Ultrastructural identification of substance P immunoreactive neurons in the main olfactory bulb of the hamster

Neuroscience Vol. 7, No. Printed in Great Britain

1I, pp.2697

0306-4522,%2/112697-08 $03.00/O Pergamon Press Ltd 0 1982 IBRO

to 2704. 1982

ULTRASTRUCTURAL IDENTIFICATION OF SUBSTANCE P IMMUNOREACTIVE NEURONS IN THE MAIN OLFACTORY BULB OF THE HAMSTER G.

D. BURD,*

B. J. DAVIS

and F. MAcaIDEst

Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545, U.S.A. Abstract-The neurons containing substance P immunoreactivity in the main olfactory bulb of the hamster are located in the glomerular layer. Their cell bodies lie in the periglomerular region and contain spherical or ovoid nuclei which lack invaginations of the nuclear membrane and tend to be positioned eccentrically in the cell body. Dendrites of these neurons extend throughout the periglomerular region and project into the glomerular neuropil. Within the glomerular neuropil, processes with substance P immunoreactivity contain agranular, spherical synaptic vesicles. Primary olfactory axons, and processes of uncertain origin which contain pleomorphic synaptic vesicles, form synaptic contacts with substance P immunoreactive processes. These ultrastructural findings confirm that the substance P immunoreactive neurons are external tufted cells. Their likely physiological properties are considered in relation to the synaptic organization in the glomerular layer of the main olfactory bulb and to the other putative neurotransmitters or neuromodulators located in this layer.

The undecapeptide, substance P, is thought to function as a neurotransmitter or neuromodulator in the central nervous system.8.26 Immunocytochemical studies have shown that neurons containing substance P immunoreactivity are widely distributed in the ner-

vous system. 5,7,12,14,16,22,23,2sfn the main olfactory bulb of the hamster, substance P immunoreactive (SPI) neurons are present in the glomerular layer.7 Neurons in this layer fall within three distinct morphological classes: periglomerular cells, superficial short-axon cells and external tufted cells.2g,35 Because the SPI neurons have relatively large cell bodies and bear processes which enter the glomerular neuropil, we have suggested that these neurons are external tufted cells.’ The purpose of the present investigation was to examine the ultrastructural features of the SPI neurons in the main olfactory bulb and to identify the class(es) of neurons associated with this immunoreactivity. EXPERIMENTAL

PROCEDURES

Seven adult male hamsters (Mesocricetus auratus) were anesthetized with sodium pentobarbital and perfused transcardially with a solution containing 2% paraformaldehyde and 0.15% picric acid in 0.15 or 0.1 M phosphate buffer.7.38 The brains were stored overnight in fixative sol* Current address: The Rockefeller University, New York, NY 10021, U.S.A. t To whom reprint requests should be addressed at the Worcester Foundation for Experimental Biology, 222 Maple Avenue, Shrewsbury, MA 01545, U.S.A. Abbreviations: GABA, y-aminobutyric acid; PAP, peroxidase-antiperoxidase; SPI, substance P immunoreactive.

ution at 4°C. The olfactory bulbs were then sectioned at 20 pm with a Vibratome and processed with the unlabeled antibody enzyme method. 39 The sections were initally soaked for 1 h in 0.1 M phosphate buffer containing 1% normal goat serum. In some instances, this soaking solution also contained 0.02% Triton X-100. Sections were then incubated for 2448 h at 4°C in either of two rabbit antisera to substance P (purchased from ImmunoNuclear Corp., or donated by Dr S. Leeman to Dr D. Landis) which were diluted (1:200, 1:800 or 1:lOOO) with 0.1 M phosphate buffer containing 1% normal goat serum. The following procedures were carried out at room temperature, and all solutions contained 0.1 M phosphate buffer and 1% normal goat serum. After incubation in primary antisera, the sections were washed for 1.5 h, incubated for 1 h in a goat-anti-rabbit IgG (Miles Laboratories) solution (1:20), washed for 30min, incubated for 1 h in a rabbit peroxidase-antiperoxidase (PAP; Miles Laboratories) solution (l:lOO), and washed for 30min. The PAP complex was reacted with a solution containing 4&50mg of 3,3’-diaminobenzidine and 0.03% hydrogen peroxide in 100 ml of 0.1 M phosphate buffer (pH 7.4). After a brief rinse, the sections were treated with 0.05% osmium tetroxide for 1 min to enhance the reaction product. Another 1 h rinse preceded immersion in a 1% paraformaldehyde and 3% glutaraldehyde fixative solution for 2 h to overnight. The sections were then washed in buffer for 1 h, osmicated (2% osmium tetroxide) for 1 h, rinsed, dehydrated in graded alcohols and propylene Epon-Araldite, and flat-embedded cover glasses. Thin sections

oxide, infiltrated with between Teflon-coated were examined either

unstained, or stained with a uranyl acetate and lead solution,34 and were photographed with a Zeiss EM9 or a JEM 1OOCX electron-microscope. Adsorption controls were carried out on sections by mixing 0.7, 2 or 2Opg of synthetic substance P (0.5, 1.5 or 15 PM) per milliliter of diluted primary antiserum 15 min

2691

prior 10 introducing the tissue. For both of the antisera, prior adsorption with synthetic substance P eliminated all

dense reaction product. Nevertheless, large. agranular. spherical vesicles could be identified in many of tllc

immunoreactivity in the main olfactory bulb. To determine levels of background or non-specific staining, adjacent sections were processed in the usual manner except that the exposure to the primary antisera was omitted.

SPI processes in the glomerular neuropil (Figs 8.9). The synaptic contacts of these processes onto other

RESULTS Light-microscopy. The laminar distribution and gross rno~hol~gi~~ features of SPI neurons in the main offactory bulb of the hamster have been described in our previous, light-microscopic study,7 which used the antiserum purchased from ImmunoNuclear Corp. The antiserum obtained from Dr Leeman produced the same distribution of neuronal labeting. The cell bodies of SPI neurons were located in the periglomerular region of the glomerular layer. These cell bodies were fusiform or muttipofar and averaged 13 pm by 11 pm along their major and minor axes. Processes from these neurons also contained substance P immunoreactivity. The SPI processes were prevalent in the periglomerular region? and often could be observed to arborize within one and occasionafty within two or more gfomeruli (Figs 12). Electron-microscopy. The SPI neurons had spherical or ovoid nuclei which typically were located eccentrically in the cell body. Nuclear chromatin was finely dispersed throughout the nucleus, with only thin patches of dense chromatin along the perimeter of the nuclear membrane. A well defined nucleolus was present in some sections (Fig. 3). Invaginations of the nuclear membrane were not observed in any of the SPI neurons. The perikarya contained strands of rough endop~asm~~ reticulum, Golgi apparati and numerous mitochondria (Fig. 4). Somatic synaptic vesicles and free ribosome rosettes may have been present, but were obscured by the peroxidase reaction product. Occasionally, SPI somata were postsynaptic to processes containing pleomorphic vesicles. The dendrites of many neurons in the main olfactory bufb have regions which are devoid of ribosomes, contain synaptic vesicles, and appear to be presynaptic to other dendrites. Therefore, in single thin sections, it is often difficult to identify any given neuronal process as a dendrite or an axon. Neuronal processes containing substance P imrn~~or~a~t~~it~ were distributed in the perig~omerular region (Figs $6) and within the glomerular neuropif (Figs 7,8,9), but were not observed to project into the external plexiform layer or superficially into the olfactory nerve layer. All of the SPI processes encountered in the gIomerular layer were unmyelinated. Within the periglomeruiar neuropil, SPX processes and cell bodies received synaptic contacts from terminals or dendrites containing pleomorphic synaptic vesicles (Cf. Figs $6). Some of the SPI processes in the peri&merular region also contained synaptic vesicles, but the shape of these vesicles was often masked by the

dendrites in the giomerular neuropif were not observed. The SPI processes in the giomeruiar neuropil were observed to receive synaptic contacts from the primary olfactory axons (cf. Fig. 8).

Previous ~i~bt-microscopic imrnuno~yto~hern~~a~ observations’ suggested that external tufted cells in the glomerular layer of the main olfactory bulb contain substance P immunoreactivity. Our present ultrastructural findings support this interpretation. The locations and sizes of the cell bodies of these neurons and the ultrastructura~ features of their nuclei and perikarya correspond to those of external tufted cells.7,29 The intraglomerular as well as periglomerular distribution of the SPI processes, presence of agranular, spherical vesciles, and synaptic contacts from primary oifactory axons and with other processes containing pleomarphic synaptic vesicles are alf features of external tufted cell dendrites.30.“” We found no evidence that substance P immunoreactivity is present in periglomerular cells or in superficial short-axon cells. Ram& y Cajal 32 described the axons of external tufted cells as ramifying in the glomerular fayer, and as bearing a radia~l~-oriented main branch which projects through the external plexiform layer before turning caudally in the granule cell layer to exit from the olfactory bulb. At the ultrastructural level, we could not confidently distinguish axons from fine dendritic branches, and we faifed to observe SPI processes projecting deep into the external plexiform layer. It is possible that the axons are too sparsefy distributed to have been encountered. Based on our findings, it is not possible to determine whether SPI tufted cell axons are myelinated or unmyelinated. The synaptic organization of the glomerular layer has been well studied. 29.30,31.40.41fn addition to their inputs from primary olfactory receptor neurons. tufted cells in this layer receive synaptic contacts from the dendrites and axons of periglomerular cells and possibly from centrifugal fibers. In turn, the dendrites and cell bodies of external tufted cells synapse with perig~omeruIar ceil dendrites and cell bodies, and the recurrent axon coIIaterals of externaf tufted C&IS synapse on the dendrites and cell bodies of superficial short-axon cells and possibly on the dendrites of mitral and tufted cells.3o,31 Reciprocal synaptic contacts have also been observed between the intraglomerular

dendrites

of tufged celis and the dendrites

of

perigfomerular cells. 30 Based un this synaptic organization, the activity of external tufted cells may be affected by carnosine in primary olfactory axons,2 y-aminobutyric acid (GABA), dopamine,““,” and methionine-enkephalin’97 in periglomerular Cells.

Fig. 1. Light-micrograph of a substance P immunoreactive neuron with dendrites which arborize within an olfactory glomerulus. Other SPI neurons (arrowheads) are also present in the surrounding periglomerular region, x 200. Fig. 2. Light-micrograph

of a substance P immunoreactive neuron with dendritic branches which arborize within different glomeruli. x 260.

Fig. 3. Electron-micrograph of a substance P immunoreactive external tufted cell in the periglomeruiar region of the glomerular layer. This cell has an oval nucleus (N) with a prominent nucleolus (Nu). Dense reaction product fills the perikarya and a dendrite (d) extending from the ceil body. Electron-micrographs in this paper are of sections stained with uranyi acetate and lead, unless otherwise noted, The section used for this photograph was not stained. x 10,800. Fig. 4. Substance P immunoreactive external tufted cell. Note the eccentrically located nucleus (N) and prominent Golgi apparati (G). x 11,200. 2699

Fig. 5. A small portion of a substance P immunoreactive cell body (C) is illustrated. An unreactive terminal (t) containing pleomorphic synaptic vesicles forms a synaptic junction (arrowhead) with this cell body.

x 42.300.

Fig. 6. Process (p) in the periglomerular neuropil that is immunoreactive for substance P and receives a synaptic contact from a terminal (t) containing pleomorphic synaptic vesicles. The presynaptic terminal contains a synaptic vesicle that is fused with the junctional membrane (arrowhead). x 71,600.

2700

Fig. 7. Substance P immunoreactive processes (p) in the glomerular neuropil. The SPI processes contain dense reaction product and stand out against unreactive dendrites (examples at ‘d’) and olfactory axons (examples at ‘a’). The density of the axoplasm of the olfactory axons is normal and can be used as an identifying characteristic. x 13,400. Fig. 8. The low density of the immunocytochemical reaction product in these substance P immunoreactive dendrites (d) makes it possible to visualize their spherical synaptic vesicles. A dense-core vesicle (arrowhead) is present in one of these dendrites. A synaptic contact from a primary olfactory axons is also present (star). x 32,800. Fig. 9. Synaptic reaction product

vesicle morphology has been restricted

is apparent in this dendrite to the membranes of synaptic Unstained section. x 54,500. 2701

in which the immunocytochemical vesicles and a mitochondrion (m).

Substance P neurons in the main olfactory bulb

and acetylcholine,3.24*37 norepinephrine,6,‘a.’ ’ WVhortoniq6*’ O and luteinizing hormone-releasing mone13,18,27 in centrifugal afferents. Within the olfactory bulb, external tufted cells have most of their synaptic inffuence on the activity of ~rigIomerula~ cells. Therefore, SPI externaf tufted cells may be able to inffuence periglomerular cells which contain GABA, dopamine, or methionine-enkephalin. The physiological function of substance P in the olfactory buib is unknown. Many investigators believe that tufted cells have an excitatory ir&Iuence on per~giomeruIar cells,36 whieh is consistent with studies showing that substance P has an excitatory action in other neuronal systems2’ In contrast, it is generally thought that periglomerular cells inhibit the activity of tufted cells. 36 In other areas of the nervous system, it has been shown that GABA, dopamine and methionine~enkephaiin inhibit the release of substance P. GABA suppresses the potassium-evoked release of substance P from substantia nigra tissue slices.” Enkephalin also inhibits the release of substance P from dorsal root ganglion cells in culture25 and reduces the pota~~jurn-induced release of substance P from tissue slices of the trigeminal nucIeus.20 In the substantia nigra, dopami~e~containing neurons have been postulated to produce a tonic inhibitory influence on substance P-containing neurons,” It is intriguing to speculate that these three substances might have similar actions on the SPI external tufted cells in the main offactory bulb. The synaptic interactions within the gIomerular

2703

layer would be of even greater experimental interest if neuropeptides and other putative neurotransmitters coexist in single olfactory bulb neurons. Several groups of neurons in other regions of the central nervous system have been shown to contain two or three neurom~ulaand neuro~ansm~tters potentia1 tors.‘5~*’ Caexistence of GABA or dopamine with methione-enkephalin in periglomerular cells has been suggested, but remains to be demonstrated.’ A population of external tufted cells has been show to synthesize dopamine. 10*ii It has also been suggested that aspartate or glutamate serve a neurotransm~tter function in output neurons of the main olfactory bulb.4,9 Substance P thus might coexist with dopamine or with putative amino acid neurotransmitters in the external tufted cells. The numerous neuropeptides, monoam~nes and other neurotransm~tter candidates in the olfactory bulb, together with our understanding of its synaptic organization, make the olfactory bulb an excellent model system in which to study the functions of neuropeptides and their interactions with other neurotransmitter or neuromodulator substances in the central nervous system. work WCS supported by NINCDS Research Grants NS12344 and NS16367, and NSF Research Grant BNS78-04248. We thank Dr D.

Acknowledgernears-This

Landis for his suggestions and criticisms, and Dr S. Lee+ man for antiserum to substance P. A pre~~rn~na~ account ofthis work was presented at tbe Eievenrh Annuat Meeting of the Society for Neuroscience, October 198f . I

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22 Aprii 1982)