Development of adenosine deaminase-immunoreactive neurons in the rat brain

59

Developmental Brain Research, 31 (1987) 59-71 Elsevier BRD 50485

Development of adenosine deaminase-immunoreactive neurons in the rat brain E. Senba, P.E. Daddona* and J.I. Nagy Department of Physiology, Facultyof Medicine, Universityof Manitoba, Winnipeg, Man. (Canada) (Accepted 22 July 1986)

Key words: Adenosine deaminase; Adenosine; Immunohistochemistry; Ontogenesis; Rat brain; Purinergic neurotransmission; Purine metabolism

It has previously been demonstrated that neurons immunoreactive for the enzyme adenosine deaminase (ADA) have a highly restricted distribution pattem in the adult rat brain. In order to determine whether the pattern of ADA expression is equally limited during the period of brain development, the localization of ADA was investigated immunohistochemically in brains of embryonic, early postnatal and young adult rats. No immunostaining for ADA was detected on the 12th embryonic day. On embryonic day 15, ADAimmunoreactive cells were first observed in the hypoglossal motor nucleus, and on day 18 in cingulate, retrosplenial and visual cortex, in the posterior basal hypothalamus, and in the facial motor nucleus. On the 20th embryonic day ADA-immunoreactive neurons appeared in various olfactory and related systems and in the superior coUiculus. On the 1st postnatal day, immunoreactivity was intensified in all structures in which it was observed at preceeding ages and, in addition, appeared in several brainstem regions. On postnatal day 10 and 15, immunostained neurons appeared in several subcortical structures whereas the number of these decreased in the anterior olfactory nucleus and some related cortical areas. In animals 25 days of age the intensity of immunostaining continued to increase, essentially producing the adult pattern in all except olfactory areas where there was a dramatic loss of ADA-immunoreactive cells. These results show that the restricted pattern of ADA-immunostaining observed in adult rat brain is generated over a protracted period of development, various stages of which are characterized predominantly by the expression of ADA in greater abundance, at least to the extent this can be gleaned immunohistochemically, in greater numbers of neurons and to a minor degree by a decreased capacity to express this enzyme. INTRODUCTION Since the t e r m 'purinergic' neurotransmission was introduced by Burnstock 8, c o n s i d e r a b l e behavioral, electrophysiological and pharmacological evidence has been accumulated indicating an i m p o r t a n t role of adenosine and its nucleotides in the regulation of neural activity in both the central and p e r i p h e r a l nervous system 28,35. In contrast, t h e r e is a paucity of information on how the n e u r o m o d u l a t o r y actions of adenine nucleosides and nucleotides m a y be coupled to cellular metabolic events and regulated by specific cellular constituents or e n z y m e pathways related to the disposition of these purines. S o m e understanding of this may be gained through k n o w l e d g e of the distribution and localization of key enzymes which participate in neuronal purine metabolism. Such knowl-

edge is likely not only to shed light on the physiological role(s) of purines but also m y provide clues as to their precise n e u r o a n a t o m i c a l sites of action. O u r interest in recent years has b e e n focussed on determining possible functional and anatomical relationships between the central actions of adenosine and the enzyme A D A which is responsible for the catabolism of this nucleoside to inosine. W e have shown i m m u n o histochemically that certain neuronal populations in discrete areas of the rat C N S express relatively high levels of A D A 21-25. W h i l e there is as yet no definitive p r o o f linking these neurons with adenosinergic transmission o r m o d u l a t i o n , there is some indirect evidence suggesting this possibility. F o r e x a m p l e , A D A has been d e m o n s t r a t e d in some preganglionic parasympathetic neurons of the rat 3° and it has been shown electrophysiologically in the cat that the spinal

* Present address: Centocor, 244 Great Valley, Parkway, PA 19355, U.S.A.

Correspondence: J.I. Nagy, Department of Physiology, Faculty of Medicine, University of Manitoba, 770 Bannatyne Avenue, Winnipeg, Man., Canada, R3E, 0W3. 0165-3806/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

60 contingent of these neurons have an adenosinergic component t. Moreover, there appears to be a very high correlation between the distribution of A D A immunoreactive structures in the rat brain and the localization of binding sites for [3H]nitrobenzylthioinosine - - a putative ligand for adenosine uptake sites13-15,23. In view of the ubiquitous distribution of adenosine and the involvement of this nucleoside in intermediary metabolism, our finding of a heterogeneous distribution of both ADA-immunoreactive neurons and A D A activity ~s in the rat brain is contrary to the expectation that this enzyme might also be ubiquitously and homogeneously distributed in the rat CNS. In order to extend this finding, the aim of the present study was to determine whether the expression of A D A in central neurons is dependent to some extent on their functional states or cellular metabolic demands as might be reflected at various stages of ontogeny. The pattern of A D A immunostaining observed in the adult rat brain was compared with that seen during development. MATERIALS AND METHODS

Animals Sprague-Dawley rats were mated overnight, checked regularly for vaginal plugs and the litters from those successfully impregnated were used in the present study. The morning on which plugs were observed was defined as the first gestational day. Embryos at gestational days 12, 15, 18 and 20 were surgically removed from pregnant rats anesthetized with chloral hydrate and measured according to crown rump length (CRL). Fetal brains 12 and 15 days of gestation were excised and immediately placed in cold tissue fixative. Fetuses at 18 and 20 days of gestation and all older animals were perfused with fixative as described below. Postnatally, animals were examined at ages of 1, 10, 15, 25 and 60 days. Some adult animals were pretreated intraventrieularly or intracisternally with 50 to 75/ag/100 g b. wt. of colchicine and allowed to survive for two days. Tissue preparation Animals were anesthetized with chloral hydrate and perfused transcardially with ice-cold 0.1 M sodium phosphate buffer, pH 7.4, containing 0.9% sa-

line, 0.1% sodium nitrate and l(10 units of heparin. This was followed by ice-cold fixative consisting of 4% paraformaldehyde in 0.18 M sodium phosphate buffer, pH 6.9, containing 0.2% picric acid, which is a modified version of the fixative described by Zamboni and De Martino 4°. Brains were excised, placed in fresh fixative for 24 h at 4 °C and then transferred to 0.1 M phosphate buffer, pH 7.4, containing 3(1% sucrose for a further 24 h at 4 °C. Brains were cut in sagittal or coronal planes on a freezing microtome at a thickness of 20¢tm, or in the case of 12th- and 15thgestational-day tissue on a cryostat at a thickness of 15 ~m. Free-floating microtome or slide-mounted cryostat sections were collected and washed in 0.1 M sodium phosphate buffer, pH 7.4, containing 0.9% saline (PBS) for a minimum of 2 h.

Immunohistochemistry Sections were processed for immunohistochemistry by the peroxidase-antiperoxidase (PAP) method 34 using antisera to purified calf-intestinal A D A 9 essentially as previously described 21'32. All antisera were diluted with PBS containing 0.6% Triton X-100. Sections were incubated with A D A antiserum diluted 1:500 for 48 h at 4 °C, washed in PBS, and incubated with goat anti-rabbit (SternbergerMeyer) serum diluted 1:10 for 45 min at 37 °C. After washing in PBS, they were incubated with rabbit PAP (Sternberger-Meyer) diluted l:100 for 45 min at 37 °C, rinsed in 50 mM Tris-HC1 buffer, pH 7.4, (Tris buffer) for 15 min and reacted with 0.02% 3,3'-diaminobenzidine and 0.005% hydrogen peroxide in Tris buffer for 20 rain. The sections were then rinsed in several changes of Tris buffer, mounted from gelatin-ethanol in the case of free-floating sections, dehydrated and cover-slipped with Permount. For adsorption controls, a series of free-floating sections from one- and 10-day-old animals were incubated with A D A antiserum preadsorbed with purified A D A and processed as described above. RESULTS

Embryonic days 12, 15 and 18 At 12 days of gestation (CRL, 7-10 ram), no ADA-immunoreactive structures were observed in any areas of embryonic brain. At 15 and 18 days of gestation (CRL 12-14 mm and t7-22 mm, respec-

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Fig. 1. Photomicrographs of transverse (a-c) and medial sagittal (d-f) sections showing ADA-immunoreactive structures in the AON at the postnatal ages indicated, a, d: numerous intensely stained neurons are seen in the m part of AON and in the Dtr. b, c, e, f: immunostaining gradually decreases in A O N and Dtr until day 25 (e, f) when only light staining of scattered neurons is seen in the medial part of the AON. Bars = 200/~m.

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Fig. 2. Photomicrographs of transverse sections showing the developmental changes of ADA-immunoreactive structures in the "lTv (a-c) and AIv (d-f) at the ages indicated, a, d: intense immunostaining of neurons is seen in deeper layers of both structures (TFv is shown bilaterally), b, e: ADA-immunoreactivity is decreased at 10 days and at P25 is almost absent in both areas (c, f). At earlier ages immunoreactive dendritic processes are seen directed towards superficial layers. Bars = 100/~m.

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Fig. 3. Photomicrographs showing the development of ADA-immunoreactive structures in transverse sections of the PCg (a-c) and in sagittal sections of the visual cortex ( d - h ) at the postnatal ages indicated, a: in PCg, numerous intensely immunostained neurons are seen in superficial layers (arrows) and a smaller number in deeper layers (arrowhead). b, c: at P10 and up to P25 a gradual loss of immunoreactive cells is seen in superficial layers with only the deeper cells remaining at the latter age. d, e: in the occipital lobe, a few scattered and lightly stained neurons are seen in the RSpi (d) and visual cortex (e) after birth and a dramatic increase in the numbers and staining intensity of these is seen up to 25 days. f-h: higher-power micrograph of the visual cortex showing the morphological development of what at 25 days appear to be ADA-immunoreactive pyramidal neurons. Bars = 100/~m.

64 tively) ADA-immunoreactive neurons were found in hypoglossal and facial motor nuclei, the detailed developmental pattern of which is described elsewhere in the context of the peripheral projections of these neurons 29. In addition to these motoneurons, lightly immunostained neurons were observed in the medial and basal part of the cortical plate corresponding to the primordium of the ACg and PCg, RSpl and VC 26. These neurons had short dendritic processes directed towards superficial cortical layers. Small round ADA-immunoreactive cells were also observed in the posterior hypothalamus (see Fig. 4e).

Embryonic day 20 The intensity of ADA-immunostaining was increased in neurons identified at the foregoing stage. At this stage (CRL 26-31 mm), additional lightly ADA-immunostained neurons were observed in the AON, TTv, prelimbic area, IL, dorsal tenia tecta or anterior hippocampal continuation, indusium griseum, AIv and POC. Very light immunostaining of neurons having short dendritic processes were also observed in the superficial layers of the SC.

Postnatal day 1 At this stage the number and staining intensity of ADA-immunoreactive neurons increased in each of the areas described above. This was particularly evident in AON, TTv, AIv, cingulate cortex and posterior hypothalamus. In the AON, ADA-immunoreactive neurons were observed in all portions of the nucleus but were by far most numerous in pars medialis (Fig. la). In medial sagittal sections dense immunostaining is also evident in the Dtr (Fig. ld). In the TTv and AIv, intensely stained neurons located in deep layers were seen extending dendritic processes to more superficial layers (Fig. 2a, d). In some coronal sections, ADA-immunoreactive axons originating from neurons in the AIv were observed running dorsomedially or ventromedially towards the corpus callosum. Somewhat smaller immunostained neurons than those in A i r were seen scattered in the dorsal part of POC (Fig. 2d). In the cingulate cortex, ADA-immunoreactive neurons located in the cortical plate were more densely packed in the PCg (Fig. 3a) than the ACg. The PCg may correspond to area 29c and 29d 37 or to the granular and agranular retrosplenial area Is'a1.

The area dorsolateral to the PCg, where the cortical plate is wider and more sparse, may correspond to occipital area 2 (ref. 41) or area 18 (ref. 37), i.e. VC~ In sagittal sections of this area (Fig. 3f), the cortex has 3 visible layers at this age: a marginal zone, a cortical plate, and an intermediate zone 2°. Most of the ADA-immunoreactive neurons in these sections were located in the cortical plate and had short apical dendrites directed towards the marginal zone. These same features were also evident for ADA-immunostained neurons in the RSpL (Fig. 3d). In the posterior hypothalamus, ADA-immunoreactive neurons were located in the tuberomammillary nucleus 17or as defined by Bleier et al. 6, the tuberal, caudal and postmammiUary CM. At this developmental stage, these neurons were small with diameters of 6 - 8 Bm and had poorly developed dendritic arborizations (Fig. 4f). In the superior colliculus, ADA-immunoreactive neurons were observed only in deeper parts of the superficial layers where they tended to be small and densely packed. Some larger neurons intermingled with these were occasionally seen extending dendritic processes dorsally (Fig. 4a, b). In addition to the above areas, a few ADA-immunoreactive neurons were first observed at this age in the PnC, the LNP and in the medullary reticular formation between the nVsp and the NTS and between the nVsp and RL. Some lightly immunostained neurons were also observed in the superficial layer of the nVsp (see Fig. 6a). lmmunostaining in all of the above areas was abolished in sections incubated with ADA-antiserum preadsorbed with purified ADA.

Posmatal day 10 Immunostaining for A D A in neurons of most areas observed at the preceding stage was increased particularly in the hypothalamus, Sc, PnC, LPN, nVsp. Exceptions to this were the AON, Dtr, TYv and AIv where the numbers of ADA-immunoreactive neurons were decreased (Figs. lb, e, 2b, e and 7). In the PCg most ADA-positive neurons were still localized to the cortical plate (layers II and Ill), but some had taken up positions in the underlying layer V (Fig. 3b). There was a paucity of immunostained neurons in the area between these two layers. In the dorsolateral part of PCg, ADA-immunoreactive neurons were located primarily in layer V. A somewhat broader band of immunostained neurons having api-

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a P!

.f PIO

PIO

Fig. 4. Photomicrographs of transverse sections showing the development of A D A - i m m u n o r e a c t i v e n e u r o n s in the SC ( a - d ) and in the posterior h y p o t h a l a m u s lateral to the mammillary recess ( e - h ) at the embryonic and postnatal ages indicated, a, e: a small n u m b e r of A D A - i m m u n o r e a c t i v e n e u r o n s are first seen in the h y p o t h a l a m u s at E l 8 and are n u m e r o u s in the SC by P1. b, f: i m m u n o s t a i n e d neurons are seen concentrated in a narrow band in the SC and appear morphologically m o r e mature than those in the h y p o t h a l a m u s , c, d: at subsequent stages immunoreactive fibers and cells with well-developed dendrites are seen scattered throughout the SC. g, h: immunoreactive n e u r o n s in the h y p o t h a l a m u s still appear relatively i m m a t u r e at 10 days and begin to approach adult m o r p h o l o g y by 25 days. Bars a, c , - e , g, h = 100/am; b, f = 50/~m.

cally directed dendrites was observed in layer V of the laterally adjoining visual cortex (Fig. 3b). In the SC small fusiform immunoreactive cells were observed in its most superficial aspect in addition to the larger more intensely stained triangular neurons observed at this and earlier stages in deeper layers (Fig. 4c). A D A - i m m u n o r e a c t i v e neurons also appeared at this stage in the Tu, the H D B , the striatum flanking the globus pallidus, central grey area, Ent and the PnO. Immunoreactivity in all the above areas was abolished in sections incubated with antisera preabsorbed with purified ADA.

Postnatal day 15

P1

LNP

J h~

P~

a

P10

The distribution pattern of A D A - i m m u n o r e a c t i v e neurons at this stage was similar to that observed on the 10th postnatal day. However, additional areas in which immunostained neurons were now observed included the nucleus accumbens, lateral septal nucleus, parafascicular nucleus of the thalamus, I M C P C and the pontine central grey.

q

-

LNP

Postnatal day 25 At this age, the number of A D A - i m m u n o r e a c t i v e neurons decreased dramatically in TTv, Dtr, AIv, P O C and the medial part of A O N such that only a few immunostained neurons were observed in each structure. In contrast, neurons in those cortical areas described at 10 days maintained or increased their immunostaining. These cortical neurons exhibited a more matured morphology (Fig. 3c, h) and had thickened apical shafts bifurcating or arborizing in layer I and other primary dendrites extending beneath their pyramid-shaped somas (Fig. 3g, h). Axons originating from these cells could be seen descending into the white matter. From their morphological appearance at this stage, it was clear that these cells were typical pyramidal neurons 27. In the RSpl cortex, a considerable number of A D A - i m m u n o r e a c t i v e neurons were observed in layer II and III, in addition to those in layer V. In the posterior hypothalamus (Fig. 4h), SC (Fig. 4d), LNP (Fig. 5c) and other brain areas described at earlier stages, the distribution, staining intensity, and morphological development of A D A immunoreactive neurons had reached that seen in adult animals. In addition, A D A - i m m u n o r e a c t i v e neurons were first observed at this age in the bed nucleus of the anterior commissure, the medial and cen-

b P25 ~ ~: ¸LIMe

C Fig. 5. Photomicrographs of transverse sections showing the developmental changes in ADA immunoreactivity in the rostral part of the FN and the LNP parasympathetic nucleus at the ages indicated. Immunostaining of neurons in LNP is seen to increase gradually with age whereas that in motoneurons of FN progressively decreases. Bars = 100 urn.

tral amygdaloid nuclei and the IP. Colchicine pretreatment slightly intensified immunostaining of neurons in areas where these were already evident without such pretreatment, but failed to reveal A D A - i m -

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Fig. 6. Photomicrographsof transverse sections showingthe development of ADA-immunoreactivecells and fibers in the nucleus caudalis of the spinal trigeminal nucleus at the ages indicated. Immunoreactivestructures are barely detectable at P1 (a), small neurons appear by P10 (b) and are more prominent at P25. Punctate and dense fiber immunostainingis scarcelyseen at 10 days (b, d) but develops by 25 days (c, e). Bars a, b, d, e = 100/~m;c = 50/am. DISCUSSION munoreactive neurons in other regions or in areas such as TTv and AIv where these had disappeared in the course of development.

ADA-immunoreactive fibers On embryonic day 20, ADA-immunostained fibers were observed in the olfactory nerve directed toward both the main and the accessory olfactory bulb. Although olfactory glomeruli are reported to be first identifiable at 19 clays of gestation 12no immunostaining was seen in these structures at this age. The number of immunoreactive glomeruli were few on the first postnatal day and thereafter increased with age. On postnatal day 10, ADA-immunoreactive fibers were seen in the stria medularis and medial habenula, the lateral posterior thalamic nucleus and the superficial layers of the spinal trigeminal nucleus (Fig. 6d). On postnatal day 15 and 25, ADA-immunoreaetive fibers were widely distributed in brain approaching the pattern seen in adult animals. A detailed description of this pattern will be the topic of a separate report.

The 3 main findings of the present study are: (1) that just as in adult rat brain, ADA-immunoreactive neurons are not ubiquitously distributed in the developing rat CNS; (2) that the appearance of ADA-immunostaining in neurons of various brain structures and the morphologenesis of these neurons occurs at different times and rates during ontogenesis; and (3) that in many brain regions the number and staining intensity of ADA-immunoreactive neurons increased whereas in a few brain areas this decreased or disappeared. The heterogeneous distribution of ADA-immunoreactive neurons in developing rat brain and the similarity between this distribution and, for the most part, that seen in the adult suggests that the expression of A D A in neurons giving the adult pattern is not simply a remnant of its ubiquitous expression in all neurons during development but rather that the production of A D A in a unique set of neurons is genetically determined early in development. This may be taken to indicate that, in addition to its probable involvement in intermediary metabolism in both adult

68

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" -3

@-

RSpl

I:;pcQ " :.

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10 P 10 Fig. 7. Diagramatic representation of ADA-immunoreactive neurons in 10-day-old rat brain at rostral (a) and caudal (b) transverse levels. Relative location and numbers of cells are indicated by black dots.

and developing neurons 15, A D A may have another role more distinctly related to specific functional capabilities of neurons in which it is found in high concentrations. While we have previously speculated that such capabilities may include the utilization of purines as transmitters or modulators by these neurons, there is at best only circumstantial evidence for this at present 14'23'3°. The functionally diverse brain regions in which A D A - i m m u n o r e a c t i v e neurons are found appears to exclude the formulation of a single underlying anatomical principal according to which the expression

of A D A may be governed. For example, A D A - c o n taining neurons were seen in phylogenetically new 6layered visual cortex, older 3-layered tenia tecta and agranular limbic mesocortex. In other brain regions these neurons were associated with various olfactory structures, visual structures such as the superior colliculus and the retina 3t, and somatosensory structures such as the trigeminal nucleus. The developmental profile of ADA-containing neurons in these diverse structures was also quite varied and, as might be expected, conformed to the known developmental pattern of large and small neuronal populations in the

69

CNS 20'26'36. For example, in the superior colliculus, where considerable neurogenesis occurs on the 15th and 16th gestational days 3, large ADA-immunoreactive neurons having advanced stages of dendritic development were observed on P1. The cell size, morphology and developmental profile of these neurons indicate that they may be collicular ganglion cells 19'3s. Similarly, in the posterior hypothalamus, the peak period of magnocellular neurogenesis occurs on the 16th and 17th gestational days 2. These neurons begin to synthesize immunohistochemically detectable levels of A D A on the 18th day of gestation. Interestingly, however, the rate of their dendritic growth is relatively slow, despite their early appearance. In contrast, smaller neurons such as marginal horizontal cells in the most superficial region of the superior colliculus and those in the marginal and outer layers of the trigeminal substantia gelatinosa do not begin to exhibit ADA-immunoreactivity and dendritic morphogenesis until after about the first postnatal week. These observations are consistent with the development of large and small neurons in these areas 4'38. The reason for the decrease or loss of A D A - i m m u nostaining in some structures during development is currently unclear. It does appear, however, that this is unrelated to either neuronal death, since the time course of decreased immunostaining did not parallel known periods of cell death, or to rapid transport of A D A from neuronal perikarya since colchicine pretreatment of animals did not reinstate immunostaining. The diverse distribution of ADA-immunoreactive neurons was equalled by the diverse regions in which a reduction in immunostaining was observed. Thus, while the numbers of neurons immunoreactive for A D A increased in several cortical areas, there ABBREVIATIONS A ACg ADA AIv AON CM CPu CRL Dtr E Ent FN

nucleus ambiguus anterior cingulate cortex adenosine deaminase ventral agranular insular cortex anterior olfactory nucleus caudal magnocellular nucleus caudate-putamen crown-rump length dorsal transition area embryonic day entorhinal cortex facial nucleus

was a dramatic decrease of these in tenia tecta and AIv to only a few at the adult stage. Other major areas of decreased immunostaining included facial and hypoglossal motoneurons 29 and olfactory structures including A O N and TTv. We have previously determined that in facial and hypoglossal motor nuclei, ADA-immunoreactivity was restricted to those innervating perioral and tongue retractor muscles and speculated that the transient perinatal expression of A D A in these neurons may be related to their functional importance in early suckling behavior. Since it is known that olfaction also plays a vitally important role leading to this behavior 5 the observation of numerous ADA-immunoreactive structures in the olfactory bulb, peduncle and cortex in the early postnatal period may be noteworthy, particularly given the reciprocal connections that have been documented to occur between these structures 7'1°' 11,16,33,39. The temporal co-occurrence of A D A in the anatomically remote but functionally related cranial motor and olfactory sensory systems suggests a role of the substrates of A D A , namely purines, in the early functional or morphological maturation of these systems.

ACKNOWLEDGEMENTS The authors thank Lyn Poison for typing the manuscript. This work was supported by grants from the Manitoba Health Research Council (MHRC) and the Canadian Medical Research Council (MRC) to J.I.N. and by Grant CA26284 from the National Cancer Institute to P.E.D. Fellowship support to E.S. was provided by the M H R C and J.I.N. is a Scholar of the MRC. HDB HGN IC IL IMCPC IP LNP m NTS nVsp P PBS PCg

horizontal limb of the diagonal band hypoglossalnucleus inferior colliculus infralimbic area interstitial magnocellular nucleus of the posterior commissure interpeduncular nucleus lacrimo-nasopalatine nucleus medial nucleus of the solitary tract spinal trigeminal nucleus postnatal day phosphate-buffered saline posterior cingulate cortex

7(J PCM Pf PnO PnC POC R RL

postmamnfillary caudal magnocellular nucleus parafascicular nucleus pontine reticular nucleus, oral pontine reticular nucleus, caudal primary olfactory cortex red nucleus lateral reticular nucleus

REFERENCES 1 Akasu, T., Shinnick-Gallagher, P. and Gallager, J.P., Adenosine mediates a slow hyperpolarizing synaptic potential in autonomic neurons, Nature (London), 311 (1984) 62-65. 2 Altman, J. and Bayer, S.A., Development of the diencephalon in the rat. 1. Autoradiographic study of the time of origin and settling patterns of neurons of the hypothalamus, J. Comp. Neurol., 182 (1978)945-972. 3 Altman, J. and Bayer, S.A., Time of origin of neurons of the rat superior colliculus in relation to other components of the visual and visuomotor pathways, Exp. Brain Res., 42 (1981) 424-434. 4 BickneU, Jr., H.R. and Beal, J.A., Axonal and dendritic development of substantia gelatinosa neurons in the lumbosacral spinal cord of the rat, J. Comp. Neurol., 226 (1984) 508-522. 5 Blass, E.M. and Teicher, M.H., Suckling, Science, 210 (1980) 15-22. 6 Bleier, R., Cohn, P. and Siggelkow, I.K., A cytoarchitectonic atlas of the hypothalamus and hypothalamic third ventricle of rat. In P.J. Morgane and J. Panksepp (Eds.),

Handbook of the Hypothalamus, Vol. I, Anatomy of the Hypothalamus. Dekker, New York, 1979, pp. 137-220. 7 Broadwell, R.D., Olfactory relationships of the telencephalon and diencephalon in the rabbit. 1. An autoradiographic study of the efferent connections of the main and accessory olfactory bulbs, J. Comp. Neurol., 163 (1975) 329-346. 8 Burnstock, G., Purinergic nerves, Pharmacol. Rev., 24 (1972) 509-581. 9 Daddona, P.E. and Kelley, W.N., Human adenosine deaminase purification and subunit structure, J. Biol. Chem., 252 (1977) 110-115. 10 Davis, B..1. and Macrides, F,, The organization of centrifugal projections from the anterior olfactory nucleus, ventral hippocampal rudiment, and piriform cortex to the main olfactory bulb in the hamster: an autoradiographic study, J. Comp. Neurol., 203 (1981) 475-493. 11 De Olmos, J., Hardy, H. and Heimer, L., The afferent connections of the main and accessory olfactory bulb formations in the rat: an experimental HRP study, J. Comp. Neurol., 18t (1978) 213-244. 12 Farbman, A., Scholz, D., Emanuel, D. and Margolis, F., Maturation of olfactory receptor cell terminals, Taste. VII (1980) 147-150. 13 Geiger, J.D., La Bella, F.S. and Nagy, J.I., Characterization of nitrobenzylthioinosine binding to nucleoside transport sites selective for adenosine in rat brain, J. Neurosci., 5 (1985) 735-740. 14 Geiger, J.D. and Nagy, J.I., Heterogeneous distribution of adenosine transport sites labelled by 3H-nitrobenzylthioinosine in rat brain: an autoradiographic and membrane binding study, Brain Res. Bull., 13 (1984) 657-666.

RSpl SC "ll'v Tu VC VI Vm

retrosplenial cortex superior colliculus ventral tenia tecta olfactory tubercle visual cortex lateral vestibular nucleus medial vestibular nucleus

15 Geiger, J.D. and Nagy, J.l., Distribution of adenosine deaminase activity in rat brain and spinal cord, J. Neurosci., in press. 16 Haberly, L.B. and Price, J.L,, Association and commissural fiber systems of the olfactory cortex of the rat. II. Systems originating in the olfactory peduncle, J. Comp. Neurol., 187 (1978) 781-808. 17 Kohler, C., Swanson, L.W., Haglund, L. and Wu, J.-Y., The cytoarchitecture, histochemistry and projections of the tuberomammillary nucleus in the rat, Neuroscience. 16 (1985) 85-110. 18 Krettek, J.E. and Price, J.L., The cortical projections of the mediodorsal nucleus and adjacent thalamic nuclei in the rat, J. Comp. Neurol., 171 (1977) 157-192. 19 Langer, T.P. and Lund, R.D., The upper layers of the superior colliculus of the cat: a Golgi study, J. Comp. Neurot., 158 (1974) 405-436. 20 Miller, M., Maturation of rat visual cortex. I. A quantitative study of Golgi-impregnated pyramidal neurons, J. Neurocytol., 10 (1981) 859-878. 21 Nagy, J.I. and Daddona, P.E., Anatomical and cytochemical relationships of adenosine deaminase-containing primary afferent neurons in the rat, Neuroscience, 15 (1985) 799-813. 22 Nagy, J.I., Buss, M., La Bella, L.A. and Daddona, P.E., Immunohistochemical localization of adenosine deaminase in primary afferent neurons of the rat, Neurosci. Lett., 48 (1984) 133-138. 23 Nagy, J.I., Geiger, J.D. and Daddona, P.E., Adenosine uptake sites in rat brain: identification using (3H)nitrobenzylthioinosine and colocalization with adenosine deaminase, Neurosci. Lett., 55 (1985) 47-53. 24 Nagy, J.I., La Bella, L.A., Buss, M. and Daddona, P.E., Immunohistochemistry of adenosine deaminase: implications for adenosine neurotransmission, Science, 224 (1984) 166-168. 25 Nagy, J.I., La Bella, L.A. and Daddona, P.E., Purinergic mechanisms in antidromic vasodilatation and neurogenic inflammation. In L.A. Chahl, J. Szolcsanyi and F. Lembeck (Eds.), Antidromic Vasodilatation and Neurogenic Inflammation, Hungarian Akademia Kiado, Hungary, 1984, pp. 193-205. 26 Peters, A. and Feldman, M., The cortical plate and molecular layer of the late rat fetus, Z. Anat. Entwickl. Gesch., 141 (1973) 3-37. 27 Peters, A. and Kara, D., The neuronal composition of area 17 of rat visual cortex. I. The pyramidal cells, J. Comp. Neurol., 234 (1985) 218-241. 28 Phillis, J.W. and Wu, P.H., The role of adenosine and its nucleotides in central synaptic transmission, Prog. Neurobiol., 16 (1981) 187-239. 29 Senba, E., Daddona, P.E. and Nagy, J.I., Transient expression of adenosine deaminase in facial and hypoglossal motoneurons of the rat during development, J. Comp. Neurol.. in press.

71 30 Senba, E., Daddona, P.E. and Nagy, J.I., A subpoputation of preganglionic parasympathetic neurons in the rat contain adenosine deaminase, Neuroscience, in press. 31 Senba, E., Daddona, P.E. and Nagy, J.I., Immunohistochemical localization of adenosine deaminase in the retina of the rat, Brain Res. Bull., in press. 32 Senba, E., Daddona, P.E., Watanabe, T., Wu, J.-Y. and Nagy, J.I., Coexistence of adenosine deaminase, histidine decarboxylase and glutamate decarboxylase in hypothalamic neurons of the rat, J. Neurosci., 5 (1985) 3393-3402. 33 Shipley, M.T. and Adamek, G.D., The connections of the mouse olfactory bulb: a study using orthograde and retrograde transport of wheat germ agglutinin conjugated to horseradish peroxidase, Brain Res. Bull., 12 (1984) 669-688. 34 Sternberger, L.A., lmmunocytochemistry, 2nd edn., Wiley, New York, 1979. 35 Stone, T.W., Physiological roles for adenosine and adenosine 5'-triphosphate in the nervous system, Neuroscience, 6 (1981) 523-555.

36 Tsumoto, T., Suda, K. and Sato, H., Postnatal development of corticotectal neurons in the kitten striate cortex: a quantitative study with the horseradish peroxidase technique, J. Comp. Neurol., 219 (1983) 88-99. 37 Vogt, B.A. and Peters, A., Form and distribution of neurons in rat cingulate cortex: areas 32, 24 and 29, J. Comp. Neurol., 195 (1981) 603-625. 38 Watson, S.S. and Jones, D.G., Postnatal development of the superficial layers in the rat superior colliculus: a study with Golgi-Cox and Kluver-Barrera techniques, Exp. Brain Res., 58 (1985) 490-502. 39 White, Jr., L.E., Olfactory bulb projections of the rat, Anat. Rec., 152 (1965) 465-480. 40 Zamboni, L. and De Martino, C., Buffered picric acid formaldehyde: a new rapid fixative for electron microscopy, J. Cell Biol., 35 (1967) 148A. 41 Zilles, K., Zilles, B. and Schleicher, A., A quantitative approach to cytoarchitectonics. VI. The areal pattern of the cortex of the albino rat, Anat. Embryol., 159 (1980) 335-360.