for all postoperative times? Product-moment coefficients obtained for the correlation between numbers of boutons on the soma and the SD showed that the rostra1 [r(8) = 0.717, P < O.Ol], middle [r(8) = 0.587, P < 0.041, and caudal [r(8) = 0.778, P < O.OOS] levels of the NG exhibited a significant positive relation between diameter and afferentation of the soma. Because these data showed significant fluctuations in the SD among the survival times, the same relationship accrued upon extrapolation of the SD

BERNSTEIN

458

AND GANCHROW

700

540

500

MIODLE

7

14

30

45 DAYS

60 POSTOPERATIVE

90

20

151

FIG. 3. Same as in Fig. 2, of neurons in the middle portion of nucleus gracilis.

to the total surface area of the neuronal soma (see Materials and Methods). However, the degree of total innervation of the somal surface appeared to maintain itself, and to fluctuate nonsignificantly about the degree of innervation in normal animals. This relationship, of total bouton area on the soma (TBA) relative to total surface of the somal membrane (TSA) is shown in Figs. 2-4, for each level of the NG (see also Materials and Methods). The dotted line in each graph represents the TBAJTSA ratio and reveals the remarkable ability of the soma to maintain normal innervation values (73,61, and 72% in the rostral, middle, and caudal NG, respectively) in parallel with fluctuations in the TSA (continuous black line in each graph). Analyses of variance of the TBA/TSA ratio showed there were no significant differences for all experimental and normal groups in the rostra1 [F(10,46) = 0.81, P > 0.051, middle [F(10,46) = 0.97, P > 0.051, and caudal NG [F(10,46) = 1.33, P > 0.051. Multiple comparison analysis of the TBA/TSA at all NG levels showed there were no significant differences between any experimental and normal group (P > 0.05). DISCUSSION These results point to the dynamic changes in soma diameter evident at each level of the nucleus gracilis during 120 postoperative days, after deafferentation by dorsal column lesion. This is reflected by significant differences in the SD among several selected survival times as well as

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680 620 2 ;5t30 T 540

500

CAIJDAL

7

14

30

45 6’0 OAYS POSTOPERATIVE

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1'

FIG. 4. Same as in Fig. 2, of neurons in the caudul portion of nucleus gracilis.

between operated and normal animals. In general, the greatest reductions in the SD occurred within the first 7 days after low thoracic dorsal column lesion. That these changes at three levels of the NG are not necessarily occurring within the same time frame, are in part shown by the following facts: (i) reduction in the SD was not wholly coextensive during the first postoperative week; (ii) in the middle NC, the SD at 14 DPO remained smaller than that in normal and 90-DPO rats; (iii) nonsignificant correlations for changes in the SD were obtained for caudal-middle and caudal-rostra1 NG comparisons. Moreover, diameter changes apparent within 1 DPO (in the rostra1 NG) suggest that the signal of deafferentation is extremely rapid and parallels the time span within which bouton terminals may be lucent or dark and contain depleted and aggregated vesicles and abnormal mitochondrial profiles after sensory rhizotomies (24, 36); 24 h after deafferentation, attenuation of the postsynaptic wave to dorsal column stimulation occurred in the lateral cuneate nucleus (17). Similarly, SD changes in the NG during the first postoperative week after dorsal column lesion mirror the time course of a decreasing number of lemniscal terminals in the ventroposterolateral nucleus of thalamus after dorsal column nuclear lesions (31). Consistent, significant enlargement of the SD occurred at 90 DPO at all NG levels. This latter result also suggests that dynamic postsynaptic changes are in play fully 3 months after lesioning, but clearly do not occur

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in isolation from their presynaptic complement. For example, innervation of the soma also significantly increased in the caudal and middle NG at 90 DPO after dorsal column lesion (15); a similar increase in boutons on motoneuron and interneuron soma at 90 DPO occurred after hemisection (2) or axotomy (6). In general, the mutual relation of the postsynaptic cell and its afferentation is shown by significant, positive correlations between the SD and boutons on the soma at all NG levels for 120 postoperative days: that is to say, an increase or decrease in the SD is mirrored by similar changes in degree of afferentation of that same postsynaptic soma. In addition, it is clear that those postoperative times showing significant changes in the SD reported here (Tables 1, 2, and Results) may be extrapolated to estimate changes in the total surface area of the neuron (TSA) (see Materials and Methods). Similarly, those significant changes between the SD and innervation of soma may be extrapolated for the correlation between the TSA and TBA, but necessarily are higher correlations because both latter computations use the obtained values for the SD [v(8) = 0.923,0.820,0.866 in rostral, middle, and caudal NG, respectively: P < 0.005 at all NG levels]. The availability of “free” surface membrane on the soma is suggested by the subtotal innervation of soma at all survival times and in normal animals, at all levels of the NG (see TBA/TSA values in Figs. 2-4). Ultrastructural studies of the dorsal column nuclei confirm the relatively small number of axosomatic synapses per somatic profile (28, 32,35); -69% of the surface area of a ventral horn cell is covered by synaptic contacts (18). Though the number of somal boutons kept pace with changes in the SD for 120 postoperative days, as reflected by the significant positive correlations between innervation and the SD, a significant level of hyper- or hypoinnervation relative to the normal animals was never achieved; maximal (nonsignificant) differences in TBA/TSA from normal values were 12% (at 3 DPO) in rostral, 11% (at 2 DPO) in middle, and 12% (at 7 DPO) in caudal NG (Figs. 2-4). This retention of a critical percentage of cell surface occupied by boutons indicates a regulatory mechanism linking the quantity of presynaptic afferent fibers to postsynaptic neuronal soma; the percentage of nondegenerating axodendritic synapses in the reinnervated septum of the rat, after fimbrial lesion, never exceeded normal values of 62% (25). Is this critical degree of innervation set by local receptor properties of the somal membrane of the postsynaptic cell, so as to attract, or serve as targets for inputs? With this “lock-and-key” model, when the normal, local complement of afferentation is reestablished, additional innervation need not be mustered. However, what is not known is whether or not only the same areas on the soma, when denervated, reestablish their

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presynaptic connections. Several lines of evidence in sensory systems suggest that the possibility for reafferentation may best be mediated by the availability of intact, “free” membrane: (i) denuded postsynaptic sites are evident for at least 6 months after dorsal rhizotomies, in the NG (27,28); (ii) glial processes often intervene between degenerating, presynaptic boutons and the postsynaptic target cell after retrogasserian rhizotomy, in the spinal trigeminal nucleus (36); (iii) the amount of extracellular space near denuded contact sites does not exceed -0.2 pm after dorsal rhizotomies, in the lateral cuneate nucleus (24). Reatferentation after a lesion does not imply that the new synaptic contacts are permanent. Significant differences occur in innervation of somas in the NG for 120 postoperative days, after low thoracic cord lesion (15); similar innervation dynamics are reported at 90 postoperative days on motoneuron soma rostra1 to the hemisected thoracic cord (2, 3), or after axotomy (6, 34). Moreover, viewed as an inherent property of the central nervous system, the sheer ability to reinnervate after a lesion, though to levels not significantly different from normal, may not ensure restitution of function compatible with the normal operating activity of the dorsal column system. Perhaps establishing new synapses or adhesions on “free” membrane are transient events, in that it is precisely these intact areas which may lack requisite receptor properties to guarantee prolonged anatomic and functional contact. Rather, the balance among these synapses in the intact population which are functionally mediative may serve to facilitate nondisrupted behavior. For example, significant deficits in components of limb movement and position, and deficits in tactile discrimination are apparent only if about 90% of the dorsal columns is destroyed [in the cat (8); in the rat (14)]. In the study reported here, 39 of 44 rats with lesions sustained a 90 to 100% bilateral destruction of the dorsal column, yet the degree of reinnervation was not significantly different from normal. Similarly, the degree to which “normal” behavior is mainlained may require a threshold complement of synapses appropriate to the function of a nucleus or system. Rats exhibit increased deficits in hind limb position and movement (compared with prelesion and early postlesion recoveries) after as little as 50% dorsal column lesion, when tested at 90 and 120 DPO (14). This concept of critical fiber ratios and threshold for expressing and maintaining “normal” behavior has been previously, exquisitely explicated in neurobiology (11, 20). REFERENCES 1. BERNSTEIN, J. J., M. R. WELLS, AND M. E. BERNSTEIN. 1978. Spinal cord regeneration and axonal sprouting in mammals. Pages 407-420 in S. WAXMAN, Ed., Physiology and Pathobiology of Axons. Raven Press, New York.

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