Cellular events induced in the molecular layer of the piriform cortex by ablation of the olfactory bulb in the mouse

Brain Research, 134 (1977) 13-34 ~) Elsevier/North-Holland Biomedical Press

13

C E L L U L A R EVENTS I N D U C E D IN T H E M O L E C U L A R L A Y E R OF T H E P I R I F O R M C O R T E X BY A B L A T I O N OF T H E O L F A C T O R Y BULB 1N T H E MOUSE

VERNE S. CAVINESS, Jr., MARGARET G. KORDE and ROGER S. WILLIAMS Departments o[' Neurology and Neuropathology, Massachusetts General Hospital and Harvard Medical School, Boston, Mass. 02115, and the Eunice Kennedy Shriver Center for Mental Retardation, Inc., Waltham, Mass. 02154 (U.S.A.)

(Accepted January 7th, 1977)

SUMMARY Cellular events associated with degeneration of the projection of the olfactory bulb to the molecular layer of the piriform cortex of the mouse have been studied with rapid-Golgi and Fink-Heimer impregnations and with the electron microscope. Four classes of axon terminals : s-l, s-d, f-l, and f-d, are differentiated on the basis of whether the synaptic vesicles are spherical or flattened and whether the axoplasm is lightly or darkly stained. The majority of s-I terminals, the predominant class in sublamina Ia of the molecular layer, degenerate after bulb ablation. Degeneration of axon terminals is associated with dilation and, eventually, degeneration of segments of dendrites in la. Both s-I and s-d terminals contribute to a partial reconstitution of the neuropil of la during the weeks after bulb ablation.

INTRODUCTION Axon terminals of the principal afferent systems to the molecular layer of the piriform cortex of rodents and other macrosomatic mammals are concentrated in complementary laminaea,4, 3a. The olfactory bulb projects via the lateral olfactory tract to an outer zone, sublamina Ia. Terminals of the association system of the piriform cortex are found in a subjacent lamina, Ib. Those of the commissural system are also distributed in Ib in the rat. These are concentrated in a narrow laminar zone in the depths of Ib where their distribution overlaps that of the ipsilateral association system aa. Because the cells of origin of these three afferent systems are widely separated from each other, the molecular layer of the piriform cortex is favorable for study of the synaptology of different afferent systems by ultrastructural and experimental methods.

14 The present study is an analysis of the synaptic patterns in the molecular layer of the rostral piriform cortex of the mouse and of the cellular events which occur in this layer consequent to ablation of the olfactory bulb and the rostral e~d of the anterior olfactory nucleus. Observations drawn from electron micrographs are correlated with those from Fink-Heimer and rapid-Golgi impregnations. The study is concerned. principally, with the ultrastructural features of synaptic patterns of the ol factory bulb projection. This aspect of the study is preliminary to comparative studies o f the synaptology of this afferent system in Reeler, a mutation in mice which causes malposition of neurons in the piriform cortex and other cortical structures 5,~ 1. The most salient observations are in accord with those of a similar study by Westrum ~u in the rat. The present analysis is complementary to the earlier study of Westrum in that it provides additional information on synaptic patterns in the intact animal as well as on patterns of synaptic modification after destruction of the olfactory bulb. In addition the present study documents cytologic features of the pathologic process associated with axonal degeneration which appears to be both more abrupt and more destructive in the mouse than in the rat. MATERIALS AND METHODS The molecular layer of the rostral piriform cortex of the adult mouse has been examined in Fink-Heimer and rapid-Golgi impregnations and by electron microscopy at graded intervals, one day to a half year or more, after ablation of the olfactory bulb and the rostral end of the anterior olfactory nucleus (Table 1). In 3 animals which had survived for 6-8 months, secondary lesions were delivered to the stumps of the olfactory peduncle. After a postoperative survival of two days, two of the animals with secondary ablations were perfused and processed by the method of Fink and Heimer while the third was perfused and processed for study by electron microscopy. In all animals the molecular layer of the piriform cortex of the unoperated hemisphere was also examined as a control. Adult mice of the C57B1/6J or the C3H/HeJ strains, or hybrid animals resulting from cross breeding between these strains 6, were used in

TABLE 1 The numbers of animals processed by the Fink-Heimer (F-H) or Golgi (G) methods or for electron microscopy (EM) are tabulated according to survival times after primary olfactory bulb ablation. In two of the animals processed by the Fink-Heimer method and one processed for electron microscopy, secondary ablations, followed by a 2-day survival, were performed more than 6 months after the primary ablation. Hours

F-H G EM

Days

Months

24

39-42

2

3

4

5

7

11

l

2

2 6

6 6 4

4

4

5 5 5

4 5 5

2 5 2

3 5 2

3 4

2

6- I2

2

2 6 6

2

15 all studies. Both primary and secondary lesions were delivered by suction through a burr hole in the frontal bone under Equi-Thesin or Avertin anesthesia. Rapid-Golgi 26 and Fink-Heimer 12 impregnations were performed by methods which have been described previously 11. All animals were perfused transcardially under general anesthesia for 10 rain with 4~,, paraformaldehyde buffered to pH 7.3 with phosphate buffer 31. The separated lesioned and unlesioned cerebral hemispheres of each animal were processed together when used for rapid-Golgi impregnations.

Electron microscopy Anesthesized animals were fixed by transcardial perfusion for 15 rain with graded concentrations (2.5 ~-1.25 ~ glutaraldehyde, 2 ~ - 1 ~o paraformaldehyde) offormaldehyde-glutaraldehyde mixture zz buffered with Sorenson's phosphate buffer (pH 7.4-7.5). The brains were left undissected overnight in fixative at 4 °C. After the brains were dissected from the skull, blocks from both the lesioned and unlesioned hemispheres were taken from the rostral piriform cortex at the base of the olfactory peduncle. Specimens were postfixed in 2 ~ osmium tetroxide buffered with phosphate. Some were stained en bloc with 2 ~ maleate-buffered uranyl acetate. After dehydration, blocks were embedded in Epon-Araldite, sectioned at a thickness of 1 /~m and stained with 1 ~ toluidine blue. Suitable blocks were trimmed so as to include the lateral olfactory tract, subjacent molecular layer and the superficial pyramidal cell layer of the piriform cortex. Thin sections were stained on the grid with uranyl acetate and lead citrate. RESULTS

Structure of the lateral olfactory tract and molecular layer The lateral olfactory tract of the mouse at the level of the rostral piriform cortex is dominated by myelinated axons whose sheaths range in outside diameter from 0.5 to 2.5 # m (Figs. IA, 2A). The myelin sheaths, composed of 4-16 lamellae, surround axons with diameters ranging from 0.45 to 2.2 #m. Most of the myelinated axons course in the rostral-caudal direction though a few, particularly at an intermediate depth in the lateral olfactory tract, are coronally aligned. Unmyelinated axons, 0.1-0.5 # m in diameter, lie between fascicles of unmyelinated fibers. These and a few small myelinated fibers penetrate the underlying molecular layer. Layer I in the mouse, as in the rat 33, may be subdivided into superficial (Ia) and deep (lb) sublaminae on the basis of a greater concentration of astrocytic nuclei in la. Subjacent to the lateral olfactory tract where layer I is widest, la is also widest and constitutes approximately 50 ~ of the total width of the molecular layer. Spinebearing apical dendrites of pyramidal neurons whose somata lie in the cellular layers of the piriform cortex, particularly in layer l l, ascend through the molecular layer and may penetrate the lateral olfactory tract. Sublamina Ia has a fine grain texture due in part to the rich terminal arborization of the apical dendrites of the pyramidal cells (Fig. 1A). Sublamina lb is a coarser weave of the larger more proximal segments of the apical dendrites of the pyramidal cells. Somata of polymorphic neurons,

16

Fig. 1. Lateral olfactory tract (LOT), molecular layer (ML) and pyramidal cell layer (11) of the rostral piriform cortex of the normal mouse (A), and 42 h (B), 48 h (C) and 23 days (D) after olfactory bulb ablation. Vertical arrows indicate the boundary between la and lb. Oblique arrows i~1 A mark the somata of polymorphic neurons in the molecular layer and LOT. A, B and Dr toluidi,ae Hue stained, I / t m sections. C, Fink Heimer preparation. A, B, C, D, 400.

Fig. 2. The lateral olfactory tract (A) and sublaminae la (B) and lb (C) of the piriform cortex of the normal, unoperated hemisphere (left) and the operated hemisphere (right) 48 h after olfactory bulb ablation. S-I terminals are abundant in la and an s-d terminal is seen in Ib of the unoperated side. On the operated side myelinated axons of the LOT are dilated. In la, a dilated axon terminal (t) is presynaptic to a dendritic spine. Other axon terminals (arrows) are undergoing the dark degenerative change. An unchanged f-d terminal (crossed arrow) is presynaptic to a dilated dendrite. There is 11o cytologic change in lb. x 14,000.

18

Fig. 3. S u b l a m i n a la o f the piriform cortex of an u n o p e r a t e d hemisphere. S-1 terminals are labeled in A a n d B, an s-d terminal in A, f-1 terminals in C a n d an f-d terminal in B. A a n d B, ~ 25,000; C, x 40,000.

19 varying widely in diameter, are scattered sparsely at all levels of the molecular layer and may lie within the lateral olfactory tract (Fig. 1A). The tangentially or obliquely aligned dendrites of these cells ramify throughout the molecular layer. Their larger proximal dendritic segments are particularly conspicuous against the generally fine grain texture of sublamina Ia. Sublamina la is a rich synaptic bed. Approximately 25-30°/,, of its volume is constituted of axon terminals and preterminals (Fig. 2B). The remainder is dominated by unmyelinated axons, dendritic branches and dendritic spines. Glial cytoplasm is conspicuous only in the vicinity of capillaries. Approximately 90~,/~ of the axon terminals in la contain spherical vesicles, 40-55 nm in diameter, which are uniformly dispersed in a lightly staining axoplasm (Figs. 2B, 3A, B). All such terminals, designated s-I, form asymmetric, type Iv,9, synapses, principally with dendritic spines and terminal dendritic branches but occasionally with larger dendritic branches. These synaptic junctions are generally curvilinear with the presynaptic axon membrane most often convex, the postsynaptic membrane concave. The largest of these terminals, as much as 2/~m in diameter, terminate exclusively on large dendritic spines, in general upon a single though occasionally upon two spines. These large terminals and the spines they engage may be deeply interdigitated, and 2-4 synaptic junctions between a single terminal and spine, either in the lateral aspect or on the apex of an interdigitation, may be seen in a single plane of section. Smaller terminals, 0.4-1.0 #m in diameter, with identical spherical vesicles and axoplasm, may contact either a single dendritic spine or dendritic branch through a single synaptic junction. A second class of axon terminal, designated s-d, is encountered which contains spherical, 20-35 nm vesicles densely packed in a darkly staining axoplasm (Fig. 3A). This type of axon terminal, approximately 6 ~i; of the terminals in Ia, also forms asymmetric synapses. In la these generally engage only a single dendritic spine or dendritic branch through a single synaptic junction. The synaptic junctions are also curved but in this instance it is the axonal membrane which tends to be concave. Finally, approximately 4 j°/oof axon terminals in la are crowded with polymorphous, flattened vesicles ranging from 20 to 40 nm in their greatest diameters. These tend to cluster near the presynaptic membrane. In about half of these, designated f-1 (Fig. 3C), the background axoplasm is lightly impregnated. In the remainder, designated f-d (Fig. 3A, B), it is darkly impregnated. These terminals form symmetric, type II 7,t7, synapses with larger dendrites and, rarely, the somata of polymorphic neurons lying within the molecular layer. Apposed membrane densities are generally not curved. Axon terminals with 40-55 nm spherical vesicles dispersed in lightly staining axoplasm (s-l) as well as those with 20 35 nm vesicles in a darkly staining axoplasm (s-d) are also present in sublamina |b (Fig. 2C). As in Ia these form only type I synapses, principally with dendritic spines but also with dendritic branches. In Ib, s-1 terminals are less abundant than in |a, and are all of small diameter. The axo-spinous junctions involve only a single spine engaged via a single synaptic junction. By contrast s-d terminals in lb are much more numerous and larger than in la and may engage multiple spines, each through a single synaptic junction (Fig. 2C). Sublamina Ib also contains axon terminals with polymorphous, flattened vesicles dispersed in

20 either lightly or darkly staining axoplasm. These f-l and and f-d terminals, like similar terminals in la, form only type II synaptic junctions with dendritic shafts or neuronal somata.

Acute ej]ects of ablations upon neuronal elements Destruction of the olfactory bulb is followed by an expansion in the width of the ipsilateral lateral olfactory tract and sublamina Ia (Figs. 1B, 4). Tissue expansion is limited at the Ia, Ib boundary and does not involve sublamina Ib. There is no alteration in the width of Ia of the piriform cortex contralateral to the olfactory bulb lesion. The tissue expansion reflects dilation of both axons and their terminals as well as dilation of segments of dendrites which lie within the lateral olfactory tract and in sublamina Ia. It is not evident until 39 h after bulb ablation at which time dilated axons and dendrites are readily visible in 1 #m plastic sections, Golgi impregnations,

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8

9

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SURVIVAL

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20

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TIME

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50

60

(DAYS).

Fig. 4. Above: graphic representation of ratio of width of LOT and Ia to the width of LOT and entire molecular layer (la and Ib) as function of survival time after olfactory bulb ablation. Measurements were determined from 1 # m toluidine blue stained sections by means of a Zeiss camera lucida drawing tube at a magnification of × 250. They were estimated at the point of maximum width of the LOT. Below: the intensity of axon terminal degeneration in la as a function of survival time after olfactory bulb ablations is estimated as maximum (max) or minimum (min) from Fink-Heimcr preparations ( e ) and electron micrographs (El).

2l and in Fink-Heimer impregnations. Dilation is sustained through 48 h after bulb ablation. Subsequently, dilated structures collapse, and the width of Ia and the lateral olfactory tract contract. A minimum width, substantially less than the normal width, is reached by the eleventh postoperative day. In the succeeding two months there is partial reconstitution of the width of Ia but not of the lateral olfactory tract. Underlying these grossly evident tissue changes are degenerative, reactive and regenerative processes which involve virtually all cellular elements of the lateral olfactory tract and sublamina Ia including axons, dendrites, glia and other supporting elements. Axons

A light dusting of degenerating axon terminals is impregnated in the molecular layer of the piriform cortex by the method of Fink-Heimer as early as 24 h after ablation of the majority of the olfactory bulb and the rostral tip of the anterior olfactory nucleus. The density of impregnation increases dramatically by 39 h to reach a peak sustained over 5 days (Fig. 1C). Subsequently, terminal argyrophilia decreases sharply and is no longer present 11 days after bulb ablation. At all survival times after surgery, the density of impregnated degenerating terminals is very much greater in sublamina Ia than in sublamina Ib. Degenerating terminals in sublamina la undergo two quite different sequences of morphologic transformation: on the one hand, a dilation associated with loss of intraaxonal organelles (Fig. 5A, C), on the other hand, a contraction associated with increased electron density of intra-axonal contents (Fig. 5B, C). Between 24 and 36 h after bulb ablation it is the first of these transformations which is predominant. Initially, at 24 h the vesicles in scattered terminals have become variable in size, irregular in shape, decreased in number and may be clustered at the presynaptic membrane density. By 36 h many terminals show a more dramatic reduction in vesicle number associated with dilation, a change which is sustained through 48 h. Subsequently, these terminals, largely devoid of organelles, collapse. Terminals undergoing the dark transformation are rare at 24 h, relatively common at 39 h and most abundant between 48 and 72 h after bulb ablation. Initially there is dilation of synaptic vesicles and mitochondria. Subsequently, there is contraction of volume associated with increased electron density of intra-axonal contents and loss of membranous detail. Whether undergoing the degenerative reaction associated with dilation or the dark reaction, actively degenerating terminals are substantially decreased in numbers beyond 72 96 h after bulb ablation. They are only rarely found in presynaptic relationship to a dendritic spine or branch beyond 11 days after ablation; that is, beyond the time that degenerating terminals may be impregnated by the method of Fink and Heimer. Only terminals undergoing the dark transformation are identified with confidence in Ib. Whether in la or Ib degenerating terminals are invariably found to be presynaptic to type I synapses when the postsynaptic density can be identified in the electron micrograph (Fig. 5). The distinctions which permit differentiation of s-1 and s-d axon terminals which form type I synapses are, however, effaced by degenerative changes. Because the majority of terminals which from type I synapses in Ia degener-

22

Fig. 5. Early (A) and advanced (C) cystic change and dark change (B) in degenerating axon terminals (t) in Ia 48 h after bulb ablation. In C, there are also terminals undergoing the dark change (arrows~ and these are partially surrounded by the cytoplasm of a macrophage. A type I postsynaptic density appears to have been engulfed into a dendrite (* in C). An unchanged f-I terminal (E) is surrounded by dark degenerating terminals (arrow) at the same survival time. In D, survival time 5 (lays, degenerating axons of the LOT characterized by splitting of myelin sheaths and granular density of axoplasm, are embedded in reactive glial processes (a). A, :.:i 31,000; B, 7. 40,000; C, : 17,000; l), : 25,000: E, "< 14,000.

23 ate and because s-1 terminals are presynaptic to about 9 0 ~ of these synapses, it is clear that these are affected by bulb ablation. Those s-I terminals which form large interdigitated axo-spinous junctions in la virtually disappear. The majority of small s-I terminals in sublamina Ia must also degenerate though a few do persist unchanged. S-d terminals also persist in la and appear not to be decreased in number, so they may not degenerate after bulb ablation. The ablations do not produce a substantial change in number in either s-1 or s-d terminals in lb. Because these appear to undergo only the dark reaction they cannot be classified as s-1 or s-d once they are recognizable as degenerating terminals. Many unmyelinated axons in Ia and in the lateral olfactory tract also dilate and may achieve diameters of 1.5-3 #m. In general the tempo parallels that of dilation of axon terminals. At 39 h this change is most prominent at the interface of the lateral olfactory tract and sublamina la, but by 48 h it is generalized throughout the lateral olfactory tract. Until 24 39 h after ablation, changes in the myelinated axons of the lateral olfactory tract are subtle, limited principally to an increased density of impregnation of axoplasm and microtubules. Subsequently, the axoplasm of many becomes densely granular (Fig. 5D). The axons of others become dilated with associated loss of microtubules and dilation and splitting of mitochondrial membranes (Figs. 1B, 2A, 5D). After 48 h the dilated myelinated axons, devoid of organelles, collapse and may become flattened or folded against other tissue elements. Dilated axons are no longer evident in the lateral olfactory tract later than I 1 days after bulb ablation (Fig. 1D), although the collapsed myelin sheaths, often with splitting or effacement of lamellar detail, may persist in the tissue as late as 4 months after bulb ablation.

A Fig. 6. Camera lucida drawings of Golgi impregnated pyramidal ceils of the piriform cortex in an unoperated hemisphere (A) and 2 days (B) and 6 months (C) after olfactory bulb ablation (details in text). × 500.

24

Fig. 7. Micrographs of apical processes of pyramidal cells in la of the piriform cortex from an unoperated hemisphere (A) and from operated hemispheres 48 h (B) and 6 months (C) after bulb ablations. A-C, "< 800. Inserts in A - C are higher power micrographs of dendritic segments in their respective fields. Arrows in A and C designate spines common to micrographs at the two magnifications. Inserts A-C, ~-,~2200.

25

d d

,/

Fig. 8. Dilated dendritic segments (d) in la identified by postsynaptic densities (arrows) in A E. Typical s-d terminals persist in A, f-I terminals in B, C, and D. The postsynaptic density in E is occluded by a macrophage (m). B and D 42 h, A and C 48 h and E 5 days after bulb ablation. A and E, 31,200; B, C and D, >,~ 25,000.

26 Dendrites Segmental fusiform dilation and beading of dendrites traversing la and entering the lateral olfactory tract is evident, to some extent, in 1 #m plastic sections as early as 39 h after bulb ablation. In Golgi impregnations dendritic dilation is found to be most widespread at 48 h after the ablation (Figs. 6B, 7B). It is sustained through 5 days but then decreases in prominence. Beyond 11 days after surgery, beading is demonstrable in Golgi impregnations only in terminal dendritic segments lying within the lateral olfactory tract where these changes persist as late as 187 days after bulb ablation. Changes involve, principally, the apical dendrites of pyramidal cells but also the dendrites of polymorphic cells whose somata lie within the molecular layer. In either case only dendritic segments within Ia and the lateral olfactory tract are altered (Figs. 6B, 7B and 8). Proximal dendritic segments in Ib (Fig. 2C) and the cell bodies and basal dendrites lying within the cellular layers of the piriform cortex remain unchanged. During the acute stage of dilation, electron micrographs show loss of microtubules within dilated segments of dendrites but relative preservation of tubules within undilated segments (Fig. 8B, D). Cisterns of the smooth endoplasmic reticulum and, to a lesser extent, mitochondria are also dilated. Dendritic spines may appear bulbous and flattened, and there may be either an increase or decrease in flocculent fibrillae material within the spines (Fig. 8C). Postsynaptic densities remain readily identifiable on the membranes of dilated dendrites and spines (Fig. 8). it is of note that both dendritic segments which are dilated as well as those which are not dilated may be contacted by either degenerating or normal appearing axon terminals. Occasionally, a postsynaptic density, with degenerating presynaptic structures still attached, appears to have been engulfed into the cytoplasm of a dendrite (Fig. 5C) as described in the deafferented ventral cochlear nucleus of the rat by Gentschev and Sotelo 15. Reaction of non-neuronal tissue elements An increase in the volume of astrocytic cytoplasm at the margins of capillaries is first evident in electron micrographs at 39 h after bulb ablation. Progressively over 48-72 h, astrocytes, distinguished by their high concentration of cytoplasmic glycogen and fibrils, as well as macrophages entering the zone of degeneration come to contain ingested degenerating membranous fragments. Occasionally, an astrocyte or microglial cell (Fig. 8E) is seen in apposition to a type I postsynaptic density on a dendritic thorn or dendritic branch. Astrocytic expansion appears to continue through the weeks subsequent to active tissue degeneration so that within a month the cytoplasm of these cells may constitute as much as 30 ~ of the total area of the lateral olfactory tract or o f l a as estimated from representative electron micrographs (Fig. 5 D). Eventually astrocytic and microglial cytoplasm contains fragments of myelin sheaths which retain a periodic lamellar structure (Fig. 9F). Multiple reduplicated membranous and cytoplasmic glial envelopes come to surround segments of dendrites or axodendritic synapses (Fig. 9E). Others invest wide caliber capillaries which appear in the region

27 of tissue degeneration during the weeks after the lesion. Oligodendral cells persist in the tissue through the weeks following lateral olfactory tract degeneration and may also contain ingested degenerating tissue fragments. There is little evidence of regeneration of myelin sheaths around axons of normal appearance by these cells.

Cellular growth and repair In the months following bulb ablation, the lateral olfactory tract and la remain contracted to a volume which is 80°J~ or less of normal (Figs. 1D, 4) and as much as 30 % of this volume is constituted of glial cells. Evidently the complement of dendritic and axonal neuronal elements is very much decreased. In Golgi impregnations some of the apical dendritic shafts of pyramidal cells appear to taper and terminate as though truncated as they enter or ascend through la (Fig. 6C) (see also, refs. 13, 14). Dendrites which traverse the full width of la are often buckled in pleats, and they may follow an extended tangential course along the interface of the scar of the lateral olfactory tract and la (Figs. 6C, 7C). Only infrequently does a terminal dendritic branch appear to persist within the scar of the lateral olfactory tract itself. The dendritic segments which persist in the zone of degeneration are restored to a more or less normal diameter though many appear thickened and irregular in outline in Golgi impregnations. Variably, the spine density appears reduced in an irregular segmental fashion along the course of a dendrite. Filamentous rather than club-shaped spines appear to be preferentially reduced in number. Surviving spines are abnormally large and bulbous in outline (Fig. 7C). The irregularity of dendritic contour is also readily documented in electron micrographs. Dendritic cytoplasm is of increased electron density and appears to contain increased quantities of both rough and smooth endoplasmic reticulum. Axon terminals with spherical vesicles reappear in la in increasing numbers during the weeks following the ablations. Although these form only type 1 synapses which, with rare exceptions, are with dendritic spines and branches, the synaptic pattern which emerges after the ablations is anomalous. Both s-l and s-d terminals reappear in la. The s-d terminals, in the minority in the normal animal, become the predominant terminal class at long postoperative survivals. In specimens surviving at least two months after total or near total ablation of the olfactory bulb, s-d terminals are 60-70o/o of all terminals with spherical vesicles. Small terminals similar to those in la before surgery are present subsequent to surgery but, atypically, may contact as many as 4 spines in a clover leaf arrangement (Fig. 9E). Both s-I and s-d terminals also may be seen to form large glomerular structures which may be seen in a single section to contact 4 6 or more dendritic spines in series, each through a single synaptic junction (Fig. 9A, B). The s-1 glomeruli are seen only when there are a few surviving axons in the LOT~ however, Although the axo-spinous contact may be interdigitated in the s-I glomeruli, the degree is very much less than that typical of large interdigitated axo-spinous engagements in la of the normal animal. These glomerular terminals appear first at 11 days, reach their peak prevalence between 28 and 58 days and remain abundant through 187 days after surgery. Even more remarkable anomalies encountered in Ia during the months after bulb ablation have included single examples

Fig. 9. Anomalies in the neuropil o f la of the piriform cortex at extended survival times after oll'actor~ bulb ablation. A illustrates an s-I (sl-g) and B an s-d (sd-g) glomerular axon terminal. An s-1 terminal forms a type I axosomatic synapse in C (arrow and insert). A n s-I terminal forms an axo-axonic type 1 synapse with another axon terminal which contains both spherical and potymorphic vesicles in D~ This terminal, in turn, forms a type 1 axodendritic synapse. In E, 4 dendritic spines engage a single s-I terminal. The configuration is surrounded by a multilaminate glial envelope. Laminated myelin fragments (arrows) persist in the cytoplasm of a macrophage (m) and an astrocyte ,~a), A and B, 2 m o n t h ; C and D, 23 day; E, 11 day; and F, 6 m o n t h survival times. A, B, insert (74 ~! , 25~000: B, x 17,000; D, • 50,000; F, • 31,200

29 of a type I synapse between an s-1 terminal and the soma of a polymorphic cell (Fig. 9C), and an s-1 type I axo-axonal synapse in series with a type I axo-dendritic synapse (Fig. 9D). Although the majority of vesicles and the axoplasm of the latter terminal are typical of those found in s-1 terminals, there are, in addition, a few flattened vesicles. Terminals with polymorphous flattened vesicles, both the f-1 and f-d terminals, are present in Ia in the months after surgery. They are still less than 1 0 ~ of total terminals with f-1 and f-d terminals about equal in numbers. Their synaptic patterns appear unaltered. No changes are observed in terminals or synaptic patterns in lb. In two animals, large secondary lesions of the remnant of the olfactory peduncle, delivered 3-4 months after the initial ablation, gave rise to only sparse terminal degeneration in Ia in Fink-Heimer preparations. Only scattered degenerating terminals, all undergoing the dark change, were found in a single animal examined with the electron microscope after such a secondary lesion. These were in presynaptic relation to type I synapses, at times with as many as 4 spines. The majority of terminals in Ia, both s-I and s-d, were unaffected by the secondary lesions. DISCUSSION

The molecular layer of the rostral piriform cortex of the mouse contains 4 classes of axon terminals, differentiated in terms of shape and size of the synaptic vesicles and in terms of the electron density of the axoplasm: (1) s-I terminals: spherical vesicles 40-55 nm in diameter of moderate packing denstiy in a lightly staining axoplasm; (2) s-d terminals: spherical vesicles 20-35 nm in diameter densely packed in a darkly staining axoplasm; (3) f-I terminals: polymorphous flattened vesicles in lightly staining axoplasm; (4) f-d terminals: polymorphous flattened vesicles in darkly staining axoplasm. As is generally the case in other structures of the nervous system 32, those with spherical vesicles form asymmetric, type I7,17 synapses with dendritic spines and branches whereas those with polymorphous vesicles form symmetric, type II synapses with dendritic trunks and neuronal somata. The s-1 terminals comprise at least 90 % of the axon terminals in la and are abundant. They may form large interdigitated axo-spinous engagements with one or two spines in la. The s-d terminals may form large minimally interdigitated glomerular junctions with several spines in Ib. Both s-I and s-d terminals also form smaller engagements with single spines or restricted portions of dendritic branches in both Ia and Ib. Similar synaptic patterns are probably characteristic of the molecular layer of the piriform cortex of the rat. Terminals identical in morphology and with the same synaptic patterns as s-1 terminals in the mouse are also the predominant class of axonal terminal in the molecular layer of the rat 43. Though not identified as belonging to a separate class, a terminal having the characteristics of the s-d terminals in the mouse is also illustrated in Fig. 2 of the study of Westrum 43. The study of Westrum also describes terminals with polymorphous flattened vesicles though are not differentiated in terms of the density of staining of their axoplasm. The lesions employed in the present experiments in the mouse, like those in the

30 similar study in the rat by Westrum 4a, destroy most of the olfactory bulb and the rostral tip of the A O N as well. These lesions induced degeneration of most axon terminals in Ia and a few scattered terminals in lb as well. The olfactory bulb is known to project only to Ia of the molecular layer in the mouse 11 as in other macrosomatic mammalsa,9,21,2< aa. For this reason only the degenerating terminals encountered in Ia in the present experiments may be assumed to be terminals of the bulb projection. In the rabbit 4, terminals of the AON projection are concentrated in lb along with those of the cortico-cortical association system of the piriform cortex. This projection of the AON has not been demonstrated in the rat with HRP injections into the piriform cortex la, but the scattered degenerating terminals encountered in lb in the present experiments appear to identify such a projection in the mouse. Whether in Ia or Ib, only those axon terminals which form type i synapses with dendritic spines and branches are caused to degenerate by these lesions (see also, ref. 43). Large and small s-1 terminals, the principal terminal class in la, are largely cleared from this sublamina by the lesions and are therefore the principal if not exclusive terminal form of the bulb projections. S-d terminals are not common in la and their numbers appear not to be decreased by the lesions. Admittedly, however. degenerating terminals of this class would not have been recognized and so they are not clearly established as belonging to a projection system other than thai of the bulb. The scattered terminals of the AON projection which degenerate in lb are recognized as degenerating terminals only if undergoing the dark change. They cannot, therefore, be classified as s-I or s-d terminals. Apical dendrites of pyramidal cells of the piriform cortex contribute the major complement of dendritic spines to the neuropil in la and lb and cells of this class are, therefore, the principal targets of the projection of the olfactory bulb and the anterior olfactory nucleus. However, the dendrites of polymorphic cells whose somata lie within the molecular layer of the piriform cortex may also be richly invested with spines. Collateral branches of the lateral olfactory tract have been observed to form connections with dendrites of such cells in embryonic mice s and the polymorphic as well as the pyramidal cells may be targets of the olfactory bulb or the AON projection in the adult animal.

Pathological consequences of olfactory bulb ablation Within 24 h of bulb ablation terminal argyrophilia appears in Fink-Heimer preparations and degenerative changes in axon terminals may be identified in electron micrographs. The most prominent initial manifestation of degeneration is a decrease in the number of vesicles in axon terminals. Subsequently, between 28 and 48 h, there is dilation of axon terminals and preterminals and of segments of dendrites within sublamina Ia. There is an associated dilation of endoplasmic reticulum and loss of microtubules within dilated structures. By 36 h, and increasingly prominent through 3 days after bulb ablation, other degenerating axon terminals become contracted and darkly staining. Whether becoming dilated or contracted, active degeneration of terminals appears to be largely completed within 5 days after bulb ablation. Degenerating fragments are rapidly ingested by phagocytic ceils and only infrequently are

31 identified in presynaptic postion more than II days after bulb ablation. Terminal degeneration is no longer impregnated by the Fink-Heimer method beyond this time. The dilation of axons and dendrites occurring in Ia during the active phase of degeneration of axon terminals must reflect changes in membrane permeability to water and electrolytes. Similar changes, usually of lesser degree, have also been encountered as a consequence of a variety of other acute and chronic pathologic processes including axon degeneration 1,15,16,2a,a(~,39. Such a change may also occur in dendrites as a result of high frequency stimulation of a principal afferent system 4z. The dilation of structures observed in la in the present experiments is clearly related to the process of axon degeneration. The phenomenon occurs only ipsilateral to the bulb ablation and is sharply confined to sublamina la. Furthermore, dilation is evident only between 36 and 72 h after bulb ablation, that is, during the peak time of disintegration of degenerating axon terminals. To some extent the permeability changes may be due to a direct toxic effect of a variety of ionic or macromolecular substances released into the tissue by axon degeneration. These might include, for example, glutamate 19,4t, a plausible candidate for the transmittor in this afferent system 2,'~°, or calcium 37,a8. On the other hand, it should be emphasized that some degree of shift of fluid from the extra- to intracellular compartments occurs inevitably despite optimum perfusion fixation of tissue 4a. Alterations induced in the osmolarity or ionic constitution of tissue in la in the course of axon degeneration may interfere with tissue perfusion. This would compromise tissue fixation and thereby alter, indirectly, the permeability of cellular membranes (see also refs. 15, 29).

Restitution of neuropil after axon terminal degeneration The ablations performed in these experiments virtually eliminate the principal afferent system to la. They destroy only a trivial fraction of afferents to lb of the piriform cortex. The pathologic processes excited by terminal axon degeneration appear to lead to destruction of a large portion of the dendritic complement of the neuropil in la (see also refs. 34, 46). Surviving dendrites may be irregular in outline and modified in structure. They may be depleted of spines or occluded by glial processes. Despite these changes, there is restitution o f a synaptic bed in la during the weeks after bulb ablation. The restitution is only partial and very much less in extent than has been observed to occur in la when similar ablations are carried out in developing hamstersl°, ~4 or rats 21,44,45. Presumably this difference reflects not only the greater potential for axonal and dendritic growth in the developing nervous system but also the degree of neuropil destruction and glial reaction encountered in the adult mouse after the olfactory bulb ablations conducted in these experiments. Only axon terminals with spherical vesicles appear to contribute to the restitution of the synaptic bed in la. There is no evidence that terminals with flattened vesicles come to occupy wide postsynaptic densities vacated by terminals with spherical vesicles though this has been observed to occur in other regions of the nervous system 15,27. Both s-I and s-d terminals contribute to the restitution of the neuropil in la but the synaptic pattern they generate is anomalous. Whereas s-d terminals are less than 10 0,i of terminals in la in the normal animal, they comprise more than half of all terminals in the layer several months after surgery.

32 The reconstitution of the synaptic bed appears to reflect principally, a remodeling of presynaptic elements, that is, the axon terminals. Terminals of both s-I and s-d classes become very much larger than normal after the ablations, forming glomerular bags which may synapse with a succession of 4-6 or more spines, each with a single synaptic j unction. Normally in l a only the s-I terminals engage a s many as two spines, though several synaptic junctions may connect a single spine and s-l terminal. Other observations suggest that there has been some remodeling o f postsynaptic structures as well. For example as many as 4 spines may engage a single small axon terminal whereas the ratio of these small terminals to spines is normally 1:1. Such arrangements might reflect growth or other structural modifications of denervated spines (see also discussion in Raisman and Field35). Even more dramatic, though rare, modifications of postsynaptic structures are exemplified by a single type i axo-axonic synapse in series with a type I axodendritic synapse and a type I axosomatic synapse upon the somatic membrane of a polymorphic cell. In each instance a characteristic postsynaptic membrane density was wider than the presynaptic density. In la of the normal animal the somata of polymorphic cells and axon terminals are not found in postsynaptic position in type I synapses. Collateral sprouting of axons from surviving axons of the olfactory bulb and rostral AON projections evidently make only a small contribution to the reconstitution of the neuropil in Ia. Large secondary lesions of the distal olfactory peduncle induce degeneration of only a trivial portion of the terminals in the reconstituted neuropil of Ia. Further, the large s-l glomerular bags which contact multiple spines are observed only if a few axons survive in the LOT. The major complement of terminals probably arises more caudally from the association system of the piriform cortex. This system has been found to reinnervate dendrites of Ia very effectively when bulb ablations are performed in developing animals a°,44,45. A small number of terminals of this system co-exist with terminals of the bulb projection in Ia o f the adult animal aa and could assume postsynaptic sites vacated by degenerating terminals as described by Gentschev and Sotelo 15 and Raisman and Field 35. Possibly the commissural system of the rostral piriform cortex 4,az or other as yet unidentified afferent systems which have no distribution in Ia of the normal animal penetrate this subtamina and form heterotypical connections, a phenomenon which has been observed to occur in other regions of the nervous system ~,2s,4°. ACKNOWLEDGEMENTS The authors wish to thank Drs. M. LaVail and C. Richards for valuable discussion and suggestions during the course of this study. This study was supported in part by USPHS Grant H D 04147 and 1 R01 NS 12005-02, National Institutes of Health, Bethesda and the Joseph P. Kennedy, Jr., Memorial Foundation.

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