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Autoradiographic analysis of [3H]dopamine and [3H]dopa uptake in the turtle olfactory bulb
0306-4522/83/040705-lOW3.00/0
Neuroscience Vol. 8, No. 4, pp. 705 to 715, 1983 Printed in Great Britain
Pergamon Press Ltd IBRO
AUTORADIOGRAPHIC ANALYSIS OF C3H]DOPAMINE AND CjH]DOPi UPTAKE IN THE TURTLE OLFACTORY BULB N. HALASZ’, M. C. NOWYCKY and G. M. SHEPHERD Sections of Neuroanatomy and Neurosurgery, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510, U.S.A. Abstract-Uptake and retention of exogenous tritiated dopamine and L-dopa was observed within turtle olfactory bulb slices. In the more superficial layers, periglomerular and superficial tufted cells, as well as their processes, and intraglomerular dendrites were recognized as labeled. Within the deeper part of the bulb, some labeled cells between the tanycytes, as well as nerve fibers and terminals within the granule cell layer, are reported. The results confirm the presence of specific intrinsic dopaminergic cells within the reptilian olfactory bulb. Recent studies combining histofluorescence, immu nocytochemistry and autoradiographic uptake12*1s have provided evidence for the presence of dopaminergic cells within the rat olfactory bulb. Most of these cells appear to be among the periglomerular (PG) cells, a type of short-axon cell related to the olfactory glomeruli. The dopaminergic cells, like other PG cells,
are involved in local synaptic circuits in the glomerular layer, both through dendro-dendritic synapses within the glomeruli, and axo-dendritic synaptic connections to neighboring glomeruli. There is physiological evidence for inhibitorylO and excitatory’ interactions mediated by the PG cells through these circuits, but the contribution of dopaminergic PG cells to these interactions is not known. In order to investigate cell properties and local circuit interactions, we have recently introduced an isolated preparation of the turtle olfactory bulb.23,25 Two questions were addressed in the present study. First, is there evidence for the presence of dopaminergic neurons in the olfactory bulb of this species? We have analyzed the uptake and retention of exogenous tritiated dopamine (DA) and dopa by the isolated olfactory bulb. Elsewhere, we have summarized briefly the main points of our autoradiographic results24 and also demonstrated the presence of the enzyme tyrosine hydroxylase within the assumed dopaminergic cells of the turtle olfactory bulb.i4 In a companion paper, we report evidence for the physiological actions of DA. EXPERIMENTAL
PROCEDURES
Histology. Turtles were decapitated, and the bulbs were removed from the skull, put into oxygenated turtle Ringer’s
1Present address: Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, H-6701 Szeged, P.O. Box 521, Hungary. Abbreoiations: DA, dopamine; EPL, external plexiform layer; GL, glomerular layer; GRL, granule cell layer; PC?, periglomerular cells.
solution36 and freed from most of the connective tissue under a dissection microscope. The bulbs were taken whole or cut into dorsal and ventral halves (hemi-slices) and preincubated for 60min at room temperature (22°C) in the same Ringer’s solution. They were then incubated in C3H]DA ([7,8-3H]dopamine; specific activity, 43 Ci/mmol), or [‘H]dopa (L-3,4 dihydroxy [ring 2,5,6-3H]phenylalanine; specific activity, 43 Ci/mmol, same supplier, the Radiochemical Centre, Amersham, England) dissolved in Ringer’s solution. Incubation lasted either 10 or 60min with concentrations of these drugs of either 5 x 1Om6 or 5 x lo-’ M. The Ringer’s solution was oxygenated during the incubation period. Half of the samples were incubated for an additional hour with 10e3 M unlabeled (‘cold’) dopamine (dopamine HCI, Sigma). The results showed no significant differences related to differences in concentration of label in the bath, incubation of the hem&slices in ‘cold’ DA, or the longer incubation time. The 60 min incubations, however, resulted in a slightly enhanced density of label, together with a higher level of background. After incubation, the whole bulbs or hemi-slices were fixed by immersing them in an ice-cold solution of fixative (2.5% glutaraldehyde, 1% paraformaldehyde in a 0.1 M phosphate buffer solution) for 60min, cut into smaller pieces, fixed for an additional hour in the same fixative, and rinsed overnight in a 0.1 M phosphate buffer solution. Postfixation was carried out for 2 h in 1 or 2% 0~0, in 0.1 M phosphate buffer solution, then rinsed in buffer, dehydrated and embedded in Epon or EM bed-812 (EMS, Fort Washington, PA), and polymerized for 16 h. All fixation steps were carried out at 4°C. After hardening, 0.554pm thick sections were cut from the bulbs and put onto glass slides for light-microscopic autoradiography. The slides were dipped in undiluted melted emulsion (Kodak NTB-2) and exposed in lighttight boxes for 16-60 days at 4”C, in the presence of anhydrous CaCI,, then developed with Kodak D19 developer. The autoradiograms were usually studied without counterstaining of the sections, in order to enhance the visibility of the grains, Areas of blocks showing typical labeling patterns were identified by observation of the autoradiograms under light-microscopy, and trimmed and sectioned for further electron-microscopic autoradiography. The electron-microscopic autoradiographic technique used in this study was
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M. <‘. Nowyck> and Ci hf. Shepherd
described in detail in an earlier paper.” We have used Ilford L4 emulsion and Amidol developer after 4-28 days exposure time. The electron-microscopic autoradiograms were photographed with a Philips EM 20. For conventional electron-microscopic studies. turtles were perfused via their common carotid-brachial arteries with 400-500 ml ice-cold fixative (containing 2.5”” glutaraldehyde and 10; paraformaldehyde) during 30 min; the bulbs were removed, cut into smaller pieces, postfixed in the same fixative for 2 h then in 2% 0~0, solution for 1 h (all these steps were carried out at 4°C). dehydrated and embedded in Epon. Semi-thin (1-2 pm thick) sections (stained with a O.l’,, toluidine blue solution). as well as silver- and gold-colored fine sections (consecutive to the semi-thin sections, and contrasted with lead citrate) were made, and compared with the autoradiograms of incubated hemi-slices.
shown onal
in the photomicrograph cell
bodies,
presumably
in Fig. those
I. Large of
mitral
neurcells.
the external plexiform layer (EPL) and the granule cell layer (GRL). Other large cells were present in the EPL: with a distribution similar to mammalian tufted cells. In the absence of information about the distribution of their axons or axon collaterals and their projection sites, we will tentatively refer to them as tufted cells. Periglomerular cells (PG) were more frequent at the deeper aspect of the glomerular layer (CL), while the superficial part of this layer was covered by olfactory nerve fascicles and multiple layers of connective tissue. About half of the total thickness of the bulb consisted of the GRL. There is a large ventricle in the turtle olfactory bulb: its surface is limited by tanycytes. were
observed
at
the
border
between
RESULTS Histology
The histological layers and main cell types of the olfactory bulb can be recognized under low power, as
All of our experiments, including the uptake of both C3H]DA and C3H]dopa, produced the most dense silver deposits in two areas: the GL and the
Fig. 1. Light-microscopic survey view of the turtle olfactory bulb laminae. Perfused animal, 2 pm thick epoxy section, toluidine blue stain. EPL, external plexiform layer; GL, glomerular layer; GRL, granule cell layer; ON, olfactory nerve layer; asterisks, olfactory ventricle. Several mitral and tufted cell bodies are labeled with arrowheads. Bar, 50 pm. Fig. 2. [3H]dopamine
uptake
into various
structures,
as shown
on a light-microscopic
autoradiogram.
Olfactory bulb hemi-slice, 60min incubation in 5 x lO-6 M DA solution, 2pm thick epoxy resin secticin, 28 days exposure, Kodak D 19 developer, no counterstaining. Most of the label appears in the glomerular region, Labeled structures, the ventricular
and is located in the glomeruler neuropil as well as in cells around the glomeruli. smaller in size, appear throughout the granule layer, especially in close proximity to surface (asterisk). Other abbreviations are similar to those in Fig. 1. Bar, 50 ltm.
Fig. 3. High power photomicrograph of a detail of the glomerular region. Light-microscopic autoradiogram, 28 days exposure, unstained epoxy section. Labeled cells (arrowheads) and their processes are shown. Note dense patches of grains within the glomerular neuropil. EPL, external plexiform layer: ON. olfactory nerve layer. Bar, 50 pm. Figs 4-5. Electron-microscopic autoradiographs of two labeled periglomerular ceils (PGC) and surrounding structures. Olfactory bulb hem&slices, C3H]DA incubation, lead citrate contrast, 7 days exposure time, Amidol developer. Note the accumulation of grains over the cell cytoplasm and nuclei; neighboring dendrites (d) and olfactory nerve fascicles (ON) remained unlabeled. Bar, 2 pm. Figs 67. C3H]dopamine uptake into structures within the glomerular neuropil. EM autoradiograms, lead citrate contrast, 7 days exposure time. Heavy deposits of silver grains are seen over the terminal enlargements of pale dendritic profiles(d), surrounded by the more electron-dense olfactory nerves (ON). Other dendrites (d) were not labeled over background level. Bar, 1 pm. The unlabeled profile (dm) in Fig. 6 contains few neurotubules and vesicles, and is characteristic of mitral and tufted cell dendrites. Fig. 8. Light-microscopic autoradiogram of labeled structures after 10 min incubation of hemi-slice in a 10m6 M solution of dopamine. The 2 pm-thick epoxy resin section was taken parallel with the granule cell layer of the slice, 28 days exposure time. Labeled fibers and terminal enlargements can be seen (arrows) throughout the layer. The label was also retained by blood vessel walls (c). Bar, 100 pm. Figs 9-10. Electron-microscopic autoradiograms taken from the granule layer. The accumulation of [“HIDA was observed over certain fibers and terminals of unknown origin. These structures, in addition to their ‘clear’ synaptic vesicle content, usually contained a few dense-core vesicles. c, capillary. Bars, 1pm. Fig. Il. labeling deposits
Light-microscopic autoradiogram of hemi-slice after 60 min incubation in 10 -6 M dopa. The pattern was similar to that seen with C3H]DA (compare with Fig. 2). Note the heavy silver close to the ventricular surface (arrows). Abbreviations for the layers same as Fig. I. Asterisks, olfactory ventricle. Bar, 50 pm.
Fig. 12. Detail of the ventricular surface, showing a labeled cell with a thin rim of cytoplasm, surrounding the nucleus. This cell is surrounded by unlabeled tanycytes (TC), extending their microvilli (mv) and cilia (ci) into the ventricular lumen. Bar. 1 {lrn.
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Dopamine in turtle olfactory bulb deep GRL. In general, with C3H]DA there was more distinct label over the glomeruli, PG cells and structures within the granule layer. In comparison, C3H]dopa gave a stronger labeling over some cell bodies close to the ventricular surface, more diffuse label in GL, and almost no labeling within the granule layer, deeper than 50 pm from the ventricular surface. Figures 2-7 show the main features of c3H]DA uptake by constituents of the glomerular layer. Heavy accumulation of grains was present throughout the glomerular neuropil (Figs 2, 3). However, closer observation showed that the distribution was not homogeneous. Under the light-microscope, unlabelled areas were present, particularly in the superficial parts of the glomeruli, where the dendrites of bulbar neurons are intermingled with groups of olfactory axons (Fig. 3). At the deeper aspect of the glomeruli, where the overall density of the grains was less, continuity of labeled thick processes with labeled PG cell bodies was seen (Fig. 3). In addition, labeled cells of the same or even larger size were occasionally seen in the outer third of the EPL. Under electron-microscopy there was good preservation of ultrastructural characteristics. In particular, mitochondria and synaptic contacts were well preserved. The main departures from normal fine structure were variations in sizes of synaptic vesicles in the olfactory nerve terminals and the occasional presence of vacuoles, some of which appeared as dilatations of the endoplasmic reticulum in perikarya or dendrites. The labeled cells in the periglomerular area were recognized as neurons with soma diameters of approximately 10pm (Figs 4, 5). These showed typical features of PG cells, similar to those in mammals,28 including one or more stacks of rough endoplasmic reticulum, rare Golgi stacks and vesicles, and the presence of free ribosomes in the rim of cytoplasm surrounding the nucleus. Synaptic vesicles were occasionally seen within the cytoplasm of these labeled cells, but no synaptic contacts of either polarity were observed. The labeled cells in the superficial EPL had larger cell bodies (up to 14pm in diameter). They were recognized as tufted cells, based on the presence of multiple stacks of rough endoplasmic reticulum a higher number of mitochondria, and well-developed Golgi system. They also contained occasional small vesicles, but did not make synaptic contacts. Accumulation of grains was also observed over large (2-3 pm diameter) dendritic profiles (Figs 6, 7), which were much paler under electron-microscope than the olfactory nerves surrounding them. These labeled dendrites were presumed to be the processes of the labeled PG cells, and their ultrastructural characteristics resembled those in mammals.2g Most of these profiles contained a large number of small (diameter 3&50 nm) synaptic vesicles of the clear type, and occasional dense-core vesicles (diameter 8&120 nm). Neurotubules were prominent within the thinner shafts connected to the varicose enlargements
(Fig, 6), similar to the mitral secondary dendrites and tufted cell dendrites in the EPL. Fasciculi of the olfactory nerve fibers, or the terminal enlargements of the axons, also showed only a background rate of labeling. In the EPL, grain accumulation was observed only over a few cell bodies in the superficial part, as mentioned above. In the GRL, a number of labeled fibers (or their varicose terminal enlargements) and also a few labeled cells were seen (Figs 2, 8-12). Most of the labeled fibers were seen running parallel with the layers, so they appeared as dots in frontal sections (Fig. 2), and more elongated structures in sections taken parallel with the granule processes (Fig. 8). In electron-microscopic autoradiograms of hemi-slices, incubated with C3H]DA or C3H]dopa, accumulation of grains was observed over thin (diameter O.>l pm) fibers (Fig. 9) or small terminal enlargements (Fig. 10); their origin is unclear. Capillaries and small blood vessels close to the sliced surface were also heavily labeled (Fig. 8). In the deepest aspect of the bulb, labeled structures about the size of very large terminals or small cells were clearly visible in the light-microscopic autoradiograms (Fig. 2, C3H]DA; Fig. 11, C3H]dopa). Under electron-microscopy a few labeled fibers were also seen. Labeled cells were numerous and easily recognized (Fig. 12, C3H]dopa). These cells had an extremely thin rim of cytoplasm around their nuclei, even thinner than in PG or granule cells. Rough endoplasmic reticulum, mitochondria and free ribosomes were observed in the cytoplasm. The cytoplasm as well as the nuclear plasm appeared relatively electron-lucent when compared to the density of neighboring tanycytes. The tanycytes showed almost no uptake of C3H]DA or c3H]dopa. Most of the tanycytes were seen to reach the ventricular surface, bearing a bushy borderline of microvilli and cilia extending into the ventricular lumen while none of the labeled cells mentioned above reached the ventricular surface. No synaptic vesicles or specialized junctions were observed in relation to the labeled cells. These cells have not been identified yet as either neuronal or non-neuronal. DISCUSSION The structure of the olfactory bulb of reptiles generally resembles that of other vertebrate bulbs,’ having similar layering, a well pronounced internal plexiform layer, and somewhat more scattered mitral cells than the higher vertebrates. According to our preliminary electron-microscopic studies (N. Hal&z, et al., unpublished observations), examples of the main synaptic configurations described in the mammalian olfactory bulb (for review, see ref. 34) can be recognized in the turtle olfactory bulb as well, thereby implying that similar synaptic circuits may exist. The early fluorescence-microscopic observations of Dahlstrom and co-workers5 demonstrating the pres-
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N. Halis7. M. C‘. Nowycky and 6. M. Shepherd
ence of a few catecholaminergic structures within the glomerular region of the rabbit olfactory bulb have been subsequently supported by studies of loading of the periglomerular cells with exogenous norepinephrine2’ or dopa2’ and further extended by US~~.‘~.~” with conventional cytochemical techniques as well as light- and electron-microscopic autoradiography of C3H]norepinephrine and [3H]serotonin uptake and immunochemistry. Similar labeling patterns were observed following C3H]DA uptake by periglomerular cells of the rat bulb.31 In lower vertebrates, catecholaminergic structures have been observed in the frog olfactory bulb.3*4,27 but no data have been published previously showing the presence of similar catecholaminergic system within the reptilian bulb. Certain biochemical data have also supported the existence of a catecholaminergic system in the mammalian olfactory bulb, by revealing a substantial amount of endogenous DA.83’7322*26 Samples taken from different olfactory bulb layers revealed that the highest level of endogenous DA was found in the glomerular layer.ig Norepinephrine has been localized to fibers in the deeper layers, within the EPL and GRL.5,12*35 No noradrenergic cell bodies have been demonstrated in the olfactory bulb. This localization is also supported by biochemical data, demonstrating that after bulbectomy there is an accumulation of norepinephrine within more central areas.6”5’16 In the rat we did not find evidence for extrabulbar projections of dopaminergic cells,12*r3 and similar conclusions were drawn by Edwards and co-workers.6 On the other hand, Hirsch16 described the presence of such a bulbofugal pathway running to certain limbic areas. In addition. a weak bulbopetal pathway may also exist, as was described by Fallon & Moore.’ However, in the rat, the dopaminergic system seems to be mostly an intrinsic bulbar system, restricted to local interneurons within the periglomerular area and superficial EPL, as was observed very recently by our serial analysis of immunohistochemical sections at the electron-microscope level.’ 1 For fine structural localization of the autoradiographic label within the superficial layers of the turtle olfactory bulb we have applied the morphological criteria for the determination of the constituents of the rat olfactory bulb,1~2s~2g supplemented with some data from perfused turtle bulbs. According to these
criteria, most of the labeled cells were recognized 211 periglomerular or superficial tufted neurons. while the intraglomerular label was largely confined to dendrite-like processes, These dendrites (containing ;I large number of synaptic vesicles) might belong to periglomerular cells. while other unlabeled dendrites resembled mitral dendrites based on their ultrastructural characteristics. Thus, the labeling pattern in the turtle olfactory bulb appears to be similar to that of the rat: periglomerular cells and their processes retained the largest proportion of the labeled catecholamine. Another similarity to the rat was the presence of a few labeled superficial tufted cells in the vicinity of the glomeruli. With immunohistochemistry. weakly fluorescent large cells, lying at the depth of the mitral and granule cells, were also visualized.14 Our present autoradiographic studies, however, did not show any label at this level comparable to the immunohistochemical results. The distribution and frequency of labeled fibers after C3H]DA uptake within the granule layer were similar to the previous C3H]noradrenaline studies in the rat,‘* however further experiments are needed in order to recognize clearly their relation to other structural constituents. The labeled neuronal processes may be some of the central fibers contacting the granule cells within this layer.30 As yet we are not able to speculate which morphological correlates of the labeled (C3H)dopa and C3H]DA) deep cells might be present in the mammalian olfactory bulb. No synaptic connections could be found between these and neighboring structures. If the labeled cells have the capability of uptake, storage and release of endogenous DA as well as label, they may serve more as a bridge between the ventricular surface and granular layer than as an interneuronal connection, similar to the proposed role for tanycytes at the median eminence.32,33 which are thought to transport catecholamines from the CSF deeper into the tissue. Acknowledgements-This work was supported by research grants NS-07609 and F32-NS06159 from the National Institute of Neurological and Communitive Disorders and Stroke, and BNS 78816545 from the National Science Foundation. We thank G. Collins for expert assistance
REFERENCES I. Andres K. H. (1965) Der Feinbau des Bulbus olfactorius der Ratte unter besonderer then Verbindungen, Z. Zellforsch mikrosk. Anal. 65, 530-561.
Beriicksichtigung der synaptis-
2. Andres K. H. (1970) Anatomy and ultrastructure of the olfactory bulb in fish, amphibia, reptiles, birds and mammals. In Taste and Smell in Vertebrates (Eds Wolsteinholme G. E. W. & Knight D. J.), pp. 1777194. Churchill, London. 3. Bartels W. (1971) Die Ontogenese der aminnhaltigen Neuronensysteme in Gehirn von RUM temporariu. Z. Zeflforsch. mikrosk. Anat. 116. 94-l 18. 4. Braak H. (1970) Biogene Amine in Gehirn von Frosch (Rana esculenta). Z. Zellfbrsch. mikrosk. Anat. 106, 269-308. 5. Dahlstrdm A., Fuxe K., Olson L. & Ungerstedt U. (1965) On the distribution and possible function of monoamine nerve terminals in the olfactory bulb of the rabbit. Life Sci. 4, 2071-2074.
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6. Edwards D. A., Schlosberg A. J., McMaster S. E. & Harvey J. A. (1977) Olfactory system damage and brain catecholamines in the rat. Brain Res. 121, 121-130. 7. Fallon J. H. & Moore R. Y. (1978) Catecholamine, innervation of the basal forebrain-III. Olfactory bulb, anterior olfactory nuclei, olfactory tubercle and piriform cortex. J. camp. Neuro[. 180, 533-544. 8. Ferriero D. & Margolis F. L. (1975) Denervation in the primary olfactory pathway of miceII. Effects on carnosine and other amine compounds. Bruin Res. 94, 75-86. 9. Freeman W. J. (1974) Relation of glomerular neuronal activity to glomerular transmission attenuation. Brain Res. 65, 91-107. IO. Getchell T. V. & Shepherd G. M. (1975) Short-axon cells in the olfactory bulb: dendrodendritic synaptic interactions. J. Physiol., Lond. 251, 523-548. Il. Hal&z N., Johansson O., Hijkfelt T., Ljungdahl A. & Goldstein M. (1981) Immunohistochemical identification of two types of dopamine neuron in the rat olfactory bulb as seen by serial sectioning. J. ~~~~oc~~fo~.10, 251-259. 12. Hal& N., Ljungdahl A. & HGkfelt T. (1978) Transmitter histochemjstry of the rat olfactory bulb-If. Fluorescence histochemical, autoradiographic and electron-microscopic localization of monoamines. Brain Res. 154, 2533271. 13. Hal&z N., Ljungdahl A., Hokfelt T., Johansson O., Goldstein M., Park D. & Biberfeld P. (1977) Transmitter histochemistry of the rat olfactory bulb--I. Immunohistochemical localization of monoamine synthesizing enzymes. Support for intrabulbar, periglomerular dopamine neurons. Brain Res. 126, 45.5-474. 14. Hal&z N., Nowycky M., Hokfelt T., Shepherd G. M., Markey K. & Goldstein M. (1982) Dopaminergic periglomeruIar cells in the turtle olfactory bulb. &&I Res. Bull. In press. 15. Harvey J. A., Scholfield C. N., Graham L. T. & Aprison M. H. (1975) Putative transmitters in denervated olfactory cortex. J. Neurochem. 24, 445-449. 16. Hirsch J. D. (1980) Opiate and muscarinic ligand binding in five limbic areas after bilateral olfactory bulbectomy. Brain Res. 198. 271-283. 17. Hirsch J. D. & Margolis F. L. (1980) Influence of unilateral olfactory bulbectomy on opiate and other binding sites in the contralateral bulb. Bruin Res. 199, 39-47. 18. Hokfelt T., Hal&z N., Ljungdahl A., Johansson O., Goldstein M. & Park D. (1975) Hist~hemic~ support for a dopaminergic mechanism in the dendrites of certain periglomerular cells in the rat olfactory bulb. Neurosci. Letc. 1, 85-80. 19. Jaffe E. H. & Cuello A. C. (1980) The distribution of catecholamines, glutamine decarboxylase and choline acetyltransferase in layers of the rat olfactory bulb. Brain Res. 186, 232-237. 20. Lichtensteiger W. (1966) Uptake of norepinephrine in periglomerular cells of the olfactory bulb of the mouse. Nurure, Land. 210,955-956. 21. Lidbrink P.: Jonsson G. & Fuxe K. (1974) Selective reserpine-resistant accumulation of catecholamines in central dopamine neurons after DOPA administration. Brain Res. 67,439-456. 22. Margolis F. L., Roberts N., Ferriero D. & Feldman J. (1974) Denervation in the primary olfactory pathway of mice: biochemical and morphological effects. Brain Res. 81, 469-483. 23. Mori K. & Shepherd G. M. (1979) Synaptic excitation and long-lasting inhibition of mitral cells in the in vitro turtle olfactory bulb. Brairl Res. 172, 155-159.
24. Nowycky M. C., Hal&z N. & Shepherd G. M. (1981) Studies of dopamine as a neurotransmitter in the turtle olfactory bulb. Sot. Nearos~i, Abs. 7, 573. 25. Nowycky M. C., Waldow U. C Shepherd G. M. (1978) Electrophysiological studies in the isolated turtle brain Sot. Neurosci. Abs 4, 583. 26. Palkovits M., Zilborszky L., Brownstein 27. 28.
29. 30. 31. 32.
33. 34. 35.
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M. J., Fekete M., Herman J. P. & Kanyicska B. (1979) Distribution of norepinephrine and dopamine in cerebral cortical areas of the rat. Brain Res. Bull. 4, 593-601. Parent A. (1975) The monoaminergic innervation of the telencephalon of the frog, Rana pipiens. Brain Res. 99, 35-47. Pinching A. J. & Powell T. P. S. (1971) The neuron types of the glomeruli of the olfactory bulb. J. CeIt Sci. 9,30>345. Pinching A. J. & Powell T. P. S. (1971) The neuropil of the glomeruli of the olfactory bulb. J. Ceil Sci. 9, 347-377. Price J. L. & Powell T. P. S. (1970) An electron-microscopic study of the termination of the efferent fibers to the olfactory bulbs from the cerebral hemisphere. J. Ceil Sci. 7, 157-187. Priestley J. V., Kelly J. S. & Cue110 A. C. (1979) Uptake of C3H]dopamine of the rat olfactory bulb: an autoradiographic study. Brain Res. 165, 149-155. Scott D. E., Dudley G. K. & Knigge K. M. (1974) The ventricular system in neuroendocrine mechanisms-II. fn vine monoamine transport by ependyma of the median eminence. Ceil Tissue Res. 154, l-16. Scott D. E., Scott P. M. & Krobisch-Dudley G. (1978) Ultrastructural localization of radiokbeled t_-dopa in the endocrine hypothalamus of the rat. Cell Tissue Res. 195, 29-43. Shepherd G. M. (1972) Synaptic organization of the mammalian olfactory bulb. Physiol. Rev. 52, 864-917. Swanson L. W. & Hartman B. K. (1975) The central adrenergic system. An immunofluoreszence study of the location of cell bodies and their efferent connections in the rat utilizing dopamine-hydroxylase as a marker. J. camp. Neural. 163.467-505. Waldow U., Nowycky M. C., & Shepherd G. M. (1981) Evoked potential and single unit responses to olfactory nerve volleys in the isolated turtle olfactory bulb. Brain Res. 211, 267-283. (Accepted 15 September 1982)
Neuroscience Vol. 8, No. 4, pp. 705 to 715, 1983 Printed in Great Britain
Pergamon Press Ltd IBRO
AUTORADIOGRAPHIC ANALYSIS OF C3H]DOPAMINE AND CjH]DOPi UPTAKE IN THE TURTLE OLFACTORY BULB N. HALASZ’, M. C. NOWYCKY and G. M. SHEPHERD Sections of Neuroanatomy and Neurosurgery, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510, U.S.A. Abstract-Uptake and retention of exogenous tritiated dopamine and L-dopa was observed within turtle olfactory bulb slices. In the more superficial layers, periglomerular and superficial tufted cells, as well as their processes, and intraglomerular dendrites were recognized as labeled. Within the deeper part of the bulb, some labeled cells between the tanycytes, as well as nerve fibers and terminals within the granule cell layer, are reported. The results confirm the presence of specific intrinsic dopaminergic cells within the reptilian olfactory bulb. Recent studies combining histofluorescence, immu nocytochemistry and autoradiographic uptake12*1s have provided evidence for the presence of dopaminergic cells within the rat olfactory bulb. Most of these cells appear to be among the periglomerular (PG) cells, a type of short-axon cell related to the olfactory glomeruli. The dopaminergic cells, like other PG cells,
are involved in local synaptic circuits in the glomerular layer, both through dendro-dendritic synapses within the glomeruli, and axo-dendritic synaptic connections to neighboring glomeruli. There is physiological evidence for inhibitorylO and excitatory’ interactions mediated by the PG cells through these circuits, but the contribution of dopaminergic PG cells to these interactions is not known. In order to investigate cell properties and local circuit interactions, we have recently introduced an isolated preparation of the turtle olfactory bulb.23,25 Two questions were addressed in the present study. First, is there evidence for the presence of dopaminergic neurons in the olfactory bulb of this species? We have analyzed the uptake and retention of exogenous tritiated dopamine (DA) and dopa by the isolated olfactory bulb. Elsewhere, we have summarized briefly the main points of our autoradiographic results24 and also demonstrated the presence of the enzyme tyrosine hydroxylase within the assumed dopaminergic cells of the turtle olfactory bulb.i4 In a companion paper, we report evidence for the physiological actions of DA. EXPERIMENTAL
PROCEDURES
Histology. Turtles were decapitated, and the bulbs were removed from the skull, put into oxygenated turtle Ringer’s
1Present address: Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, H-6701 Szeged, P.O. Box 521, Hungary. Abbreoiations: DA, dopamine; EPL, external plexiform layer; GL, glomerular layer; GRL, granule cell layer; PC?, periglomerular cells.
solution36 and freed from most of the connective tissue under a dissection microscope. The bulbs were taken whole or cut into dorsal and ventral halves (hemi-slices) and preincubated for 60min at room temperature (22°C) in the same Ringer’s solution. They were then incubated in C3H]DA ([7,8-3H]dopamine; specific activity, 43 Ci/mmol), or [‘H]dopa (L-3,4 dihydroxy [ring 2,5,6-3H]phenylalanine; specific activity, 43 Ci/mmol, same supplier, the Radiochemical Centre, Amersham, England) dissolved in Ringer’s solution. Incubation lasted either 10 or 60min with concentrations of these drugs of either 5 x 1Om6 or 5 x lo-’ M. The Ringer’s solution was oxygenated during the incubation period. Half of the samples were incubated for an additional hour with 10e3 M unlabeled (‘cold’) dopamine (dopamine HCI, Sigma). The results showed no significant differences related to differences in concentration of label in the bath, incubation of the hem&slices in ‘cold’ DA, or the longer incubation time. The 60 min incubations, however, resulted in a slightly enhanced density of label, together with a higher level of background. After incubation, the whole bulbs or hemi-slices were fixed by immersing them in an ice-cold solution of fixative (2.5% glutaraldehyde, 1% paraformaldehyde in a 0.1 M phosphate buffer solution) for 60min, cut into smaller pieces, fixed for an additional hour in the same fixative, and rinsed overnight in a 0.1 M phosphate buffer solution. Postfixation was carried out for 2 h in 1 or 2% 0~0, in 0.1 M phosphate buffer solution, then rinsed in buffer, dehydrated and embedded in Epon or EM bed-812 (EMS, Fort Washington, PA), and polymerized for 16 h. All fixation steps were carried out at 4°C. After hardening, 0.554pm thick sections were cut from the bulbs and put onto glass slides for light-microscopic autoradiography. The slides were dipped in undiluted melted emulsion (Kodak NTB-2) and exposed in lighttight boxes for 16-60 days at 4”C, in the presence of anhydrous CaCI,, then developed with Kodak D19 developer. The autoradiograms were usually studied without counterstaining of the sections, in order to enhance the visibility of the grains, Areas of blocks showing typical labeling patterns were identified by observation of the autoradiograms under light-microscopy, and trimmed and sectioned for further electron-microscopic autoradiography. The electron-microscopic autoradiographic technique used in this study was
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M. <‘. Nowyck> and Ci hf. Shepherd
described in detail in an earlier paper.” We have used Ilford L4 emulsion and Amidol developer after 4-28 days exposure time. The electron-microscopic autoradiograms were photographed with a Philips EM 20. For conventional electron-microscopic studies. turtles were perfused via their common carotid-brachial arteries with 400-500 ml ice-cold fixative (containing 2.5”” glutaraldehyde and 10; paraformaldehyde) during 30 min; the bulbs were removed, cut into smaller pieces, postfixed in the same fixative for 2 h then in 2% 0~0, solution for 1 h (all these steps were carried out at 4°C). dehydrated and embedded in Epon. Semi-thin (1-2 pm thick) sections (stained with a O.l’,, toluidine blue solution). as well as silver- and gold-colored fine sections (consecutive to the semi-thin sections, and contrasted with lead citrate) were made, and compared with the autoradiograms of incubated hemi-slices.
shown onal
in the photomicrograph cell
bodies,
presumably
in Fig. those
I. Large of
mitral
neurcells.
the external plexiform layer (EPL) and the granule cell layer (GRL). Other large cells were present in the EPL: with a distribution similar to mammalian tufted cells. In the absence of information about the distribution of their axons or axon collaterals and their projection sites, we will tentatively refer to them as tufted cells. Periglomerular cells (PG) were more frequent at the deeper aspect of the glomerular layer (CL), while the superficial part of this layer was covered by olfactory nerve fascicles and multiple layers of connective tissue. About half of the total thickness of the bulb consisted of the GRL. There is a large ventricle in the turtle olfactory bulb: its surface is limited by tanycytes. were
observed
at
the
border
between
RESULTS Histology
The histological layers and main cell types of the olfactory bulb can be recognized under low power, as
All of our experiments, including the uptake of both C3H]DA and C3H]dopa, produced the most dense silver deposits in two areas: the GL and the
Fig. 1. Light-microscopic survey view of the turtle olfactory bulb laminae. Perfused animal, 2 pm thick epoxy section, toluidine blue stain. EPL, external plexiform layer; GL, glomerular layer; GRL, granule cell layer; ON, olfactory nerve layer; asterisks, olfactory ventricle. Several mitral and tufted cell bodies are labeled with arrowheads. Bar, 50 pm. Fig. 2. [3H]dopamine
uptake
into various
structures,
as shown
on a light-microscopic
autoradiogram.
Olfactory bulb hemi-slice, 60min incubation in 5 x lO-6 M DA solution, 2pm thick epoxy resin secticin, 28 days exposure, Kodak D 19 developer, no counterstaining. Most of the label appears in the glomerular region, Labeled structures, the ventricular
and is located in the glomeruler neuropil as well as in cells around the glomeruli. smaller in size, appear throughout the granule layer, especially in close proximity to surface (asterisk). Other abbreviations are similar to those in Fig. 1. Bar, 50 ltm.
Fig. 3. High power photomicrograph of a detail of the glomerular region. Light-microscopic autoradiogram, 28 days exposure, unstained epoxy section. Labeled cells (arrowheads) and their processes are shown. Note dense patches of grains within the glomerular neuropil. EPL, external plexiform layer: ON. olfactory nerve layer. Bar, 50 pm. Figs 4-5. Electron-microscopic autoradiographs of two labeled periglomerular ceils (PGC) and surrounding structures. Olfactory bulb hem&slices, C3H]DA incubation, lead citrate contrast, 7 days exposure time, Amidol developer. Note the accumulation of grains over the cell cytoplasm and nuclei; neighboring dendrites (d) and olfactory nerve fascicles (ON) remained unlabeled. Bar, 2 pm. Figs 67. C3H]dopamine uptake into structures within the glomerular neuropil. EM autoradiograms, lead citrate contrast, 7 days exposure time. Heavy deposits of silver grains are seen over the terminal enlargements of pale dendritic profiles(d), surrounded by the more electron-dense olfactory nerves (ON). Other dendrites (d) were not labeled over background level. Bar, 1 pm. The unlabeled profile (dm) in Fig. 6 contains few neurotubules and vesicles, and is characteristic of mitral and tufted cell dendrites. Fig. 8. Light-microscopic autoradiogram of labeled structures after 10 min incubation of hemi-slice in a 10m6 M solution of dopamine. The 2 pm-thick epoxy resin section was taken parallel with the granule cell layer of the slice, 28 days exposure time. Labeled fibers and terminal enlargements can be seen (arrows) throughout the layer. The label was also retained by blood vessel walls (c). Bar, 100 pm. Figs 9-10. Electron-microscopic autoradiograms taken from the granule layer. The accumulation of [“HIDA was observed over certain fibers and terminals of unknown origin. These structures, in addition to their ‘clear’ synaptic vesicle content, usually contained a few dense-core vesicles. c, capillary. Bars, 1pm. Fig. Il. labeling deposits
Light-microscopic autoradiogram of hemi-slice after 60 min incubation in 10 -6 M dopa. The pattern was similar to that seen with C3H]DA (compare with Fig. 2). Note the heavy silver close to the ventricular surface (arrows). Abbreviations for the layers same as Fig. I. Asterisks, olfactory ventricle. Bar, 50 pm.
Fig. 12. Detail of the ventricular surface, showing a labeled cell with a thin rim of cytoplasm, surrounding the nucleus. This cell is surrounded by unlabeled tanycytes (TC), extending their microvilli (mv) and cilia (ci) into the ventricular lumen. Bar. 1 {lrn.
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Dopamine in turtle olfactory bulb deep GRL. In general, with C3H]DA there was more distinct label over the glomeruli, PG cells and structures within the granule layer. In comparison, C3H]dopa gave a stronger labeling over some cell bodies close to the ventricular surface, more diffuse label in GL, and almost no labeling within the granule layer, deeper than 50 pm from the ventricular surface. Figures 2-7 show the main features of c3H]DA uptake by constituents of the glomerular layer. Heavy accumulation of grains was present throughout the glomerular neuropil (Figs 2, 3). However, closer observation showed that the distribution was not homogeneous. Under the light-microscope, unlabelled areas were present, particularly in the superficial parts of the glomeruli, where the dendrites of bulbar neurons are intermingled with groups of olfactory axons (Fig. 3). At the deeper aspect of the glomeruli, where the overall density of the grains was less, continuity of labeled thick processes with labeled PG cell bodies was seen (Fig. 3). In addition, labeled cells of the same or even larger size were occasionally seen in the outer third of the EPL. Under electron-microscopy there was good preservation of ultrastructural characteristics. In particular, mitochondria and synaptic contacts were well preserved. The main departures from normal fine structure were variations in sizes of synaptic vesicles in the olfactory nerve terminals and the occasional presence of vacuoles, some of which appeared as dilatations of the endoplasmic reticulum in perikarya or dendrites. The labeled cells in the periglomerular area were recognized as neurons with soma diameters of approximately 10pm (Figs 4, 5). These showed typical features of PG cells, similar to those in mammals,28 including one or more stacks of rough endoplasmic reticulum, rare Golgi stacks and vesicles, and the presence of free ribosomes in the rim of cytoplasm surrounding the nucleus. Synaptic vesicles were occasionally seen within the cytoplasm of these labeled cells, but no synaptic contacts of either polarity were observed. The labeled cells in the superficial EPL had larger cell bodies (up to 14pm in diameter). They were recognized as tufted cells, based on the presence of multiple stacks of rough endoplasmic reticulum a higher number of mitochondria, and well-developed Golgi system. They also contained occasional small vesicles, but did not make synaptic contacts. Accumulation of grains was also observed over large (2-3 pm diameter) dendritic profiles (Figs 6, 7), which were much paler under electron-microscope than the olfactory nerves surrounding them. These labeled dendrites were presumed to be the processes of the labeled PG cells, and their ultrastructural characteristics resembled those in mammals.2g Most of these profiles contained a large number of small (diameter 3&50 nm) synaptic vesicles of the clear type, and occasional dense-core vesicles (diameter 8&120 nm). Neurotubules were prominent within the thinner shafts connected to the varicose enlargements
(Fig, 6), similar to the mitral secondary dendrites and tufted cell dendrites in the EPL. Fasciculi of the olfactory nerve fibers, or the terminal enlargements of the axons, also showed only a background rate of labeling. In the EPL, grain accumulation was observed only over a few cell bodies in the superficial part, as mentioned above. In the GRL, a number of labeled fibers (or their varicose terminal enlargements) and also a few labeled cells were seen (Figs 2, 8-12). Most of the labeled fibers were seen running parallel with the layers, so they appeared as dots in frontal sections (Fig. 2), and more elongated structures in sections taken parallel with the granule processes (Fig. 8). In electron-microscopic autoradiograms of hemi-slices, incubated with C3H]DA or C3H]dopa, accumulation of grains was observed over thin (diameter O.>l pm) fibers (Fig. 9) or small terminal enlargements (Fig. 10); their origin is unclear. Capillaries and small blood vessels close to the sliced surface were also heavily labeled (Fig. 8). In the deepest aspect of the bulb, labeled structures about the size of very large terminals or small cells were clearly visible in the light-microscopic autoradiograms (Fig. 2, C3H]DA; Fig. 11, C3H]dopa). Under electron-microscopy a few labeled fibers were also seen. Labeled cells were numerous and easily recognized (Fig. 12, C3H]dopa). These cells had an extremely thin rim of cytoplasm around their nuclei, even thinner than in PG or granule cells. Rough endoplasmic reticulum, mitochondria and free ribosomes were observed in the cytoplasm. The cytoplasm as well as the nuclear plasm appeared relatively electron-lucent when compared to the density of neighboring tanycytes. The tanycytes showed almost no uptake of C3H]DA or c3H]dopa. Most of the tanycytes were seen to reach the ventricular surface, bearing a bushy borderline of microvilli and cilia extending into the ventricular lumen while none of the labeled cells mentioned above reached the ventricular surface. No synaptic vesicles or specialized junctions were observed in relation to the labeled cells. These cells have not been identified yet as either neuronal or non-neuronal. DISCUSSION The structure of the olfactory bulb of reptiles generally resembles that of other vertebrate bulbs,’ having similar layering, a well pronounced internal plexiform layer, and somewhat more scattered mitral cells than the higher vertebrates. According to our preliminary electron-microscopic studies (N. Hal&z, et al., unpublished observations), examples of the main synaptic configurations described in the mammalian olfactory bulb (for review, see ref. 34) can be recognized in the turtle olfactory bulb as well, thereby implying that similar synaptic circuits may exist. The early fluorescence-microscopic observations of Dahlstrom and co-workers5 demonstrating the pres-
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N. Halis7. M. C‘. Nowycky and 6. M. Shepherd
ence of a few catecholaminergic structures within the glomerular region of the rabbit olfactory bulb have been subsequently supported by studies of loading of the periglomerular cells with exogenous norepinephrine2’ or dopa2’ and further extended by US~~.‘~.~” with conventional cytochemical techniques as well as light- and electron-microscopic autoradiography of C3H]norepinephrine and [3H]serotonin uptake and immunochemistry. Similar labeling patterns were observed following C3H]DA uptake by periglomerular cells of the rat bulb.31 In lower vertebrates, catecholaminergic structures have been observed in the frog olfactory bulb.3*4,27 but no data have been published previously showing the presence of similar catecholaminergic system within the reptilian bulb. Certain biochemical data have also supported the existence of a catecholaminergic system in the mammalian olfactory bulb, by revealing a substantial amount of endogenous DA.83’7322*26 Samples taken from different olfactory bulb layers revealed that the highest level of endogenous DA was found in the glomerular layer.ig Norepinephrine has been localized to fibers in the deeper layers, within the EPL and GRL.5,12*35 No noradrenergic cell bodies have been demonstrated in the olfactory bulb. This localization is also supported by biochemical data, demonstrating that after bulbectomy there is an accumulation of norepinephrine within more central areas.6”5’16 In the rat we did not find evidence for extrabulbar projections of dopaminergic cells,12*r3 and similar conclusions were drawn by Edwards and co-workers.6 On the other hand, Hirsch16 described the presence of such a bulbofugal pathway running to certain limbic areas. In addition. a weak bulbopetal pathway may also exist, as was described by Fallon & Moore.’ However, in the rat, the dopaminergic system seems to be mostly an intrinsic bulbar system, restricted to local interneurons within the periglomerular area and superficial EPL, as was observed very recently by our serial analysis of immunohistochemical sections at the electron-microscope level.’ 1 For fine structural localization of the autoradiographic label within the superficial layers of the turtle olfactory bulb we have applied the morphological criteria for the determination of the constituents of the rat olfactory bulb,1~2s~2g supplemented with some data from perfused turtle bulbs. According to these
criteria, most of the labeled cells were recognized 211 periglomerular or superficial tufted neurons. while the intraglomerular label was largely confined to dendrite-like processes, These dendrites (containing ;I large number of synaptic vesicles) might belong to periglomerular cells. while other unlabeled dendrites resembled mitral dendrites based on their ultrastructural characteristics. Thus, the labeling pattern in the turtle olfactory bulb appears to be similar to that of the rat: periglomerular cells and their processes retained the largest proportion of the labeled catecholamine. Another similarity to the rat was the presence of a few labeled superficial tufted cells in the vicinity of the glomeruli. With immunohistochemistry. weakly fluorescent large cells, lying at the depth of the mitral and granule cells, were also visualized.14 Our present autoradiographic studies, however, did not show any label at this level comparable to the immunohistochemical results. The distribution and frequency of labeled fibers after C3H]DA uptake within the granule layer were similar to the previous C3H]noradrenaline studies in the rat,‘* however further experiments are needed in order to recognize clearly their relation to other structural constituents. The labeled neuronal processes may be some of the central fibers contacting the granule cells within this layer.30 As yet we are not able to speculate which morphological correlates of the labeled (C3H)dopa and C3H]DA) deep cells might be present in the mammalian olfactory bulb. No synaptic connections could be found between these and neighboring structures. If the labeled cells have the capability of uptake, storage and release of endogenous DA as well as label, they may serve more as a bridge between the ventricular surface and granular layer than as an interneuronal connection, similar to the proposed role for tanycytes at the median eminence.32,33 which are thought to transport catecholamines from the CSF deeper into the tissue. Acknowledgements-This work was supported by research grants NS-07609 and F32-NS06159 from the National Institute of Neurological and Communitive Disorders and Stroke, and BNS 78816545 from the National Science Foundation. We thank G. Collins for expert assistance
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Beriicksichtigung der synaptis-
2. Andres K. H. (1970) Anatomy and ultrastructure of the olfactory bulb in fish, amphibia, reptiles, birds and mammals. In Taste and Smell in Vertebrates (Eds Wolsteinholme G. E. W. & Knight D. J.), pp. 1777194. Churchill, London. 3. Bartels W. (1971) Die Ontogenese der aminnhaltigen Neuronensysteme in Gehirn von RUM temporariu. Z. Zeflforsch. mikrosk. Anat. 116. 94-l 18. 4. Braak H. (1970) Biogene Amine in Gehirn von Frosch (Rana esculenta). Z. Zellfbrsch. mikrosk. Anat. 106, 269-308. 5. Dahlstrdm A., Fuxe K., Olson L. & Ungerstedt U. (1965) On the distribution and possible function of monoamine nerve terminals in the olfactory bulb of the rabbit. Life Sci. 4, 2071-2074.
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6. Edwards D. A., Schlosberg A. J., McMaster S. E. & Harvey J. A. (1977) Olfactory system damage and brain catecholamines in the rat. Brain Res. 121, 121-130. 7. Fallon J. H. & Moore R. Y. (1978) Catecholamine, innervation of the basal forebrain-III. Olfactory bulb, anterior olfactory nuclei, olfactory tubercle and piriform cortex. J. camp. Neuro[. 180, 533-544. 8. Ferriero D. & Margolis F. L. (1975) Denervation in the primary olfactory pathway of miceII. Effects on carnosine and other amine compounds. Bruin Res. 94, 75-86. 9. Freeman W. J. (1974) Relation of glomerular neuronal activity to glomerular transmission attenuation. Brain Res. 65, 91-107. IO. Getchell T. V. & Shepherd G. M. (1975) Short-axon cells in the olfactory bulb: dendrodendritic synaptic interactions. J. Physiol., Lond. 251, 523-548. Il. Hal&z N., Johansson O., Hijkfelt T., Ljungdahl A. & Goldstein M. (1981) Immunohistochemical identification of two types of dopamine neuron in the rat olfactory bulb as seen by serial sectioning. J. ~~~~oc~~fo~.10, 251-259. 12. Hal& N., Ljungdahl A. & HGkfelt T. (1978) Transmitter histochemjstry of the rat olfactory bulb-If. Fluorescence histochemical, autoradiographic and electron-microscopic localization of monoamines. Brain Res. 154, 2533271. 13. Hal&z N., Ljungdahl A., Hokfelt T., Johansson O., Goldstein M., Park D. & Biberfeld P. (1977) Transmitter histochemistry of the rat olfactory bulb--I. Immunohistochemical localization of monoamine synthesizing enzymes. Support for intrabulbar, periglomerular dopamine neurons. Brain Res. 126, 45.5-474. 14. Hal&z N., Nowycky M., Hokfelt T., Shepherd G. M., Markey K. & Goldstein M. (1982) Dopaminergic periglomeruIar cells in the turtle olfactory bulb. &&I Res. Bull. In press. 15. Harvey J. A., Scholfield C. N., Graham L. T. & Aprison M. H. (1975) Putative transmitters in denervated olfactory cortex. J. Neurochem. 24, 445-449. 16. Hirsch J. D. (1980) Opiate and muscarinic ligand binding in five limbic areas after bilateral olfactory bulbectomy. Brain Res. 198. 271-283. 17. Hirsch J. D. & Margolis F. L. (1980) Influence of unilateral olfactory bulbectomy on opiate and other binding sites in the contralateral bulb. Bruin Res. 199, 39-47. 18. Hokfelt T., Hal&z N., Ljungdahl A., Johansson O., Goldstein M. & Park D. (1975) Hist~hemic~ support for a dopaminergic mechanism in the dendrites of certain periglomerular cells in the rat olfactory bulb. Neurosci. Letc. 1, 85-80. 19. Jaffe E. H. & Cuello A. C. (1980) The distribution of catecholamines, glutamine decarboxylase and choline acetyltransferase in layers of the rat olfactory bulb. Brain Res. 186, 232-237. 20. Lichtensteiger W. (1966) Uptake of norepinephrine in periglomerular cells of the olfactory bulb of the mouse. Nurure, Land. 210,955-956. 21. Lidbrink P.: Jonsson G. & Fuxe K. (1974) Selective reserpine-resistant accumulation of catecholamines in central dopamine neurons after DOPA administration. Brain Res. 67,439-456. 22. Margolis F. L., Roberts N., Ferriero D. & Feldman J. (1974) Denervation in the primary olfactory pathway of mice: biochemical and morphological effects. Brain Res. 81, 469-483. 23. Mori K. & Shepherd G. M. (1979) Synaptic excitation and long-lasting inhibition of mitral cells in the in vitro turtle olfactory bulb. Brairl Res. 172, 155-159.
24. Nowycky M. C., Hal&z N. & Shepherd G. M. (1981) Studies of dopamine as a neurotransmitter in the turtle olfactory bulb. Sot. Nearos~i, Abs. 7, 573. 25. Nowycky M. C., Waldow U. C Shepherd G. M. (1978) Electrophysiological studies in the isolated turtle brain Sot. Neurosci. Abs 4, 583. 26. Palkovits M., Zilborszky L., Brownstein 27. 28.
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M. J., Fekete M., Herman J. P. & Kanyicska B. (1979) Distribution of norepinephrine and dopamine in cerebral cortical areas of the rat. Brain Res. Bull. 4, 593-601. Parent A. (1975) The monoaminergic innervation of the telencephalon of the frog, Rana pipiens. Brain Res. 99, 35-47. Pinching A. J. & Powell T. P. S. (1971) The neuron types of the glomeruli of the olfactory bulb. J. CeIt Sci. 9,30>345. Pinching A. J. & Powell T. P. S. (1971) The neuropil of the glomeruli of the olfactory bulb. J. Ceil Sci. 9, 347-377. Price J. L. & Powell T. P. S. (1970) An electron-microscopic study of the termination of the efferent fibers to the olfactory bulbs from the cerebral hemisphere. J. Ceil Sci. 7, 157-187. Priestley J. V., Kelly J. S. & Cue110 A. C. (1979) Uptake of C3H]dopamine of the rat olfactory bulb: an autoradiographic study. Brain Res. 165, 149-155. Scott D. E., Dudley G. K. & Knigge K. M. (1974) The ventricular system in neuroendocrine mechanisms-II. fn vine monoamine transport by ependyma of the median eminence. Ceil Tissue Res. 154, l-16. Scott D. E., Scott P. M. & Krobisch-Dudley G. (1978) Ultrastructural localization of radiokbeled t_-dopa in the endocrine hypothalamus of the rat. Cell Tissue Res. 195, 29-43. Shepherd G. M. (1972) Synaptic organization of the mammalian olfactory bulb. Physiol. Rev. 52, 864-917. Swanson L. W. & Hartman B. K. (1975) The central adrenergic system. An immunofluoreszence study of the location of cell bodies and their efferent connections in the rat utilizing dopamine-hydroxylase as a marker. J. camp. Neural. 163.467-505. Waldow U., Nowycky M. C., & Shepherd G. M. (1981) Evoked potential and single unit responses to olfactory nerve volleys in the isolated turtle olfactory bulb. Brain Res. 211, 267-283. (Accepted 15 September 1982)