Somatostatin-like immunoreactivity in non-pyramidal neurons of the human entorhinal region

Summary The distribution of somatostatin-immunoreactive cells and processes throughout the human entorhinal region and subjacent white matter was examined either by the unlabelled antibody-enzyme method or by the avidin-biotin method. The brain slices were obtained at autopsy with a short post-mortem delay. The majority of somatostatin immunoreactive nerve cells was found in the inner principal layer and subjacent white matter. In addition, individually scattered immunoreactive neurons were observed in both the outer principal layer and lamina dissecans. The immunoreactive perikarya varied in shape and ranged in size from 10 to 30 m. Without exception the neurons could be classified as belonging to the group of non-pyramidal neurons. Each neuron gave rise to a few thick dendrites and a thin axon with a beaded appearance. In the adult human brain, the pattern formed by lipofuscin granules deposited in the nerve cells can be considered characteristic for the type of the neuron. Therefore, immunoreactive perikarya were documented, destained of chromogen and restained to demonstrate lipofuscin pigment and basophilic substance. It became evident from these studies that the previously immunoreactive cells were characterized by a large rounded and eccentrically located nucleus, sparse basophilic substance and, in most cases, a lack of lipofuscin granules. A few of the immunoreactive cells were laden with coarse pigment granules. The findings permit classification of entorhinal somatostatin-immunoreactive neurons as either non-pigmented or pigment-laden non-pyramidal neurons.Dedicated to Prof. Dr. J. Lang, Würzburg, on the occasion of his 65th birthdayA portion of the results has been presented at the annual meeting of the European Neuroscience Association 1986 in Marseille, France (Friederich-Ecsy et al. 1986)     

Neuronal types in the striatum of man

Summary Nerve cells of the human striatum were investigated with the use of a newly developed technique that reveals the pattern of pigmentation of individual nerve cells by means of transparent Golgi impregnations of their cell bodies and processes. Five types of neurons are distinguished:Type I is a medium-sized spine-laden neuron with an axon giving off a great number of collateral branches. The vast majority of the cells in the striatum belong to this type. Numerous intensely stained lipofuscin granules are contained in one pole of the cell body and may also extend into adjacent portions of a dendrite.Type II is a medium-sized to large neuron with long intertwining dendrites decorated with spines of uncommon shape. A distinguishing feature of this cell type is the presence of somal spines. This cell type is devoid of pigment or contains only a few tiny lipofuscin granules.Type III is a large multipolar neuron. The cell body generates a few rather extended dendrites that are very sparsely spined. The finely granulated pigment is evenly dispersed within a large portion of the cytoplasm.Type IV is a large aspiny neuron with rounded cell body and richly branching tortuous dendrites. The axon branches frequently in the vicinity of the parent soma. Large pigment granules are concentrated within a circumscribed part of the cell body close to the cell membrane.Type V is a small to medium-sized aspiny neuron. The dendrites break up into a swirling mass of thin branches. More than one axon may be given off from the soma. The axons branch close to the soma into terminal twigs. Cells of this type contain numerous large and well-stained lipofuscin granules.Each of the cell types has a characteristic pattern of pigmentation. The different varieties of nerve cells in the striatum can therefore be distinguished not only in Golgi impregnations but also in pigment-Nissl preparations.     

Neuronal types in the lateral geniculate nucleus of man

Summary Nerve cell types of the lateral geniculate body of man were investigated with the use of a transparent Golgi technique that allows study of not only the cell processes but also the pigment deposits. Three types of neurons have been distinguished:Type-I neurons are medium-to large-sized multipolar nerve cells with radiating dendrites. Dendritic excrescences can often be encountered close to the main branching points. Type-I neurons comprise a variety of forms and have a wide range of dendritic features. Since all intermediate forms can be encountered as well, it appears inadequate to subdivide this neuronal type. One pole of the cell body contains numerous large vacuolated lipofuscin granules, which stain weakly with aldehyde fuchsin.Type-II and type-III neurons are small cells with few, sparsely branching and extended dendrites devoid of spines. In Golgi preparations they cannot be distinguished from each other. Pigment preparations reveal that the majority of these cells contains small and intensely stained lipofuscin granules within their cell bodies (type II), whereas a small number of them remains devoid of any pigment (type III). Intermediate forms do not occur.     

On the structure of the human striate area. Lamina IVcβ

Summary Layer IVc of the human striate area consists mainly of a great number of small spinous local circuit neurons which store numerous characteristic lipofuscin granules. Since the neurons of the neighbouring layers are almost devoid of pigment deposits the boundaries of lamina IVc are easily traceable. Hence, the pigment granules can be used as internal markers to unequivocally identify these small pigmented spinous local circuit neurons of lamina IVc in ultrathin sections. They have a large spherical nucleus surrounded by a narrow cytoplasmic rim poor in organelles, and very scarcely receive axosomatic symmetric synapses.Within layer IVc four types of synaptic boutons can be distinguished. Type-1-boutons are large, contain a few and loosely arranged round vesicles and make asymmetric synaptic contacts with dendrites and dendritic spines. The type-2-boutons which are also large are filled with densely packed round vesicles which accumulate at the presynaptic membrane. The large type-3-boutons are characterized by elongated vesicles and symmetric synaptic contact zones. These boutons generate several fingerlike protrusions. Small profiles which contain elongated vesicles and form symmetric synaptic contacts, are most probably parts of these protrusions. The large amount of small boutons with round vesicles and asymmetric synaptic contact zones are tentatively described as type-4-boutons although it is far from certain that they represent a uniform class. The presumable origins of the different types of boutons are discussed.Supported by the Deutsche Forschungsgemeinschaft (Br. 634/1)Dedicated to Prof. Dr. med. H. Leonhardt in honor of his 60th birthday     

LIPOFUSCIN (AGING) PIGMENT GRANULES OF THE NEWBORN HUMAN LIVER

We have observed pigmented cytoplasmic granules, with the characteristic staining properties of lipofuscin (ceroid, "wear-and-tear") pigment, in newborn human liver. The pigment is found at the periphery of the lobule in hepatocytes and some bile ductular cells. It is acid-fast, PAS-positive after diastase digestion, slightly argyophilic and sudanophilic, and markedly Schmorl's- and peroxidase positive in paraffin sections. Difficult to see in sections stained with hematoxylin and eosin, the pigment can be detected in unstained sections. The granules also resemble lipofuscin found in adult tissues, in their ultra-structural and enzymatic properties. They are polymorphic, contain granular material of moderate and high electron opacity, and are delimited by a single membrane. Acid phosphatase and β-glucuronidase activities are visualized in the newborn granules, identifying them as lysosomes. The granules also contain copper and, to a much lesser extent, iron. The accumulation of lipofuscin pigment in lysosomes in many tissues correlates well with aging, and this process has been interpreted as a reflection of cellular degeneration or wear-and-tear. However, the presence of lipofuscin granules as a constant component of neonatal liver suggests that they are not a measure of cellular senescence.     

Neurons and their synaptic organization in the visual cortex of the rat

Summary Cells in the visual cortex (area 17) of adult rats were impregnated by the rapid Golgi method and characterized by light microscopy. Selected cells were then sectioned for electron microscopy and their cytological characteristics and the pattern of synapses on their cell bodies and dendrites were studied Twelve classical pyramidal cells from layers II–VI, two pyramid-like cells from layer VI, two inverted pyramidal cells from layers V and VI, ten spine-free non-pyramidal cells from layers II–VI and two spinous non-pyramidal cells from layer IV were examined.The cytoplasmic features of the identified cells, where these could be discerned, corresponded to those previously reported for the different cell types in conventionally prepared tissue. Pyramidal Cells received exclusively type 2 synaptic contacts on their cell bodies, type 1 contacts on their dendritic spines and a mixture of synaptic types (type II predominating) on their shafts, where synaptic density was relatively low. This pattern of synaptic contacts was consistent for all portions of the dendritic tree; inverted pyramidal cells and pyramid-like cells showed the same synaptic organization as classical pyramids. The axon collaterals of pyramidal cells established type I contacts with dendritic spines (or, rarely, shafts) of unknown origin. Non-Pyramidal Cells received both type 1 and type 2 contacts (the former predominating) on their cell bodies and dendrites. The spinous variety also received type I contacts on their dendritic spines. Axon terminal of spine-free non-pyramidal cells established type II synaptic contacts with dendritic shafts of unknown origin. The similarity in synaptic organization between the spine-free and spinous non-pyramidal cells examined in this study suggest that the latter correspond to the sparsely spinous stellate cells rather than to the spinous stellate cells of cat and monkey visual cortex.We thank the Medical Research Council for financial support     

Neuronal types and their percent distribution within the magnocellular nuclei of the human basal forebrain

Three neuronal types constituting the magnocellular nuclei of the human basal forebrain have been differentiated with the aid of preparations stained for both Nissl material and pigment deposits: type I = large multipolar neurons contain loosely packed and faintly stained lipofuscin granules occupying a large portion of the cell body; type II = large spindle-shaped neurons reveal a densely packed accumulation of coarse and intensely stained lipofuscin granules, and type III = small nerve cells, scattered among these large neuronal components, with only a small number of faintly stained lipofuscin granules. The determination of the projection areas of the somata of the three neuronal types has led to a distribution pattern with three peaks. The ratio of the nerve cell types has been evaluated: 73.6% type I; 8.6% type II, and 17.8% type III neurons.     

The locus coeruleus of cat

Summary The locus coeruleus of cat is populated by two types of neurons: medium sized ones, with plump cell bodies and relatively short dendrites; and small ones, with triangular bodies and relatively long dendrites. The former type is regarded here as typical of the centre, whereas the second type could simply represent displaced neurons from the adjacent griseum centrale. Electron microscopy failed to reveal any outstanding richness in pigment granules in kittens up to five weeks old. Very characteristic somatic appendages were found, mostly in the medium sized neurons. These somatic spines communicate with the perikaryon by means of a narrow neck region. A complex, multilayered, glial sheath surrounds the cells. This glial sheath is pierced by the somatic appendages, which are not surrounded by glia and make contact with axonal knobs. Typical dendritic spines appear to be absent. Axodendritic synapses are made on medium sized dendritic trunks. By and large, most of the synaptic vesicles present in the centre are of the small, clear-centered type. However, dense core vesicles extremely variegated in size and appearance were found, both in presynaptic and postsynaptic profiles. The possibility that dense core vesicles should be regarded as atypical lysosomes rich in by-products of the metabolism of catecholamines (melanine) has been considered.Supported by grant MA 4183 from the Medical Research Council of Canada.     

The lipofuscin in neuroglial cells of the optic nerve of Formosan rock-monkey (Macaca cyclopis)

The ultrastructure of lipofuscin granules in neuroglial cells of the optic nerve of the Formosan Rock-Monkey was investigated by electron microscopy. In the cytoplasm of astroglial cells, numerous irregular lipofuscin granules were characterized by the presence of large lipid droplets, small electron-dense pigment granules, and some lamellar structures. The lipofuscin granules of the oligodendroglial cells were composed largely of dense, coarse pigment granules, multilinear structures, and a few small lipid droplets. The lipofuscin granules in microglial cells were characterized by numerous lipid droplets in various sizes, small electron-dense pigment granules, and prominent lamellar structures. It was reported that the lipofuscin granules are wear-and-tear materials and products from the cells in lower functional activity. However, our observations suggest that the presence of lipofuscin granules in the neuroglial cells of the optic nerve is likely a characteristic product of active phagocytosis.     

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     

Mast cells in rat dermis and jejunal lamina propria show a five-fold difference in unit granule volume

Summary The cytoplasmic granules of mast cells have a periodic multimodal size distribution in which the volumes of individual granules are integral multiples of the intermodal distance, a volume defined as the unit granule or ₁. In this study, we used two 3-month-old male rats to analyze two classical mast cell subpopulations, dermal connective tissue-type mast cells and jejunal lamina propria mucosal mast cells, for the morphometric characteristics of their cytoplasmic granules. Both and the mean volume of individual cytoplasmic granules were much smaller in dermal than in jejunal mast cells (ratios of 1:5.5 and 1:4.2, respectively), but dermal mast cells contained 150% more granules per cell than did jejunal mast cells. The two types of mast cells did not differ significantly in total cell volume, nucleus volume, aggregate volume of cytoplasmic granules per cell or numbers of unit granules comprising a granule of mean volume. These findings add unit granule volume to the list of phenotypic characteristics which express significant variation in anatomically distinct populations of mast cells.     

Ultrastructural study of neurotensin immunoreactivity in the superficial laminae of the dorsal horn of the rat

Neurotensin immunoreactivity was identified in cell bodies, dendrites, spines, axons, terminals and varicosities in superficial laminae of rat spinal cord with the electron microscope. Unlabeled terminals synapsed with neurotensin-immunoreactive cell bodies, dendrites and spines. Presynaptic terminals contained round or pleomorphic vesicles and generally made symmetrical contacts with medium-sized neurotensin-containing dendrites in outer lamina II, and asymmetrical or symmetrical contacts with large and small dendrites and spines in inner lamina II. Neurotensin immunoreactive axons were unmyelinated, and their terminals were presynaptic to unlabeled dendrites and spines in laminae I and II. Terminals contained small, round, clear vesciles (31 nm) and occasional large granular vesicles (78 nm). Contacts in outer lamina II were evenly distributed among dendrites of various sizes and spines, whereas the majority of labeled terminals in inner lamina II made contacts onto small dendrites and spines. These findings indicate that neurotensin effects in rat spinal cord are mediated by axodendritic synapses, and that neurotensin cells at the inner and outer borders of lamina II contact dendrites of efferent neurons or other interneurons in the dorsal horn.     

Neurotransmitters alter the numbers of synapses and organelles in photoreceptor terminals in the lamina of the housefly, Musca domestica

Various organelles in the lamina terminals of housefly photoreceptors exhibit daily rhythms having a circadian basis. These include changes in the numbers of photoreceptor tetrad and L2 feedback synapses, and longitudinal movements of screening pigment. Circadian information has previously been suggested to spread from the clock to the lamina via widefield cells expressing either 5-hydroxytryptamine or pigment-dispersing hormone-like immunoreactivity. We examined the action of these neuromodulators, and other candidate neurotransmitters, 4 h after injecting either the transmitter or a control into the medulla. We counted electron microscope profiles of organelles that normally exhibit circadian changes, and two types of invagination into photoreceptor terminals, capitate projections and inter-receptor invaginations. No single substance mediated the changes observed. Injected pigment-dispersing hormone peptide decreased the number of pigment granules, implicating this peptide in screening pigment migration, but produced no changes in synapse-related organelles. α-Aminobutyric acid exclusively decreased the number of L2 feedback synapses. Responses to other transmitters were specific, and often large, but generally not statistically significant. Histamine, for example, may decrease the number of tetrads, possibly by direct autoregulation. The results suggest that there is likely to be more than one effector in the circadian pathways to the lamina. Accepted: 11 August 1998     

Constituents of connective tissue around the capillary of chick pecten oculi

The constituents of the connective tissues around the capillary of the chick pecten oculi were examined electron microscopically by HCl-collagenase and HCl-elastase methods. The basal lamina like membrane below the endothelial cell of the pecten capillary was digested by collagenases I, II and IV and elastase, and may be a false basal lamina. The basal lamina of cells with pigment granules which surround the capillary was digested by collagenase IV and elastase, and contained type IV collagen. Fibrils between the basal lamina like membrane of the pecten capillary endothelium and the basal lamina of the cells with pigment granules were digested by collagenases I, II and IV, and elastase. Thus, these fibrils are composed of many kinds of collagen. Elastase may be responsible for the breakdown of most collagens as well as elastin.     

Age changes in the centrally and peripherally located sensory neurons in rat

Summary Lipofuscin pigment formation and distribution in the Mes. N.5 neurons, trigeminal and spinal ganglia of male Wistar rats of 2, 14, 32 and 49 months as an indication of aging has been investigated. These intraneuronal pigment granules are found as early as 2 months in all the cells, and continue to accumulate in all the cells in varying amounts until the first year of life. The different rate at which lipofuscin accumulates probably shows the difference in the maturation of the functionally related cells. At later stages the obvious findings are complex pigment body formation and localization of the pigment bodies either at one pole as seen in the Mes. N. 5 neurons or arranged submembranously parallel to the long axis of the cells in the ganglia. The vacuolated lipofuscin pigment bodies are bound by a double limiting membrane and among the vacuoles are found tubular membranous structures resembling residual mitochondrial substructures. These findings suggest a mitochondrial origin of lipofuscin, rather than a lysosomal. The intracellular pigment bodies seen in the perineuronal satellite cells of peripheral ganglia appear to be signs of removal of lipofuscin from the ganglion cells. Acknowledgements. We wish to thank Mr. J. Kirchhoff, Miss E. Heyder, Mr. W. Dresp and Mrs. M. del C. Weinrichter for the technical assistance, Mr. R. Dungan and Mrs. S. Ruelke for the photographic work. We are grateful to the DAAD and the Universitätsbund of the University of Göttingen for the financial assistance. This work was supported by the Deutsche Forschungsgemeinschaft, Grant No. G1 28, 16/17.DAAD fellow on leave from the Department of Anatomy, A.I.I.M.S., New Delhi 16, India.     

Anatomical evidence for interactions between somatostatin neurites in lamina II of the rat spinal cord.

Light microscopic analysis of adult and 10-14-day-old rat spinal cords suggested that somatostatin-immunoreactive (SOM-I) fibers apposed SOM-I cell bodies in lamina (L) II. Electron microscopic analysis of these relationships at both ages showed the presence of direct appositions between SOM-I fibers and SOM-I cells. However, synapse formation between SOM-I fibers and cells was observed only in the young rat. Similarly, synapses between SOM-I fibers and SOM negative cell bodies were only found in the young animal. Adjacent SOM-I perikarya directly contacted each other, but, again, membrane specializations were evident only in the young rat. Within L I of the adult dorsal horn, a SOM-I fiber directly apposed an unlabeled cell body. Despite analysis of serial sections through the apposition, no synaptic contacts were observed.     

Ultrastructural and immunohistochemical study of endocrine cells in the midgut of the cockroach Blaberus craniifer (Insecta,Dictyoptera)

Summary The midgut of Blaberus craniifer is principally made up of columnar epithelial cells which are derived from small regenerative cells found grouped in nidi. Between them, small sparsely granulated cells with clear cytoplasm can be observed lying on the basal lamina. Mainly based on the size, shape and texture of their secretory granules, at least ten types of such endocrine cells have been identified. Five cell types contain a uniform population of dense granules: (1) medium-sized, round to oval granules; (2) small elongated granules; (3) large irregular granules; (4) oval granules with a highly osmiophilic core; (5) oval, haloed granules. Five others are characterized by a heterogeneous population of granules: (6) small, round to oval, variably electron-dense granules; (7) oval medium-sized granules of variable electron density; (8) large irregular granules of variable electron density; (9) small dense granules and large vesicles with filamentous material; (10) small dense granules and very large pale vesicles.In addition, near the regenerative cells, large cells characterized by very large, irregular, dense granules (up to 4 m), lack contact with the lumen, and reach the basal lamina only by slender cytoplasmic processes.Several antisera raised against mammalian peptides and amine were used to reveal axonal fibers and endocrine cells. Serotonin-like immunoreactivity is localized in a profuse innervation of the muscle layers that surround the epithelium, whereas cholecystokinin and methionine-enkephalin antisera stain a more moderate number of axonal fibers. Cholecystokinin-, methionine-enkephalin-, substance P-, vasoactive intestinal peptide-, somatoliberin-, and gonadoliberin-like immunoreactivities were detected in endocrine cells of the epithelium. While most of the cells appear pyramidal, oval, fusiform or bowl-shaped, and seem to lack contact with the lumen, cells reaching it have been detected reacting with antisera to cholecystokinin, substance P, vasoactive intestinal peptide, somatoliberin and gonadoliberin.     

Zinc deficiency leads to lipofuscin accumulation in the retinal pigment epithelium of pigmented rats

Background

Age-related macular degeneration (AMD) is associated with lipofuscin accumulation whereas the content of melanosomes decreases. Melanosomes are the main storage of zinc in the pigmented tissues. Since the elderly population, as the most affected group for AMD, is prone to zinc deficit, we investigated the chemical and ultrastructural effects of zinc deficiency in pigmented rat eyes after a six-month zinc penury diet.

Methodology/Principal Findings

Adult Long Evans (LE) rats were investigated. The control animals were fed with a normal alimentation whereas the zinc-deficiency rats (ZD-LE) were fed with a zinc deficient diet for six months. Quantitative Energy Dispersive X-ray (EDX) microanalysis yielded the zinc mole fractions of melanosomes in the retinal pigment epithelium (RPE). The lateral resolution of the analysis was 100 nm. The zinc mole fractions of melanosomes were significantly smaller in the RPE of ZD-LE rats as compared to the LE control rats. Light, fluorescence and electron microscopy, as well as immunohistochemistry were performed. The numbers of lipofuscin granules in the RPE and of infiltrated cells (Ø>3 µm) found in the choroid were quantified. The number of lipofuscin granules significantly increased in ZD-LE as compared to control rats. Infiltrated cells bigger than 3 µm were only detected in the choroid of ZD-LE animals. Moreover, the thickness of the Bruch''s membrane of ZD-LE rats varied between 0.4–3 µm and thin, rangy ED1 positive macrophages were found attached at these sites of Bruch''s membrane or even inside it.

Conclusions/Significance

In pigmented rats, zinc deficiency yielded an accumulation of lipofuscin in the RPE and of large pigmented macrophages in the choroids as well as the appearance of thin, rangy macrophages at Bruch''s membrane. Moreover, we showed that a zinc diet reduced the zinc mole fraction of melanosomes in the RPE and modulated the thickness of the Bruch''s membrane.     

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

The catecholaminergic innervation of the hypothalamic paraventricular nucleus (PVN) of the rat was studied 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-beta-hydroxylase (DBH)-immunopositive axons were most numerous in the periventricular zone and the medial parvocellular subnucleus of PVN. Labeled terminal boutons 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.     

Non-peptidergic primary afferents are presynaptic to neurokinin-1 receptor immunoreactive lamina I projection neurons in rat spinal cord

ABSTRACT: BACKGROUND: Pain-related (nociceptive) information is carried from the periphery to the dorsal horn of the spinal cord mostly by two populations of small diameter primary afferents, the peptidergic and the non-peptidergic. The peptidergic population expresses neuropeptides, such as substance P and calcitonin gene-related peptide, while the non-peptidergic fibers are devoid of neuropeptides, express the purinergic receptor P2X3, and bind the isolectin B4 (IB4). Although it has been known for some time that in rat the peptidergic afferents terminate mostly in lamina I and outer lamina II and non-peptidergic afferents in inner lamina II, the extent of the termination of the latter population in lamina I was never investigated as it was considered as very minor. Because our preliminary evidence suggested otherwise, we decided to re-examine the termination of non-peptidergic afferents in lamina I, in particular with regards to their innervation of projection neurons expressing substance P receptors (NK-1r). We used retrograde labeling of neurons from the parabrachial nucleus combined with lectin IB4 binding and immunocytochemistry. Samples were examined by confocal and electron microscopy. RESULTS: By confocal microscopy, we studied the termination of non-peptidergic afferents in lamina I using IB4 binding and P2X3 immunoreactivity as markers, in relation to CGRP immunoreactivy, a marker of peptidergic afferents. The number of IB4 or P2X3-labeled fibers in lamina I was higher than previously thought, although they were less abundant than CGRP-labeled afferents. There were very few fibers double-labeled for CGRP and either P2X3 or IB4. We found a considerable number of IB4-positive fiber varicosities in close apposition to NK-1r-positive lamina I projection neurons, which were distinct from peptidergic varicosities. Furthermore, we confirmed at the ultrastructural level that there were bona fide synapses between P2X3-immunoreactive non-peptidergic boutons and neurokinin-1 receptor-positive lamina I dendrites. CONCLUSIONS: These results indicate the presence of direct innervation by non-peptidergic nociceptive afferents of lamina I projection neurons expressing NK-1r. Further investigations are needed to better understand the role of these connections in physiological conditions and chronic pain states.