The CVS strain of rabies virus as transneuronal tracer in the olfactory system of mice

146

13rain Research, t~19 ( 19931 14~- 151~ © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.1111

BRES 19091

The CVS strain of rabies virus as transneuronal tracer in the olfactory system of mice Liliane Astic a

a, D i a n e Saucier b, Patrice Coulon c, Florence Lafay c and A n n e F l a m a n d c

Laboratoire de Physiologie Neurosensorielle, Universitd Claude-Bernard ~Lyon 1, l/illeurbanne (France), h Universit~ de Moncton, Centre universitaire de Shippagan, Shippagan (Canada) and CLaboratoire de G~n~tique des Virus, CNRS, Gif-sur-Yvette (France)

(Accepted 9 March 1993)

Key words: Tract-tracing; Intranasal inoculation; Rhabdovirus; Viral propagation; Main olfactory system

The sequential distribution of transneuronally infected neurons was studied in the olfactory pathway of mice after unilateral inoculation of the challenge virus standard (CVS) strain in the nasal cavity. A first cycle of viral multiplication was observed in a subpopulation of receptor cells scattered in the main olfactory epithelium and in the septal organ. No viral spread from cell body to cell body was reported even in later stages of infection. The second round of viral replication which took place in the ipsilateral main olfactory bulb at 2 and 2.5 days post-inoculation (p.i.), involved second order neurons and periglomerular cells, known to be directly connected with the axon terminals of receptor cells. Also reported as a result of a second cycle of viral replication, was surprisingly the spread of CVS at 2 and 2.5 days p.i. in bulbar interneurons located in the internal plexiform layer and in the superficial granule cell layer, as well as that of 2 ipsilateral cerebral nuclei, the anterior olfactory nucleus and the horizontal limb of the diagonal band. From day 3, a rapid spread of CVS was suggested by detection of virus in all ipsilateral direct terminal regions of the second order neurons and in most tertiary olfactory projections. The locus coeruleus, a noradrenergic nucleus which sends direct afferents to the olfactory bulb, never appeared immunoreactive. In spite of a certain inability of CVS to infect some neuron types, the virus appears relevant to provide new information regarding the complex network of olfactory-related neurons into the CNS.

INTRODUCTION Live n e u r o t r o p i c viruses have r e c e n t l y r e c e i v e d att e n t i o n as p o t e n t i a l t r a n s n e u r o n a l tracers. O n e o f the m a j o r a d v a n t a g e s of viruses over m o r e c o n v e n t i o n a l t r a n s s y n a p t i c t r a c e r s such as, w h e a t - g e r m agglutinin ( W G A ) , W G A - H R P or t e t a n u s toxin C - f r a g m e n t , is that t h e signal c o u l d b e a m p l i f i e d by viral r e p l i c a t i o n at e a c h s t e p of n e u r o n a l transfer, so t h a t r a t h e r small p r o j e c t i o n s m a y be r e c o g n i z a b l e . A m o n g t h e few n e u r o t r o p i c viruses a l r e a d y t e s t e d as p o s s i b l e neur o a n a t o m i c a l tracers, r a b i e s virus has r e c e i v e d up to d a t e only little a t t e n t i o n 8'9'14't5. This virus is a R N A e n v e l o p e d r h a b d o v i r u s having only o n e e x t e r n a l glycop r o t e i n which is r e s p o n s i b l e for t h e a t t a c h m e n t o f t h e virus to t h e host-ceU a n d t h e n to the viral tropism. R a b i e s virus is a c y t o p l a s m i c virus which, like the o t h e r viruses, can only r e p l i c a t e in t h e cell b o d y o f t h e n e u r o n . It has b e e n shown t h a t C V S c o u l d travel a n t e r o g r a d e l y a n d r e t r o g r a d e l y a l o n g axons at a flow

r a t e o f a b o u t 1 m m p e r h o u r 6. In cell cultures, the r a b i e s cycle has b e e n e s t i m a t e d to 24 h; this cycle is faster in n e u r o n s , b u t it s h o u l d not be s h o r t e r t h a n 12 h ( C o u l o n ' s p e r s o n a l observation). Since t h e w o r k of S a b i n in 1938 33, the p e r i p h e r a l olfactory system has b e e n o f t e n u s e d as r o u t e of e n t r y o f viruses into t h e brain. This sensory system p r e s e n t s t h e a d v a n t a g e o f having d e n d r i t e s o f its p r i m a r y neurons in d i r e c t c o n t a c t with t h e e x t e r n a l e n v i r o n m e n t a n d its axons e n d i n g in the c e n t r a l n e r v o u s system (CNS). I n a p r e v i o u s study 15, we have c o m p a r e d the c h a l l e n g e virus s t a n d a r d ( C V S ) strain a n d t h e avirulent m u t a n t A v O 1 for t h e i r ability to p e n e t r a t e a n d p r o p a g a t e in t h e C N S o f mice a f t e r i n t r a n a s a l instillation. D a t a have shown s o m e restrictiveness o f A v O 1 replication in t h e CNS. R e l y i n g on t h e fact that C V S d o e s n o t s e e m to infect c e n t r a l glial cells 4°, we a t t e m p t in this study to investigate t h e p r e c i s e i d e n t i t y a n d s e q u e n c e o f t h e n e u r o n s involved in t h e t r a n s n e u r o n a l s p r e a d o f t h e virus a l o n g t h e olfactory p a t h w a y following C V S

Correspondence: L. Astic, Laboratoire de Physiologie Neurosensorielle, Universit6 Claude Bernard/Lyon I, 69622 Villeurbanne cedex, France. Fax: (33) 78.94.95.85.

147 intranasal inoculation. To do so, animals were sacrificed at 12-h intervals which roughly corresponded to each viral cycle. Results provide further information regarding the connectivity in the olfactory system as well as some new insights into the use of rabies virus as neuroanatomical tracer. MATERIALS AND METHODS The challenge virus standard (CVS) strain was grown in BHK-21 cells and the concentration of viruses from the cell culture supernatants was made according to Coulon et ai. 6. Experiments were performed on 6-week-old female OF1 mice (IFFA-CREDO, St. Germain-sur-l'Arbresle, France). Animals were anesthetized with equithesin (3 mg/kg) and maintained on their back during the inoculation. Three/.d of CVS virus suspension, titre 4 × 107 PFU, in buffer (Tris-HCl pH 7.4 25 raM, NaCl 150 raM, KCI 5 mM, NaEHPO 4 0.7 m M ) + E D T A 1 mM were instillated in the right nostril using a Hamilton microsyringe connected to a stretched catheter. At the end of inoculation, mice were remained on their back for 15 rain. Mice were killed at intervals (1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6 and 7 days) following intranasal CVS inoculation. After deep anesthesia, animals were perfused intracardially with 0.1 M phosphate-buffered saline (PBS) pH 7.4 followed by 2% paraformaldehyde in PBS and then 10% sucrose in PBS using a peristaltic pump. Nasal cavities and brains including olfactory bulbs were dissected and stored in 10% sucrose in PBS for at least 24 h at 4°C. Serial frontal sections of the nasal cavity and the brain were cut at 30 /zm with a cryostat and collected on gelatin-coated slides. In mice killed between days 1 and 2.5 following inoculation, all brain sections were treated for immunofluorescence. In other animals, brain sections were either all treated for immunofluorescence or divided in 3 series, one for immunofluorescence, one for immunoper-

oxidase and one left untreated. All sections of the nasal cavity were treated for immunofluorescence. For the immunofluorescent reactions, sections were permeabilized in PBS + 0.3% Triton X-100 for 30 min at room temperature, washed 3 times in PBS and then incubated in fluorescein isothiocyanate-conjugated anti-nucleocapsid antibodies (Pasteur Production) diluted 1 : 100 in PBS for 24h at 4°C. After rinsing with tap water, the slides were mounted in Fluoprep (bioM6rieux, France) or Elvanol (TrisPO4 50 mM, polyvinyl alcohol 20%, glycerol 20%, pH 8.2) and then examined with an UV microscope. For the immunoperoxidase reactions, sections were permeabifized in PBS+ 1% Triton X-100 for 30 min at room temperature, then washed 3 times with ELISA III buffer (NaCl 150 raM, Tris-HC1 50 mM, EDTA 1 mM, Tween-20 0.05%, bovine serum albumin 0.1% pH 7.4). After a 3-rain incubation in 0.3% H202, sections were washed 3 times in ELISA III and then incubated in a rabbit antirabies antibody (diluted 1:300 in ELISA III) for 24 h at 4°C. Sections were washed 3 times in ELISA III and incubated in antirabbit biotinylated antibody (ABC kit 'Elite', Vector Labs.) diluted 1:200 in ELISA III for 1 h at 37°C. The signal was amplified by incubating the sections in a mixture of avidin and biotinylated peroxidase for 20 rain at 37°C. Sections were washed and then incubated for 1 rain in 0.1% diaminobenzidine (DAB, Sigma) in 0.1 M Tris-HCl (pH 7.4) to which an equal volume of 0.02% H 2 0 2 was added. The reaction was stopped by rinsing with tap water. Tissue sections were counterstained with Giemsa, rinsed, dried and mounted with DPX. For identification of brain structures, the atlases of Lehmann 17 and of Paxinos and Watson 2s were used.

RESULTS

Penetration of CVS in the olfactory epithelium Unilateral inoculations of CVS strain induced primary cellular infection exclusively in the ipsilateral

Fig. 1. Darkfield photomicrographs of CVS-infected olfactory receptor cells in the mouse nasal cavity at 2 days post-inoculation (p.i.). A: overview of immunoreactive receptor cells (arrows) distributed along the septal organ. Bar = 100 pro. B-C: higher magnification photomicrographs showing positive receptor cells with their labeled dendrite (solid arrow in B) or axonal process (arrowhead in C). Bar in B = 25/zm and in C = 50/~m. OE = olfactory epithelium; S = septum.

148 nasal fossa in about 70% of the nasal cavities studied, thus allowing to study viral propagation along the ipsiand contralateral olfactory pathway. In the other cases, some scarce infected cells were also detected in the non-instillated cavity. As far as the 2 fossae communicate by the septal window, the inoculum may have reached the contralateral side. In these cases, only the distribution of infected neuroreceptors in the sensory sheet was analyzed. The first round of viral multiplication in the nasal cavity occurred in the receptor cells of the main olfactory epithelium. One day after inoculation of 4 x 107 PFU of virus, the antigen was detected in few receptor cells in which only the cell body was immunofluorescent. At 2 days post-inoculation (p.i.), the number of labeled cells increased noticeably and may reach 2- to 7 x 103, which value remained relatively constant for the next 2 days. This value is comparable to that reported in our previous study for a same concentration of virus inoculated ts. As shown in Fig. 1, positive receptor cells were generally non-adjacent and their dendritic and axonal processes appeared well labeled. Infected cells were reproducibly found mainly in the lower part of the different turbinates, in the ventral part of the septal wall and in the septal organ. When free horseradish peroxidase (HRP) was instilled in-

TABLE

stead of rabies virus, one could observed that epithelial areas showing the heaviest HRP labeling were similar to those infected by the virus (data non shown). At days 6 or 7, when CVS-infected mice died, positive receptor cells were still noted in the main olfactory epithelium, but their number was reduced to about 1 x 10 s infected cells. Very few infected receptor cells were occasionally observed in the sensory epithelium of the vomeronasal organ. The virus also penetrated in the trigeminal endings since the Gasser ganglion was positive at 2 days p.i.. The respiratory epithelium never appeared infected, even in later stages of infection.

Viral infection in the olfactory bulb The first neurons detected as infected by immunofluorescence in the main olfactory bulb (MOB) were observed 48 h after instillation. This agrees with the occurrence of a first cycle of infection in the olfactory epithelium before the virus reached the bulb. At 2 and 2.5 days p.i., the labeling features were quite similar. Sparse positive fibers were observed in the olfactory nerve layer in the ipsilateral bulb and some glomeruli exhibited a fluorescence of punctiform aspect, suggesting the presence of viral nucleocapsids in the axon endings of receptor cells. Only few

1

Number of infected neurons detected by immunofluorescenee in the olfactory bulb, the anterior olfactory nucleus and the horizontal limb of the diagonal band after unilateral intranasal CI,'S inoculation -: no virus detected; nd: not done; AOB: accessory olfactory bulb; AON: anterior olfactory nucleus; HDB: horizontal limb of the diagonal band; pg: p e r i g l o m e r u l a r cells; I P L : i n t e r n a l p l e x i f o r m layer; G R L : g r a n u l e cell layer; ipsi: ipsilateral; contra: c o n t r a l a t e r a l .

Days postinoculation

Main olfactory bulb

AOB

pg & external tufted cells ipsi

other tufted cells & mitral cells

contra

ipsi

IPL & superficial GRL interneurons

contra

ipsi

L5

.

.

.

.

.

.

( n = 2)

.

.

.

.

.

.

2 ( n = 5)

. -

.

.

.

.

.

.

.

.

contra

contra

ipsi

11

contra

contra

.

1

.

ipsi

HDB

AON ipsi

-

1

.

.

.

3

-

-

1

-

nd

-

1

-

-

4

-

15

-

3

10

-

-

-

8

-

5

-

28

-

3

14

-

3

-

26

-

17

-

2,5

7

-

1

-

nd

nd

3

1

22

-

( n = 5)

7 7

. -

-

1

-

2

-

3 ( n = 4)

3,5 ( n = 1)

.

.

.

.

2

-

3

-

-

1

-

2

-

10

-

1

-

6

-

-

29

-

9

-

18

-

-

-

8

-

-

20

-

18

-

37 49 53 64

-

39 17 12 22

1 -

38 35 47 42

-

3

-

++*

+

++

+

1

-

+ +

+

+ +

+

_

_

+ +

+

+ +

+

4

-

++

+

++

+

57

8

5

1

54

+-t-

-t-

++

+

* F r o m d a y 3, i n j e c t e d c e l l s i n A O B a n d H D B w e r e o n l y e s t i m a t e d .

149 periglomerular cells a n d / o r external tufted cells appeared immunoreactive (Table I). An example is shown in Fig. 2A. As far as these two types of neurons have

their cell body located in the periglomerular region, it is quite difficult to make clear distinction between both and they were then pooled together. At this stage of

Fig. 2. A: darkfield photomicrograph showing a CVS-infected periglomerular or external tufted cell (arrow) at 2 days p.i. Bar = 100/~m. B-C: Examples of infected cells located in the internal plexiform layer (ipi) with their dendritic arborizations extending tangentially to the mitral cell layer (m) at 2.5 and 3 days p.i., respectively. Immunofluorescence reaction in B and immunoperoxidase reaction in C. These infected neurons can be identified as horizontal cells. Bar in B = 100 p,m and in C = 50/~m. D: brightfield photomicrograph exhibiting infected periglomerular a n d / o r external tufted cells at 3 days p.i. with their dendritic processes largely ramifying in the glomeruli. Bar = 50 p~m. epl = external plexiform layer; g = glomernlus.

150 infection, dendritic processes of these cells were rarely labeled. Antigen was also detected in few neurons located in the internal plexiform layer (IPL) or inframitral cell layer and in the superficial granule cell layer (GRL) (Fig. 2B). With the immunoperoxidase method, the large-bodied neurons in the IPL could be seen with their dendritic arborizations widely extending tangentially to the mitral cell layer (Fig. 2C). Weak im-

munoreactivity was occasionally observed in middle tufted and mitral ceils in which only the cell body was labeled. At day 3, the infection was still confined to the ipsilateral bulb in 3 of the 4 mice studied. The number of previously infected neurons consistently increased (Table I). As shown in Fig. 2D, the dendritic arborizations of the periglomerular and/or external tufted cells

Fig. 3. A: darkfield photomicrograph exhibiting a well-immunoreactive mitral cell with its primary dendrite penetrating into a glomerulus at 4 days p.i. Bar = 50/.Lm. B: brightfield photomicrograph showing at 5 days p.i. the extent of viral infection in the mitral cell layer (m). One can note absence of infection of the granule cell layer (gr). Bar = 100/xm. epl = external plexiform layer; g = glomerular layer.

151

Fig. 4. A: darkfield photomicrograph of the horizontal limb of the diagonal band (HDB) showing 2 heavily infected neurons at 2 days p.i. B: overview of HDB in which numerous neurons appear infected at 4 days p.i.. For A and B, Bar = 100 ~,m.

.....~: iil¸il iil Fig. 5. High magnification photomicrographs of CVS-infeeted neurons presenting a Golgi-like appearance at 3 days p.i.. A: positive cells in the posterior part of the anterior olfactory nucleus. B: a positive neuron in the piriform cortex showing spines (arrows) onto its dendritic arborizations. For A and B, Bar = 50/~m.

152 may extend largely within the immunoreactive glomeruli and differences in labeling intensity can be noted between adjacent infected cells. Otherwise, the infection of mitral cells was generally restricted to the cell body. Very few positive neurons (less than 6) could be occasionally observed in the deep granule cell layer, particularly when elements in the upper part of the layer were consistently infected. At 4 days p.i., the number of infected periglomerular cells, second order neurons and neurons located in the IPL and the superficial GRL increased noticeably in the ipsilateral bulb. At this stage of infection, positive mitral cells appeared generally well labeled, with their primary dendrite ending in the glomeruti. An example is shown in Fig. 3A. In the contralateral bulb, second order neurons, periglomerular cells and few neurons in the superficial GRL appeared infected. Few positive elements in the contralateral bulb have been already observed at 3.5 days p.i.. From day 5 onwards, nearly all the mitral ceils in the ipsi- and contralateral bulb appeared immunoreactive (Fig. 3B), whereas the granule cell layer rather remained free from labeling, except for few superficial positive neurons. In the accessory olfactory bulb, only few infected periglomerular cells were occasionally observed (Table I), even in later stages of infection.

pallidum and the medial forebrain bundle (MFB) area deep to the OT, appeared consistently infected from 3 days p.i. The vertical limb of the diagonal band (VDB)

TABLE

II

Infection of the cerebral structures after unilateral intranasal CVS inoculation -,

no virus

intensity

areas

performed

was

Switzer

Positive

reaction

from

of immunoreactivity. according

+

to

+ + +

Classification

to Shipley

and

represent

of projection

Adamek

(1984)

and

et al. (1985).

Days post-inoculation 2 n=5

2.5 n=5

3 n=4

4 n=3 + + +

Direct olfactory bulb projections Anterior

+

+

+ +

tecta

olfactory

-

-

+ +

Piriformcortex

-

-

+/+

~

+ + / + + +

Olfactory

tubercle

-

-

+/+

+

+ +

cortex

-

-

+

-~ ÷

-

-

+

+ +

-

-

+

+ +

Taenia

Entorhinal Cortical and

nucleus

amygdala

+ + +

(anterior

posterolateral)

Ventral

agranular

insular

neocortex

Afferent projections to the bulb other than terminal regions of the LOT Horizontal

limb of the

diagonal

band

+

+

+ +

+ +

Ventral

pallidum

-

-

+ +

+ +

Vertical

limb of the

diagonal Lateral Raphe

band

-

-

+

+ +

hypothalamus

-

-

+

+

-

-

+

+ +

-

-

-

+

-

-

-

+

nuclei

and

Viral infection of the higher olfactory structures and other related cerebral structures As early as 2 days p.i., few positive cells were observed ipsilaterally in two cerebral nuclei, the anterior olfactory nucleus (AON) and the horizontal limb of the diagonal band (HDB) (Table I; Fig. 4A). Positive neurons in the HDB were generally well labeled. Similar features of infection were noted at 2.5 days p.i. (Table I). At day 3, all ipsilateral areas receiving direct projections from the MOB were positive, but differences in labeling intensity were noted between infected structures. Thus, many immunoreactive cells were observed in all subdivisions of the AON, in the taenia tecta and in the anterior part of both the olfactory tubercle (OT) and the piriform cortex (PC). But, only few positive neurons were found in the caudal part of the OT and the PC, in the olfactory amygdaloid nuclei and in the entorhinal cortex (EC) (Table II). Most infected cells presented a Golgi-like appearance with dendritic arborizations of individual neurons on which dendritic spines could be seen. Examples of Golgi-like labeled neurons are shown in Fig. 5. Among the afferent projections to the MOB, the HDB (Fig. 4B) and a region including the ventral

detected;

increasing

Nucleus Zona

(dorsalis

medialis) of the LOT

incerta

Locus

coeruleus

.

.

.

.

Tertiary olfactory projections Endopiriform

nucleus

(anterior

part)

Hippocampus Ventral

(CA1,

and

CA3)

lateral

amygdaloid

Lateral

preoptic

Sub-median

-

+

+ + +

-

-

+

+ +

-

-

+

+ +

-

-

+

+ / + +

-

-

+

+

-

-

+

+

-

-

-

+

-

+

+ +

+

+ + +

orbital

neocortex Other

-

nuclei area

thalamic

nucleus Medio-dorsal

thalamic

nucleus

Other infected cerebral structures Magnocellular

preoptic

nucleus Septal

-

area

Medullary

reticular

Messencephalic

area

-

-

+

trigeminal -

-

+

+

Substantia

nuclei nigra

-

-

+

+

Cingulate

cortex

-

-

+

+

-

-

+

+ / + +

Claustrum

-

-

-

+ +

Bed

-

-

-

+ +

-

-

-

+ +

Caudate-Putamen

-

-

-

+

Accumbens

-

-

-

+

-

-

-

+

-

-

-

+

Raphe

magnus

nucleus

Supraoptic

nucleus nucleus

Central

gray

Ventral

tegmental

area

153 also exhibited immunoreactive cells (Table II). These structures were unambiguously infected via the retrograde route, since they send non-reciprocial inputs to the M O B 7'37'39. On the other hand, scattered positive cells were found in most of the basal forebrain structures which receive inputs from the terminal regions of the lateral olfactory tract (LOT). These included endopiriform nucleus (anterior part), hippocampus (CA1, CA3), lateral preoptic area and lateral hypothalamus (Table II), this last structure being known to also send afferents to the MOB. Presence of few positive neurons were also noted in both the insular cortex, which is thought to be directly connected with the MOB, and the lateral and ventro-lateral orbital cortex, which is expected to receive direct input from PC and indirect input from OT and endopiriform nucleus, via medio-dorsal and submedian thalamic nuclei 35'39. Antigen was also seen in other brain areas which are not known to have direct connections with the olfactory pathway, as for example, the magnoceUular preoptic nucleus, the septal area (medialis and lateralis), the medullary reticular area and the ventral tegmental area.

Fig. 6. Darkfield photomicrograph of the entorhinal cortex which appears heavily immunofluorescent at 4 days p.i. Bar = 100/zm.

The contralateral infection at 3 days p.i. was restricted to the AON and the HDB. Well labeled neurons were observed in all subdivisions of the contralateral AON, but they were less numerous than in the ipsilateral one. At day 4, the infection largely spread out ipsilaterally, invading all direct terminal regions of the LOT which appeared mildly to heavily labeled (Table II). An example is shown in Fig. 6. All tertiary olfactory projection areas and all structures which send afferent inputs to the MOB became involved. Only the locus coeruleus remained free from labeling, even at the latest stage of infection. Many additional brain regions not directly connected with the olfactory pathway, became newly infected (Table II).

DISCUSSION Results of this study confirm that rabies virus intranasally inoculated is able, like other neurovirulent viruses, to invade the brain by transfer along the olfactory pathway. Animals being sacrificed at intervals of 12 h, this therefore allowed one to provide details about the cell types that were sequentially involved in the transneuronal spread of CVS at each viral cycle. The highly neurotropic nature of CVS is confirmed by the viral infection of the sensory epithelium, excluding that of the respiratory mucosa. A same selective affinity for olfactory epithelium has been reported for vesicular stomatitis virus (VSV), another rhabdovirus TM, whereas necrosis of both the respiratory and olfactory epithelia was observed in mouse hepatitis virus (MHVJHM) infection3. The occurrence of only few occasional positive cells in the vomeronasal organ (VNO) let suppose that this organ which is isolated in a small cavity at the base of the septal wall, could be less accessible to viral particles, all the more that animals were maintained on their back during the instillation. On the other hand, the possibility of a restricted tropism of CVS for VNO receptor cells cannot be ruled out. If it was the case, the difference of permissivity for rabies virus noted between receptor cells of the VNO might be somehow related to differences in molecular properties between the two cell populations, as already shown in studies using antibodies or lectins 2,12. In the main olfactory epithelium, only a small subset of receptor cells appeared CVS infected. Immunoreactive cells represent less than 1% of the neuronal population which could be estimated to about 9.5 × 10 6 according to data of Benson et al. 4 and Meisami 25. The low percentage of infected primary neurons may be

154 related in part to viral particle trapping during their passage through the mucus layer covering the epithelium. This suggestion is supported by the observation of a better success rate of HSV1 infection after an injection of atropine 24, a substance which may decrease nasal secretions 1. It could be also asked if the occurrence of viral material in only a subpopulation of receptor cells might be related to differences in CVS permissivity between primary neurons. Nonetheless, what is important to note here is the fact that the number of infected receptor cells was sufficient to follow the spread of CVS along the olfactory pathway and bring out most of the synaptically linked cell populations described with conventional neuroanatomical tracers. More, from our previous study 15, it has been concluded that the olfactory route is at least 100 times more efficient to invade the CNS than that follows by the virus after an injection in the forelimb. Absence of patches of infected receptor cells even after 4 or 5 days p.i., indicates that cells which are contiguous to receptor cells primarily infected, did not become necessarily infected probably because there was no budding of virions in the cell body. This was also observed in the dorsal root ganglion after intramuscular inoculation of CVS 6. Absence of any lateral viral spread reinforces the assumption that only one cycle of viral replication should take place at the receptor cell level and also argues for a strictly transneuronal transport of CVS. This feature may represent an advantage over the more commonly used HSV1. It has been observed that after an intranasal instillation of HSV1, immunoreactive cells present a patchy distribution in the olfactory epithelium and in the bulb, thus suggesting a viral spread from cell body to cell body in addition to the transneuronal transport 24. Following the first cycle of CVS multiplication in the receptor cells, the virus entered the brain, via olfactory nerve fibers, and spread intracerebrally by replicating along some specific chains of olfactory-related neurons. The second step of CVS replication took place in the main olfactory bulb and resulted in the infection at 2 and 2.5 days p.i. of both second order neurons and periglomerular cells, known to be directly connected with the axon terminals of receptor cells ~9. Also reported at 2 and 2.5 days p.i., was the infection of interneurons located in the IPL and the superficial GRL, which might suggest that these neurons have been secondarily infected by virions produced from axon endings of the receptor cells. The possibility of a tertiary infection of the I P L / G R L interneurons, via second order neurons and periglomerular cells, has to be excluded since all these bulbar neurons appeared immunoreactive after a same delay p.i. This is a sur-

prising result since it has never been shown with conventional neuroanatomical tracers that cellular processes of interneurons located in the I P L / G R L region might penetrate into the glomerular layer and consequently into the glomeruli. About identification of the cell types involved, several classes of interneurons have been described within this bulbar region by Golgi studies, on the basis of the laminar location of the cell somata and the organization of the cellular arbors 3°'34. With the typical tangential orientation of their dendrites to the mitral cell layer, positive neurons observed in the IPL can be easily identified as horizontal cells. According to Schneider and Macrides 34, axons of horizontal cells can project superficially toward the glomerular layer, but they were never traced into individual glomeruli. Identification of the few infected cells in the superficial G R L is more puzzling. Because most sections were treated for immunofluorescence, the fine structure of dendritic arborizations of these cells was quite difficult to visualize. Nonetheless, according to their size and their distribution, it could be assumed that superficial G R L neurons are clearly not granule cells. They are probably Blanes cells or Golgi cells, 2 types of short axon interneurons associated with the G R L 34. It has been reported that dendrites and axons of these 2 types of interneurons may ramify in the external plexiform layer, but they were never seen penetrating into the glomerular layer. The infection of 2 cerebral nuclei, the A O N and the HDB, in concomitance with that of second order neurons at 2 days p.i., was another unexpected result. As d e m o n s t r a t e d for positive interneurons in the I P L / G R L region, the early infection of the AON and the HDB could only be the result of a second step of viral replication occurring in these nuclei. This excludes the possibility that the AON has been anterogradely infected, via axons of mitral and tufted cells, which should imply a third step of viral multiplication. Both the AON and the HDB appear to have been retrogradely infected via their afferent projections to the MOB. On the basis that these nuclei were secondarily infected, one can expect that they might have neuron terminals ending within individual glomeruli. Even though this projection has never been described for the AON, we have actually few indirect clues regarding such a possibility. It has been demonstrated that the A O N is the unique olfactory direct projection area to the MOB which sends afferents in the glomerular layer, the other centrifugal olfactory nuclei projecting almost exclusively in the G R L 19'21'26'31'35. As regards the HDB, it has been shown that this structure which is the unique source of cholinergic innervation of the bulb 5,13'2°'41, sends fibers in the glomerular layer ~7.

155 From the 3rd day post-inoculation, a rapid spread of CVS was reported in all direct terminal regions of the second order neurons and in most tertiary olfactory projections which have been described in a review by Switzer et al. 39. One can note, however, that at 3 days p.i., the density of infected neurons appeared higher in the AON and the anterior piriform cortex (PC) than in the more caudal targets of the bulb. This result is probably the reflection of the organization of these projections, the AON and the anterior PC receiving projections from tufted and mitral cells and the posterior olfactory cortex receiving inputs only from mitral cells 22'36. Thus, the occasional infection of few mitral cells, compared to that of external tufted cells at 2.5 days p.i., may explain the more restricted infection noted in the caudal parts of the olfactory cortex. At 4 days p.i., in addition to the labeling of olfactory-related structures, CVS also spread in many other cerebral structures via a complex network of interconnections. Data show that among the nuclei sending direct afferents to the bulb, the locus coeruleus (LC) was the only structure showing no CVS labeling, even in later stages of infection. The fact that this nucleus, which represents the unique source of noradrenergic (NA) fibers to the olfactory bulb ~3, was not infected, supports the idea that NA terminals are non-permissive to the virus. Data also bring out a rather low ability of rabies virus to infect serotonergic (5-HT) fibers coming from raphe. These nuclei exhibited only a weak immunoreactivity at 3 days p.i., whereas it is well known that raphe send massive projections to the glomerular layer and more, the major targets of these 5-HT fibers could be likely the dendrites of second order neurons and periglomerular cells or possibly the receptor cell terminals themselves 23. Thus, as for some receptor cell populations in the olfactory epithelium and the VNO, some neuron types in the CNS also appeared few or non-permissive to rabies virus. Ability of viruses to infect certain populations of neurons more efficiently than others has been reported in recent studies 1°,16,32,38. This might be regarded as a potential limitation for using viruses as neuroanatomical tracers. Viral transneuronal method should be rather regarded as a useful complementary tool to the conventional neuroanatomical tracers, especially when viruses spread is strictly transneuronal without any cell-to-cell infection. For neuronal pathways whose synaptically linked circuits are well described like the olfactory system 39, neurotropic viruses should then provide further information regarding rather small projections. Thus, in this study, the early infection of both the I P L / G R L interneurons and the AON and HDB neurons strongly suggest that these cells may have terminals ending

within glomeruli. At this point, one can wonder if these neuron terminals might be in direct contact with axon endings of receptor cells. Data actually available do not seem to agree with such a possibility since it is rather suggested that the olfactory axons occupy presynaptic positions within a glomerulus 1~'29. One of the first steps to answer this question will be to determine if viral particles released into the extracellular space could be taken up by nearby neuronal processes. As for many other transneuronal markers, it appears that transmission electron microscopy studies should be useful to clarify the events taking place at the synaptic level. Acknowledgments.

This work was supported by the Centre National de la Recherche Scientifique (LP A2431 and URA 180), and by the Ministb.re de la Recherche et de la Technologic (Contract 88-1896).

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