Richard M. Kriebel

Cytology of brain stem neurons projecting to the caudal neurosecretory complex: an HRP-electron microscopic study.

Brain stem projections to the neurons in the caudal neurosecretory complex (CNC) of Poecilia sphenops (molly) have been studied with HRP retrograde tracing. Using light microscopic procedures, HRP-labelled neurons were located in the reticular nucleus of the medulla (RMN) and in the vicinity of the nucleus of the medial longitudinal fascicle (NMLF). The present study was undertaken to examine the cytology of the brain stem neurons that project to the caudal neurosecretory complex using combined HRP-electron microscopic methods. Cells in the midbrain NMLF containing HRP-filled profiles were located bilaterally close to the midline and just beneath the ependyma. HRP reaction product was found additionally in the neurosecretory cells of the midbrain dorsal tegmental magnocellular nucleus (DTMN) located dorsal to the NMLF. Cells containing HRP-labelled profiles were also seen in the RMN and in neurons dorsal-lateral to the RMN. This latter group of neurons contained small dense core vesicles in addition to HRP labelled dense bodies.

Caudal neurosecretory system synaptic morphology following deafferentation: An electron microscopic degeneration study

The caudal neurosecretory system of Poecilia sphenops (molly) is an isolated population of neurosecretory cells located in the caudal most aspect of the teleost spinal cord. The structure of this neuroendocrine system is favorable for studies on the synaptic control of neurosecretory mechanisms. Little is known about the detailed synaptology of the system. Morphological and electrophysiological reports have shown that the caudal neurosecretory system is linked to higher brain centers by descending spinal projections. To examine the synaptology of the descending synaptic input, surgical deafferentation was performed by microsuction removal of a segment of spinal cord rostral to the caudal system. The degeneration of axon terminals was studied at various times following deafferentation and compared to control synaptology. Based on vesicle content and morphology, three axon terminal types were found in the caudal neurosecretory system. These terminals formed axosomatic, axodendritic, and axoaxonic synaptic contacts. Following deafferentation, axon terminals with dense-cored vesicles and boutons with round clear vesicles degenerated as evidenced by the electron dense dark reaction and the electron lucent reaction respectively. This suggested that at least two different types of axon terminals arise from the descending projection to the caudal neurosecretory system and that two different neurotransmitters may be influencing the neurosecretory activity of these cells.

Brain stem innervation of the caudal neurosecretory system

SummaryThe innervation of the caudal neurosecretory system of Poecilia sphenops (black molly) was studied by use of the retrograde horseradish peroxidase (HRP) method. The structure of the caudal neurosecretory system in this species was well suited for application of HRP procedures. Acrylamide/HRP gel implants were placed in the nucleus of the caudal neurosecretory system. Two neuronal groups which contained HRP filled cells were found in the brain stem. Bilateral projections originate from the dorsal tegmentum of the midbrain and the reticular nucleus of the medulla.

Glutaminase Immunoreactivity and Enzyme Activity Is Increased in the Rat Dorsal Root Ganglion Following Peripheral Inflammation

Following inflammation, primary sensory neurons in the dorsal root ganglion (DRG) alter the production of several proteins. Most DRG neurons are glutamatergic, using glutaminase as the enzyme for glutamate production, but little is known about glutaminase following inflammation. In the present study, adjuvant-induced arthritis (AIA) was produced in rats with complete Freunds adjuvant into the hindpaw. At 7 days of AIA, DRG were examined with glutaminase immunohistochemistry, Western blot immunoreactivity, and enzyme activity. Image analysis revealed that glutaminase was elevated most in small-sized neurons (21%) (P < 0.05). Western blot analysis revealed a 19% increase (P < 0.05) in total glutaminase and 21% in mitochondrial glutaminase (P < 0.05). Glutaminase enzyme activity was elevated 29% (P < 0.001) from 2.20 to 2.83 moles/kg/hr. Elevated glutaminase in primary sensory neurons could lead to increased glutamate production in spinal primary afferent terminals contributing to central sensitization or in the peripheral process contributing to peripheral sensitization.

Monoamines in the caudal neurosecretory complex: Biochemistry and immunohistochemistry

Monoaminergic inputs to the caudal neurosecretory complex (CNc) of Poecilia latipinna have been identified using histofluorescence and immunohistochemical techniques. The present study was undertaken to identify specific monoamines and determine the relative contribution of indolamines and catecholamines in supraspinal and intrinsic innervation of the nucleus. The CNc was deafferented by transecting the spinal cord rostral to the CNc. Ten days subsequently, CNc of spinal-transected and control fish were processed for either biochemical or immunohistochemical analysis. Norepinephrine and serotonin were detected in pooled samples of control CNc. Following deafferentation, the content of both monoamines was diminished. Using immunohistochemical labeling for serotonin or for the catecholamine-synthesizing enzymes, tyrosine hydroxylase (TH) or dopamine-beta-hydroxylase (DBH), the number of monoamine fibers was decreased in deafferented CNc compared to control. A substantial serotonergic innervation remains after deafferentation, as evidenced by serotonin-positive neurons and heavy, varicose fibers. Occasional TH/DBH-positive cells and fibers remain after deafferentation. These data suggest that both norepinephrine and serotonin are associated with descending supraspinal projections, while serotonin predominates as the intrinsic monoamine.

Telencephalic projections to the goldfish hypothalamus: An anterograde degeneration study

In this study, large areas of goldfish telencephalon were ablated including rostral nucleus preopticus periventriculare (rNPP), and degenerating axons were traced by a modified Fink and Heimer procedure. The lesioning procedure ablated large regions of area dorsalis telencephali pars medialis, centralis, and dorsolateral complex; and completely removed area ventralis telencephali pars dorsalis, ventralis, and lateralis. In addition, the supracommissural nucleus and rNPP were lesioned specifically because both nuclei have been thought to be involved in courtship behavior and endocrine control of reproduction. This investigation demonstrated extensive fiber projections from telencephalic nuclei and/or rNPP to the hypothalamus. Lesioned telencephalon and/or rNPP projected bilaterally to nucleus preopticus and the suprachiasmatic nucleus and unilaterally to the following tuberal nuclei: nucleus anterior tuberis, and the lateral hypothalamic nucleus. A much larger fiber projection to the inferior lobe nuclei was also observed with a large contralateral as well as ipsilateral input.

Ultrastructural changes in the urophysis of Mollienesia sphenops following adaptation to seawater

SummaryThe urophysis or neurohemal contact site of the caudal neurosecretory system of Mollienesia sphenops, the black molly, was studied in animals adapted to an artificial seawater environment. This species of fish was chosen for these studies because of its known ability to osmoregulate and its adaptability to the laboratory aquarium. The urophysis of freshwater acclimated mollys contained an abundance of neurosecretory granules. However, in fish subjected to a seawater environment for one week the number of neurosecretory granules was significantly decreased. In addition, there was an increase in blood cell infiltration of the urophysis.

Terminal processes of serotonin neurons in the caudal spinal cord of the molly, Poecilia latipinna, project to the leptomeninges and urophysis.

SummaryThe caudal neurosecretory complex of poeciliids has previously been shown to be innervated by extranuclear and intrinsic serotonergic projections. In the present study, immunohistochemical techniques were used to characterize fibers originating from serotonin neurons intrinsic to the caudal spinal cord. Bipolar and multipolar neurons were oriented ventromedially, and contained numerous large granular vesicles. Three types of serotonergic fibers were distinguished based on their distribution and morphology. Intrinsic Type-A fibers branched into varicose segments near the ventrolateral surface of the spinal cord and contacted the basal lamina beneath the leptomeninges. Type-B fibers coursed longitudinally to enter the urophysis, where they diverged and terminated around fenestrated capillaries. Labelled vesicles in Type-A and Type-B terminals were the same size as those in labelled cells and in unlabelled neurosecretory terminals in the urophysis. Type-C small varicose fibers branched within the neuropil of the caudal neurosecretory complex. Serotonin may be secreted into the submeningeal cerebrospinal fluid, the urophysis, and the caudal vein by Type-A and Type-B fibers, whereas, Type-C fibers may be processes of serotonergic interneurons in the neuroendocrine nucleus. The possibility that urotensins I and II or arginine vasotocin were colocalized in the processes of the intrinsic serotonin neurons was investigated immunohistochemically. The negative results of these experiments suggest that serotonin-containing neurons may represent a neurochemically distinct subpopulation in the caudal neurosecretory complex.

Brainstem location of serotonin neurons projecting to the caudal neurosecretory complex

Serotonergic fibers in the caudal neurosecretory complex (CNc) of poeciliids originate from neurons within, and extrinsic to this spinal cord nucleus. In the present study, retrograde tracing and immunofluorescence techniques were combined to localize extrinsic serotonergic projection neurons. The entire spinal cord and brain were sectioned after Fast Blue (FB) or horseradish peroxidase (HRP) was implanted in the CNc. No HRP or FB filled neurons were found in the spinal cord. Retrogradely filled neurons were found bilaterally in dorsolateral and ventromedial reticular nuclei, and the dorsal midbrain tegmentum. Fusiform cells in the medullary fasciculus longitudinalis medialis filled with FB but not HRP. Serotonin immunopositive neurons were found surrounding the third ventricle, in the raphe and in medullary reticular nuclei. Double labelled neurons in the medial reticular nucleus were determined to be the source of serotonergic projections to the CNc. Reticular projection nuclei are strategically situated to receive visceral sensory input from rhombencephalic cranial nerves. These putative pathways may provide an anatomical substrate by which visceral sensory information is transmitted to the CNc.

Biogenic amine localization in cardiac ganglion intrinsic neurons: Electron microscopic histochemistry of SIF cells

The parasympathetic cardiac ganglion in the mudpuppy, N. maculosus, contains postganglionic nerve cells and intrinsic neurons, many of which are small intensely fluorescent (SIF) cells. Several bioactive substances have been localized in the intrinsic nerve cells which may have integrative effects at synapses within the ganglion. Ganglionic intrinsic neurons can be identified electron microscopically by the presence of numerous cytoplasmic granular vesicles 80-120 nm in diameter. Throughout the ganglion there are bundles of unmyelinated fibers some of which are filled with granular and agranular vesicles and axosomatic terminals with similar vesicles synapsing on principal parasympathetic nerve cells. To understand the aminergic contribution to ganglionic synaptic circuitry the chromaffin reaction was used. The intrinsic neurons (i.e., SIF cells) were readily identified by their characteristic intracellular granule population. All intrinsic nerve cells identified showed granules which were positively labelled by the chromaffin reaction. Granular vesicles in synaptic profiles on principal cells (P cells) were also labelled indicating a direct aminergic synaptic innervation to these cells. The cell bodies of intrinsic neurons, ensheathed with supportive glial-like cellular processes, rarely received synapses. Elemental microanalysis was used to verify the chromium content of the electron dense product within the granular vesicles. These studies demonstrated direct aminergic synaptic input to at least a subpopulation of principal parasympathetic cells in the cardiac ganglion of mudpuppy.