Localization of preganglionic neurons of the accessory ciliary ganglion in the midbrain: HRP and WGA-HRP studies in the cat

Localization of preganglionic neurons of the accessory ciliary ganglion (ACG), including ectopic intraocular ganglion cells, was investigated in the cat with the aid of horseradish peroxidase (HRP) and HRP-conjugated wheat germ agglutinin (WGA-HRP) methods. When HRP or WGA-HRP was injected into the anterior and posterior chambers of the eye, no retrogradely labeled cells were found in the visceral oculomotor nuclei, although most neurons of the ACG and the main ciliary ganglion (CG) were intensely labeled. When a microsyringe needle was inserted into the ciliary body, the tracer diffused into the suprachoroid lamina and the intraocular ganglion cells, and a small number of labeled neurons appeared in the midplane between each side of the somatic oculomotor nuclei. After injection into the ACG, many labeled neurons were observed in the anteromedian nucleus, Edinger-Westphal nucleus, and midplane between the somatic oculomotor nuclei, their ventral continuations of the ventral tegmental area, and the periaqueductal gray. HRP/WGA-HRP injection into the CG labeled cells in all these areas and in the lateral border zones of the anteromedian, Edinger-Westphal and somatic oculomotor nuclei, and their ventral continuations of the ventral tegmental area. These findings indicate that the visceral oculomotor neurons which project to the ACG tend to be located more medially than those to the CG.     

Visual acuity losses in pigeons with lesions of the nucleus of Edinger-Westphal that disrupt the adaptive regulation of choroidal blood flow

Choroidal blood flow (ChBF) in birds is regulated by a neural circuit whose components are the retina, the suprachiasmatic nucleus, the medial division of the Edinger-Westphal nucleus (EWM), the ciliary ganglion, and the choriod. We have previously shown that lesions of EWM appear to result in pathological alterations in the retina. To determine whether EWM lesions also lead to altered visual functions, we have examined the effects of EWM lesions on visual acuity in pigeons. Bilateral lesions of EWM were made electrolytically, and visual acuity for high-contrast, square-wave gratings was determined behaviorally about 1 year later and compared to that of a group of pigeons that had received sham lesions of EW about 1 year prior to acuity testing. Because lesions targeting EWM invariably resulted in damage to the adjoining lateral part of the Edinger-Westphal nucleus (EWL), which controls pupillary constriction and accommodation, two additional control groups were studied. In one such control group, bilateral lesions in the area pretectalis (AP), which innervates the pupillary control part of EWL and thereby controls pupillary constriction, were made and the effects on visual acuity determined about 1 year later. In the second such control group, the effects of acute accommodative and pupillary dysfunction on acuity were studied in pigeons made cycloplegic. The accuracy of all lesions was later confirmed histologically. The mean acuities of birds with AP lesions (9.1+/-1.4 cycles/deg) and sham lesions (7.1+/-1.5 cycles/deg) were not significantly different from normal, based on published normative data on pigeons. In contrast, pigeons with lesions that completely destroyed EW bilaterally showed visual acuity (2.7+/-0.1 cycles/deg) that was well below the acuity of the sham and AP-lesion control groups. The acuity of the cycloplegic pigeons (4.8+/-0.3 cycles/deg) and one pigeon with a nearly complete bilateral EWL but a unilateral EWM lesion (6.4 cycles/deg) indicated that only about half of the loss with a bilateral EW lesion could be attributed to accommodative dysfunction. Thus, bilateral destruction of EWM appears to have led to a loss in visual acuity. This conclusion suggests that disruption of adaptive neural regulation of ChBF may impair visual function. Destruction of EWM was, however, associated with damage to the somatic components of the oculomotor and trochlear nuclei. The possibility cannot be excluded that such damage also contributed to the acuity loss.     

Alpha and beta subunits of CaM-kinase II are localized in different neurons in chick ciliary ganglion

The ciliary ganglion of the chicken contains only two types of neurons. Using monoclonal antibodies against the alpha and the beta subunits of Ca2+/calmodulin-stimulated protein kinase II (CaMPK-II) we found that the alpha-subunit was localized to the choroid neurons while beta subunit was associated with the ciliary neurons. As both neurons receive their inputs from the oculomotor nerve, while their postganglionic axons leave via different nerves, the ciliary ganglion of the chicken is a neuronal system in which the functional differences between alpha and beta CaMPK-II homopolymers in the regulation of synaptic transmission can be investigated.     

A phosphorylation site mutant of Schizosaccharomyces pombe cdc2p fails to promote the metaphase to anaphase transition

The mammalian cornea receives a dense sensory innervation and a modest sympathetic innervation. The purpose of the current study was to determine if the rat cornea is also innervated by parasympathetic nerves. In the first set of experiments, unilateral combined sympathetic and sensory ocular denervations were performed in adult rats by surgical removal of the superior cervical ganglion and intracranial transection of the trigeminal ophthalmomaxillary nerve. Completeness of the denervation procedure was verified postmortem by a variety of macroscopic and immunohistochemical methods. Five to twelve days later, the corneas were serially sectioned tangential to the ocular surface and processed immunohistochemically with antibodies against the pan-neuronal markers, protein gene product 9.5 (PGP-9.5) and peripherin. In every animal a small, but constant, population of corneal and limbal immunoreactive fibers were unaffected by the surgical denervations and were concluded to derive from parasympathetic ganglia. In the second set of experiments, the origins of the rat corneal innervation were determined by applying the neuroanatomical tracer, wheat germ agglutinin-horseradish peroxidase (WGA-HRP) to the central cornea. Two to four days later, the trigeminal, superior cervical, ciliary, accessory ciliary and pterygopalatine ganglia were sectioned and analysed for the presence of HRP-labeled neurons. Examination of the corneal application site and associated ocular tissues revealed no evidence of tracer spread into neighbouring structures. Small numbers (0-6 per animal) of HRP-labeled neurons were observed in the ipsilateral ciliary and accessory ciliary ganglia of most animals. The results of these carefully controlled studies provide strong anatomical evidence of a modest parasympathetic innervation of the rat cornea.     

Projections of the nucleus of the basal optic root in pigeons (Columba livia) revealed with biotinylated dextran amine

The nucleus of the basal optic root (nBOR) of the accessory optic system is known to be involved in the analysis of the visual consequences of self-motion. Previous studies have shown that the nBOR in pigeons projects bilaterally to the vestibulocerebellum, the inferior olive, the interstitial nucleus of Cajal, and the oculomotor complex and projects unilaterally to the ipsilateral pretectal nucleus lentiformis mesencephali and the contralateral nBOR. By using the anterograde tracer biotinylated dextran amine, we confirmed these projections and found (previously unreported) projections to the nucleus Darkshewitsch, the nucleus ruber, the mesencephalic reticular formation, and the area ventralis of Tsai as well as ipsilateral projections to the central gray, the pontine nuclei, the cerebellar nuclei, the vestibular nuclei, the processus cerebellovestibularis, and the dorsolateral thalamus. In addition to previous studies, which showed a projection to the dorsomedial subdivision of the contralateral oculomotor complex, we found terminal labelling in the ventral and dorsolateral subdivisions. Individual fibers were reconstructed from serial sections, and collaterals to various nuclei were demonstrated. For example, collaterals of fibers projecting to the vestibulocerebellum terminated in the vestibular or cerebellar nuclei; collaterals of fibers to the inferior olive terminated in the pontine nuclei; many individual neurons projected to the interstitial nucleus of Cajal, the nucleus Darkshewitsch, and the central gray and also projected to the nucleus ruber and the mesencephalic reticular formation; collaterals of fibers to the contralateral nucleus of the basal optic root terminated in the mesencephalic reticular formation and/or the area ventralis of Tsai; neurons projecting to the nucleus lentiformis mesencephali also terminated in the dorsolateral thalamus. The consequences of these data for understanding the visual control of eye movements, neck movements, posture, locomotion, and visual perception are discussed.     

Secondary vestibulo-oculomotor projections in larval sea lamprey: anterior octavomotor nucleus

The ventral octavolateral area of lampreys contains three nuclei: the anterior, intermediate and posterior octavomotor nuclei, formed of large neurons that are contacted by thick primary vestibular fibres. We used horseradish peroxidase (HRP) or fluorescein-dextran-amine (FDA) labelling to study the projections of the anterior octavomotor nucleus (AON) in the larval sea lamprey, Petromyzon marinus. The tracers were injected either in the AON, the oculomotor nucleus or the rostralmost spinal cord. HRP injection in the AON labelled thick axons that coursed to the basal mesencephalic tegmentum, where most decussate and project to the oculomotor nucleus and the third Müller cell. Electron microscopy confirmed that AON axons contact with the contralateral third Müller cell and with oculomotor neurons. Some AON axons run in the mesencephalic tegmentum and the ventral diencephalon. An AON axon was observed to run close to the axon of the contralateral third Müller cell, establishing what appeared to be en passant contacts. HRP injection in the AON also revealed commissural fibres projecting to the contralateral octavolateral area. HRP or FDA injections in the oculomotor nucleus labelled both large and small neurons of the AON, mostly contralateral to the injection site, as well as of cells in the intermediate octavomotor nucleus, mainly ipsilateral. HRP injection in the AON or in the rostral spinal cord did not reveal any projections from the AON to the spinal cord. Our results indicate that the pattern of octavo-oculomotor connections in the lamprey is different from that observed in other vertebrates.     

Oculomotor nuclear pathology in amyotrophic lateral sclerosis

We examined the oculomotor and/or trochlear nuclei of 27 amyotrophic lateral sclerosis (ALS) patients and 10 controls by histological and immunohistological methods. Their neurons were relatively well preserved. In 7 of 22 sporadic ALS patients (including 3/3 ALS with ophthalmoplegia) and in 4 of 5 ALS patients with dementia, some morphological changes similar to those in anterior horns (Bunina bodies, ubiquitin-positive skein-like inclusions, Lewy body-like inclusions, conglomerate inclusions and spheroids) were rarely, but clearly seen. These changes were not observed in controls. Our results suggest that the oculomotor and trochlear nuclei in ALS patients are slightly affected in a manner similar to that in the anterior horns, but the degree is less than that necessary for development of ophthalmoplegia in the majority of ALS patients.     

Nonuniformity in the linear network model of the oculomotor integrator produces approximately fractional-order dynamics and more realistic neuron behavior

The oculomotor integrator is a network that is composed of neurons in the medial vestibular nuclei and nuclei prepositus hypoglossi in the brainstem. Those neurons act approximately as fractional integrators of various orders, converting eye velocity commands into signals that are intermediate between velocity and position. The oculomotor integrator has been modeled as a network of linear neural elements, the time constants of which are lengthened by positive feedback through reciprocal inhibition. In this model, in which each neuron reciprocally inhibits its neighbors with the same Gaussian profile, all model neurons behave as identical, first-order, low-pass filters with dynamics that do not match the variable, approximately fractional-order dynamics of the neurons that compose the actual oculomotor integrator. Fractional-order integrators can be approximated by weighted sums of first-order, low-pass filters with diverse, broadly distributed time constants. Dynamic systems analysis reveals that the model integrator indeed has many broadly distributed time constants. However, only one time constant is expressed in the model due to the uniformity of its network connections. If the model network is made nonuniform by removing the reciprocal connections to and from a small number of neurons, then many more time constants are expressed. The dynamics of the neurons in the nonuniform network model are variable, approximately fractional-order, and resemble those of the neurons that compose the actual oculomotor integrator. Completely removing the connections to and from a neuron is equivalent to eliminating it, an operation done previously to demonstrate the robustness of the integrator network model. Ironically, the resulting nonuniform network model, previously supposed to represent a pathological integrator, may in fact represent a healthy integrator containing neurons with realistically variable, approximately fractional-order dynamics.     

Intracellular insulin binding in Tetrahymena pyriformis

Insulin, a classic vertebrate hormone, produces alterations in cellular metabolism and growth in the ciliate Tetrahymena pyriformis, as well as an increase in insulin binding upon subsequent exposure, a phenomenon known as hormonal imprinting. An antibody to a peptide corresponding to the alpha-subunit of the human insulin receptor (amino acid residues 657-670) was used to investigate the location and to partially characterize immunoreactive proteins in insulin-exposed and non-insulin-exposed cells (control). Confocal microscopy revealed immunofluorescent labeling of cilia, nuclei, vesicles and an oblong structure of unknown nature. Labeling of nuclei, mitochondria and ciliary microtubules was seen with immunoelectron microscopy. Labeling was absent on the cell and ciliary membranes by immunoelectron microscopy. Polyacrylamide gel electrophoresis revealed several differences in protein composition between control and insulin-exposed ciliary membrane extracts, especially in the 30-50 kDa range. Immunoblotting revealed 2 reactive proteins in whole cell lysates but none were detected in ciliary membrane extracts or wheat germ agglutinin affinity column eluates of T. pyriformis whole cell preparations. Based on these findings it is unlikely that a cell surface structure similar to a mammalian insulin receptor exists in T. pyriformis.     

Renin-angiotensin system in an infant with malignant renovascular hypertension

The clinical and electromyographic signs of the misdirection syndrome after oculomotor palsy are described. Besides the previously described findings we found an increase of intraocular pressure dependent on the direction of gaze. In three young patients (16-26 years), we were able to demonstrate an aberrant reinnervation of the ciliary muscle, which was responsible for an increasing myopia in adduction. These observations supplement the previous concept of the misdirection syndrome.     

Neuroendocrine and autonomous mechanisms underlying thermoregulation in cold environment

This review focuses on the central regulation of thermoregulatory responses with special attention to the participation of thyrotropin-releasing hormone (TRH) in both autonomous and endocrine responses to a cold environment. Besides a direct projection of TRH neurons from paraventricular nuclei (PVN) to the median eminence, and the subsequent activation of the thyroid axis, there are direct projections from the PVN to the autonomic preganglionic neurons controlling autonomous responses. There projections convey information to peripheral targets involved in thermogenesis through the dorsal vagal complex and the spinal cord, for parasympathetic and sympathetic neurotransmissions respectively. Furthermore, cold exposure increases TRH mRNA levels in the PVN but also in dorsal motor and caudal raphe nuclei, thus providing strong evidence for a functional link between autonomous and neuroendocrine systems involved in thermoregulation. The review also focuses on neuroendocrine regulation of cold-induced TRH/TSH release associated with modifications in somatostatin release, with special reference to the participation of several central neurotransmitters (catecholamines, serotonin or GABA) or the influence of sex steroids.     

Second-order vestibular neuron morphology of the extra-MLF anterior canal pathway in the cat

Second-order vestibular neurons form the central links of the vestibulo-oculomotor three-neuron arcs that mediate compensatory eye movements. Most of the axons that provide for vertical vestibulo-ocular reflexes ascend in the medial longitudinal fasciculus (MLF) toward target neurons in the oculomotor and trochlear nuclei. We have now determined the morphology of individual excitatory second-order neurons of the anterior semicircular canal system that course outside the MLF to the oculomotor nucleus. The data were obtained by the intracellular horseradish peroxidase method. Cell somata of the extra-MLF anterior canal neurons were located in the superior vestibular nucleus. The main axon ascended through the deep reticular formation beneath the brachium conjunctivum to the rostral extent of the nucleus reticularis tegmenti pontis, where it crossed the midline. The main axon continued its trajectory to the caudal edge of the red nucleus from where it coursed back toward the oculomotor nucleus. Within the oculomotor nucleus, collaterals reached superior rectus and inferior oblique motoneurons. Some axon branches recrossed the midline within the oculomotor nucleus and reached the superior rectus motoneuron subdivision on that side. Since these neurons did not give off a collateral toward the spinal cord, they were classified as being of the vestibulo-oculomotor type and are thought to be involved exclusively in eye movement control. The signal content and spatial tuning characteristics of this anterior canal vestibulo-oculomotor neuron class remain to be determined.     

Inhibition effect of shengma-gegen-tang on measles virus in Vero cells and human peripheral blood mononuclear cells

The nucleotide sequence of gene 6 encoding the rotavirus major capsid protein VP6 of EDIM strain (EW) was determined and compared to that of 20 previously reported strains with known subgroup specificities. Multiple alignments of amino acid sequences exhibited a high level of sequence conservation (87 to 99.2%). Site-specific mutagenesis experiments were undertaken to localize regions involved in subgroup specificity. Amino acid positions 305, 315, and a region 296-299 (or 301 for equine strain H-2) were identified as contributing to subgroup epitopes. A single amino acid mutation at position 305 or 315 was sufficient to change the subgroup specificity of EW VP6 protein from non I/II to subgroup I- or subgroup II-like, respectively. Mutation at these sites may be another important mechanism for subgroup variation, along with gene reassortment.     

A nuclear membrane-associated DNA complex in cultured mammalian cells capable of synthesizing DNA in vitro

A DNA-nuclear membrane complex has been isolated by two different methods from the nuclei of cultured mouse fibroblast (3T3) cells. One method, utilizing the detergent sarkosyl (sodium lauroyl sarkosinate), yields a DNA-nuclear membrane complex (the M band), which contains virtually all of the DNA in the nuclei. However, treatment of the M band by sonication, vortexing, or freeze-thaw reduces the amount of DNA in the complex by approximately 50-80%, depending upon the phase of the cell cycle from which the complex was extracted. The remaining DNA is tightly bound to the nuclear membrane and resists further shearing procedures. Over 90% of the choline-labeled phospholipid present in nuclei is also found in these sheared M bands. The percentage of DNA associated with the nuclear membrane varies during the cell cycle and correlates well with the onset, continuation, and cessation of DNA synthesis. Thus, although DNA-membrane complexes can be detected throughout the cell cycle, the percentage of DNA bound to membrane increases during late G1 and S and decreases during G2. In addition, there are distinct qualitative differences in the type of DNA present in the membrane fraction, with a more highly d(A-T) rich DNA being present in confluent (G0) cells than in cells during the S phase. This d(A-T) rich DNA may be related to the mouse satellite DNA identified by others. The M band can be separated into two DNA-nuclear membrane subfractions by centrifugation through a continuous sucrose gradient. The relative proportions of these two subfractions depend upon the percentage of sarkosyl present in the M band prior to centrifugation, with complete removal of sarkosyl resulting in a very large increase in the sedimentation velocity of the complex and in the formation of only one fraction. Evidence that this is a complex of DNA with membrane is given by the finding that DNA is dissociated from the complex with Pronase, deoxycholate, or high levels of sarkosyl. Removal of virtually all of the DNA with DNase from this rapidly sedimenting complex does not dissociate any of the phospholipid which still sediments rapidly as a single band. A second method, which yields a DNA-membrane fraction from nuclei, utilizes sedimentation of lysed nuclei to equilibrium in CsCl density gradients. This low-density CsCl fraction contains only 10-15% of the total DNA, but contains most of the nascent DNA, which may be chased into a membrane-free fraction. The DNA-membrane fraction from CsCl gradients possesses properties in common with the M-band fraction and can be converted into an M band. DNA membrane complexes from sucrose gradients, as well as the crude M-band preparation and a non-membrane-associated DNA fraction from nuclei can synthesize DNA in vitro without the addition of an external DNA template or DNA polymerase. In contrast to the activity in the non-membrane-associated DNA fraction, the membrane-associated polymerase activity is strongly stimulated by adenosine triphosphate and is unaffected by ethidium bromide...     

A release mechanism for stored ATP in ocular ciliary epithelial cells

Purines can modify ciliary epithelial secretion of aqueous humor into the eye. The source of the purinergic agonists acting in the ciliary epithelium, as in many epithelial tissues, is unknown. We found that the fluorescent ATP marker quinacrine stained rabbit and bovine ciliary epithelia but not the nerve fibers in the ciliary bodies. Cultured bovine pigmented and nonpigmented ciliary epithelial cells also stained intensely when incubated with quinacrine. Hypotonic stimulation of cultured epithelial cells increased the extracellular ATP concentration by 3-fold; this measurement underestimates actual release as the cells also displayed ecto-ATPase activity. The hypotonically triggered increase in ATP was inhibited by the Cl--channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) in both cell types. In contrast, the P-glycoprotein inhibitors tamoxifen and verapamil and the cystic fibrosis transmembrane conductance regulator (CFTR) blockers glybenclamide and diphenylamine-2-carboxylate did not affect ATP release from either cell type. This pharmacological profile suggests that ATP release is not restricted to P-glycoprotein or the cystic fibrosis transmembrane conductance regulator, but can proceed through a route sensitive to NPPB. ATP release also was triggered by ionomycin through a different NPPB-insensitive mechanism, inhibitable by the calcium/calmodulin-activated kinase II inhibitor KN-62. Thus, both layers of the ciliary epithelium store and release ATP, and purines likely modulate aqueous humor flow by paracrine and/or autocrine mechanisms within the two cell layers of this epithelium.     

Differential c-fos expression in the nucleus of the solitary tract and spinal cord following noxious gastric distention in the rat

c-Fos has been used as a marker for activity in the spinal cord following noxious somatic or visceral stimulation. Although the viscera receive dual afferent innervation, distention of hollow organs (i.e. esophagus, stomach, descending colon and rectum) induces significantly more c-Fos in second order neurons in the nucleus of the solitary tract and lumbosacral spinal cord, which receive parasympathetic afferent input (vagus, pelvic nerves), than the thoracolumbar spinal cord, which receives sympathetic afferent input (splanchnic nerves). The purpose of this study was to determine the contribution of sympathetic and parasympathetic afferent input to c-Fos expression in the nucleus of the solitary tract and spinal cord, and the influence of supraspinal pathways on Fos induction in the thoracolumbar spinal cord. Noxious gastric distention to 80 mmHg (gastric distension/80) was produced by repetitive inflation of a chronically implanted gastric balloon. Gastric distension/80 induced c-Fos throughout the nucleus of the solitary tract, with the densest labeling observed within 300 microns of the rostral pole of the area postrema. This area was analysed quantitatively following several manipulations. Gastric distension/80 induced a mean of 724 c-Fos-immunoreactive nuclei per section. Following subdiaphragmatic vagotomy plus distention (vagotomy/80), the induction of c-Fos-immunoreactive nuclei was reduced to 293 per section, while spinal transection at T2 plus distention (spinal transection/80) induced a mean of 581 nuclei per nucleus of the solitary tract section. Gastric distension/80 and vagotomy/80 induced minimal c-Fos in the T8-T10 spinal cord (50 nuclei/section), but spinal transection/80 induced 200 nuclei per section. Repetitive bolus injections of norepinephrine produced transient pressor responses mimicking the pressor response produced by gastric distension/80. This manipulation induced minimal c-Fos in the nucleus of the solitary tract and none in the spinal cord. It is concluded that noxious visceral input via parasympathetic vagal afferents, and to a lesser extent sympathetic afferents and the spinosolitary tract, contribute to gastric distention-induced c-Fos in the nucleus of the solitary tract. The induction of c-Fos in the nucleus of the solitary tract is significantly greater than in the viscerotopic segments of the spinal cord, which is partially under tonic descending inhibition, but is not subject to modulation by vagal gastric afferents. Distention pressures produced by noxious gastric distention are much greater than those produced during feeding, suggesting that c-Fos induction in the nucleus of the solitary tract to noxious distention is not associated with physiological mechanisms of feeding and satiety. The large vagal nerve-mediated induction of c-Fos in the nucleus of the solitary tract following gastric distension suggests that parasympathetic afferents contribute to the processing of noxious visceral stimuli, perhaps by contributing to the affective-emotional component of visceral pain.     

Primitive neuroectodermal tumors of bone and soft tissue: histological subclassification and clinicopathologic correlations

Recent reports of Ewing's sarcoma (EW) and extraskeletal Ewing's sarcoma (EEW) support the hypothesis that these tumors are neuroectodermal in origin. Primitive neuroectodermal tumors (PNET) of bone (32 cases) and soft tissue (25 cases) including those previously categorized as EW in 27 cases and EEW in 15 cases were carefully studied histologically, immunocytochemically and morphometrically, focusing on tumor cell differentiation. This study attempts to subclassify these tumors on the basis of the size of tumor cells and nuclei, their variations (uniformity or diversity), arrangement of tumor cells (rosette or non-rosette), focal differentiation to larger ganglion-like cells, and staining intensity for neural markers. All tumors were histologically subclassified as small, medium or large cell types, three basic subtypes (rosette type, abortive rosette type, non-rosette type) and four complementary subtypes (fibrillary type, non-fibrillary type, angiomatoid type, ganglion cell type). Classic EW or EEW is consistent with small or medium, non-rosette, non-fibrillary type tumors, previously described large cell EW with large, non-rosette, fibrillary or non-fibrillary type tumors, and classic neuroectodermal tumor with small or medium, rosette, fibrillary type tumors, according to the present subclassification. Clinicopathologic correlations with the different subtypes are discussed. Long-term survival, more than 5 years, was seen in patients with small cell type, and those younger than 14 years of age.     

Reduced levels of acetylcholine receptor expression in chick ciliary ganglion neurons developing in the absence of innervation

Chick ciliary ganglion neurons receive innervation from a single source, the accessory oculomotor nucleus (AON), and nicotinic ACh receptors (AChRs) mediate chemical synaptic transmission through the ganglion. Previous experiments examining the developmental expression of AChRs in embryonic chick ciliary ganglion neurons in situ have shown that AChR levels increase substantially in the neurons at the time of innervation. Prior to synapse formation, few AChRs are detected in the neurons. In the present experiments, the role of presynaptic inputs in inducing an increase in AChRs was established by examining AChR levels in ciliary ganglion neurons that have been deprived of innervation by surgical ablation of the AON prior to synapse formation. AChR levels were dramatically reduced in neurons of input-deprived ganglia as compared to control innervated neurons at all developmental stages examined from embryonic day (ED) 5 to ED 12 as determined by indirect immunocytochemical labeling of frozen ganglion sections with the anti-AChR monoclonal antibody mAb 35, and light microscopy. In contrast, neuronal somata of input-deprived and control ganglia had equivalent levels of immunolabeling for three other components, a transmembrane glycoprotein of synaptic vesicles, SV2, and two microtubule-associated proteins, MAP 1B and MAP 2, from ED 5 up to ED 10. The results demonstrate that presynaptic inputs specifically increase the levels of AChR expression in developing neurons. In addition, changes in the levels of immunolabeling for AChRs, SV2, MAP 1B, and MAP 2 in neuronal somata after ED 10 demonstrate that other major developmental events also influence the levels of these components in neurons. Declines in the intensity of AChR, SV2, MAP 1B, and MAP 2 immunolabeling within a subset of neuronal somata in both operated and control ganglia at ED 10 and 12 coincide with the period of neuronal cell death. Increases in AChR labeling in the rest of the neuronal population of input-deprived ganglia at ED 12 suggest that, in addition to innervation, synapse formation with the peripheral target tissue influences AChR levels in developing neurons in situ.     

Video-microscopy for analysis of molecular dynamics in cells

Real-time analysis of molecular dynamics in living cells was studied by developed video-microscopes. Two new detective methods were reported, one is for analysis of ciliary movement and the other is the qualitative analysis of exocytosis of insulin-containing granules with a video-enhanced light/fluorescent microscope. For analysis of ciliary movement, glass beads were migrated in the flow. The migration speed parallel to the flow produced by ciliary beating was used as an index of the beating activity. When tracheal epithelium isolated from mouse was incubated with ambroxol, and expectorant known to activate ciliary beat frequency, the floating speeds of glass beads were changed with 1 min of incubation. The results suggest that the present method is useful not only for screening of expectorants but also for the study of molecular mechanisms underlying ciliary beat of tracheal epithelium. Visualization of the moment of the release of contents from insulin-containing granules was achieved using video-enhanced fluorescent microscopy in MIN6 cells of mouse insulinoma cell line. A fluorescent amino acridine dye, quinacrine, was found to be incorporated into low-pH secretory granules, including insulin, in the cells. The granules which incorporated quinacrine emitted a slightly blue-green fluorescence. Upon stimulation with glucose, release of the quinacrine fluorescence from granules were observed. The present method would be useful for quantitative analysis of secretion of insulin from MIN6 cells as well as pancreatic beta-cells.