Synaptic contact curvature: effects of differential rearing on rat occipital cortex

Developmental Brain Research, 4 (1982) 253-257

253

Elsevier Biomedical Press

Synaptic contact curvature: effects of differential rearing on rat occipital cortex JANICE M. WESA, FEN-LEI F. CHANG, WILLIAM T. GREENOUGH* and ROGER W. WEST Departments of Psychology and Anatomical Sciences and Neural and Behavioral Biology Program, University of lllinois at Urbana-Champaign, IL 61820 (U.S.A.) and ( R. IV. W.) Department of Psychology, Memorial University, St. Johns, Nfld (Canada)

(Accepted February 2nd, 1982) Key words: synaptic curvature - - enriched environment - - synaptic efficacy - -

electron microscopy - - rat - - visual cortex

Synaptic contact curvature has been proposed as an index of synaptic efficacyor maturity. In this report, members of 11 triplet sets of hooded rats were reared for 30 days postweaning in environmental complexity (EC), social cages (SC), or isolation cages (IC). Synapses from EC visual cortex showed the greatest presynaptic concavity while the IC visual cortical synapses showed the least. This result both suggests that curvature may reflect a functional aspect of the synapse and raises the possibility that other measures, such as synaptic size and density could be confounded by differential curvature. A number of structural characteristics of synapses have been proposed as potential substrates of efficacy change. Parameters such as synaptic size, number of vesicles and sub-synaptic plate perforations have been suggested because they differ in animals given differential experience7,9,12,ts, ~2-24. Others, such as structure of paramembrane densities and invaginations of presynaptic by postsynaptic processes, have yet to receive this sort of support in most cases, but remain attractive because they change with development or because they conceptually fit with current theory regarding structure-function relationships 14,t°,ll,2L Recently, Dyson and Jones 11 proposed that the curvature of the synaptic contact might be a structural index of synaptic efficacy or maturity, since they found a developmental trend from presynaptically convex (termed 'negative' by Dyson and Jones) to increasing proportions of flat and presynaptically concave (termed 'positive') contacts in rat cortex. Rats reared in environmental complexity (EC) differ in both behavior and a number of brain measures from those reared in social cages (SC) or in-

dividual cages (IC). In addition to being thicker and heavier 20 the occipital cortex contains more glial cells, more extensively branched dendritic fields, larger synapses, and a higher frequency of subsynaptic plate perforationsS,9,14,15,24. These differences presumably reflect differences in the opportunities the environments offer for sensory, motor and social activity, and associated information processing and storage, mediated via different neuronal activity. Thus differential rearing provides one way of assessing whether a given brain parameter is affected by a procedure known to affect other brain and behavioral measures. As a test of the Dyson and Jones 11 proposal, we have, therefore, measured the curvature of synapses in the occipital cortex of differentially reared rats. Twelve littermate triplet sets of male Long-Evans hooded rats (descendants of stock from Simonsen Laboratories, Gilroy, CA) were assigned at weaning (22-25 days of age) to 3 different environments such that each set had a representative in each condition. The EC rats were housed in a group of 12 in a large cage (80 × 80 × 90 cm) in which a new set of toys was

* To whom reprint requests should be addressed at: Department of Psychology, 603 E. Daniel St., University of Illinois, Champaign, IL 61820, U.S.A. 0165-3806/82/0000-0000/$02.75 © Elsevier Biomedical Press

254 arranged each day, and additionally the group was allowed 30 min exploration in different toy-equipped 1.2 sq.m field. IC rats were housed in individual standard sheet metal laboratory cages (20 × 25 × 20 cm). SC rats were housed in pairs in similar laboratory cages (20 × 25 x 40 cm). After 30 days, all rats were anesthetized with sodium pentobarbital 72-79 mg/kg) and perfused intracardially with 15 ml of 0.54 ~ dextrose in 0. I M sodium phosphate buffer at pH 7.4 followed by 150 ml of 0 . 5 ~ glutaraldehyde and 4 ~ paraformaldehyde in the same bufferdextrose (BD) solution (both at room temperature). A 1 sq. mm column of occipital cortex, 2 mm lateral to the midline and 2 mm anterior to the posterior pole with one corner cut for orientation (area 17 of Krieg 17) was dissected and immersed in 4 °C perfusate (30 min), transferred to 4 °C 2 ~ OsO4 in BD (2 h), then warmed to room temperature in BD (30 min), then dehydrated through increasing concentrations of ethanol and propylene oxide and embedded in Epon. One littermate set was eliminated due to inadequate quality of tissue fixation; thus 33 subjects (11 sets) were used. The block was trimmed to a very narrow (approximately 0.2 ram) coronal plane face extending through layers I to VI. When silver-grey sections were obtained and successfully mounted on 400 mesh grids, an adjacent 2 /~m section was taken, mounted on a glass slide, stained with para-phenyene diamine, and used, along with a wider, pretrimming coronal 2/~m section, to determine the position of cortical layers. Ultrathin sections were stained with lead citrate/uranyl acetatO 9. Eight micrographs were taken and printed at a final magnification of 41,800 × from each of layers I, II1, and IV semirandomly by spinning the stage controls to any stopping point within the layer boundaries and taking micrographs unless the frame fell upon a soma or blood vessel. The micrographs were coded to prevent experimenter bias, and all transversely sectioned, complete synapses which had a clear presynaptic membrane, cleft, and postsynaptic membrane were measured. Curvature of the postsynaptic membrane coincident with the region containing the postsynaptic thickening (PST) was assessed on coded micrographs (to prevent experimenter bias) using a summagraphics data tablet interfaced to an LSI 11/02 computer. The computer calculated a curvature

measure which was the inverse radius of the circle passing through the points at the ends of the PST and the point in between which was most distant from a straight line (chord) between the end points using the formula of Cooke et al. 6 : r -- a/2 ~ b2/8a and I/r ~- 8a/(4a 2 + b 2) (see Fig. 1B). The direction of curvature (presynaptically convex or concave) was recorded separately (Fig. 1). Synapses with subsynaptic plate perforations (SSPPs), defined as discontinuities in the postsynaptic thickening of 0.05 /,m or longer is were analyzed separately. The individual segments of the postsynaptic thickenings of SSPP synapses were also analyzed. Dyson and Jones (ref. I l) did not include SSPP synapses or their components in their measures. Synapses with complex curvatures (e.g. those with curvature in one direction and then the other along the PST; invaginations of the presynaptic component by the postsynaptic process) were excluded from both non-SSPP and SSPP synapse calculations. All data analyses were performed using the analysis of variance and related programs in the SOUPAC package of the University of Illinois Digital Computer Laboratory. Synapses were treated as independent measures nested within Jitter × treatment 7": layer. Results for non-SSPP and SSPP synapses are presented in Table I. Curvature of non-SSPP synapses differed significantly across differential rearing conditions (F(2,2635) -- 3.05; P < 0.05). ECs showed the greatest presynaptically concave curvature and ICs the least. Posthoc Newman-Keuls tests indicated that only the difference between EC and IC was statistically significant. There was no significant difference across cortical layers. Considerable variance in curvature occurred across littermate sets (F(10,2635) ~ 9.16; P < 0.001). There was also a significant treatment × litter interaction (F(20,2635) ~: 7.67; P <~ 0.001). Within littermate sets, however, EC rats' values (in unweighted means across layers) exceeded the others in the majority of cases (8/11 EC IC; 7/11 EC ~> SC)while SC vs IC comparisons were more even (6/11 SC > IC). For SSPP synapses, individual values were highly variable, and none of the main effects or interactions were statistically significant. The components of these synapses were presynaptically convex for all groups. The trend was for EC components to be less presynaptically convex than SC and, in turn, SC less

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Curvature Fig. 1. Micrographs showing presynaptically convex (A), and presynaptically concave (B) synapses. In the Cooke et al. 6 terminology, A is negatively curved, and B is positively curved. In A the calibration bar is 0.2 pm for both micrographs; in B, the lengths used for calculating radius of curvature are also illustrated; A is the distance from the most distant point on the postsynaptic membrane to the line connecting the membrane at the two end points of the postsynaptic thickening (the chord), and B is the chord length. The inverse of the radius is presented in Table I, such that a larger number indicates greater curvature. C: percentage distribution of synapses falling into equal interval curvature categories for EC and IC subjects. Zero is fiat, towards right, increasing presynaptically concave curvature; towards left, increasing presynaptically convex curvature. TABLE I M e a n curvatures (inverse radius) o f n o n - S S P P and S S P P synapses, -4- standard errors o f the mean

All curvatures are presynaptically concave, as in Fig. lB. Units are/~m -1. Numbers in parentheses are the total number of synapses measured for each mean. Non-SSPP

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EC

1.02 :~ 0.12 (931)

0.52 ± 0.18 (95)

SC IC

0.82 q- 0.15 (902) 0.74 4- 0.15 (901) P < 0.05

0.97 -4- 0.28 (82) 0.57 4- 0.27 (53) N.S.

p r e s y n a p t i c a l l y convex t h a n IC, b u t this was n o t statistically significant (F(2,829) = 1.55; P ~ 0.10). Fig. 1C shows the percentage distributions o f E C a n d I C n o n - S S P P synapses b y c u r v a t u r e categories

c o m p a r a b l e to those presented by D y s o n a n d Jones (ref. 11) b u t using an equal interval scale. The curvature differences between these groups reflect a relatively general tendency t h r o u g h o u t the distribution, r a t h e r t h a n a sharp increase in the frequency o f presynaptically concave synapses in the E C group. There are 2 possible sources o f artifact in the d a t a indicating differential p r e s y n a p t i c concavity. First, the e l i m i n a t i o n o f SSPP synapses c o u l d be r e m o v i n g a larger n u m b e r o f less p r e s y n a p t i c a l l y concave synapses f r o m the E C p o p u l a t i o n , since this type o f synapse occurs m o r e frequently in E C rats 15. W e therefore p e r f o r m e d an anlysis on the c o m b i n e d SSPP a n d n o n - S S P P categories, (still excluding those for which n o single curvature could be determined, the n u m b e r o f which was c o m p a r a b l e a m o n g groups). T h e g r o u p means r e m a i n e d similar to those

256 in Table I, although the greater variability of the SSPP synapse curvatures reduced statistical significance (F(2,2865) = 2.72; P < 0.07). Thus curvature differences are not an artifact of differential exclusion of synapses. Second, since PSTs in some layers are of different size in EC and IC ratsg, 24, it is possible that unreliable curvature measurement of different numbers of smaller synapses could differentially affect the groups. We therefore removed from the nonSSPP population all synapses with a chord length (Fig. I B) of less than 10 measured m m (at 41,800 ×, approximately 0.24/~m). Differences between groups remained comparable to those in Table I, with the same pattern of statistical significance (F(2,1206) = 5.06; P < 0.01 ; Newman-Keuls tests indicated that EC was different from both SC and IC with P < 0.05). We therefore conclude that there is a general tendency for non-SSPP synapses to be more presynaptically concave in animals reared in more complex environments, regardless of their size. We have confirmed the Dyson and Jones 11 report that smaller synapses are more curved (chord length × absolute value of curvature r == --0.21, P 0.01). In contrast to Dyson and Jones 1~, our curvature distribution (Fig. 1C) had relatively more flat synapses (the distribution for layer I only which was Dyson and Jones' sampling region was similar to the distribution in Fig. 1C). Possible reasons for this discrepancy might be either that we were more conservative in judging whether a synapse was curved, or that there were variations due to different fixation and dehydration procedures. We also found that the individual components of SSPP synapses tended to be presynaptically convex. Average curvatures of these components were --0.54 for EC, --0.94 for SC and --0.96 for IC (where negative sign indicates presynaptic convexity). This finding offers an intriguing explanation for the differences found between resuits using osmicated tissue and EPTA stained material. Jones and Devon ~6 found that the curvature measures of tissue from animals treated with different pentobarbitone doses were consistently more

negative from the EPTA material and did not offer any explanation. It is possible that the curvature measures from the EPTA material also included the SSPP components, since the membranes indicating synaptic continuity were not stained, and the average curvatures were moved toward greater presynaptic negativity as compared to the curvature measures from the osmicated material. This finding also points to the need for caution when comparing results from osmicated and EPTA-stained material. There remains the question of the functional meaning of synapse curvature, if any. Dyson and Jones 11 suggested that presynaptic convexity might be associated with non-functional (or less active) synapses, since synapses both in younger animals and in anesthetized animals tend toward presynaptic convexity. The relative lability of this parameter to short-term events such as anesthesia would seem to argue against its value as an indicator of long-term functional state. However, all animals in our study were anesthetized at the time of tissue fixation and experientially-associated differences were still observed. Curvature change could be a cause or consequence of other changes which increase or decrease net surface area of the synaptic contact, or could result from interactions between traditional tissue fixation procedures and some structural feature(s) of synapses. While we cannot suggest an interpretation based upon current theories of synaptic structurefunction relationships, we feel curvature should be measured in future studies of activity effects upon synapse structure both because of its potential for contributing to an understanding of plastic synaptic change and because of its potential to confound other measures, such as of size and density of synapses (see Greenough and ChanglZ).

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3 Bailey, C. H., Thompson, E. B., Castellucci, V. F. and Kandel, E. R., Ultrastructure of the synapses of sensory neurons that mediate the gill withdrawal reflex in Aplysia, J. NeuroeytoL, 8 (1979) 415-444. 4 Bloom, F. E., The formation of synaptic junctions in developing rat brain. In G. D. Pappas and D. P. Purpura (Eds.), Structure and Fanction of Synapses, Raven Press,

This study was supported by N S F BNS 77-23660. We thank T. Blaise Fleischmann and Timothy J. DeVoogd for histological assistance and Harris D. Schwark for comments on the manuscript.

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