P.E. Daddona

The organization and hypothalamic projections of the tuberomammillary nucleus in the rat: An immunohistochemical study of adenosine deaminase-positive neurons and fibers

The intense immunohistochemical reaction for the enzyme adenosine deaminase displayed by neurons in the tuberomammillary nucleus in the rat was used to study the distribution and morphology of cells comprising this nucleus, their fiber fields within the posterior hypothalamus and their projection pathways from the hypothalamus. Neurons immunoreactive for adenosine deaminase were found along ventricular and basal aspects of the hypothalamus from the level of the dorsomedial nucleus to the caudal pole of the mammillary body. Approximately 4500 neurons were seen on each side of the brain. Positive neurons showed a complex distribution, largely avoiding nuclear boundaries within the posterior basal hypothalamus and mammillary body. This distribution is mapped in detail and a nomenclature based on topography is introduced so that different regions of the cell distribution may be discussed more easily. Reactive neurons showed a Golgi-like staining which allowed careful study of their morphology. In general, neurons were large, with major axes of from 22 to 30 micron, and bipolar in shape. A second, smaller cell type, 14-16 micron in diameter was also seen and, although often less intensely stained, it was considered a constituent of tuberomammillary nucleus of the hypothalamus as well. Stained dendritic arbours extended considerable distances from the parent cell bodies and branched regularly. Dendrites showed very sparse spines and had an apparently scalloped surface. Features suggestive of varicose segments of dendrites were also noted. The long, smooth dendrites of positive neurons were often seen to aggregate into bundles which avoided nuclear boundaries and tended to collect adjacent to basal and ventricular surfaces of the posterior hypothalamus. Varicose fibers immunoreactive for adenosine deaminase formed a dense network within the hypothalamus. These fibers were considered to derive from the positive neurons in the tuberomammillary nucleus and were similar to adenosine deaminase-immunoreactive fibers seen throughout much of the rest of the brain. The density of this type of positive fiber was, however, much greater within the hypothalamus. The region of the posterior basal hypothalamus also contained relatively sparse populations of adenosine deaminase-positive fibers, apparently distinct from this network. These consisted of a field of fine fibers in the median division of the medial mammillary nucleus and a few large varicosities in the dorsolateral part of the median eminence.(ABSTRACT TRUNCATED AT 400 WORDS)

On the innervation of trigeminal mesencephalic primary afferent neurons by adenosine deaminase-containing projections from the hypothalamus in the rat

The localization and sources of adenosine deaminase-containing structures in the mesencephalic nucleus of the trigeminal nerve of the rat was studied using indirect immunofluorescence or immunoperoxidase immunohistochemical staining techniques for adenosine deaminase in combination with retrograde fluorescent tracing or lesion methods. The majority of large mesencephalic neurons were engulfed by a dense adenosine deaminase-immunoreactive plexus. Immunostaining was often punctate surrounding neuronal profiles or sometimes had the appearance of varicose fibers coursing over the neuronal surface. Occasionally, immunostained axons were found travelling towards and contacting mesencephalic neurons. Mesencephalic neuronal somas surrounded by immunofluorescence staining for adenosine deaminase were simultaneously labelled with fast blue after injections of this dye into the temporalis or masseter muscles of mastication. Injections of fast blue into the mesencephalic nucleus resulted in fast blue labelling of adenosine deaminase-immunoreactive neurons in the tuberal, caudal and postmammillary caudal magnocellular nuclei of the hypothalamus. Ablation of these hypothalamic nuclei caused a near total depletion of adenosine deaminase-immunostained fibers in the mesencephalic nucleus including those associated with mesencephalic neurons. It is concluded that adenosine deaminase-containing neurons in the posterior hypothalamus innervate mesencephalic primary sensory neurons, which are known to convey proprioceptive input to trigeminal motor nuclei controlling jaw muscles. The possibility is considered that the hypothalamus, via a direct action on these sensory neurons, may exert automatic control over jaw movements related to aggressive attack, defensive or feeding behavior. In addition, it appears that mesencephalic neurons may provide an ideal model system for electrophysiological investigations of the neurotransmitter(s) utilized by adenosine deaminase-containing hypothalamic projections.

Anatomical and cytochemical relationships of adenosine deaminase-containing primary afferent neurons in the rat.

The distribution of adenosine deaminase-containing neurons and fibers in the spinal cord and medulla was examined and the relationship of dorsal root ganglia neurons containing this enzyme to those containing somatostatin, substance P, fluoride-resistant acid phosphatase (FRAP) and 5-nucleotidase was determined using immunohistochemical and histochemical methods. In the spinal cord adenosine deaminase-immunoreactive fibers and neurons were confined to layer I and IIo. A similar localization of these was observed in the spinal trigeminal nucleus. In adult animals treated neonatally with capsaicin adenosine deaminase-positive fibers were totally depleted in layer IIo but only partially depleted in layer I. Analysis of lumbar sensory ganglia revealed that small type-B neurons immunoreactive for adenosine deaminase were also immunoreactive for somatostatin but not substance P. In addition, adenosine deaminase-positive neurons lacked histochemical reaction-product for FRAP and exhibited the lowest activity of 5-nucleotidase. Examination of the neuronal populations containing the two phosphatase enzymes showed that a proportion of neurons exhibiting 5-nucleotidase activity were devoid of FRAP activity. It is concluded that dorsal root ganglia neurons immunoreactive for adenosine deaminase and somatostatin constitute a single subpopulation of type-B ganglion cells separate from those containing substance P or FRAP. It appears that the lack of coexistence of adenosine deaminase with either FRAP or 5-nucleotidase cannot be attributed simply to a coexistence of the two latter enzymes since some 5-nucleotidase-positive neurons lacking FRAP were also devoid of adenosine deaminase-immunoreactivity. Insofar as these three enzymes may contribute to the regulation of transmission processes in primary sensory neurons, our results indicate a minimal functional relationship between adenine nucleoside and nucleotide degrading enzymes in these neurons. In addition, FRAP appears to have some functional independence from 5-nucleotidase.

Subcellular, regional and immunohistochemical localization of adenosine deaminase in various species

Immunohistochemical and subcellular fractionation techniques were employed to compare the cellular and subcellular localization of adenosine deaminase (ADA) in various brain regions of several mammalian species. A relatively restricted distribution of ADA-immunoreactive neurons in rat brain was previously reported. Mouse brain exhibited a pattern similar in many respects to rat and, in addition, contained intensely immunostained neurons in lateral habenula and hippocampus. Glial immunostaining was absent or very light in rat but evident in mouse. Prominent immunoreactive fibers and neurons were observed in hamster spinal cord and anterior hypothalamus, respectively. ADA-immunostaining in guinea-pig was localized to presumptive fibers in the superficial layers of spinal cord dorsal horn and to glial cells throughout the brain. Demonstration of specific immunostaining in rabbit was not possible. ADA activity was far more heterogeneously distributed in rat and most brain areas in guinea-pig and rabbit contained up to 5-fold and 10-fold higher levels of activity, respectively, compared with rat. Crude synaptosomal (P2) fractions of rat cortex contained a greater proportion of ADA activity than those of rabbit cortex. Within rat, relatively high activity was found in P2 fractions of whole hypothalamus, cerebellum, and hippocampus. ADA activity was greater in P2 fractions of rat anterior compared with whole hypothalamus and the greatest proportion of the enzyme in this fraction was localized to purified synaptosomes. The large variations in the activity and cellular location of ADA in the animals examined suggest species differences in mechanisms governing adenosine metabolism in brain and possible differences in the relationships between cellular metabolism, ADA and the neuroregulatory role of adenosine in the CNS.

A subpopulation of preganglionic parasympathetic neurons in the rat contain adenosine deaminase

Immunohistochemical staining and retrograde fluorescent tracing techniques were used to demonstrate the presence of adenosine deaminase in preganglionic parasympathetic neurons. Both brainstem and sacral spinal cord parasympathetic nuclei were found to contain a subpopulation of neurons immunoreactive for adenosine deaminase. Immunostaining of preganglionic neurons in brainstem was restricted to a group of cells which were shown by retrograde tracing with Fast Blue to project exclusively to the sphenopalatine ganglion. This group was defined as the lacrimo-nasopalatine parasympathetic nucleus. Neurons in all other cranial preganglionic centers were devoid of adenosine deaminase immunoreactivity. In spinal cord adenosine deaminase-immunoreactive neurons were found in the intermediolateral gray matter in the region of the sacral parasympathetic nucleus. Injections of Fast Blue into the pelvic ganglion labeled large numbers of neurons in this nucleus, only some of which contained adenosine deaminase. The majority of neurons immunoreactive for adenosine deaminase were also shown to be immunoreactive for choline acetyltransferase in both brainstem and sacral parasympathetic nuclei. The present results show that a subclass of preganglionic parasympathetic neurons are among the few structures in the central nervous system that express what appear to be high levels of adenosine deaminase. This observation together with evidence suggesting that purines serve as neurotransmitters in some sacral parasympathetic neurons supports the notion that adenosine deaminase may constitute a marker for adenine nucleoside and/or nucleotide neurotransmission.

Distribution, morphology and habenular projections of adenosine deaminase-containing neurons in the septal area of rat

Adenosine deaminase (ADA) was localized within several types of neurons within the septum and in septal efferent projections to the habenula by immunohistochemical, biochemical, retrograde tracing and lesion methods. Numerous ADA-immunoreactive (ADA-IR) neurons were observed in the septofimbrial nucleus, the triangular septal nucleus and the bed nucleus of the anterior commissure, while considerably fewer numbers were seen in the lateral septal area. Based on their size, shape and dendritic features, 4 morphologically distinct types of ADA-IR neurons were recognized in these septal structures. In addition, fine, non-varicose, ADA-IR fibers appeared to emanate from the postcommissural cell groups and these coalesced within the stria medullaris, continued caudally within this fiber bundle, and gave rise to a dense field of very fine immunoreactive elements within a restricted zone of the dorsal half of the medial habenula. Comparisons of the habenular localization of ADA-IR and enkephalin-IR elements showed that fibers labelled for either ADA or enkephalin occupied distinct, non-overlapping regions within the dorsal half of the medial habenula. After injections of Fluoro-gold (FG) into the medial habenula, the majority of ADA-IR neurons in the septofimbrial nucleus, triangular septal nucleus, and the bed nucleus of the anterior commissure were retrogradely labelled with this fluorescent tracer, whereas no ADA-positive FG-labelled neurons were observed in the lateral septal region. Unilateral transections of the stria medullaris caused substantial depletions of ADA-immunoreactivity and reduced enzymatically determined ADA activity by up to 80% in the medial habenula on the lesioned compared with the contralateral control side. These results demonstrate that ADA-IR neurons in the septum are heterogeneously distributed and that populations of positive neurons within the postcommissural septal nuclei give rise to dense, focal projections to the medial habenula. These projections appear to be restricted to a portion of the medial habenula known to contain substance P-IR neurons and are subregionally segregated from enkephalin-positive septohabenular projections ending within this same portion. In addition to pointing out a unique capacity for adenosine catabolism within some septal neurons, possibly related to purinergic neuromodulation, the results indicate the utility of ADA-immunohistochemistry for the delineation of anatomical relationships between the septum and the medial habenula.

Further observations on the relationship between adenosine deaminase-containing axons and trigeminal mesencephalic neurons: An electron microscopic, immunohistochemical and anterograde tracing study

The somas of primary afferent neurons in the mesencephalic nucleus of the trigeminal nerve in rat have a dense investment of axons immunoreactive for the enzyme adenosine deaminase. We previously suggested that these axons may originate from adenosine deaminase-immunoreactive neurons located in the tuberomammillary nucleus of the hypothalamus [Nagy et al. (1986) Neuroscience 17, 141-156]. Anterograde tracing and immunohistochemical techniques were used to investigate this possibility further. In addition, the appearance of adenosine-immunoreactive axons and the nature of their interactions with mesencephalic neurons was examined ultrastructurally. After injections of either Phaseolus vulgaris-leucoagglutinin or wheat germ agglutinin-horseradish peroxidase into the region of the tuberomammillary nucleus, punctate deposits of anterogradely transported tracer, detected by immunoperoxidase methods, were seen surrounding mesencephalic neurons. In sections immunostained for tracer and adenosine deaminase by double immunofluorescence, some fibres in the periaqueductal gray matter and around Mes V somas were found to be labelled for both the lectin and the enzyme. Ultrastructurally, only a single morphological class of adenosine deaminase-immunoreactive axons adjacent to, or indenting the cytoplasmic membranes of, large somas in the mesencephalic nucleus could be recognized; they were varicose and contained relatively large immunoreactive vesicles ranging in diameter from 45 to 70 nm. Occasionally, thin processes of these axons could be traced back to small adenosine deaminase-positive neuronal cell bodies located not within the tuberomammillary nucleus, but rather, within the periaqueductal gray matter. In serial ultrathin sections, membrane specializations resembling synaptic junctions were sometimes seen at points where mesencephalic somas were in contact with adenosine deaminase-immunoreactive terminals. Somas within the mesencephalic nucleus also formed such junctions with non-immunoreactive boutons which were morphologically different from, and often seen in close proximity to, those containing adenosine deaminase. These results indicate that in addition to possible afferents from the tuberomammillary nucleus, primary sensory somas within the mesencephalic nucleus are also associated with axonal processes originating from adenosine deaminase-positive neurons located within the periaqueductal gray matter. The infrequent synaptic contacts between these somas and adenosine deaminase-positive axons, despite their close anatomical arrangement, is suggestive of a diffuse endocrine or neurocrine type of axonal relationship with mesencephalic somas or with the n

Adenosine deaminase-containing neurons in the olfactory system of the rat during development

The development, distribution and olfactory bulb projections of neurons immunoreactive for the enzyme adenosine deaminase (ADA) were studied in olfactory systems of embryonic, early postnatal and young adult rats. On embryonic day (E) 12, ADA-immunoreactivity first appeared in the placode of the olfactory epithelium. On E15, ADA-immunoreactive olfactory receptor and precursor cells gave rise to immunostained axons projecting to the olfactory bulb. Numerous immunostained glomeruli were observed on postnatal day (P) 1. After P25, immunoreactivity within receptor cells and glomeruli decreased. In prenatal and early postnatal animals, ADA-immunoreactive neurons were observed in the anterior olfactory nucleus (AON), dorsal transition area, ventral taenia tecta, primary olfactory cortex (POC), entorhinal cortex and ventral agranular insular cortex. After P25 to P30, these neurons lost their immunoreactivity, except those in the medial AON where light immunostaining persisted. In contrast, ADA-immunostaining of neurons in the horizontal limb of the diagonal band (HDB) and olfactory tubercle increased throughout development. About 70 to 75% of the ADA-immunoreactive neurons in the AON, a small number of those in the POC and about 75% of the ADA-immunoreactive non-cholinergic neurons in the HDB were found to project to the olfactory bulb. The functions of ADA in the olfactory system may be related to the precocious development of, and/or purinergic neurotransmission within, this system.

Distinct adenosine deaminase-containing inputs to the substantia nigra from the striatum and tuberomammillary nucleus

Immunohistochemical, neuroanatomical and lesion methods were used to investigate the projections of adenosine deaminase immunoreactive (ADA-IR) neurons in the striatum (caudate/putamen) and hypothalamus to the substantia nigra (SN). Striatal ADA-IR neurons were distributed within two zones; anteriorly in the medial and ventromedial extreme of the head and body of the striatum, and posteriorly in the tail of the striatum. The posterior hypothalamus contained ADA-positive neurons which were confined to the tuberomammillary nucleus (TM). The SN was devoid of ADA-positive neurons, but contained two distinct types of ADA-IR fiber terminations. One type was confined to bands located at the ventrolateral and dorsomedial borders of the pars reticulata and consisted of fine puncta. The other type was distributed throughout the SN and consisted of long, beaded fibers. Injections of the retrograde tracer Fluoro-gold (FG) into the SN gave rise to FG-labelling of significant numbers of ADA-IR neurons in both the striatum and TM. Medial SN injections preferentially labelled ADA-IR neurons in the anterior striatum and lateral SN injections labelled posterior ADA-IR striatal neurons. Kainic acid lesions of the anterior medial striatum selectively abolished the punctate ADA-IR band in the medial SN and left the long, ADA-IR nigral fibers in an apparently hypertrophied state. Despite depletion of ADA-IR neurons in the striatum by kainic acid, ADA activity increased significantly at striatal lesion sites. The results suggest that the SN receives two topographically segregated fine terminal fields from striatal ADA-IR neurons, and a substantial innervation from ADA-IR neurons in the TM as well. These findings add to the heterogeneous chemical composition of nigral afferents and are discussed in the context of adenosine neuromodulatory mechanisms in the striatonigral system.

Adenosine deaminase-'like' immunoreactivity in cerebellar Purkinje cells of rat.

The localization of adenosine deaminase (ADA) throughout the rat CNS was investigated immunohistochemically with 5 different affinity purified polyclonal antibody preparations against ADA. Except in the cerebellum, identical immunostaining patterns of ADA-immunoreactivity were observed with all 5 antibodies. In the cerebellum, Purkinje cells and presumptive swellings on their axons were immunostained by only one of the 5 antibodies. Preabsorption with purified ADA abolished immunostaining with all 5 antibodies. Biochemical tests showed that the Km for substrate, the specific activity of immunoprecipitable enzyme and the isozymic characteristics of cerebellar ADA was not different from ADA in whole brain or several other brain regions examined. The atypical immunohistochemical behavior of cerebellar ADA, despite its biochemical similarity to ADA elsewhere in the brain, suggests that the enzyme in the cerebellum has some unique features which must be taken into account when considering its possible role in regulating the neuromodulatory actions of adenosine in the cerebellar cortex.