Vasoactive intestinal polypeptide-immunoreactive neurons in the main olfactory bulb of the hedgehog (Erinaceus europaeus)

Neuroscience Letters, 98 (1989) 19 24 Elsevier Scientific Publishers Ireland Ltd.

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NSL 05924

Vasoactive intestinal polypeptide-immunoreactive neurons in the main olfactory bulb of the hedgehog

(Erinaceus europaeus) L. L 6 p e z - M a s c a r a q u e , R . M . Villalba and J.A. de Carlos Instituto de Neurobiologia Santiago Ramdn y Cajal, CSIC, Madrid (Spain)

(Received 21 September 1988~ Revised version received 7 November 1988; Accepted 7 November 1988) Key words." Olfactory bulb; Vasoactive intestinal peptide: Hedgehog; Glomerular layer: External plcxiform layer; Immunocytochemistry

Vasoactive intestinal polypeptide (VIP) immunoreactivity was localized by the indirect antibody enzyme method (PAP technique) in the main olfactory bulb of the hedgehog. Most VIP-immunoreactivc cells were located in the glomerular layer and throughout the external plexiform layer. Fewer cells were observed in the granule cell layer. At the morphological level they exhibit the characteristics of periglomerular, external tufted, superficial short axon, horizontal and Van Gehuchten cells. It should be mentioned that another specific neuronal type was found in the inner third of the external plexiform layer, which is not described in other animals. These results revealed that a high number of intrinsic neuronal types of the olfactory bulb of the hedgehog display a strong VIP immunoreactivity.

The structure of the main olfactory bulb (MOB) of the hedgehog has been the subject of some studies [5, 11], and its cytoarchitecture reveals an organization similar to that of other mammals [! 1]. Little is known, however, about its detailed neurochemical features. The purpose of this study is to describe the distribution and morphology of vasoactive intestinal polypeptide-immunoreactive (VIP-I) neurons in the MOB of this animal. Our findings raise the possibility that VIP may play a role in the intrinsic circuitry of the MOB. Five hedgehogs were used in this study. Animals were deeply anaesthetized [5] and perfused with saline solution followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4. MOBs were then removed and postfixed for 3~4 h, at room temperature, in the same fixative and left in 30% sucrose buffered solution at 4"C. They were frozen-sectioned coronally at 40/2m and collected in cold PB. Free floating sections were processed according to the peroxidase-antiperoxidase (PAP) technique [19]. Sections were rinsed in phosphate-buffered saline (PBS), and incubated for 1 h in normal goat serum (1:20) in PBS containing 0.2% Triton X-100 (TBS). Sections were incubated with VIP antiserum (1:5000) in TBS 48 h at 4 C . After washing in Correspondence." L. Ldpez-Mascaraque, Instituto Cajal, Velfizques, 144, 28006 Madrid, Spain.

0304-3940;89/$ 03.50 ~" 1989 Elsevier Scientific Publishers Ireland Ltd.

20 PBS, sections were incubated in a sheep anti-rabbit IgG (Miles; 1:50) in TBS, for 1 h at room temperature. The sections were again washed in TBS and incubated in a rabbit PAP polyclonal antibody (Sigma; 1:1000) in PBS, for 1.5 h at room temperature. Sections were thoroughly washed with PBS and placed for 10 rain in 0.06% diaminobenzidine (DAB, Sigma) dissolved in PBS containing 0.01% hydrogen peroxide. Later, sections were washed in PBS, mounted on slides and, in order to intensify the staining, treated for 15 s in 0.1% osmium tetroxide, dehydrated and coverslipped. Antiserum specificity was tested with preabsortion experiments using 1 nmol of synthetic VIP per ml of diluted antisera preabsorbed overnight at 4'C, Specificity of the immunocytochemical method was established by incubating some sections with PBS or non-immune rabbit serum instead of the primary antibody. These sections showed no immunostaining. The localization and morphology of VIP-I neurons has been studied in different layers of the MOB of the hedgehog. Most of VIP-I neurons were stained in a Golgilike fashion, with extensive detail of their processes, exhibiting a wide variety of sizes, shapes and dendritic patterns. The cell bodies were found primarily throughout the external plexiform layer (EPL), glomerular layer (GL) or in the border G L / E P L and, to a lesser extension in the granule cell layer (GCL). They showed soma sizes, averaging 7--15/~m of diameter for the largest neurons and 5-9 ktm for the smaller ones. In the G L we found a large number of small and medium size VIP-I neurons (Fig. I A-C). Medium size neurons were noted adjacent to the glomeruli with a process generally oriented towards the surface of the MOB (Fig. 1C). These neurons were spineless with ovoid or round somas. Small neurons in the G L were found surrounding the glomeruli (Fig. I B) and could be recognized as periglomerular cells. In the border of this layer (GL/EPL) we observed some neurons with dendrites running parallel to the surface of the MOB (Figs. I D, 2A and 3A). In this layer we observed a significant number of VIP-! neurons with 3 to 5 varicose dendrites (Fig. 3B) which arborized close to the cell body which may correspond to short axon cells. The largest VIP-I neurons had their cell bodies in the upper portion of the EPL extending vertically oriented dendrites towards the glomeruli (Fig. 2B). This type of cells are presumably external tufted cells. Other large fusiform neurons had the soma throughout the EPL (Figs. 1A (arrows), 2C and 3C), Most dendritic arborizations were restricted to the basal portion of the soma. We also distinguish the axonal processes (Fig. 2C, arrow) arising from the lateral part of the cell body and extending toward the mitral cell layer (MCL). Several VIP-! neurons were localized adjacent to the MCL and within the inner third o f the EPL. These cells have dense dendritic arborizations in the'border G L / E P L (Figs. 2D and 3D). They have ellipsoidal cell bodies and a feature of their dendrites was the appearance of boutons along finecalibrated terminal dendritic collaterals. Fewer VIP-I neurons were present in the GCL. We could distinguish some neurons in the upper third of this layer (Fig. 2E) with round somatas, and a long basal process. This process forms a richly ramified terminal complex in the inner part of GCL. Similar to that neuronal type was found to different levels of the EPL (Fig. 2F). The major difference between the present findings in the MOB of the hedgehog

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Fig. I. Photomicrographs showing VIP-immunoreactive neurons. A: superficial layers of the main olfactory bulb including the glomeruli (gl). Arrowheads show a specific type of neuron through the external plexiform layer. B: high-power magnification showing a glomerulus surrounded by presumed periglomerular cells. C: medium-size neurons in the glomerular layer with processes oriented towards the pial surface. D: horizontal neuron in the border glomerular layer/external plexiform-layer. Scale bar = 30 lira.

and those in the MOB of the rat [6], is the presence of a large number of VIP-I periglomerular cells and the existence of large VIP-I neurons in the inner third of the EPL. According to morphological studies [17] 3 classes of interneurons are localized in the EPL: Van Gehuchten cells, superficial short-axon cells and periglomerular cells. These interneurons have been identified in our study as VIP-I neurons. Earlier studies [8] showed that a single neuronal type of the MOB contains different neuroactive substances. Periglomerular cells were described as containing tyrosine hydroxylase (TH) [3, 9], 7-aminobutyric acid (GABA) [16], methionine-enkephalin [4], and enkephalin [1]. Within these neuronal populations, a colocalization of both GABA and TH in ~t single neuron has been reported [7]. The VIP-I superficial short-axon cells have been identified because their morphology is similar to those stained by the Golgi method [17]. These cells, in the rat. are known to contain neuropeptide Y [6, 18]. NADPH-diaphorase and somatostatin [18] and a colocalization of GABA and TH [7]. With respect to Van Gehuchten cells, they have been described as neurons with an apparent lack of axon, and to be localized in the IPL, MCL and mainly in the EPL [12, 17]. Gall et al. [6] suggested that they could correspond to VIP-I neurons~

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Fig. 2. VIP-immunoreactive neurons in the main olfactory bulb of the hedgehog. A: horizontal cell in the border glomerular layer/external plexiform layer. B: large neuron in the upper portion of the external plcxiform layer. C: large fusiform neuron in the external plexiform layer. Arrowhead points the axonal process. D: neuron in the inner third of the external plexiform layer. E: neuron located in the upper third of the granule cell layer (GCL). F: neuron located in the superficial part of the external plexiform layer (EPL), with a richly ramified descendent process. Scale bar = 30/tm. a n d in the h a m s t e r [3], as T H - I neurons. In o u r s t u d y we have seen a large n u m b e r o f V I P - I cells with the m o r p h o l o g i c a l characteristics described to V a n G e h u c h t e n cells [12, 17]. H o w e v e r , we have d i s t i n g u i s h e d m a n y o f these cells e m i t t i n g a x o n a l processes. M o r e o v e r , we have identified a type o f V I P - I neuron, which a c c o r d i n g to its m o r p h o l o g i c a l characteristics [13], m a y c o r r e s p o n d to external tufted cells. It has been r e p o r t e d t h a t s o m e o f the e x t e r n a l tufted cells serve as i n t e r n e u r o n s r a t h e r t h a n p r o j e c t i o n n e u r o n s [20]. I m m u n o h i s t o c h e m i c a l a n d b i o c h e m i c a l analyses have shown that a large n u m b e r o f these n e u r o n s synthetize s u b s t a n c e P [2, 4, 10]. The relatively large, intensely stained n e u r o n s in the i n n e r third o f the E P L were

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Fig. 3. Different types of VlP-immunoreactiveneurons in the superficial layers of the main olfactorybulb of the hedgehog. A: horizontal oriented cell in the glomerular layer/external plexiform layer border. B: superficial short axon cells in the external plexiform layer (EPL). C: a presumably Van Gehuchten cell. D: cells located in the inner third of the external plexiform layer with profused dendritic arborization in the border glomerular layer/external plexiform layer. Scale bar- 30/~m.

not described in morphological or immunohistochemical studies. Despite the elaborate branching pattern of their dendrites, these cells could not correspond to the tufted cells on the basis of the lack of a dendritic tuft entering the glomerulus [13, 15]. Occasionally, we found in the G C L some VIP-I neurons which we could not characterize, similar to those reported in the rat [6]. In conclusion, the results presented in this study provide evidence for the existence of an extensive VIP-I neuronal system, mainly in the upper layers of the MOB of the hedgehog. VIP-I neurons and terminals were mostly present around the blood vessels, suggesting that VIP may participate in the regulation of blood flow [14]. None of the VIP-I neurons described in this study exhibited characteristics of projecting cells and therefore may possibly represent different types of local circuit neurons within the MOB. Overall, the macrosmatical character of the hedgehog and the existence of a large variety of neuronal types [1 1], makes it an important model for the study of the olfactory system neurochemistry from a comparative point of view. The authors are greatly indebted to Drs. F. Valverde and J. Rodrigo, for the critical reading of the manuscript and valuable suggestions and Dr. J.M. Polak (Hammersmith Hospital, London) for providing us, with the VIP-antiserum. The technical

24 assistance of M. L6pez and the English revision byDr.

L. F r a n i c a r e g r a t e f u l l y ac-

k n o w l e d g e d . T h i s i n v e s t i g a t i o n h a s b e e n s u p p o r t e d by p r o j e c t s n u m b e r s 154 a n d 177 f r o m the C o n s e j o S u p e r i o r d e I n v e s t i g a c i o n e s C i e n t i f i c a s ( C S I C ) a n d by G r a n t s 86/ 708, 8 7 / 1 6 9 4 a n d 8 7 / 1 6 9 6 f r o m t h e F o n d o d e I n v e s t i g a c i o n e s S a n i t a r i a s de la S e g u r i dad Social (FISSS). l Bogan, N., Brecha, N., Gall, C. and Karten, H.J., Distribution of enkephalinqike immunoreactivity in the rat main olfactory bulb, Neuroscience, 7 (1982) 895-906. 2 Burd, G.D., Davis, B.J. and Macrides, F., Ultrastructural identification of substance P immunoreactive neurons in the main olfactory bulb of the hamster, Neuroscience, 7 (1982) 2697-2704. 3 Davis, B.J. and Macrides, F., Tyrosine hydroxilase immunoreactive neurons and fibers in the olfactory system of the hamster, J. Comp. Neurol., 203 (1983) 427-440. 4 Davis, B.J., Burd, G.D. and Macrides, F., Localization of methionine-enkephalin substance P and somatostatin immunoreactivities in the main olfactory bulb of the hamster, J. Comp. Neurol., 204 (1982) 377-383. 5 De Carlos, J.A., L6pez-Mascaraque, L. and Valverde, F., Connections of the olfactory bulb and nucleus olfactorius anterior of the hedgehog (Erinaceus europaeus): Fluorescent tracers and HRP study, J. Comp. Neurol., 279 (1989) (in press). 6 Gall, C., Seroogy, K.B. and Brecha, N., Distribution of VIP- and NPY-like immunoreactivities in rat main olfactory bulb, Brain Res., 374 0986) 389-394. 7 Gall, C., Hendry, S.H.C., Seroogy, K.B., Jones, E.G. and Haycock, J.W., Evidence for coexistence of GABA and dopamine in neurons of the rat olfactory bulb, J. Comp. Neurol., 266 (1987) 307-318. 8 Halfisz, N. and Shepherd, G.M., Neurochemistry of the vertebrate olfactory bulb, Neuroscience, 3 (1983) 579-619. 9 Halfisz, N., Johansson, O., H6kfelt, T., Ljundahl, A. and Goldstein, M., Immunohistochemical identification of two types ofdopamine neurons in the rat olfactory bulb as seen by serial sectioning, J. Neurocytol., l0 (1981) 251 259. l0 Kream, R.M., Davies, B.J., Kawano, T., Margotis, F.L. and Macrides, F., Substance P and catecholaminergic expression in neurons of the hamster main olfactory bulb, J. Comp. Neurol., 222 (1984) 140 154. l l L6pez-Mascaraque, L., De Carlos, J.A. and Valverde, F., Structure of the olfactory bulb of the hedgehog (Erinaceus europaeus): description of cell types in the granular layer, J. Comp. Neurol., 253 (1986) 135~ 152. 12 Macrides, F. and Davis, B.J., The olfactory bulb. In P.C. Emson (Ed.), Chemical Neuroanatomy, Raven, New York, 1983, pp. 391-426. t3 Macrides, F., Schoenfeld, T.A., Marchand, J.E. and Clancy, A.N., Evidence for morphologically, neurochemically and functionally heterogeneous classes of mitral and tufted cells in the olfactory bulb, Chem. Sens., 10 (1985) 175-202. 14 McCulloch, J. and Edvinsson, L., Cerebral circulatory and metabolic effects on vasoactive intestial polypeptide, Am. J. Physiol., 231 (1976) H449-H456. 15 Orona, E., Rainer, E.C. and Scott, J.W., Dendritic and axonal organization of mitral and tufted cells in the rat olfactory bulb, J. Comp. Neurol., 226 (1984) 346-356. 16 Ribak, C.E., Vaughn, J.E., Saito, K., Barber, R. and Roberts, E., Glutamate decarboxylase localization in neurons of the olfactory bulb, Brain Res., 126 (1977) 1-18. 17 Schneider, S.P. and Macrides, F., Laminar distribution of interneurons in the main olfactory bulb of the adult hamster, Brain Res. Bull., 3 (1978) 73-82. 18 Scott, J.W., McDonald, J.K. and Pemberton, J.L., Short axon cells of the rat olfactory bulb display NADPH-diaphorase activity, neuropeptide Y-like immunoreactivity, and somatostatin-like immunoreactivity, J. Comp, Neurol., 260 (1987) 378-391. 19 Sternberger, L., Immunohistochemistry, 2nd edn., Wiley, New York, 1979. 20 Switzer, R.C., De Olmos, J. and Heimer, L., Olfactory system. In G. Paxinos (Ed.), The Rat Nervous System: Forebrain and Midbrain, Vol. l, Academic, Kensington, N.S.W., Australia, 1985, pp. I 31.