Electron microscopy of the development of synaptic patterns in the ventrobasal complex of the rat

Brain Research, 135 (1977) 197-215 © Elsevier/North-Holland Biomedical Pr:,.-s

197

Research Reports

ELECTRON MICROSCOPY OF THE DEVELOPMENT OF SYNAPTIC PATTERNS IN THE VENTROBASAL COMPLEX OF THE RAT

M. A. MATTHEWS and C. L. FACIANE Department of Anatomy, Louisiana State University Medical Center, New Orleans, La. 70119 (U.S.A.)

(Accepted February 17th, 1977)

SUMMARY

Electron microscopy has been employed to analyze morphological alterations of components of synaptic complexes of the developing ventrobasal (VB) nuclear complex of the rat at 7, 12, 15, 20 and 60 days postnatal (dpn). Dendrites of immature VB ,eurons seen at 7 dr are characterized by a thick, irregular shaft with large bulbous expansions from which filopodia are elaborated. S~naptic development is minimal at this stage, but occasional boutons are found in association with both the shaft and filopodia of dendrites. By 12 dpn dendritic maturation is considerably advanced and specialized postsynaptic structures begin to appear as synaptic populations increase and diversify. Synaptic boutons form both simple (single junction) and compound (multiple junctions) complexes with dendritic elements of varying size. Lemniscal afferents can be distinguished during this period on the basis of a large presynaptic element forming multiple junctional specializations with one or more dendritic spinous protrusions and partially ensheathed with a glial lamella. With further development (15 dpn and older) termina!s of lemniscal afferents increase in size, complexity and numbers of junctional contacts with the postsynaptic component. Some populations of smaller boutons increase further in density in temporal association with the maturation of intrinsic circuitry and descending cortical and subcortical afferents34. Based upon the appositional relations and spatial arrangement of the pre- and postsynaptic members, synaptic complexes were classified and counted to analyze developmental alterations in population density, and these data, together with a qualitative analysis of synaptogenesis in the developing VB, are briefly discussed in relation t.~ somatosensory impulse propagation, local integration and putative mechanisms of somatotopy.

198

INTRODUCI~ON Patterns of somesthetic projection to the ventrobasal (VB) complex of the 'rat have been estabfished by means of electrophysiological recording of peripheral receptive fields and subsequent histological reconstructions of recording locations within the VBe,7. Lemniscal and antero-lateral terminals have been described 17,1s, and the relationship of the former to e specffic boumn type within the synaptic glomerulus is tentatively establishedse. Locations of termination of other kinds of afferent input, i.e. from cortex, the brain stem or thalamic reticular formation as, and probable recurrent collaterals from some thalamocortical relay cells, are yet to be precisely specified in relation to different types of VB neurons in the rat. However general arrangements of the synaptic organization of a variety of thalamic relay nuclei in several species have been examined and functional interpretations proposed3,S,to,x~,15,~4,~s,aT,3s.46. An analysis of synaptogenesis in the VB complex should provide valuable information concerning the appearance of different classes of boutons within an established temporal framework and in relation to particular postsynaptic components. These data, together with observations of developmental alterations in the complexity of interrelationships between the pre- and postsynaptic members, could be correlated with subsequent electrophysiologic examinations of these complex central internuncial systems in a series of neonate through increasingly more mature animals to determine the mechanisms involved in the ontogeny of specific neuronal connections and related iacremental changes in functional sophistication of a thalamic relay nucleus. The present communication will constitute a quantitative study of developmental changes in synaptic population density within the rat VB complex in association with a descriptive analysis of the sequence of development of synaptic specializations, maturation of the synaptic glomerulus, and modifications in relative proportions of specified categories of non-glomerular synaptic complexes. MATERIALS AND METHODS

(1) Animals Male and female Sprague-Dawley rats were used in this study. Variations in growth rate among different litters were minimized by rejecting litters of 5 pups or less from the study and removing pups from large litters until an optimal range of 6-8 animals was achieved.

(2) Fixation, tissue processing Groups of 5 pups taken from at least two different litters were sacrificed at postnatal ages of 7, 12, 15, 20 and 60 days for a total of 25 animals. Each pup was weighed and only those of an average weight compared with their littermates were used because disturbances of somatic development can be accompanied by alterations in CNS maturation 5. After anesthesia was induced via an intraperitoneal injection of ketamine hydrochloride (20 mg]kg), three animals from each group were perfused according to a method described by Peters 96, in which a dilute mixture containing

199 1.25 % glutaraldehyde-1% formaldehyde-0.01% CaCi2 in 0.1 M sodium cacodylate buffer is initially passed through the systemic vasculature for 15 min followed by a more concentrated solution of 5% glutaraldehyde-4% formaldehyde-0.01% CaCI2 in 0.1 M cacodylate buffer for an additional 15 min. The remaining two animals were perfused with a mixture of 4 % formaldehyde-0.5 % glutaraldehyde-0.01% CaCI2 in 0.1 M cacodylate buffer. After a 12 h "in situ" fixation, the brain was removed, the thalamus exposed and a series of coronal secticns 0.2-0.4 mm in thickness were taken through the entire thalamus. These serial sections were separated and placed in numbered vials wherein subsequent buffer washes, osmication, dehydration and Epon infiRration were carried out. Semithin 1.5 nm sections, of sufficient size to encompass the entire coronal profile of a hemithalamus, were stained with Paragon or toluidine blue and examined to verify the location of the ventrobasal complex based on landmarks given by a number of investigators4,7,~l, 13. Thin sections were stained with lead citrate 40 and aqueous uranyl acetate and examined in a Philips EM 300.

(3) Differential quantitative analysis of synaptogenesis Thin sections were taken from a selected location within VB which was comparable throughout the entire series of animals. Because the quality of fixation in the VB appeared superior in those animals sacrificed with the two stage perfusion procedure, all quantitative analyses were performed upon sections taken from this material. From such sections, 35-40 random, non-overlapping fields were photographed at an initial magnification of × 4400 and two copies of each micrograph were subsequently printed at x 12,500. Two investigators examined a copy of each field, located and classified each synaptic complex according to pre-established criteria, which will be dealt with in Results, and tabulated each type of bouton together with the number of individual junctions established by e~.ch bouton. For this quantitative analysis a clear plastic overlay, into which was etched a squared grid with divisions of 6.25 mm × 6.25 mm, was superimposed upon each print. With a print magnificat.ion of x 12,500 each division encompassed 0.25 sq.nm of tissue section. After subtracting portions of the field occupied by cell bodies, large fascicles of axons and blood vessels, the precise number of synaptic junctions within a known area of neuropil could be directly read from the print. The amount of variation between the two sets of data seldom exceeded 1-2 % thus confirming the efficacy of the classification system and method of surface area determination. To further eliminate bias resulting from variations in the surface area of neuropil depicted within each micrograph, the numerical data were corrected proportionally to reflect the number of junctions in a standardized surface area of 280 sq.nm. A total number of 11,730 synaptic junctions was recorded throughout this study within an area of 54,320 sq.nm. RESULTS

(I) Preliminary observations The ventrobasal complex receives afferent input from a variety of extrinsic sources, the most important of which inciude the ascending lemniscal and antero-

200 lateral systems together with descending fibers arising in the cerebral cortex ano nucleus reticularis thalamig.le.14,ee,eg.31.32. Add!tionally, intrinsic circuitry involved in integrative function contributes substantially to the synaptic architecture of this region of the diencephalon ae. The Scheibe!s, employing several variants of the rapid Golgi technique, have been able to identify patterns of termination of afferents from a number of specific sources a3 and found these to be present in recognizable, albeit abbreviated, form in neonatal rats a4. Lemniscal afferents are described by these investigators as initially displaying a "calyx-like terminal which, with increasing age, undergoes extension and division resulting in the appearance of distinctive bushy arbors with clusters of nodules which form the presynaptic element of the glomerular complex of VB." Antero-lateral and non-specific afferents are usually seen as smaller caliber axons displaying a diffuse branching pattern and st.-.~.il terminals. Descending afferents have distinctive preterminal branching patterns characteristic of their source and end as individual or small clusters of terminal boutons.

(11) Elimination of some interpretative errors Series of sections through the ventrobasal nucleus were carefully evaluated in an attempt to detect regional differences in the progress of development of synaptic complexes. While distinctive gradients were not obvious either in the present analysis or from analyses of Golgi impregnated VB neurons reported elsewhere ~, a wide region of the VB complex midway between its rostrocaudal extent and along the lateral border of the pars dorsomedialis was always evaluated for qualitative and quantitative descriptions in corresponding samples from all 5 groups of animals in order to minimize subtle heterogeneity in patterns of synaptic distribution which might affect subsequent quantitative classifications.

(i!!) Qualitative electron microscopy (A) Sew,nth postnatal day. The neural matrix of VB characteristically displays numerous large, pale dendritic profiles containing a few small mitochondria, tubules

Fig. I. This longitudinally sectioned dendrite displays a large bulbous dilation at one end. Clusters of polyribosomes (ps) and a few neurotubules (nt) constitute the principal organelles within this structure. A coated vesicle can be seen within the center of the large open arrow which points to an inset depicting this structure (small open arrow) at higher magnifcation. The occasional synapses (arrows) visible in the field have few vesicles and minimal densification of the apposed pre- and postsynaptic membrane, x i 1,045. Scale indicates Inm. Fig. 2. Two synaptic complexes (shown with arrows) are associated with this dendritic profile. One is located along the dilated shaft while the other engages a thin filopodium (f) arising from the main body of the dendrite at the lower left. x 8836. Scale indicates I rim. Fig. 3. Several immature synaptic profiles are shown. Some are indicated with arrows. Postsynaptic elements in 7 day VB are generally small to :aedium sized dendrites, some of which contain multivesicular bodies (mvb). x I 1,045. Scale indicates 1 rim. Fig. 4. A presumptive dendritic growth cone is enclosed within the oval. Note the large collection of vacuoles, small profiles of granular ER and mitochondria, x 13,254. Scale indicates 1 nm. Fig. 5. A postsynaptic profile (enclosed within oval) era:raining ribosome clusters (arrow) but an abundance of granular electron-dense material may represent dendritic degeneration, x 13,254. Scale indicates 1 nm.

201

202 and scattered dusters of polyribosomes seen as aggregations of from 4 to 6 individual particles (Figs. 1, 2 and 3). Metubranous cisternae of endoplasmic reticulum occur as isolated, short profiles (Fig. 2) which are often moderately dilated. Multivesicular bodies and coated vesicles30 can be found in some profiles (Fig. 1); however, morphological continuities between the latter structures and the neuronal plasma membrane, suggestive of early formation of" postsynaptic specializations in some developing neurons, were not observed. Fortuitous longitudinal sections reveal large dilations of the dendritic shaft (Fig. I) from which extend delicate branchlets (Fig. 2) undoubtedly corresponding to the filopodia described by Ram6n y Cajal 99, Morest ~°.91 and Scheibel et al. a4, as typical of immature dendrites. Synaptic complexes are infrequently encountered within the VB at 7 days postnatal. Most are characterized by small collections of spherical vesicles approximately 500 nm in diameter and generally engage an isolated dendritic profile between 1 and 3 nm in diameter (Figs. 1 and 3). Some contacts are est~tblished on terminal expansions or the filopodia of the growing dendrite (Fig. 2). Dendrite growth cones, characterized by a concentration of vacuoles of varying size, mitochondria and small profiles of granular endoplasmic reticulum, are often distributed about the neuropil (Fig. 4). These are indicative of dendritic arborization as one expression of an intermediate phase of neuronal maturation 85. Spontaneous degeneration of a significant proportion of VB neurons, together with further population dilution resulting from an increase in volume of the developing thalamus, appears to be most prominent during the period between birth and the 6th postnatal day 19. Developmental alterations in branching patterns of afferent input show a period of active growth and arborization from birth through 10 dpn followed by a secondary reshaping of resultant terminal plexuses involving selective degeneration of some branches to achieve the mature pattern 84. This process is considered to continue throughout the first postnatal month, A profile suggestive of postsynaptic degeneration is shown in Fig. 5, characterized by the accumulation of a finely granular electron-dense material within a dendritic element. However, it must be noted that Skoff and Hamburger 35 have described similar structures in embryonic chick spinal cord and consider them to be viable dendrites. Presynaptic degeneration was not detectable but, considering the limitations of sample size imposed by electron microscopy, such a random, infrequent occurrence would rarely be seen within a given section. (B) Twelflhpostnatalday.Further progression ofsynaptogenesisresultsin a much more dense population which shows a greater degree of variety and complexity. Numerous large boutons, containing a concentration of spherical vesicles and a few dense-core vesicles, become evident by this age. Many of these form multiple junctions with several individual post-synaptic profiles (Fig. 6) while larger dendritic elements engage varying numbers of separate boutons both along the shaft and spinous protrusions. A definite population of small presynaptic elements is readily observed throughout the ventrobasal complex and typically form asymmetrical junctions with a postsynapti c member less thari I nm in maximal diameter. Some large de~drites display distinctive synaptic complexes in which the pre-

203 synaptic bouton is invaginated by and participates in junctional complexes with one or two spinous protrusions emanating from the main dendrite (Fig. 9, inset). Such boutons often form additional junctions with the adjacent dendritic shaft. The spinous protrusions usually lack organelles whereas the parent dendrite may display a concentration of mitochondria, together with polyribosomes, tubular profiles and vacuoles. A few of these synaptic complexes are associated with a glial process (Fig. 9, inset) which appears to partially sheath the bouton. (C) Fifteenth postnatal day. A further increase in synaptic population density characterizes this period of development. The synaptic complexes displaying a small postsynaptic member and observed in significant numbers at 12 days postnatal are found to be much more numerous at 15 days (Fig. 10). An increased incidence of larger boutons, with several junctional specializations on a number of separate postsynaptic elements, can also be found in the VB complex during this period of development. However, the most distinctive feature of the synaptic pattern within the nucleus is the unique and complex arrangement of the synaptic glomerulus (Fig. 11), an entity found in a variety of relay nuclei and which in the rat VB complex appears to have achieved a level of maturity at 15 days of age almost comparable to that of the adult. The large presynaptic component always contains an abundance of clear spherical vesicles 40-50 nm in diameter, collections of round to oval mitochondria, and occasional dense-core vesicles. Two to 5 spinous protrusions arising from an adjacent large dendrite indent or deeply penetrate the bouton forming characteristic asymmetrical junctions with a markedly thickened postsynaptic density and a plaque of dense extracellular material within the junctional cleft. Most glomeruli are encapsulated by a multilamellated glial capsule (Fig. I l), which forms a cup-like structure about the central bouton and its associated dendritic specializations. Individual layers of the glial capsule are normally attenuated with the exception of a slightly thickened free edge somewhat remniscent of the free edge of the myelin sheath at a node of Ranvier, although the glial process forming the glomeralar capsule has been traced to a nearby astrocyte36. Boutons which exhibit an exceedingly dense population of mixed flattened and round vesicles are sometimes found in the vicinity of large glomeruli (Fig. 11). These often form an asymmetrical contact with a smooth surface of the dendrite on the side opposite to the location of the glomerulus. Because of their relative rarity, this variety of bouton was not included as a separate category in the quantitative portion of this study. (D) Twenty and 60 dpn. Analysis of the ventrobasal complex reveals few striking qualitative differences in synaptic architecture over that demonstrated at 15 dpn with the exception of the fact that many synaptic glomeruli exhibit an increased incidence of junctional complexes related to the single presynaptic element. After a careful survey of the VB throughout all postnatal ages examined in this study, presynaptic dendrites, of the type described for the dorsal lateral geniculate nucleus of several species and the ventrobasal complex of the cat, do not appear to be present in the rat VB. Such structures are known to represent a significart morphological and functional component associated with the glomerulus in other relay nuclei. Comparison of

:204

205 mature glomeruli in the rat VB with those examined in other species reveals a reduced complexity of this structure in the present model.

(IV) Quantitative electron microscopy Distinctive trends with respect to synaptic categories were apparent upon qualitative examination of electron micrographs representative of each group of animals. Therefore, rather than quantifying the data only to express developmental changes in overall synaptic population density, it was felt that a more meaningful analysis should reflect numerical alterations in specific types of complexes together with morphological changes apparent within individual synaptic units as maturation progresses. For each synapse encountered the presynaptic element was initially classified as either simple axo-dendritic, characterized by a single junctional complex or compound axo-dendritic, characterized by multiple junctional complexes, in order to account for variations in the morphology of the postsynaptic component, each initial category was further subdivided with the following result. Type ,4 - - a simple axo-dendritic complex displaying a single junction with a postsynaptic dendritic profile greater than 1 nm in largest diameter. If several individual boutons established contact with a single dendrite, each bouton was counted as a single type A complex and tabulated. Type B -a simple axo-dendritic complex which exhibited a single junction and whose postsynaptic element was less than I nm in largest diameter. Type C - - a compound axodendritic unit demonstrating two or more junctions with one dendrite. Lemniscal boutons appear to represent the majority of those complexes placed within this category. Type D - - a compout~d axo-dendritic complex displaying two or more junctions with two or more different dendritic profiles, it is acknowledged that such profiles could arise from a single parent dendrite. Several interesting trends become evident upon examination of Tables ! and l l and Fig. 12.

Fig. 6. The illustrated synaptic complexes (arrows) are characterized by multiple junctional specializations on the presynaptic component (arrows). × 11,045. Scale indicates I nm. Fig. 7. This dendrite profile (two-headed arrow) should be compared with those shown in Figs. I and 2. Note the concentration of large mitochondria and distinctive spinous protrusions (p). Two synapses can be seen on both the shaft and one protrusion. The small vertical arrow in the left of the figure denotes a symmetrical (Gray type II) junction along the shaft while the vertical arrow in the center shows an asymmetrical (Gray type I) junction with the spinous protrusion, x I 1,045. Scale indicates I nm. Fig. 8. Two presynaptic components form junctional complexes with a single protrusion (p). Note the thickened postsynaptic membrane at this point which forms a Gray type I (asymmetrical) densification of the receptive surface. An arrow indicates a small Gray type !! synapse at the dendritic shaft.

× 23,392. Scale indicates 0.5 nm. Fig. 9. An immature synaptic giomerulus is shown (upper arrow). A spinous protrusion (p) has invaginated into and formed a synaptic specialization with the bouton. A second bouton (lower arrow) lies adjacent to the shaft of:he large dendrite and engages smaller, isolated dendritic profiles. ~: 15,595. Scale indicates 1 nm. The inset illustrates the continuity between a spinous protrusion and the shaft of the dendrite. Additionally, two, symmetrical, non-synaptic filamentous specializatiops can be seen along the appositional surfaces of the presynaptic terminal and the main shaft of the dendrite (short arrows). Note also the glial process (long arrow) which partially encapsulates the presynaptic component.

206

207 TABLE

I

Mean number o f synaptic junctions within an established standard area of 280 sq.nm

A = Simple axo-dendritic; single junction with dendrite profile greater than 1 nm in largest diameter. B = Simple axo-dendritic; single junction with dendrite profile less than 1 nm in largest diameter. C = Compound axo-dendritic; multiple junction of single bouton with single dendrite (lemniscal). D ----Compound axo-dendritic; multiple junction of single bouton with different dendrites, n -- Number of electron micrographs used in sample. Age o f animal (dpn)

Total area sampled (280 n m × n)

Total junctions mean S.E.

A

B

C

D

n=40

11200

305.2 7.63 0.919

209.33 5.01 0.706

12,.53 0.31 I) 061

6.13 0.15 0.073

12

n = 39

10920

426.99 10.95 0.504

752.51 19.29 0.428

254.03 6.51 0.~.82

50.00 t .43 O.164

15

n : 40

11200

787.06 18.49 0.828

2350.29 54.84 !.388

243.46 5.68 0.731

157.60 3.68 0.473

20

n=35

9800

829.85 23.7 1.364

1465.06 41.86 1.826

159.91 4.57 0.822

176.44 5.04 0.221

60

n ::40

11200

1813.60 45.34 3.814

1514.00 37.85 2.907

177.44 4.44 0.654

47.92 1.20 0.301

7

(1) Type A complexes are initially the m o s t abundant synaptic entity observed within VB. This is not surprising in view o f the fact that a Golgi analysis of this nucleus at the same age demonstrated that most VB neurons have rele.tively short, stubby dendrites which are large in proportion to perikaryal size ~9. Electron microscopy further confirmed the preponderance of large dendritic profiles during this time of development. Type B boatons also represent a substantial proportion o f the synaptic population in part because o f specialized contacts between presynaptic elements and the filopodia o f i m m a t u r e dendrites. Fig. 10. In the upper right of the micrograph a large presynaptic element is shown to engage several, separate dendrite profiles (arrow). To the left lies a large dendrite (D) which forms synaptic contacts with three large boutons. Numerous small boutons, associated with small, individual dendritic profiles less than I nm in diameter are scattered about the neuropil. × 13,254. Scale indicates !nm. Fig. I 1. A large synaptic glomerulus is shown. Seven distinct profiles of dendritic spinous protrusions penetrate the associated bouton to varying depths. A glial sheath partially encapsulates the complex and the inset at the lower left illustrates the multilamellate nature of this sheath in some examples. Note the large boutons characterized by a dense population of mixed flattened and round ve~icles (F). Fortunate plan¢~ of section reveal these to be associated with the glomerulus. × 29,236. Scale indicates I rim. The inset at the upper right is subdivided into three panels. The upper two panels represent high magnification micrographs of F boutons. A mixed population of vesicles is evident and should be compared with the round vesicles found within most other boutons as shown in the lower panel.

2O8 20 18 . ~,,

16

o

14

m

12

m

10

"5 O ,., e-

E

8

*"

6 4

2 0

7

N

12 15 Age d Animal (Post-natal day)

JB XJ

Z0

h 60

Fig. 12. A histogram depicting the relationship between proportions of type C (lenmiscal) boutons and postnatal age. The asterisk denotes that values of lenmiscal boutons are expressed as a percentage of the total number of simple and compound boutons establishingjunctions with a single dendrite. (2) By 12 dpn, an approximate 3-fold increase in the total number of synaptic junctions is seen. Type C boutons achieve their highest mean value per 280 sq.nm (Fig. 12) while the number ofjunctions/bouton (Multiple Junctional Index (MJI)) for this type of synaptic complex is essentially unchanged (Table II). However, the value of 2.68 for 12 dpn is undoubtedly more reliable than the MJI determined for type C boutons at 7 dpn since very few of these terminals were encountered in the youngest animals. Type B complexes also display a prominent increase in population during this period possibly reflecting the initial establishment of intrinsic circuitry and the maturation of other afferent input from a variety of sources. Dramatic changes in the patterns of dendritic arborization of Golgi impregnated VB neurons cause these TABLE !! Ratio of synaptic junctions/bouton (multiple junctional index) Age of animal (dpn)

7 12 15 2O 60

C (meen S.E.)

D (mean S.E.)

2.91 i 211 2.68 0.943 2.83 0.711 4.25 0.776 5.75 0.834

2.01 1.006 2.78 0.632 2.37 0.804 2.06 0.691 2.00 0.413

209 cells to resemble adult forms even at 12 days of age. Further changes were confined to apparent increases in cell volume and subtle refinements of dendritic excrescencesX9. A spatiotemporal and functional relationship between the maturation of afferent input (particnlarly primary afferents) and concomitant rapid advances in postsynaptic dendritic development has been proposed by Moreste0.2x and seems consistent with our results. O) While populations of type C boutons remain relatively stable after 12 dpn, the MJI reflects an increasing complexity in this kind of synapse which exteads well into the young adult. Thus, lengthening and secondary branching of dendritic spinous protrusions, together with wobable sprouting of additional protrusions from the main shaft of the dendrite, may underlie the increased incidence of synaptic junctional specializations found within the type C synaptic unit at 20 and 60 dpn. Type B boutons achieve their highest population density at 15 days of age as further maturation and arborization of intrinsic neurons as well as corticofugal, and other extrinsic afferents, continues to take place. Type A boutons also graduafly increase in number with further maturation and undoubtedly also represent a significant proportion of extrinsic cortical and subcortical afferents. Following visual cortical ablation a percentage of similar synaptic profiles, engaging dendrites of small and medium diameter scattered about the extraglomerular neuropil, quickly degenerately. (4) The gradual decline in the mean number of type B boutons after 15 dpn is subject to a number of interpretations. First, the reshaping of terminal arborizations of descending thalamo-petal afferents34 may involve an outright loss of boutons of this category. However, growth and enlargement of the nucleus together with an increased incidence of interposed non-neuronal processes might also serve to dilute the synaptic population. Second, in order for a synaptic profile to be included in the counts, a recognizable pre- and postsynaptic component and a distinct synapt.ic junction was required. Anker and Cragg x have indicated that some of these features of small synapses may not be displayed in a given section thus causing a proportion to be overlooked with a resultant negative bias of the data. This bias would only be significant in determination of synaptic density/unit volume of tissue. Finally, it is likely that some profiles designated as type A boutons could in reality be a type D synapse in which additional junctions are located above or below the plane of section. Since no other criteria were found to distinguish between the two groups, developmental fluctuations in their respective values are to be cautiously interpreted.

( V) Summarization of synaptic sequences Fig. 13 is an idealized schematic representation of the sequence of development of pre- and postsynaptic components of the ventrobasal circuitry as analyzed with the electron microscope. At 7 dpn (I) most dendrites of VB neurons are short, thick appendages of irregular outline displaying scattered ribosomal clusters, short profiles of granular ER, and vesicles of varying size, some of which correspond with the coated vesicles described in detail by Rees et al.30. Thin filopodia extend primarily from the

.,b

..

~----=

.. :-

...-

....

--..~

:

~

.... ~ ~ . ' . "

~ . . ~ . :

.: ~ / : .

.L,~f"

B

•_ - ' _ . :

e

O0

•"

...-

~.

.:

0

sp

III B A

• °

,.f

D O

O

O

Cp

~o

® Fig. 13. A drawing representative of three stages of dendritic and synapti¢ maturation. I indicates a typical dendritic shaft and associated boutons at 7 dpn. 1I illustrates those dendritic and presynapti¢ elements commonly found at 12 dpn. III shows the most mature pattern occurrinB at 15 dpn and older, cv, coated vesicles; f, filopodia; mvb, multivesicular body; A, type A bouton; B, type B bouton; C, type C (lemniscal) bouton; D, type D bouton; gc, glial capsule; sp, spinous protrusion; F, bouton with mixed round and flattened vesicle.

211 bulbous tips of such dendrites although Golgi analysis19.20,21 reveals the presence of filopodia in association with dilations of the shaft. Type A and type B boutons form simple, symmetrical junctions with the shaft and filopodia of the dendrite. The analysis of dendrites at 12 dpn (ll) reveals a more mature pattern characterized by the presence of larger mitochondria, fewer vesicles and a thinner shaft of less irregular diameter interrupted by small digital processes engaging type A and B boutons. The most important observation is the appearance of the type C bouton which repre~nts the maturation of lemniscal afferents. Most of these complexes are partially ensheathed by a single-layered glial process. By 15 dpn and thereafter (lid the lemniscal type C bouton has achieved a maximal degree of morphological complexity characterized by the invagination of multiple dendritic spinous protrusions into the large presynaptic element. The associated glial capsule is composed of 2 or 3 astrocytic lamellae. Spacek and Lieberman36 have indicated that with maturation of the VB thalamocortical relay neuron, several lemniscal boutons could become functionally associated with each neuron. Type A, B and D boutons become increasingly associated with dendritic profiles as maturation occurs and various components of the intrinsic and extrinsic circuitry form their proper relationships. As stated by Spacek and Lieberman, the unusual dendro-dendritic, somato-dendritic complexes and highly elaborated synaptic glomeruli of the visual system in most mammals s and the VB complex of the cat 2s do not appear to be present in the VB of the rat. However, the large axonal bags, which display mixed populations of round and fiat vesicles and are found in the immediate vicinity of some glomeruli, may represent part of a less sophisticated inhibiting mechanism controlling signal specificity. DISCUSSION

Proper interpretation of somatosensory stimuli requires a multi-level relay system through which propagated signals are subjected to integration and modification at each stage by an increasingly complex intrinsic circuitry responsive to input from cortical and subcortical nuclear groups. Maturation of phylogeneticaily newer, long axoned circuits which link each nucleus in the chain occurs earlier in development than those neurons with exclusively local synaptic connections. Thus, the earliest cortical responses to cutaneous stimulation, which in the rat occur at 7 dpn, are carried into the VII complex by lemniscal afferents2. Although Golgi studies indicate the presence within the VB of this type of terminal in neonatal animals, the establishment of functional connections with thalamo-cortical projection neurons is delayed, possibly awaiting the maturation of post-synaptic receptive surfaces. In the present study, type A complexes, which predominate at 7 days age, must therefore include some lemniscal afferents despite the fact that features characteristic of the synaptic glomerulus (large presynaptic member, multiple junctional specializations, highly elaborated postsynaptic component and glial capsule) were almost never encountered. Instead, most synaptic complexes observed during thi~ period of development display a paucity of vesicles, minimal thickening of junctional membranes and a small area of functional

212 apposition. Armstrong-James~ has demonstrated that the nucleus gracilis of the 7 day rat can consistently follow stimulus repetition rates of 3~see, whereas, at thalamocortical levels, stimulus frequencies of greater than 5 or 6/min will result in a fatigue of this portion of the relay. Recruiting and augmenting phenomena within the Sx neocortex can be evoked by direct thalamic stimulation as early as 3 dpn 4t and will tolerate a stimulus frequency of 8-10/sec. This suggests that the limiting factor is at the thalamic level. Although these data are minimal and should be repeated in animals of a greater age range to specify developmental temporal patterns in response characteristics, the paucity and immaturity of lemniscal and other types of terminals may represent one important factor which could tentatively explain the unusually rapid decrease in transmission ability with repetitive stimulation. The 20-fold increase in type C boutons between 7 and 12 dpn and the concomitant elaboration of specialized dendritic spinous protrusions associated with initial maturation of the glomerulus would be expected to herald the appearance of powerful, short-latency EPSPs in the VB principal neurons following an afferent volley in the medial lemniscus, particularly if more than one lemniscal afferent terminated upon each VB neuron 36. With the establishment of additional junctional complexes and a definite glial capsule about each glomerulus as development proceeds, further potentiation of EPSPs to produce the characteristic repetitive discharge of principal neurons would be expected to result. Mature VB neurons have been shown to follow lemniscal volleys with external stimulus frequencies as high as 120/sec9+~. Another characteristic of the immature somatosensory system is the exceedingly large size of the peripheral receptive fields, whereas in the adult animal single columns of cortical neurons are uniquely responsive to movements of individual vibrissae45, or relatively limited cutaneous areas of the body. Such a precise somatotopy has also been demonstrated in the ventrobasal complex itself such that small, pre:ise groups of units, occupying a distinctive, cylindrical volume of VB tissuep were each activated solely by individual vibrissae and many VB units were found to respond to movement of only one hair 42.4a. Because of the similarity between VB and cortical figurines6,~,45, it seems reasonable to assume that morphological substrates for mechanisms of the nature of surround inhibition might develop in a similar temporal and spatial sequence. The relatively extensive arborization of terminals of long ascending thalamic afferents (either lemniscal or anterolaterai) in adult animals would require some means of signal modifcation to produce a precision and specificity of transmission through VB. Perl et al. ~5 tested for the mechanism of surround inhibition and found it to be present in dorsal column nuclei for ordinary hair responses. Studies addressing themselves to this question in rat VB appear to be lacking at the present time. Spacek and Lieberman a6 have postulated that lemniscal afferents synapse upon both principal (thalamocortica!) neurons and Golgi type 11 intrinsic neurons. The latter element may then postsynaptically hyperpolarize a number of principal neurons in the area thus constituting a system of feed-forward inhibition. Principal neurons receiving a smaller number of excitatory terminals for a specific lemniscal afferent would be more readily affected by this kind of system. Those boutons containing a mixed population of round and flat vesicles must

213 certainly correspond to the F-boutons described elsewhere 3~, and it seems reasonable to assume that some of these elements represent terminals of Golgi type II interneurons and would therefore mediate the inhibitory mechanism. Their proximity to the synaptic glomeruli of lemniscal afferents would offer an advantage in this regard. Cortical ablation results in the loss of only a small proportion of boutons corresponding to our type A and B categories thus, many of these must arise from thalamic intrinsic neurons or subcortical structures. It is difficult to assess the relative physiological importance of these two sources in modulating activity within the VB but it is suggested that the appearance of large numbers of type B boutons at 15 dpn and subsequent increases in population density of type A synapses could correspond with and possibly form the substrate for the physiological maturation of thalamic somatotopy. ACKNOWLEDGEMENTS We wish to thank Mrs. Yamuna Narayanan for helpful discussions concerning synaptic classification and quantitative procedures employed in this study, Mr. Garbis Kerimian, for printing the micrographs and Mrs. Jo Ann Richard and Mrs. Suzan Orazio for typing the manuscript. We are especially pleased to acknowledge Mr. Raymond Calvert of the Learning Resources Center for preparing the illustration used in Fig. 13. This work was supported by a grant from the Edward G. Schlieder Foundation and N.I.H. Grant RR 05376-11.

REFERENCES

1 Anker, R. L. and Cragg, B. G., Estimation of the number of synapses in a volume of nervous tissue from counts in thin sections by electron microscopy, J. NeuroeyWl., 3 (1974) 725-735. 2 Armstrong-James, M., The functional status and columnar organization of single ceils responding to cutaneous stimulation in neonatal rat somatosensory cortex Sj, J. Physiol. (Lond.), 246 0975) 501-538. 3 Campos-Ortega, J. A., Glees, P.. and Neuhoff, V., Ultrastructural analysis of individual layers in the lateral geniculate body of the monkey, Z. Zellforsch., 87 (1968) 82-100. 4 Davidson, N., The projection of afferent pathways on the thalamus of the rat, J. comp. Neurol., 124 (1965) 377-390. 5 Dobbing, J., Effects of experimental undernutrition on development of the nervous system. In N. S. Scrimsh-.," and J. E. Gordon (Eds.), Mabmtrition, Learning and Behavior, M.1.T., Cambridge, Mass., 1968, pp. 181-202. 6 Donaldson, L., Hand, P.J. and Morrison, A.R., Cortical-thalamic relationships in the rat, Exp. NeuroL, 47 (1975) 448-458. 7 Emmers, R., Organization of the first and the second somesthetic regions ($1 and Szl) in the rat thalamus, J. comp. Neurol., 124 (1965) 215-228. 8 Famiglietti, E. V. and Peters, A., The synaptic glomerulus and the intrinsic neurons in the dorsal lateral geniculate nucleus of the cat, J. comp. Nearol., 144 (1972) 285-334. 9 Garey, L. J. and Powell, T. P. S., An experimental study of the termination of the lateral geniculocortical pathway in the cat and monkey, Proc. roy. Soe. B, 179 (1971) 41-63. 10 Guillery, R. W., The organization of synaptic interconnections ;n the laminae of the dorsal lateral geniculate nucleus of the cat, Z. Zellforsch., 96 (1969) 1-38. 11 Gurdjian, E. S., The diencephalon of the albino rat, J. eomp. NeuroL, 43 (1927) 1-114. 12 Jones, E.G. and Powell, T. P. S., Electron microscopy of synaptic glomeruli in the thalamic relay nuclei of the cat, Proc. roy. $oe. B, 172 (1969) 153-171.

214 13 Lashley, K. $., Thalamo-cortical connections of the rats brain, .:. camp. Nearol., 75 (1941) 67-121. 14 LcGros Clark, W. E., An experimental study of the thalanfic ~onnections in the rat, Phil. Trans. B, 222 (1932) 1-28. 15 LeVay, S., On tl~ neurons and synapses of the lateral geniculate nucleus of the monkey, and the effect of eye enuclcation, Z. Zellforsch., 113 (1971) 396-419. 16 Liclmman, A. R. and Webs~'r, K. E., Aspects of the synaptic organization of intrinsic neurons in the dorsal lateral geniculate nucleus. An ultrastructuml study of the norms! and of the experimentally deaffcrentcd nucleus in the rat, J. Neurocytol., 3 (1974) 677-710. 17 Lund, R. D. and Webster, K. E., Thalami¢ affcrents from the dorsal column nuclei: an experimental anatomical study in the rat brain, J. camp. Nearol., 130 (1967) 301-312. 18 Lurid, R. D. and Webster, K. E., Thalami¢ afferents from the spinal cord and trigeminal nuclei: an experimental anatomical study in the rat brain, J. camp. Neural., 130 (1967) 313-328. 19 Matthews, M.A., Narayanan, C.H. and Y., and Ong¢, M. F. St., Neuronal maturation and synaptogenesis in the rat ventrobaral complex: alignment with developmental ¢han~-s in rate a~ :1 severity of axon reaction, J. camp. Neural. (1977) In lmmS. 20 Mortar, D. K., The differentiation of cerebral dendrites: a study of the post-migratory neuroblast in the medial nucleus of the trapezoid body, 7.. Anat. Entwickl.-Gesch., ]28 (1969) 271-289. 21 Morest, D. K., The growth of dendrites in the mammalian brain, Z. Anat. £ntwickl.-Gesch., 128 (1969) 290-317. 22 Nauta, W. J. H. and guypers, H. G. J. M., Some ascending pathways in the brain stem:: ficular formation. In H. H. Jasper, R. T. CAmtello, L. D. Proctor, R. S. Knighton and W. C. Noshay (Eds.), Reticular Formation o f the Brain, Little, Brown and Co., Boston, Mass., 1958, pp. 3-30. 23 Pappas, G. D., Cohen, E. B. and Purpura, D. P., Fine structure of synapti¢ and non-synaptic neuronal relations in the thalamus of the cat. In D. P. Purpura and M. D. Yabr (Eds.), The Thalamus, Columbia University Press, New York, 1966, pp. 47-75. 24 Pecci-Saavedra, J., Vaccarezza, O. L. and Reader, T. A., Ultrastructure of cells and synapses in the parvoccllular portion of the cebus monkey lateral geniculate nucleus, Z. Zellforsch., 89 (1968) 462-477. 25 Perl, E. R., Whitlock, D. G. and Gentry, J. R., Cutaneous projection to ~cond-order neurons of the dorsal column system, J. Neurophysiol., 25 (1962) 337-358. 26 Peters, A., The fixation of central nervous tissue and the analysis of electron micrographs of the neuropil, with special reference to the cerebral cortex. In W. J. H. Nauta and S. O. E. Ebbesson (Eds.), Contemporary Research Methods in Neuroanatomy, Sprinf~r, Berlin, 1970, pp. 56-76. 27 Poggio, G. F. and Mountcastle, V. B., The functional properties of ventrobasal thalami¢ neurons studied in unanesthetized monkeys, J. Neurophyslol., 26 (1963) 77~-806. 28 Ralston, H. J. and Herman, M. M., The fine structure of neurons and synapses in the ventrobasal thalamus of the cat, Brain Research, 14 (1969) 77-97. 29 Ram6n y Cajal, S., Histolagie du Systime Nerveux de rHomme et des Vertdbrds, Vol. 2, Maloine, Par'is, 1911. 30 Rces, R. P., Bunge, M. B. and Bunge, R. P., Morphological chant's in the neuritic growth cone and target neuron during synapticjunction development in culture, J. CelIBiol., 68 (1976) 240-263. 31 Rinvik, E., Organization of thalamic connections from motor and somatosensory cortical areas in the cat. In J. Frigyesi et al. (Eds.), Corticothalamic Projections and Sensorimotor Activities, Raven Press, New York, 1972. 32 Scheibel, M.E. and Scheibel, A. B., Structural substrates for integrative patterns in the brain stem reticular core. In H. H. Jasper, R. T. CAmtello, L. D. Proctor, R. S. Knighton and W. C. Noshay (Eds.), Reticular Formation of the Brain, Little, Brown and Co., Boston, Mass., 1958, pp. 31-55. 33 Scheibel, M. E. and Scheibel, A. B., Patterns of organization in specific and non-specific thalamic fields. In D. P. Purpura and M. D. Yahr (Eds.), The Thalamus, New York: Columbia University Press, New York, 1966, pp. 13-46. 34 Scheibel, M. E., Davies, T. L. and Scheibel, A. B., Ontogenetic development of somatosensory thalamus. I. Morphogenesis, Exp. Neural., 51 (1976) 392-406. 35 Skoff, R. P. and Hamburger, V., Fine structure of dendritic and axonal growth cones in embryonic chick spinal cord, J. comp. Neurol., 153 (1974) 107-148. 36 Spacek, J. and Lieberman, A. R., Ultrastructure and thrce-dimensional organization of synaptic glomeruli in rat somatosensory thalamus, J. Anat. (Lond.), 117 (1974) 487-516. 37 Szentdgothai, J., Hamori, J. and TOmb~l, T., Degeneration and electron microscope analysis of the synaptic glomeruli in the lateral geniculate body, Exp. Brain Res., 2 (1966) 283-301.

215 38 Szent~gothai, J., Neuronal and synaptic architecture of the lateral geniculate nucleus. In R. Jung (Ed.), Handbook of Sensory Physiology, Vol. VIII, 3B, Central Processing of Visual Information, Part B, Springer, Berlin, 1973, pp. 141-176. 39 T6mb~l, T., Short neurons and their synaptic relations in the specific thalamic nuclei, Brain Research, 3 (1969) 307-326. 40 Venable, J. H. and Coggeshall, R., A simplified lead citrate stain for use in electron microscopy, l. Cell Biol., 25 (1965) 407-408. 41 Verley, R. and Axelrad, H., Postnatal ontogenesis of potentials elicited in the cerebral cortex by afferent stimulation, NeuroscL Lett., 1 (1975) 99-104. 42 Waite, P. M. E., Somatotopic organization of vibrissal responses in the ventrobasai complex of the rat thalamus, J. Physiol. (Lond.), 228 (1973) 527-540. 43 Waite, P. M. E., The responses of cells in the rat thalamus to mechanical movements at the whiskers, J. Physiol. (Lond.), 228 (1973) 541-561. 44 Waller, W. H., Topographical relations of cortical lesions to thalamic nuclei in the albino rat, J. comp. Neurol., 60 (1934) 237-269. 45 Welker, C., Microelectrode delineation of fine grain somatotopic organization of Sml cerebral neocortex in albino rat, Brain Research, 26 (1971) 259-275. 46 Wong-Riley, M. T. T., Neuron and synaptic organization of the normal dorsal lateral geniculate nucleus of the squirrel monkey, Saimiri sciureus, J. comp. Neurol., 144 (1972) 25-60.