Adrenergic innervation of corticotropin releasing factor (CRF)-synthesizing neurons in the hypothalamic paraventricular nucleus of the rat. A combined light and electron microscopic immunocytochemical study

Corticotropin releasing factor (CRF), a neuropeptide synthesized in the parvocellular subnuclei of the hypothalamic paraventricular nucleus (PVN), takes part in the regulation of different stress evoked responses of the organism. In order to elucidate the role of the central adrenergic system in the regulation of these CRF-synthesizing neurons, a novel ultrastructural immunocytochemical dual localization technique was utilized. Phenylethanolamine-N-methyltransferase (PNMT), a specific enzyme marker for the central adrenaline system, and CRF-immunoreactive elements were simultaneously visualized in hypothalamic sections. PNMT-immunoreactive axon terminals established synaptic connections with somata, dendrites and spinous structures of CRF-producing neurons. This morphological finding indicates that the central adrenergic system directly influences CRF-synthesizing neurons in the PVN and provides basis for a more definitive pharmacological manipulation of this system.     

Reciprocal connections between the red nucleus and the trigeminal nuclei: a retrograde and anterograde tracing study.

An anterograde biocytin and a retrograde WGA-colloidal gold study in the rat can provide information about reciprocal communication pathways between the red nucleus and the trigeminal sensory complex. No terminals were found within the trigeminal motor nucleus, in contrast with the facial motor nucleus. A dense terminal field was observed in the parvicellular reticular formation ventrally to the trigeminal motor nucleus. The parvicellular area may be important for the control of jaw movements by rubrotrigeminal inputs. On the other hand, the contralateral rostral parvicellular part of the red nucleus receives terminals from the same zone in the rostral part of the trigeminal sensory complex, where retrogradely labelled neurones were found after tracer injections into the red nucleus. Such relationships could be part of a control loop for somatosensory information from the orofacial area.     

HRP-labeled masticatory neurons in the rat trigeminal mesencephalic nucleus: a light and electron microscopic study

The neurons innervating the muscles of mastication were labeled retrogradely with horseradish peroxidase (HRP) which was injected into each muscle of mastication of the rats. The TMB-HRP labeled neurons were for light microscopic and DAB-HRP labeled neurons for electron microscopic study. Many HRP-labeled mesencephalic neurons were observed in the trigeminal mesencephalic nucleus (TMEN) after HRP injection in jaw-closing muscles (JCM). On the other hand, no labeled neurons were found following the application of HRP to the lateral pterygoid and the anterior belly of the digastric muscles, with the exception of a very few from the mylohyoid muscle. The latter three muscles were jaw-opening muscles (JOM). The mesencephalic neurons of each JCM in the TMEN were rather randomly distributed, although they were concentrated more in the caudal region of this nucleus. These neurons were typically unipolar, with spherical to oval perikarya. Each neuron had a single process which coursed caudolaterally to join the mesencephalic tract of the trigeminal nerve. Ultrastructurally, mesencephalic masticatory neurons had a rather regular nucleus locating either centrally or eccentrically in the perikaryon, which is rather plump. The cytoplasm was endowed with very well developed Golgi apparatus and rough endoplasmic reticulum. Neurofilaments, varying in number, intermingled mostly with the Golgi apparatus in the cytoplasm. Somatic spines were frequently observed; however, synapses abutting upon the soma were few. Macula adherens-like structures were occassionally encountered in the contact zone between two cells.     

Immunohistological detection of degenerating CRF-immunoreactive nerve fibers in the median eminence after lesion of paraventricular nucleus of the rat. A light and electron microscopic study

Corticotropin releasing factor (CRF)-immunoreactive neurons were detected in the paraventricular nuclei (PVN) of the rat brain, using both the traditional and the recently developed silver-gold intensified PAP methods at light and electron microscopic levels. The latter technique was more sensitive, compared to the classical PAP method, and proved to be highly specific at the ultrastructural level. The immunolabeled perikarya showed smooth or rough contoured fusiform or multipolar shape. Bilateral surgical destruction of PVN caused a gradual decrease in the number of CRF-immunopositive fibers of the median eminence. Following the second post-operative week, CRF-immunoreactivity practically disappeared from this area. In the case of unilateral lesion of PVN, the diminution of immunoreactivity was restricted to the ipsilateral side of the median eminence-pituitary stalk region. Applying the silver-gold intensified PAP method to electron microscopy, the detection of immuno-labeled degenerating fibers became possible, among morphologically similar, densely degenerating, but unlabeled, profiles. This study reports that CRF fibers to the capillary system of the median eminence of the rat originate principally from PVN.     

Amygdalofugal axon terminals immunoreactive for L-aspartate or L-glutamate in the nucleus accumbens of rats and domestic chickens: a comparative electron microscopic immunocytochemical study combined with anterograde pathway tracing

Several studies have shown that L-aspartate (Asp) is present in synaptic vesicles and released exocytotically from presynaptic terminals, possibly by Ca²⁺-dependent corelease of Asp and L-glutamate (Glu). It has been demonstrated that both excitatory amino acids (EAAs) are released from the rat striatum as part of corticostriatal neurotransmission. The single or colocalized occurrence of Asp and Glu in specific synaptic boutons of the chicken medial striatum/nucl. accumbens has been demonstrated by our group using ultrastructural immunocytochemistry. However, evidence for the presence of EAAs in any specific striatal pathway was only circumstantial. Here, we report on the distribution of Asp and Glu in specific synaptic terminals of the amygdalostriatal pathway, both in rat and chicken brains, combining anterograde tracing with postembedding immunogold labeling of Asp or Glu. Immunoreactivity for Asp and Glu was observed in amygdalofugal terminals with asymmetrical synaptic junctions (morphologically representing excitatory synapses) in both species. The postsynaptic targets were either dendritic spines or small dendrites, whereas axosomatic or axo-axonic connections were not observed. Ultrastructurally, the synaptic terminals immunoreactive for Asp were indistinguishable from those immunoreactive for Glu. The findigs are consistent with an Asp?CGlu corelease mechanism, with a distinct synaptic contingent, evolutionarily conserved in the amygdalostriatal pathway.     

A light and electron microscopic study of glutamate receptors in the monkey subthalamic nucleus

The distribution of glutamate receptors in the monkey subthalamic nucleus was studied using affinity purified polyclonal antibodies to GluR1, phosphorylated GluR1, GluR2/3, NMDAR1, mGluR1a and mGluR5. Intense staining for both the unphosphorylated and the phosphorylated forms of the AMPA receptor subunit GluR1 was observed in the cell bodies and proximal dendrites of neurons in this nucleus. In comparison to GluR1, less intense staining for GluR2/3 was observed in the cell bodies and processes. NMDAR1 immunoreactivity was present in cell bodies and large numbers of small diameter dendrites. Light staining was observed in cell bodies with mGluR1a and no staining was observed on cell bodies with mGluR5. The neuropil, however, contained many processes that were labeled for mGluR1a or mGluR5. Electron microscopy showed that label was present in cytoplasmic locations in cell bodies and dendrites, in addition to components of the synaptic region, in sections stained for GluR1, GluR2/3 and NMDAR1. In contrast, very lightly labeled or unlabeled cell bodies but labeled dendrites and axon terminals, was observed in sections stained for mGluR1a and mGluR5. In addition to neural processes, occasional astrocytic processes were also labeled for mGluR5. Of the immunogold particles that were associated with components of the synaptic region, label for ionotropic glutamate receptors was mostly present on postsynaptic densities, whilst that for metabotropic glutamate receptors was mostly present in a perisynaptic location. The ratio of GluR1/GluR2 messenger RNAs has been reported to increase in the aged hippocampus (PAGLIUSI, S. R., GERRARD, P., ABDALLAH, M., TALABOT, D. & CATSICAS, S. (1994) Neuroscience 61, 429–433.), and it is possible that a similar change in the ratio of GluR1 and GluR2 may occur in neurons of the subthalamic nucleus with age. It is postulated that this could result an increase in calcium permeability via AMPA receptors, and an enhancement of excitatory transmission in this nucleus.     

Chemically defined collateral projections from the pons to the central nucleus of the amygdala and hypothalamic paraventricular nucleus in the rat

Triple fluorescence labelling was employed to reveal the distribution of chemically identified neurons within the pontine laterodorsal tegmental nucleus and dorsal raphe nucleus which supply branching collateral input to the central nucleus of the amygdala and hypothalamic paraventricular nucleus. The chemical identity of neurons in the laterodorsal tegmental nucleus was revealed by immunocytochemical detection of choline-acetyltransferase or substance P; in the dorsal raphe nucleus, the chemical content of the neurons was revealed with antibody recognizing serotonin. The projections were defined by injections of two retrograde tracers, rhodamine-and fluorescein-labelled latex microspheres, in the central nucleus of the amygdala and paraventricular nucleus, respectively. Neurons projecting to both the central nucleus of the amygdala and the paraventricular nucleus were distributed primarily within the caudal extensions of the laterodorsal tegmental nucleus and dorsal raphe nucleus. Approximately 11% and 7% of the labelled cells in the laterodorsal tegmental nucleus and dorsal raphe nucleus projected via branching collaterals to the paraventricular nucleus and central nucleus of the amygdala. About half of these neurons in the laterodorsal tegmental nucleus were cholinergic, and one-third were substance-P-ergic; in the dorsal raphe nucleus, approximately half of the neurons containing both retrograde tracers were serotonergic. These results indicate that pontine neurons may simultaneously transmit signals to the central nucleus of the amygdala and paraventricular nucleus and that several different neuroactive substances are found in the neurons participating in these pathways. This coordinated signalling may lead to synchronized responses of the central nucleus of the amygdala and paraventricular nucleus for the maintenance of homeostasis. Interactions between different neuroactive substances at the target site may serve to modulate the responses of individual neurons.     

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.     

Rapid anterograde and retrograde tracing from mesenteric nerve trunks to the guinea-pig small intestine in vitro

A novel technique for rapid anterograde labelling of cut axons in vitro was used to visualise the peripheral branches of mesenteric nerve trunks supplying the guinea-pig small intestine. Biotinamide, dissolved in an artificial intracellular solution, was applied to the cut ends of the mesenteric nerves and the tissue was maintained in organ culture overnight. Labelled nerve fibres were visualised by fluorescein isothiocyanate (FITC)-conjugated streptavidin. Intense staining of nerve fibres and terminal varicosities in the ganglia and internodal strands of the myenteric plexus was achieved up to 15 mm from the application site. Filled fibres formed baskets around some myenteric nerve cell bodies, suggesting target-specific neurotransmission. When combined with multiple-labelling immunohistochemistry for tyrosine hydroxylase (TH), calcitonin gene-related protein (CGRP) or choline acetyltransferase (ChAT), most anterogradely labelled nerve fibres, and many pericellular baskets, were found to be TH immunoreactive, indicating their postganglionic sympathetic origin. Double-labelling immunohistochemistry revealed that the postganglionic sympathetic pericellular baskets preferentially surrounded 5-hydroxytryptamine (5-HT)-handling myenteric neurons. Some biotinamide-filled fibres were CGRP immunoreactive, and are likely to originate from spinal sensory neurons. We describe for the first time many pericellular baskets labelled from the mesenteric nerves which were ChAT immunoreactive. Retrogradely filled intestinofugal nerve cell bodies were also observed, all of which had a single axon arising from a small nerve cell body with short filamentous or lamellar dendrites. Many of these cells were ChAT immunoreactive. This in vitro technique is effective in identifying the fine arrangement of nerve terminals arising from nerve trunks in the periphery.     

Light- and electron microscopic localization of vasopressin or a vasopressin-like substance in the neurons of the rat suprachiasmatic nucleus

Summary In the suprachiasmatic nucleus of the rat light microscopic immunostaining for vasopressin reveals a distribution pattern of the immunoreactive material different from that known for the supraoptic nucleus. Among non-stained neurons positive-reacting perikarya display a cap- or tiplike labeling. The area of the suprachiasmatic nucleus is marked by delicate vasopressin-positive fibers. At the ultrastructural level the reaction product, after incubation with anti-vasopressin, is localized in small elementary granules unevenly distributed over the cytoplasm. Groups of axons containing specifically labeled granules contact non-reacting fibers.Supported by the Deutsche Forschungsgemeinschaft (Grant Nr. Kr. 569/2) and Stiftung Volkswagenwerk     

A combined light and electron microscopic immunocytochemical method for the simultaneous localization of multiple tissue antigens. Tyrosine hydroxylase immunoreactive innervation of corticotropin releasing factor synthesizing neurons in the paraventricular nucleus of the rat

In order to study the morphological interrelationships between immunocytochemically identified neuronal systems, a double labelling procedure - suitable for correlative light and electron microscopic observations - is introduced. The technique is based on the consecutive use of the silver-gold (SG) intensified and non-intensified forms of the oxidized 3,3'-diaminobenzidine (DAB) chromogen in the framework of the peroxidase-antiperoxidase complex (PAP) indirect immunocytochemical procedure. The first tissue antigen is detected by the SG intensified DAB chromogen, which has a black color and high electron density. The structures containing the second antigen are visualized by the non-intensified DAB-endproduct, which is less electron-dense than the silver-gold amplified form and is brown. The metallic shield that forms around the labeled antibody sequences associated with the first antigen prevents non-specific binding of immunoglobulins used for the detection of the second tissue antigen. The application of this method for the simultaneous detection of tyrosine hydroxylase (TH)- and corticotropin releasing factor (CRF)-immunoreactive structures revealed that black colored TH-immunopositive fibers contacted brown colored CRF-synthesizing neurons in the hypothalamic paraventricular nucleus. The juxtaposition of TH- and CRF-containing elements was apparent in both thick vibratome (40 micron) and semithin (1 micron) sections. At the ultrastructural level, TH-positive terminals - labeled by silver-gold grains - were observed to establish asymmetric synapses with both CRF- and TH-immunoreactive neurons. The former finding indicates a direct, TH-immunopositive, catecholaminergic influence upon the hypothalamic CRF system, while the latter demonstrates the existence of intrinsic connections between TH-positive elements.     

Development of the crayfish retina: A light and electron microscopic study

The development of the crayfish retina was examined in embryos and first, second and third instars with both and light and electron microscope. Light microscopic observations indicate that differentiation begins at the posterior portion of the optic disc and progresses in an anterior direction. Development of screening pigment, dioptric elements, and rhabdoms all parallel this posterior to anterior gradient in the retina. Tracer studies in early embryos reveal that the retina is separated from the proximal neuropil regions by a distinct vascular space. This observation suggests that the source of new cells for the retina may not be the more proximal cell proliferation zone as previously indicated. It is proposed that mitotic activity within the retina and/or differentiation of cells from the anterior surface layer of the eye may be sources for addition of new cells to the retina. Proto-ommatidial clusters of seven retinula cells occur very early at the posterior region of the embryonic retina. Initially the receptor cells extend throughout the entire thickness of the retina, but later they withdraw from beneath the cornea to occupy only the proximal portion of the retina. Microvilli of the rhabdom arise from the centrally opposed membranes of the retinula cells in each cell cluster. Each new microvillus contains a core of fine filaments which extend out into the cytoplasm at its base. As development of the microvilli continues, the core filaments appear to be lost or altered, but the cytoplasmic bundles at the base of the microvilli persist.     

The intrinsic neuronal organisation of the nucleus of the basal optic root in the domestic chicken; a light and electron microscopic study using anterograde tracers and postembedding GABA-immunostaining

The intrinsic neuronal organisation in the nucleus of the basal optic root of chickens was investigated. The divergent connections with various areas and the functional complexity of the nucleus require a complex intrinsic structural arrangement. Therefore, an analysis of Golgi impregnated material, ultrastructure, GABA-immunocytochemistry and biotinylated dextran-amine anterograde tracer analysis of the nucleus was carried out. In the Golgi analysis, a characteristic dendritic ramification pattern of two types of putative projection neurons was observed. These neurons form dendritic nests with their overlapping dendritic terminal sections, that develop synaptic fields with the optic fibre terminals. These synaptic fields were confirmed by electron microscopy. GABA-immunopositive terminals synapse with distinct loci of the dendritic trees of projection neurons; they may therefore play an important role in the inhibitory-modulatory system of the nucleus of the basal optic root. The GABA-immunopositive terminals derive from small and/or elongated local circuit neurons which receive retinal afferents, and from myelinated fibres afferents to the nucleus of unknown origin.     

Cholinesterase activity of capillaries in the rat brain. A light and electron microscopic study

Synopsis The presence of cholinesterase activity in association with capillaries of the central nervous system was investigated in the rat by means of both light and electron microscopic methods. Throughout most of the rat brain, the smaller blood vessels stain intensely for butyrylcholinesterase activity. In some areas, such as the commissural nucleus of the vagus and parts of the medial thalamus, the capillaries possess both acetylcholinesterase and butyrylcholinesterase activity. Blood vessels in those structures which lie outside the blood-brain barrier are completely devoid of cholinesterase activity. The electron microscope reveals that reaction product occurs within the matrix of the basement membrane, in the intermembranous space of the endothelial nuclear envelope and occasionally in the endothelial granular endoplasmic reticulum. It is suggested that the presence of cholinesterase within the basement membrane of brain capillaries is evidence of the role that the basement membrane may play in transfer mechanisms.