Regeneration of vomeronasal nerves into the main olfactory bulb in the mouse

Brain Research, 216 (1981) 239-251 © Elsevier/North-Holland Biomedical Press

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R E G E N E R A T I O N OF V O M E R O N A S A L NERVES INTO T H E MAIN O L F A C T O R Y BULB IN T H E MOUSE

P. C.

BARBER

Laboratory of Neurobiology, National Institute Jbr Medical Research, The Ridgeway, Mill Hill, London NW7 1AA (U.K.)

(Accepted December 4th, 1980) Key words: regeneration -- synaptogenesis -- olfactory bulb

SUMMARY After surgical section of the vomeronasal nerves the neurosensory cells in the vomeronasal epithelium die. Electron microscopy has been used to demonstrate that their axons, and synaptic terminals in the accessory olfactory bulb degenerate and are removed by phagocytic astroglia. The vacated postsynaptic sites in the accessory bulb persist, and are not re-innervated, either by vomeronasal or olfactory axons, as long as 150 days post-operatively. However, new neurosensory cells which are produced in the vomeronasal epithelium after vomeronasal nerve section do form axons. Light and electron microscope autoradiography of axonally transported material has been used to show that some of these axons grow back into the cranial cavity and form glomeruli in the main olfactory bulb, in regions where it is damaged or de-afferented. The regenerated vomeronasal glomeruli contain synapses between vomeronasal nerve terminals and dendrites of main bulb neurons.

INTRODUCTION After axotomy, olfactory and vomeronasal receptor neurones undergo irreversible retrograde degeneration and die. They are subsequently replaced by new receptors, which arise by proliferation of a stem cell population, and restore the normal structure of the sensory epithelium4,7,8,12,1L In the case of the olfactory system, there is evidence that axons, arising from the new receptors, grow back to re-establish synaptic contacts in the glomeruli of the olfactory bulb, in the adult animalS,12, la. Such structural regeneration may be accompanied by recovery of sensory function 14.

240 In the vomeronasal epithelium the new receptors also produce axons, but in contrast to the olfactory system these axons form large entangled intra-epithelial neuromata 4. This raises the question of whether the regenerating vomeronasal axons can also grow back to the central nervous system and re-establish synaptic contact there. In the normal animal retrograde transport of horseradish peroxidase has been used to show that vomeronasal axons arising from continuously formed neurosensory cells do gain access to the central nervous system 1, but the H R P method leaves open the question of whether they form synapses. However, the present study, using an autoradiographic tracing method, demonstrates that after destruction of the vomeronasal nerves in the mouse at least some axons from new receptors grow back along the nasal septum to the olfactory bulb. These regenerating axons do not re-innervate the accessory olfactory bulb. Instead, they form glomeruli in the main olfactory bulb, and make synaptic contacts with dendrites of main bulb neurons. MATERIALS AND METHODS Twenty adult female albino mice of the Parkes strain were subjected to bilateral section of the vomeronasal nerves (VNN). The operation was performed under inhalation anaesthesia with methoxyflurane (Penthrane, Abbott Laboratories) vapour in 95 ~ oxygen/5 ~ CO2 as carrier gas. With the head immobilized in a stereotaxic holder, a micromanipulator was used to insert a two-pronged knife (constructed by grinding down a 23-gauge hypodermic needle) vertically through a dorsal craniotomy at a point 1.5 m m rostral to the frontal pole of the cerebral cortex. The knife was lowered with the prongs on either side of the midline until the tip touched the floor of the cranial cavity. It was then rotated through several complete turns about its vertical axis, completely severing the V N N bilaterally, where they lie close to the midline on the medial surfaces of the main olfactory bulbs, rostral to the accessory bulbs. In addition to severing the VNN, the operation also cut a variable number of olfactory axons and damaged the medial surfaces of the main bulbs to a variable extent. A further 2 animals were subjected to bilateral section of the V N N by a handheld knife constructed from a fragment of a razor blade and inserted through a dorsal craniotomy to a position under the olfactory bulbs and close to the cribriform plate. The knife was moved laterally across the midline, severing the V N N and a large number of fascicles of the olfactory nerves. After various survival periods, all animals were re-anaesthetized and a pledget of Gelfoam (Sterispon) soaked in a solution of [3H]proline (L-3,4-[aH]proline, 60 Ci/mmol, Radiochemical Centre, Amersham) was inserted into the lumen of the V N O on the right sideL Eight hours or 5 days later the animals were perfused through the heart with a mixture of I ~ glutaraldehyde and 1 ~ formaldehyde in 0.1 M phosphate buffer at p H 7.4. Blocks of the nasal cavities including the vomeronasal organs were decalcified in buffered 5.5 ~ EDTA, embedded in paraffin wax, and coronal sections

241 cut at 8 #m for light microscope autoradiography. The olfactory bulbs were cut into coronal blocks, postfixed in 2 ~ buffered osmium tetroxide, and embedded in Epon (TAAB, Reading). Semithin (2 /~m) and ultrathin sections were cut for light and electron microscope autoradiography. For light microscope autoradiography, sections were mounted on gelatinized glass slides, coated by dipping in Ilford G5 emulsion, exposed for 14-30 days at 4 °C and developed in Ilford Phen-X. For electron microscopic autoradiography ultrathin sections (pale gold interference colour) were mounted on glass microscope slides that had been previously coated with a thin layer of formvar (0.3 ~o) and then one of carbon (4-6 nm). The sections were then coated (by dipping) with a closely packed monolayer of Ilford L4 emulsion, left to expose at 4 °C for 2-6 months, and then developed at 20 °C in D19 for 3 min. After fixation and washing in distilled water the slides were allowed to dry in air, and then each section or ribbon of sections was scored around with a needle, and a drop of dilute hydrofluoric acid ( < 1 ~) pipetted into the groove. By means of gentle etching of the glass, the carbon film on which the sections were mounted was lifted onto the drop of acid, thus enabling it to be picked up with a platinum loop and transferred to a Petri dish of water. It was then taken from the water directly onto grids and the sections stained with uranyl acetate and lead citrate. RESULTS 4 Days after vomeronasal nerve section (n = 2 animals) Accessory olfactory bulb (AOB) Light microscopy of the AOB showed dark-staining granular deposits throughout the glomerular layer, and irregular pale staining of the vomeronasal nerve (VNN) layer (Fig. 3). This was easily distinguished from the more uniform staining of both layers in normal animals (Fig. 1). The glomerular layer appeared disorganized and shrunken with an increase in cell packing density. Electron microscopy showed degeneration of virtually all VNN terminals throughout the glomerular layer. Most degenerating terminals were swollen, and paler than normal with marked swelling of the intra-terminal vesicles (compare Figs. 2 and 4). A proportion appeared shrunken and electron dense. Glial processes in the glomerular and vomeronasal nerve layers contained electron-dense accumulations of phagocytosed debris. These ultrastructural observations are similar to previous descriptions of olfactory and vomeronasal terminal degeneration in the rat 16, mouse lz and rabbit 5. Vomeronasal nerves Light microscope autoradiography, 8 h after [3H]proline application, showed labelled vomeronasal nerves running caudally from the vomeronasal organ (VNO) in their normal position along the nasal septum and entering the cranial cavity at the cribriform plate. Labelled nerve fascicles could be distinguished on the ventromedial surface of the main olfactory bulb, rostral to the region of the lesion, but could not be followed to an obvious zone of termination.

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Fig. 1. Light micrograph~of a coronal section through the accessory olfactory bulb (AOB) in a normal mouse. 2/~m Epon section, stained with methylene blue and Azur I1. V, vomeronasal nerve layer; G, glomerular layer; E, external plexiform layer; gr, granule cell layer; Lot, myelinated fibres running through the AOB to join the lateral olfactory tract. Scale bar: 200/~m. Fig. 2, Electron micrograph taken from the glomerular layer of the AOB in the same animal as Fig. 1. Vomeronasal nerve terminals (Vt) with characteristic irregular shape, electron-dense cytoplasmic matrix, and packed with small round vesicles make asymmetric synaptic contacts (arrows) with mitral cell dendritic appendages (d). Scale bar: 1/~m.

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14 Days after vomeronasal nerve section (n -~ 2 animals) Accessory olfactory bulb Light microscopy o f the A O B showed that the V N N layer and glomerular layer had collapsed. A pale-staining glial lamina, with interspersed small dark nuclei, covered the surface o f the external plexiform layer.

Vomeronasal nerves Light microscope autoradiography, 8 h after [3H]proline application, showed labelled fascicles o f the V N N running caudally f r o m the V N O along the nasal septum, but these fascicles became progressively smaller and more difficult to recognize, and could n o t be traced into the cranial cavity.

49-150 Days after vomeronasal nerve section (n = 16 animals) Accessory olfactory bulb The A O B appeared similar in all animals examined after survivals between 49 and 150 days. The light microscopic appearance was similar to that at 14 days, with a glial lamina covering the external plexiform layer (Fig. 5). The ultrastructural appearance was o f a total disappearance o f the vomeronasal nerve layer and glomerular layer. Glia covered the surface o f the A O B with m a n y layers o f apposed and interlocking processes. Directly beneath the reduplicated glial lamellae the neuropil resembled that o f the normal external plexiform layer. Vacated postsynaptic thickenings occurred in this region in clusters on adjacent dendritic shafts 16 (Fig. 6). This suggests that at least some, and possibly all denervated postsynaptic sites would be available for reinnervation. However, there was no indication o f any reinnervation

Fig. 3. Light micrograph of a coronal section through the AOB, 4 days after transection of the vomeronasal nerves. The vomeronasal nerve layer (V) is paler than normal with the glial nuclei prominent. The glomerular layer (G) is shrunken and irregularly stained with an increase in cell packing density. The other layers of the AOB appear normal. Abbreviations as Fig. 1. Scale bar: 200 /tm. Fig. 4. Electron micrograph from the glomerular layer of the AOB 4 days after vomeronasal nerve transection (same animal as Fig. 3). Synaptic contacts (arrows) are made by degenerating vomeronasal nerve terminals (Vt) which are swollen and paler than normal (cf. Fig. 2). Synaptic vesicles are swollen and more heterogeneous in size. Synaptic terminals are separated by swollen, glycogencontaining reactive astrocytic processes (*). Scale bar: 1/~m. Fig. 5. Light micrograph of a coronal section through the AOB, 49 days after transection of the vomeronasal nerves. The vomeronasal nerve layer and glomerular layer are absent. Many small, darkstaining glial cells (arrowheads) cover the surface of the AOB. Beneath this glial lamina the other layers (E, gr) of the AOB appear normal. Abbreviations as Fig. 1. Scale bar: 200 pm. Fig. 6. Electron micrograph taken from an area just below the glial lamina covering the surface of the AOB 49 days after vomeronasal nerve transection (same animal as Fig. 5). Vacated postsynaptic thickenings (arrowheads) are seen on profiles which resemble the dendritic branches of mitral cells normally receiving afferent vomeronasal nerve terminals (cf. Fig. 2.). Three of the vacated postsynaptic sites are apposed by sheet-like astrocytic processes now devoid of glycogen granules. Scale bar: 1/~m.

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by synaptic structures resembling the terminals of either olfactory or vomeronasal axons. Vomeronasal nerves

In 14 animals, light microscope autoradiography, 5 days after [aH]proline application, allowed one or two labelled fascicles o f the V N N to be traced f r o m the V N O along the ipsilateral nasal septum (Figs. 11 and 12) and into the cranial cavity. Since the nerves had been completely transected (as evidenced by the absence o f recognizable terminals or autoradiographic label in the AOB), these labelled fascicles presumably arose f r o m new vomeronasal receptors, and represented regeneration o f the V N N into the cranial cavity. In 6 o f these 14 animals, such labelled regenerating nerve fascicles ran caudally on the medial surface o f the ipsilateral or contralateral olfactory bulb outside the olfactory nerve layer, sometimes in close relationship to b l o o d vessels in the extrabulbar connective tissue. Labelled fascicles could be traced for up to 2 m m before disappearing, or ending in n e u r o m a t o u s 4 expansions. In the remaining 8 animals autoradiographically labelled regenerating vomeronasal nerves were observed growing into the region o f the medial surface o f the main bulb where the superficial layers had been directly d a m a g e d or removed by the surgical procedures used to cut the V N N (Fig. 7). The regenerating V N N bundles were observed to end in labelled expansions which resembled glomeruli o f the accessory bulb (Fig. 8). At light microscope level these regenerated vomeronasal glomeruli could be seen to contain dendritic profiles apparently derived f r o m the subjacent mitral or tufted cells o f the main bulb external

Fig. 7. Light microscope autoradiograph of a 2-~m coronal section through the main olfactory bulb 100 days after transection of the vomeronasal nerves and 5 days after application of [3H]proline to the vomeronasal neurosensory epithelium. This section is taken at a level 1.5 mm rostral to the accessory bulb, where the medial surface of the main bulb has been damaged by the surgical procedure. On the dorsal half of the medial surface of the bulb the deafferented glomeruli are shrunken, and pale, due to absence of the dark-staining primary afferent terminals (cf. the intact lateral surfaces of the bulb, and Fig. 9). On the lower half of the medial surface of the bulb the external plexiform layer is thinned or missing, and a regular single row of giomeruli is not present. Instead, two irregular aggregations of giomeruli (arrow and double arrow) invade the external plexiform layer (E). The more dorsal aggregation (single arrow) is autoradiographically labelled, (see Fig. 8) and thus represents a regenerated vomeronasal nerve fascicle. The more ventral aggregation of giomeruli (double arrow), is not radioactively labelled (see Fig. 10) and probably represents regenerated main olfactory nerves. Scale bar." 500/~m. Fig. 8. Detail from region of single arrow in Fig. 7. Autoradiographically labelled vomeronasal nerve fascicles and glomeruli have regenerated into the main bulb external plexiform layer. Scale bar: 50 pm. Fig. 9. Detail from region of rectangle in Fig. 7. Normal glomeruli of the main olfactory bulb. Darkstaining regions consist of the olfactory nerve terminals (*). Arrowheads, periglomerular cells; E, external plexiform layer. Scale bar: 75/~m. Fig. 10. Detail from region of double arrow in Fig. 7. The external plexiform layer (E) is invaded by small, glomerular clusters of dark-staining axons which are not autoradiographically labelled and are not associated with perigiomerular cells. This configuration suggests the regeneration of main olfactory axons into damaged regions of the main bulb. Scale bar: 75/~m.

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247 plexiform layer. They could be distinguished f r o m unlabelled n o r m a l glomeruli o f the main bulb (Fig. 9) by their relatively paler staining, (characteristic o f n o r m a l accessory bulb glomeruli) by their less regular organization, and by their lack o f a distinct shell o f periglomerular cells. M o s t o f the regenerated vomeronasal glomeruli (Fig. 16) were either situated on the surface o f the main bulb external plexiform layer, or extended as an irregular aggregation into the external plexiform layer, on either the ipsilateral or contralateral side. These variations in location presumably reflect the degree o f destruction o f the main bulb layers at the site o f formation o f the regenerated glomeruli. In a few situations, where the external plexiform layer h a d been completely destroyed, vomeronasal glomeruli were seen to have formed on or in the exposed granule cell layer o f the main bulb. The regenerated vomeronasal glomeruli intermingled with adjacent unlabelled glomeruli o f the main bulb some o f which m a y have been f o r m e d as a result o f regeneration of olfactory nerves into the d a m a g e d region o f the medial surface o f the bulb (Fig. 16). Such unlabelled glomeruli exhibited the dark staining characteristic o f main olfactory glomeruli, but were smaller in size than those o f the intact parts o f the bulb, and tended to occur in irregular aggregations which invaded the external plexiform a n d / o r granule cell layers o f the bulb (Fig. 10), presumably reflecting variable local tissue damage. They also lacked a distinct shell o f periglomerular cells, since in these areas the local periglomerular cells had been destroyed by the lesioning process. In two animals, at 100 days survival, ultrathin sections were taken t h r o u g h the labelled regenerated vomeronasal glomeruli in the main bulb, and electron microscope a u t o r a d i o g r a p h y performed. In b o t h cases, labelled axons could be identified forming

Fig. 11. Dark-field autoradiograph of a coronal 8/~m paraffin section through the vomeronasal organ I00 days after transection of the vomeronasal nerves, 5 days after unilateral insertion of [SH]proline into the vomeronasal lumen (*). Arrows, autoradiographically labelled regenerated vomeronasal nerve fascicles. Scale bar." 300/~m. Fig. 12. Dark-field autoradiograph of two regenerated fascicles of the vomeronasal nerve (arrows) running along the bony nasal septum (S) 1 mm caudal to the vomeronasal organ, in the same animal as Fig. 11. Scale bar" 100/zm. Fig. 13. Light microscope autoradiograph of the medial surfaces of both olfactory bulbs, 150 days after transection of the vomeronasal and olfactory nerves by undercutting the olfactory bulbs at the cribriform plate. [3H]Proline has beeninserted into the lumen of the vomeronasal organ on the left side of the figure, 5 days before sacrifice. 2/~m Epon section, stained with methylene blue and Azur II. An autoradiographically labelled fascicle of the regenerated vomeronasal nerve is seen running on the surface of the main bulb on the left side of the figure. The nerve has formed glomeruli (arrows) in the superficial part of the glomerular layer (G). E, external plexiform layer. Scale bar: 100/zm. Fig. 14. Detail from Fig. 13. An autoradiographically labelled glomerulus (*) of the regenerated vomeronasal nerve is shown at higher power. It is in the appropriate (glomerular) layer (G) of the main bulb, has a size and shape similar to that of adjacent main bulb glomeruli, and is surrounded by periglomerular cells. This configuration suggests takeover of a pre-existing, presumably denervated, main bulb glomerulus by regenerating vomeronasal axons. E, external plexiform layer. Scale bar: 50/~m. Fig. 15. Electron microscope autoradiograph of radioactively labelled terminals of regenerated vomeronasal axons in a glomerulus in the external plexiform layer of the main bulb 100 days after transection of the vomeronasal nerves. Arrowheads, synaptic contacts. Scale bar: 1/~m.

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Fig. 16. a: light microscope autoradiograph of regenerated structures on the medial surface of a main olfactory bulb 103 days after transection of the vomeronasal nerves. 2/~m Epon section, stained with methylene blue and Azur It, b: a tracing taken from (a) to show the distribution in this lower power field of the various tissue components whose identification was determined at higher magnification. Autoradiographically labelled glomeruli (VG) formed by the regenerated vomeronasal nerve invade the external plexiform layer (E) and in 2 areas (VG*) extend deeply to contact the granule cell layer (gr). Adjacent regions of unlabelled (olfactory) glomeruli and nerve fascicles, G. The olfactory glomeruli are probably also the result of regeneration, since they are smaller than normal, arranged irregularly, and in places (G*) extend as far as the granule cell layer. The deep location of the glomeruli, and the absence of periglomerular cells suggest that in this area the lesion had caused direct superficial damage to the bulb as well as deafferentation. Scale bar: 50 l~m. terminals within the glomeruli (Fig. 15). The axons resembled those o f the n o r m a l V N N , i.e. unmyelinated, a b o u t 0 . 2 / ~ m in diameter, containing 4 or 5 microtubules, a n d organized in bundles which were ensheathed by glial processes. Their terminals were characteristically electron-dense, irregularly shaped, interwoven, contained m a n y vesicles, a n d resembled n o r m a l v o m e r o n a s a l nerve terminals. Synapses were seen between labelled terminals o f the regenerated V N N a n d dendritic profiles which p e n e t r a t e d f r o m the subjacent external plexiform layer. These profiles, which were electron-lucent, c o n t a i n e d m i t o c h o n d r i a , and the characteristic vesicles o f olfactory bulb mitral cell dendrites. T h e synapses were a s y m m e t r i c a l in type with the axon terminal always f o r m i n g the p r e s y n a p t i c element a n d occurred in regions o f neuropil f r o m which glial processes were absent.

249 Two further animals were examined at a survival time of 115 days after undercutting the olfactory bulb at the cribriform plate. This procedure was adopted in order to section the vomeronasal nerves without damage to the medial surfaces of the main bulbs, in the hope that this would allow regenerating vomeronasal axons to regrow on the surface of the main bulb with less obstruction due to scarring, and to reach the accessory bulb. However, in these cases also, there was no indication that vomeronasal axons regenerated as far as the accessory bulb. Instead, labelled bundles of the regenerated vomeronasal nerve were seen to enter the olfactory nerve layer of the main bulb at the cribriform plate, and to travel in this layer for some hundreds of # m caudal to the lesion before terminating in glomeruli on the medial surface of the main bulb (Figs. 13 and 14). These regenerated glomeruli showed the paler staining characteristic of the vomeronasal nerve but otherwise resembled adjacent unlabelled glomeruli of the main bulb. They were similar in size and shape, fitted in discretely between the adjacent glomeruli and, in contrast to vomeronasal glomeruli in the normal AOB, were outlined by a distinct shell of periglomerular cells. This appearance contrasted with the irregular organization of regenerated glomeruli formed on a damaged surface of the main bulb, and gave the impression that regenerated vomeronasal axons had 'taken over' and terminated in existing, presumably denervated, main bulb glomeruli. DISCUSSION It has been previously shown 4 that after section of the vomeronasal nerves in the mouse, the neurosensory cells of the vomeronasal epithelium undergo a process of retrograde degeneration and die. They are subsequently replaced by newly-formed neurosensory cells, which arise from a stem cell present in the edges of the epithelium 3. In the present experiments we have shown that vomeronasal nerve section results in orthograde degeneration of neurosensory cell axons and their terminals in the glomeruli of the accessory olfactory bulb. The ultrastructural appearances of the degenerative reactions resemble those previously described in the rat 16 main and accessory olfactory bulbs, and in the rabbit 5. The products of degeneration are removed by phagocytic astroglia. Subsequently, the glomerular layer effectively disappears and the external plexiform layer of the accessory bulb becomes covered by glial lamellae which are directly continuous with the external glial lamellae on the dorsal surface of the bulb. Many vacated postsynaptic sites are seen 16. In the samples examined there was no evidence for reinnervation of the denervated accessory olfactory bulb either by vomeronasal or olfactory axons, even as long as 150days after operation. However, autoradiographic labelling demonstrates that vomeronasal axons arising from newlyformed neurosensory cells in the regenerated epithelium are able to innervate the main olfactory bulb, in regions where it is damaged or deafferented. The regenerated vomeronasal axons form glomeruli-containing terminals and synapses similar to those of the normal vomeronasal nerves in both the glomerular and the external plexiform layers of the main bulb.

250 It is apparent from previous work that the presence of the specific target tissue is not necessary for growth of new vomeronasal axons in the adult animal, since this occurs as readily after olfactory bulbectomy as after simple section of the vomeronasal nerves 4. The current experiment complements previous results by indicating that denervated tissue does not exert a guiding influence or 'attraction at a distance' on regenerating vomeronasal axons. In about 50 ~ of animals, labelled regenerating fascicles of the vomeronasal nerves were observed to grow caudally in loose extrabulbar connective tissue for considerable distances (up to 2 mm) without being directed towards the deafferented accessory bulb or towards nearby deafferented main bulb. Such fascicles terminated in neuromatous expansions, or dispersed in the connective tissue. The present observations also indicate that the presence of denervated postsynaptic sites in the accessory bulb does not induce collateral sprouting by the intact olfactory axons which innervate the nearby main bulb, and does not attract growing olfactory axons which would be constantly present in the nearby main bulb as a consequence of normal neurosensory cell turnover. In contrast to the extended growth shown by axons running in extrabulbar connective tissue, those regenerating fascicles which were seen to contact the neuropil of the main bulb did not seem to continue growth, but instead terminated within the area of direct damage or denervation caused by the lesion. There was no indication that regenerating fascicles had grown caudally into intact main bulb, nor towards the denervated accessory bulb. These observations would be compatible with the view that contact of the regenerating axons with denervated tissue inhibits further axon growth and initiates the formation of synaptic terminals. At their terminations in the main bulb, regenerated vomeronasal axons were seen to form glomeruli which resembled normal glomeruli of the accessory bulb, and which contained synapses. In many cases these regenerated glomeruli were formed on surfaces of the bulb where the pre-existing glomeruli had been destroyed by the surgical procedure. This observation would imply that regenerating vomeronasal axons have the capacity to induce postsynaptic dendritic differentiations necessary for the organization of glomeruli, and to induce the formation of synapses elsewhere than at a previously vacated postsynaptic site. Such a process of synapse induction contrasts with other areas of the adult nervous system such as the superior cervical ganglion 15 or the septum 17 where it has been suggested that re-innervation could be entirely accounted for by the occupation of existing denervated postsynaptic sites. The embryonic olfactory epithelium has an inductive effect on the development of the olfactory bulb 6 and regenerating neonatal olfactory axons have been shown to be able to induce formation of synapses in an abnormal location, the frontal cortex 9A°. It seems likely from the present experiment that a similar capacity for synapse induction is present in vomeronasal axons, and persists into adult life. A recent light microscopic study in the adult mouse 11 also concludes that regenerating olfactory and vomeronasal axons are able to form glomerular structures in abnormal locations in the olfactory bulb after partial bulbectomy, supporting the present electron microscopic observations.

25l ACKNOWLEDGEMENTS T h e a u t h o r gratefully a c k n o w l e d g e s the help an d advice o f Dr. G. R a i s m a n d u r i n g all stages o f this work.

REFERENCES 1 Barber, P. C., Axonal growth by newly-formed vomeronasal neurosensory cells in the normal adult mouse, Brain Research, 216 (1981) 229-237. 2 Barber, P. C. and Raisman, G., An autoradiographic investigation of the projection of the vomeronasal organ to the accessory olfactory bulb in the mouse, Brain Research, 81 (1974) 21-30. 3 Barber, P. C. and Raisman, G., Cell division in the vomeronasal organ of the adult mouse, Brain Research, 141 (1978) 57-66. 4 Barber, P. C. and Raisman, G., Replacement of receptor neurones after section of the vomeronasal nerves in the adult mouse, Brain Research, 147 (1978) 297-313. 5 Berger, B., Degenerescence transsynaptique dans le bulbe olfactif du lapin, apres d6safferentation p6riph6rique, Acta neuropath., 24 (1973) 128-152. 6 Giroud, A., Martinet, M. and Deluchat, C., Mecanisme de d6veloppement du bulbe olfactif, Arch. Anat. Hist. Embryol., 48 (1964) 203-217. 7 Graziadei, P. P. C., Cell dynamics in the olfactory mucosa, Tiss. Cell, 5 (1973) 113-131. 8 Graziadei, P. P. C. and Delian, R. S., Neuronal regeneration in frog olfactory system, J. Cell Biol., 59 (1973) 525-530. 9 Graziadei, P. P. C., Levine, R. R. and Monti Graziadei, G. A., Regeneration of olfactory axons and synapse formation in the forebrain after bulbectomy in neonatal mice, Neurobiology, 75 (1978) 5230-5234. 10 Graziadei, P. P. C., Levine, R. R. and Monti Graziadei, G. A., Plasticity of connections of the olfactory sensory neuron: regeneration into the forebrain following bulbectomy in the neonatal mouse, Neuroscience, 4 (1979) 713-727. 11 Graziadei, P. P. C. and Samanen, D. W., Ectopic glomerular structures in the olfactory bulb of neonatal and adult mice, Brain Research, 187 (1980) 467-472. 12 Harding, J., Graziadei, P. P. C., Monti Graziadei, G. A. and Margolis, F. L., Denervation in the primary olfactory pathway of mice. IV. Biochemical and morphological evidence for neuronal replacement following nerve section, Brain Research, 132 (1977) 11-28. 13 Monti Graziadei, G. A. and Graziadei, P. P. C., Neurogenesis and neuron regeneration in the olfactory system of mammals. II. Degeneration and reconstitution of the olfactory sensory neurons after axotomy, J. Neurocytol., 8 (1979) 197-213. 14 Oley, N., Delian, R. S., Tucker, D., Smith, J. C. and Graziadei, P. P. C., Recovery of structure and function following transection of the primary olfactory nerves in pigeons, J. comp. physiol. Psychol., 88 (1975) 477-495. 15 Ostberg, A. J. C., Raisman, G., Field, P. M., Iversen, L. L. and Zigmond, R. E., A quantitative comparison of the formation of synapses in the rat superior cervical sympathetic ganglion by its own and by foreign nerves, Brain Research, 107 (1976) 445-470. 16 Pinching, A. J. and Powell, T. P. S., A study of terminal degeneration in the olfactory bulb of the rat, J. Cell Sci., 10 (1972) 585-619. 17 Raisman, G. and Field, P. M., A quantitative investigation of the development of collateral reinnervation after partial deafferentation of the septal nuclei, Brain Research, 50 (1973) 241-264. 18 Takagi, S. F., Degeneration and regeneration of the olfactory epithelium. In L. M. Beidler (Ed.), Handbook of Sensory Physiology, Vol. IV, Springer-Verlag, Berlin, 1971, pp. 75-94.