Cytoarchitectonic pattern of the hypothalamus in the cobra,Naja naja

Summary The distribution and cytoarchitectonic pattern of the magno- and parvocellular hypothalamic nuclei of the cobra, Naja naja, are described at the light-microscopic level. With respect to their tinctorial affinity to paraldehyde fuchsin (AF) as a representative of the Gomori-type of stains, the magnocellular neurons belong to the AF-positive and the parvocellular neurons to the AF-negative elements. In addition to the supraoptic and paraventricular nuclei proper, two accessory aggregations of magnocellular neurons, the nucleus retrochiasmaticus and nucleus circularis, can be identified. Although in a peculiar location, they may be regarded as subunits of the supraopticoparaventricular neurosecretory complex. As many as 22 AF-negative nuclear areas are identified in the hypothalamus of the cobra. The nucleus periventricularis hypothalami of earlier authors is subdivided into several circumscribed neuronal complexes. The nucleus arcuatus, nucleus hypothalamicus lateralis and nucleus lateralis recessus infundibuli are well developed. An attempt is made to interpret the significance of these nuclei on a comparative and phylogenetic basis.On leave from the Department of Zoology, Nagpur University, Nagpur, India     

Synaptic interaction of serotonergic axons and corticotropin releasing factor (CRF) synthesizing neurons in the hypothalamic paraventricular nucleus of the rat. A light and electron microscopic immunocytochemical study

The morphological interrelationship between the central serotonergic and hypothalamic corticotropin-releasing factor (CRF) synthesizing systems was studied in the hypothalamic paraventricular nucleus (PVN) of colchicine pretreated male rats. The simultaneous immunocytochemical localization of the transmitter and peptide employed the peroxidase-antiperoxidase complex (PAP) technique using the silver-gold intensified (SGI) and non-intensified forms of the oxidized 3,3'-diaminobenzidine (DAB) chromogen. The paraventricular nucleus received a moderate serotonergic innervation as compared with other diencephalic structures. The distribution and arborization of serotonergic axons were more prominent in the parvocellular subnuclei than in the magnocellular units of the nucleus. Serotonin containing axons formed terminal bouton and en passant type synapses with dendrites and somata of parvocellular neurons. The immunocytochemical double labelling technique revealed the overlapping of serotonergic axons and CRF-immunoreactive neurons. Vibratome (40 micron) and semithin (1 micron) sections indicated that the interneuronal communication may take place on both dendrites and cell bodies of CRF-immunoreactive neurons. Ultrastructural analysis demonstrated that serotonin-containing terminals formed axo-dendritic and axo-somatic synapses with CRF-immunoreactive neurons. These findings indicate that the central serotonergic neuronal system can influence the function of the pituitary-adrenal endocrine axis via a direct action upon the hypophysiotrophic CRF synthesizing neurons.     

Catecholamine distribution and relationship to magnocellular neurons in the paraventricular nucleus of the rat

Summary The distribution of catecholamine synthesizing enzymes within the paraventricular nucleus of the rat hypothalamus is elucidated immunocytochemically by use of antibodies to tyrosine hydroxylase, dopamine -hydroxylase, and phenylethanolamine-N-methyltransferase. Tyrosine hydroxylase-immunostained cell bodies are localized in the periventricular stratum and adjacent parvocellular regions, but rarely in magnocellular subnuclei of the paraventricular nucleus. Tyrosine hydroxylase-immunostained fibers are present in greatest density in the periventricular zone, and moderate density in the parvocellular and magnocellular subnuclei. Dopamine -hydroxylase-immunostained fibers are remarkably dense in the posterior magnocellular division of the paraventricular nucleus, especially in the dorso-lateral portion where vasopressin-containing cells predominate. Noradrenergic fiber input to these magnocellular neurons is likely since phenylethanolamine-N-methyltransferase-immunostained fibers are sparse in magnocellular subnuclei of the paraventricular nucleus. Dual immunocytochemical staining of thick and thin tissue sections demonstrates with clarity an anatomical association of dopamine -hydroxylase-immunostained fibers and magnocellular neurons. Dopamine -hydroxylase-immunostained and phenylethanolamine-N-methyltransferase-immunostained fibers are dense in the medial parvocellular component of the paraventricular nucleus; distinct features of both antisera are presented.     

Electron microscopic analysis of tyrosine hydroxylase,dopamine-β-hydroxylase and phenylethanolamine-N-methyltransferase immunoreactive innervation of the hypothalamic paraventricular nucleus in the rat

Summary The catecholaminergic innervation of the hypothalamic paraventricular nucleus (PVN) of the rat was studred by preembedding immunocytochemical methods utilizing specific antibodies which were generated against catecholamine synthesizing enzymes. Phenylethanolamine-N-methyltransferase (PNMT)-immunoreactive terminals contained 80–120 nm dense core granules and 30–50 nm clear synaptic vesicles. The labeled boutons terminated on cell bodies and dendrites of both parvo- and magnocellular neurons of PVN via asymmetric synapses. The parvocellular subnuclei received a more intense adrenergic innervation than did the magnocellular regions of the nucleus. Dopamine--hydroxylase (DBH)-immunopositive axons were most numerous in the periventricular zone and the medial paryocellular subnucleus of PVN. Labeled terminal boutens contained 70–100 nm dense granules and clusters of spherical, electron lucent vesicles. Dendrites, perikarya and spinous structures of paraventricular neurons were observed to be the postsynaptic targets of DBH axon terminals. These asymmetric synapses frequently exhibited subsynaptic dense bodies. Paraventricular neurons did not demonstrate either PNMT or DBH immunoreactivity. The fibers present within the nucleus which contained these enzymes are considered to represent extrinsic afferent connections to neurons of the PVN.Tyrosine hydroxylase (TH)-immunoreactivity was found both in neurons and neuronal processes within the PVN In TH-cells, the immunolabel was associated with rough endoplasmic reticulum, free ribosomes and 70–120 nm dense granules. Occasionally, nematosome-like bodies and cilia were observed in the TH-perikarya. Unlabeled axons established en passant and bouton terminaux type synapses with these TH-immunopositive cells. TH-immunoreactive axons terminated on cell bodies as well as somatic and dendritic spines of paraventricular parvocellular neurons. TH-containing axons were observed to deeply invaginate into both dendrites and perikarya of magnocellular neurons.These observations provide ultrastructural evidence for the participation of central catecholaminergic neuronal systems in the regulation of the different neuronal and neuroendocrine functions which have been related to hypothalamic paraventricular neurons.Supported by NIH Grant NS 19266 to W.K. Paull     

CRF-containing neurons of the rat hypothalamus

Summary The immunoreactive CRF-neurons of the rat hypothalamus have been examined immunohistochemically employing anti-rat CRF serum. These neurons are confined to the paraventricular nucleus, dorsomedial-lateral hypothalamic area, and suprachiasmatic nucleus, and are, respectively, also immunoreactive to anti-Met-enk, -alpha-MSH, and -VIP sera. Intraventricular administration of colchicine (50 g/5 l/rat) induces a dramatic enhancement of the immunostainability of the cell somata, and also accelerates the development of immunoreactivity of other stored peptides, especially in the paraventricular nucleus.The CRF-neurons respond to adrenalectomy by showing increased immunoreactivity and an increase in the number of cell bodies; in the dorsomedial-lateral area and suprachiasmatic nucleus, there is also an enhanced immunoreactivity for alpha-MSH and VIP, respectively. CRF-cells in the paraventricular nucleus become markedly hypertrophied, but do not show any enhanced immunoreactivity for Met-enk. Since the axons of the paraventricular neurons run to the median eminence, it is probable that they are involved with the endocrine control of hypophysial ACTH release. It is concluded that the CRF-containing neurons in rat hypothalamus consist of three types which are functionally and morphologically different.     

Immunohistochemical localization of neuropeptide Y in the guinea pig medulla oblongata

Summary The immunohistochemical localization of neuropeptide Y (NPY) was correlated with those of dopamine--hydroxylase (DBH) and vasoactive intestinal polypeptide (VIP) by mapping serial 7 m paraffin sections at three levels of the guina pig lower brainstem: a) area postrema, b) dorsal motor nucleus of the vagus, and c) nucleus prepositus of the hypoglossal nerve. Based on differences in transmitter expression, three populations of NPY-immunoreactive (IR) neurons were distinguished: NPY-IR catecholaminergic cells (NPY/CA), NPY-IR VIP-ergic cells (NPY/VIP), and NPY-IR cells which were not reactive to either DBH or VIP. Within these populations, size differences among neurons in characteristic locations allowed differentiation among the following subpopulations: NPY/CA neurons in the lateral reticular nucleus — magnocellular part (mean neuronal size 538 m²) and parvocellular part (318 m²)-, in the vagus-solitarius complex (433 m²), and in the dorsal strip (348 m²); NPY/VIP neurons in the vagus-solitarius complex (368 m²) and in the nucleus ovalis (236 m²). Apart from scattered NPY-IR cell bodies in the regions listed above, NPY-IR cell bodies in the lateral portion of the nucleus solitarius and in the caudal part of the spinal nucleus of the trigeminal nerve did not exhibit IR to either DBH or VIP. NPY-IR neurons in the area postrema occurred too infrequently for co-localization studies. The differential distribution of heterogeneous NPY-IR cell subpopulations may reflect the involvement of NPY in a variety of neuronal functions.Supported by the Deutsche Forschungsgemeinschaft, grant He 919/6-1     

Expression of a neuromodulin-β-galactosidase fusion protein in primary cultured neurons and its accumulation in growth cones

Cultured embryonic neurons share a number of characteristic morphological and physiological properties with their counterparts in vivo. For example, differentiating hippocampal neurons in culture develop two distinct classes of processes that serve as dendrites and axons. It has also been shown that the microtubule organization and composition in axons differs from those in dendrites, which may contribute to differential transport of macromolecules into axons or dendrites. We have expressed a neuromodulin--galactosidase fusion gene in cultured mesencephalic neurons in order to study the transport of the neurospecific protein neuromodulin into neurite growth cones. When -galactosidase alone was expressed in neurons, it was found in the cell bodies with diffuse neurite staining. In marked contrast, the neuromodulin--galactosidase fusion protein was rapidly transported into neurites and was concentrated in the growth cones. This system may provide a useful model for studying the structural domain(s) of neuromodulin that are required for transport and accumulation of neuromodulin in the growth cones of neurons.     

Hypophysiotrophic thyrotropin releasing hormone (TRH) synthesizing neurons

Summary The neuropeptide thyrotropin releasing hormone (TRH) is capable of influencing both neuronal mechanisms in the brain and the activity of the pituitary-thyroid endocrine axis. By the use of immunocytochemical techniques, first the ultrastructural features of TRH-immunoreactive (IR) perikarya and neuronal processes were studied, and then the relationship between TRH-IR neuronal elements and dopamine--hydroxylase (DBH) or phenylethanolamine-N-methyltransferase (PNMT)-IR catecholaminergic axons was analyzed in the parvocellular subnuclei of the hypothalamic paraventricular nucleus (PVN). In control animals, only TRH-IR axons were detected and some of them seemed to follow the contour of immunonegative neurons. Colchicine treatment resulted in the appearance of TRH-IR material in parvocellular neurons of the PVN. At the ultrastructural level, immunolabel was associated with rough endoplasmic reticulum, free ribosomes and neurosecretory granules. Non-labelled axons formed synaptic specializations with both dendrites and perikarya of the TRH-synthesizing neurons. TRH-IR axons located in the parvo-cellular units of the PVN exhibited numerous intensely labelled dense-core and fewer small electron lucent vesicles. These axons were frequently observed to terminate on parvocellular neurons, forming both bouton- and en passant-type connections. The simultaneous light microscopic localization of DBH or PNMT-IR axons and TRH-synthesizing neurons demonstrated that catecholaminergic fibers established contacts with the dendrites and cell bodies of TRH-IR neurons. Ultrastructural analysis revealed the formation of asymmetric axo-somatic and axo-dendritic synaptic specializations between PNMT-immunopositive, adrenergic axons and TRH-IR neurons in the periventricular and medial parvocellular subnuclei of the PVN.These morphological data indicate that the hypophysiotrophic, thyrotropin releasing hormone synthesizing neurons of the PVN are directly influenced by the central epinephrine system and that TRH may act as a neurotransmitter or neuromodulator upon other paraventricular neurons.Supported by NIH research grants NS19266 and DK34540     

Morphology of enkephalin-immunoreactive myenteric neurons in the human gut

The aim of this study was the morphological and further chemical characterisation of neurons immunoreactive for leu-enkephalin (leuENK). Ten wholemounts of small and large intestinal segments from nine patients were immunohistochemically triple-stained for leuENK/neurofilament 200 (NF)/substance P (SP). Based on their simultaneous NF-reactivity and 3D reconstruction of single NF-reactive cells, 97.5% of leuENK-positive neurons displayed the appearance of stubby neurons: small somata; short, stubby dendrites and one axon. Of these leuENK-reactive stubby neurons, 91.3% did not display co-reactivity for SP whereas 8.7% were SP-co-reactive. As to their axonal projection pattern, 50.4% of the recorded leuENK stubby neurons had axons running orally whereas in 29.4% they ran anally; the directions of the remaining 20.2% could not be determined. No axons were seen to enter into secondary strands of the myenteric plexus. Somal area measurements revealed clearly smaller somata of leuENK-reactive stubby neurons (between 259±47 m² and 487±113 m²) than those of putative sensory type II neurons (between 700±217 m² and 1,164±396 m²). The ratio dendritic field area per somal area of leuENK-reactive stubby neurons was between 2.0 and 2.8 reflecting their short dendrites. Additionally, we estimated the proportion of leuENK-positive neurons in comparison to the putative whole myenteric neuron population in four leuENK/anti-Hu doublestained wholemounts. This proportion ranged between 5.9% and 8.3%. We suggest leuENK-reactive stubby neurons to be muscle motor neurons and/or ascending interneurons. Furthermore, we explain why we do not use the term Dogiel type I neurons for this population.     

The organization of synaptic interconnections in the laminae of the dorsal lateral geniculate nucleus of the cat

Summary Four axon types occur in the lateral geniculate nucleus. Two contain vesicles with mainly round profiles and these are distinguished from each other by their size, the appearance of their contents and by the types of contact they make. The larger RLP axons are interpreted as retinogeniculate and the smaller RSD axons as corticogeniculate fibers. The other two axon types contain many irregular or flattened vesicles and these F axons are regarded as two types of intrageniculate fiber.In laminae A and A 1 encapsulated synaptic zones form around grape-like dendritic appendages. These zones contain all axon types, but RSD axons are rare. Interstitial zones lie between the encapsulated zones and contain synapses formed by many RSD axons, some F and few RLP axons. The interstitial zones continue into the central interlaminar nucleus which forms a narrow band containing no encapsulated zones and few RLP axons. Lamina B contains relatively small RLP axons, very many RSD axons and only a few small encapsulated zones.Axosomatic junctions are rare throughout the nucleus. Axo-axonal junctions occur in all laminae but mostly in the encapsulated zones; the postsynaptic element is always an F axon, RLP or RSD axons generally form the presynaptic element.Supported by Grant NB 06662 from the USPHS. The skillful technical assistance given by Mrs. E. Langer during the course of this work is gratefully acknowledged.     

Distribution of CK2, its substrate MAP1B and phosphatases in neuronal cells

Neuronal morphogenesis depends on the organization of cytoskeletal elements among which microtubules play a very important role. The organization of microtubules is controlled by the presence of microtubule-associated proteins (MAPs), the activity of which is modulated by phosphorylation and dephosphorylation. One of these MAPs is MAP1B, which is very abundant within growing axons of developing neurons where it is found phosphorylated by several protein kinases including CK2. The expression of MAP1B is notably decreased after neuronal maturation in parallel with a change in the localization of the protein, which becomes largely concentrated in neuronal cell bodies and dendrites. Interestingly, MAP1B remains highly phosphorylated at sites targeted by protein kinase CK2 in mature neurons.We have analyzed the expression and localization of CK2 catalytic subunits along neuronal development. CK2 subunit appears early during development whereas CK2 subunit appears within mature neurons at the time of dendrite maturation and synaptogenesis, in parallel with the change in the localization of MAP1B. CK2 subunit is found associated with microtubule preparations obtained from either grey matter or white matter from adult bovine brain, whereas CK2 subunit is highly enriched in microtubules obtained from grey matter. These results lend support to the hypothesis that CK2 subunit is concentrated in neuronal cell bodies and dendrites, where it associates with microtubules, thus contributing to the increased phosphorylation of MAP1B in this localization in mature neurons.     

The hypothalamus of Lacerta sicula R.

Summary An analysis of the preoptic area of the lizard, Lacerta sicula R., with the use of the Golgi method revealed that: 1)in principle, the dendritic pattern of its neurons is relatively simple; 2) the supraoptic nucleus contains large- to medium-sized bipolar or multipolar neurons together with small, usually multipolar nerve cells; 3) the preoptic periventricular gray and the paraventricular nucleus exhibit a varied neuronal typology, including large multipolar or bipolar elements, abundant CSF-contacting neurons, and some tufted elements; and 4) the lateral regions display some conspicuous multipolar neurons.With a financial contribution from Ministero della Pubblica Istruzione     

Differing immunoreactivities of somatostatin in the cortex and the hypothalamus of the rat

Summary Using an antibody against somatostatin (antiserum F), two somatostatin-immunoreactive systems, (i) a hypothalamic and (ii) an extrahypothalamic cortical system, are demonstrated in the rat. Another antiserum raised against somatostatin (antiserum BS 102) stains only the axons but not the perikarya of the hypothalamic system; the cortical somatostatin system does not react with this antiserum. The electron microscopic findings do not allow decision whether the above-mentioned hypothalamic and cortical neurons possess a common prohormonal form of somatostatin, immunoreactive only with antiserum F. They show, however, that the granules in both neuronal systems differ considerably; in the cortical neurons they measure approximately 65 nm in diameter, in the hypothalamic neurons 90–120 nm in diameter. Thus, both somatostatin systems are different and independent from one another.Supported by the Deutsche Forschungsgemeinschaft (Grant Nr. Kr 569/3) and the Stiftung Volkswagenwerk     

Location of neurons projecting to the hypophysial stalk — median eminence in ring doves (Streptopelia roseogrisea)

The median eminence/pituitary stalk represents the final common pathway for fibers from neurons that project to the pituitary gland. We have used the lipophilic fluorescent tracer 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI) to determine the location of neurons projecting to the median eminence/pituitary stalk in ring doves. The tracer can be precisely applied to fixed tissue, in areas to which it is otherwise difficult to gain access. Follwwing application of DiI to the median eminence/pituitary stalk, labeled neurons were detected in six distinct regions: the ventromedial hypothalamic nucleus, paraventricular nucleus, supraoptic nucleus, in and ventral to the lateral forebrain bundle, preoptic area, and lateral septum. Labeled fibers branched extensively in the diencephalon, particularly along the third ventricle and in the septal-preoptic area. Sparse fiber labeling occurred caudal to the tuberal hypothalamus, even though these regions were close to the application site of the tracer. Labeled cerebrospinal-fluid-contacting cells were seen in the paraventricular region of the third ventricle. The results indicate that the avian neuronal system that projects to the median eminence and neural lobe occurs in diffuse clusters largely along the midline region of the hypothalamic septal-preoptic area. The paucity of fiber staining caudal to the tuberal hypothalamic region indicates that cells of these regions do not project to the median eminence/pituitary.     

Nuclear inclusions in paraventricular nucleus neurons of the rat hypothalamus

Summary This paper deals with the ultrastructure of two types of intranuclear inclusions, microfilamentous spindle-shaped and crystalloid, present in paraventricular nucleus neurons of adult normal rats. These inclusions appear occasionally in some non-secretory neurons of the parvocellular system, but have never been seen in neurosecretory cells of the magnocellular system. The microfilamentous spindle-shaped inclusions show a close spatial relationship with the granulofibrillar body and interchromatin granules.The distribution and functional significance of such structures are discussed in the light of recent ultrastructural and biochemical studies on nuclear inclusions.     

Synaptic organization of the cabbage looper moth ocellus

Summary Chemical and electronic synapses are present in the ocellar synaptic region of the moth, Trichoplusia ni. The chemical synapses all appear to be of the conventional type. Four different chemical synaptic contacts were observed: Receptor cell axons presynaptic to receptor cell axons, receptor cell axons presynaptic to 1st order interneurons, 1st order interneurons presynaptic to receptor cell axons, and 1st order interneurons presynaptic to 1st order interneurons. Two different types of contact made by electronic synapes were observed: Contacts between receptor cell axons and 1st order interneurons, and contacts between 1st order interneurons. The significance of this synaptic arrangement for the generation of on and off responses in the 1st order interneurons is discussed.Supported by NSF Grant BMS 75-07645 and by the VPI & SU Research Division     

Immunocytochemistry of GABA in the antennal lobes of the sphinx moth Manduca sexta

Summary The ganglionated plexus of the trachea of mice was studied quantitatively with a histochemical method that stains electively the ganglion nerve cells in whole-mount preparations. The plexus lies exclusively over the muscular part of the trachea, dorsal to the muscle itself, and it varies considerably in pattern and extent between individual animals. In young adult mice the plexus contains on average 235 neurons, occurring singly or gathered in packed ganglia. The ganglion neurons are relatively small, the profile area of three quarters of them measuring between 150 and 275 m² with an average of 251 m². In ageing mice the average number of ganglion neurons is the same as in young animals; however, cell sizes are markedly increased, the average being 341 m². Among the ultrastructural features of the ganglia, is a capsule (perineurium) of very regular structure, the presence of collagen, capillaries and myelinated axons inside the ganglia, and the presence of only few and short dendrites, some of which are abutted by synapsing nerve endings.     

Immunocytochemical study by two photon fluorescence microscopy of the distribution of GABA<Subscript>A</Subscript> receptor subunits in rat cerebellar granule cells in culture

Summary. An immunocytochemical investigation of the expression of ₁, ₆, _2/3, ₂ and subunits was performed on rat cerebellum granule cells in culture by the two photon microscopy technique.The first four subunits appear to be expressed abundantly in these cells, whereas the one seems to be expressed at a lower level. Another major difference in the distribution of these subunits is that whereas ₆, _2/3 and ₂ appear only on plasma membranes ₁ and are present mainly in the cell bodies cytoplasm. Still another difference was found in that the presence of ₂ on neurites is polarized, preferentially labelling neurites with the appearance of dendrites. The subunits ₆ and _2/3 appear to label all types of neurites, with _2/3 being by far the most heavily expressed subunit type. A final distinct characteristic is that ₆ and, even more, ₂ appear to accumulate in the cytoplasmic domains immediately below the cone of emergence of neurites. This suggests a conspicuous transport of such subunits from the site of synthesis in the cell body to the site of final expression in the neurites (dendrites and axon terminals).     

Spermatogonia and the cycle of the seminiferous epithelium in the nine-banded armadillo

Summary The cycle of the seminiferous epithelium of the nine-banded armadillo can be divided into ten stages. As in most mammals, only one stage is observed per tubular cross-section. The process of spermiogenesis can be divided into thirteen steps according to the development of the acrosomal system and the flagellum. Four generations of spermatogonia are observed in the germinal epithelium: 1) stem cells, 2) type A, 3) intermediate, and 4) type B spermatogonia. The stem cell is characterized by a highly irregular nucleus and the presence of glycogen in its cytoplasm. The type A spermatogonium contains an oblong nucleus with one or two shallow infoldings of the nuclear membrane. The intermediate spermatogonium contains an ovoid nucleus characterized by one or two nuclei and heterochromatin scattered in the nucleoplasm. The nucleus of the type B spermatogonium is more spherically shaped with a centrally placed nucleolus and heterochromatin associated with the nuclear envelope.The author wishes to acknowledge the technical assistance of Teri Lane