Effects of cervical sympathectomy on vasospasm induced by meningeal haemorrhage in rabbits

This study investigates the role of cervical sympathectomy in the prevention of acute vasospasm induced by meningeal haemorrhage in rabbits. Sixteen adult English Norfolk rabbits were divided into 2 experimental groups: bilateral cervical sympathectomy of the superior sympathetic ganglion (SSSG, n=8), and bilateral SSSG and sympathectomy of the inferior sympathetic ganglion (SISG, n=8). Other 24 animals were used as controls. Basilar artery diameter was evaluated by angiography. SSSG protected the animals against developing cerebral vasospasm; SSSG associated with SISG did not increase this effect.

Experimental study of progressive facial hemiatrophy: effects of cervical sympathectomy in animals

Progressive facial hemiatrophy (Romberg's syndrome) is of unknown cause and uncertain pathogenesis. The main pathogenetic hypotheses are: sympathetic system alterations, localized scleroderma, trigeminal changes, possibly of genetic origin. To test the hypothesis of sympathetic system alterations, we designed an experimental model with ablation of the superior cervical sympathetic ganglion in rabbits, cats and dogs. All the animals were operated upon when 30 days old and were examined monthly for 1 year. During this period localized alopecia, corneal ulceration, keratitis, strabismus, enophthalmos, ocular atrophy, hemifacial atrophy and slight bone atrophy on the side of the sympathectomy were observed. Thus, cervical sympathectomy reproduces in animals the principal clinical alterations of Romberg's syndrome. Our data suggest that the sympathetic system is involved in the pathogenesis of this syndrome.

Dendritic thickness: a morphometric parameter to classify mouse retinal ganglion cells

To study the dendritic morphology of retinal ganglion cells in wild-type mice we intracellularly injected these cells with Lucifer yellow in an in vitro preparation of the retina. Subsequently, quantified values of dendritic thickness, number of branching points and level of stratification of 73 Lucifer yellow-filled ganglion cells were analyzed by statistical methods, resulting in a classification into 9 groups. The variables dendritic thickness, number of branching points per cell and level of stratification were independent of each other. Number of branching points and level of stratification were independent of eccentricity, whereas dendritic thickness was positively dependent (r = 0.37) on it. The frequency distribution of dendritic thickness tended to be multimodal, indicating the presence of at least two cell populations composed of neurons with dendritic diameters either smaller or larger than 1.8 µm ("thin" or "thick" dendrites, respectively). Three cells (4.5%) were bistratified, having thick dendrites, and the others (95.5%) were monostratified. Using k-means cluster analysis, monostratified cells with either thin or thick dendrites were further subdivided according to level of stratification and number of branching points: cells with thin dendrites were divided into 2 groups with outer stratification (0-40%) and 2 groups with inner (50-100%) stratification, whereas cells with thick dendrites were divided into one group with outer and 3 groups with inner stratification. We postulate, that one group of cells with thin dendrites resembles cat ß-cells, whereas one group of cells with thick dendrites includes cells that resemble cat a-cells.

THE RETINAL TARGETS OF CENTRIFUGAL NEURONS AND THE RETINAL NEURONS PROJECTING TO THE ACCESSORY OPTIC-SYSTEM

In birds, neurons of the isthmo-optic nucleus (ION), as well as ''ectopic'' neurons, send axons to the retina, where they synapse on cells in the inner nuclear layer (INL). Previous work has shown that centrifugal axons can be divided into two anatomically distinct types depending on their mode of termination: either ''convergent'' or ''divergent'' (Ramon y Cajal, 1889; Maturana and Frenk, 1965). We show that cytochrome-oxidase histochemistry specifically labels ''convergent'' centrifugal axons and target neurons which appear to be amacrine cells, as well as three ''types'' of ganglion cells: two types found in the INL (displaced ganglion cells) and one in the ganglion cell layer. Labeled target amacrine cells have distinct darkly labeled ''nests'' of boutons enveloping the somas, are associated with labeled centrifugal fibers, and are confined to central retina. Lesions of the isthmo-optic tract abolish the cytochrome-oxidase labeling in the centrifugal axons and in the target amacrine cells but not in the ganglion cells. Cytochromeoxidase-labeled ganglion cells in the INL are large; one type is oval and similar to the classical displaced ganglion cells of Dogiel, which have been reported to receive centrifugal input; the other type is rounder. Rhodamine beads injected into the accessory optic system results in retrograde label in both types of cells, showing that two distinct types of displaced ganglion cells project to the accessory optic system in chickens. The ganglion cells in the ganglion cell layer that label for cytochrome oxidase also project to the accessory optic system. These have proximal dendrites that ramify in the outer inner plexiform layer. Neither the target amacrine cells nor either of the displaced ganglion cells are immunoreactive for the inhibitory transmitter gamma aminobutyric acid. At least some of the target amacrine cells may, however, be cholinoceptive: we found that the antibody to the alpha-7 subunit of the nicotinic ACh receptor labels a population of cells in the INL that are similar in location, size, and the presence of labeled bouton-like structures to those we find labeled with cytochrome oxidase. This antibody also labels neurons in the ION proper but not ectopic cells. In conclusion, it appears that cytochrome oxidase may be a marker for ''convergent'' centrifugal axons and at least one of their target cells in the INL.

Toxoplasmosis: morphological and morphometric evaluation of spinal cord neurons from nonsymptomatic seropositive dogs

The aim of this work was to analyze the neuron morphology and morphometry of cervical, thoracic and lumbar areas of nonsymptomatic seropositive dogs’ spinal cord for toxoplasmosis. Twenty indefinite-breed adult dogs were used; ten dogs were healthy, with negative serology for toxoplasmosis, and were used as the control group (group 1), and ten dogs were nonsymptomatic but seropositive for toxoplasmosis (group 2). After the microtomy, with interval of 100 micrometers (µm), the histological 5-µm-thick cuts were dyed by hematoxylin-eosin and Masson's trichrome techniques. The glass slides were analyzed under light microscope to examine the neuron morphology. The parameters considered for the morphometric analysis were area, perimeter, maximum diameter, minimum diameter and shape factor of cytoplasm and nucleus of neuron. The results were statistically analyzed by Student’s t test at 5% probability level. The morphological characteristics between the two groups were similar and according to literature. The morphometric results showed that there were changes in neurons size and structure, and increase and loss of star shape were noticed in seropositive animals. The results suggest that the neurons of these dogs, yet nonsymptomatic, can have lost their conductor function.

Twelve chromatically opponent ganglion cell types in turtle retina

The turtle retina has been extensively used for the study of chromatic processing mechanisms. Color opponency has been previously investigated with trichromatic paradigms, but behavioral studies show that the turtle has ail ultraviolet (UV) channel and a tetrachromatic visual system. Our laboratory has been working ill the characterization of neuronal responses in the retina of vertebrates using stimuli in the UV-visible range of the electromagnetic spectrum. In the present investigation, we recorded color-opponent responses from turtle amacrine and ganglion cells to UV and visible stimuli and extended our previous results that UV color-opponency is present at the level of the inner nuclear layer. We recorded from 181 neurons, 36 of which were spectrally opponent. Among these, there were 10 amacrine (5%), and 26 ganglion cells (15%). Morphological identification of color-opponent neurons was possible for two ganglion cell classes (G17 and G22) and two amacrine cell classes (A22 and A23b). There was a variety of cell response types and a potential for complex processing of chromatic stimuli, with intensity- and wavelength-dependent response components. Ten types of color opponency were found in ganglion cells and by adding previous results from our laboratory, 12 types of opponent responses have been found. The majority of the ganglion cells were R+UVBG- and RG+UVB-color-opponents but there were other less frequent types of chromatic opponency. This study confirms the participation of a UV channel in the processing of color opponency in the turtle inner retina and shows that the turtle visual system has the retinal mechanisms to allow many possible chromatic combinations.

Morphological Homogeneity of Neurons: Searching for Outlier Neuronal Cells

We report a morphology-based approach for the automatic identification of outlier neurons, as well as its application to the NeuroMorpho.org database, with more than 5,000 neurons. Each neuron in a given analysis is represented by a feature vector composed of 20 measurements, which are then projected into a two-dimensional space by applying principal component analysis. Bivariate kernel density estimation is then used to obtain the probability distribution for the group of cells, so that the cells with highest probabilities are understood as archetypes while those with the smallest probabilities are classified as outliers. The potential of the methodology is illustrated in several cases involving uniform cell types as well as cell types for specific animal species. The results provide insights regarding the distribution of cells, yielding single and multi-variate clusters, and they suggest that outlier cells tend to be more planar and tortuous. The proposed methodology can be used in several situations involving one or more categories of cells, as well as for detection of new categories and possible artifacts.

Dynamic Range of Vertebrate Retina Ganglion Cells: Importance of Active Dendrites and Coupling by Electrical Synapses

The vertebrate retina has a very high dynamic range. This is due to the concerted action of its diverse cell types. Ganglion cells, which are the output cells of the retina, have to preserve this high dynamic range to convey it to higher brain areas. Experimental evidence shows that the firing response of ganglion cells is strongly correlated with their total dendritic area and only weakly correlated with their dendritic branching complexity. On the other hand, theoretical studies with simple neuron models claim that active and large dendritic trees enhance the dynamic range of single neurons. Theoretical models also claim that electrical coupling between ganglion cells via gap junctions enhances their collective dynamic range. In this work we use morphologically reconstructed multi-compartmental ganglion cell models to perform two studies. In the first study we investigate the relationship between single ganglion cell dynamic range and number of dendritic branches/total dendritic area for both active and passive dendrites. Our results support the claim that large and active dendrites enhance the dynamic range of a single ganglion cell and show that total dendritic area has stronger correlation with dynamic range than with number of dendritic branches. In the second study we investigate the dynamic range of a square array of ganglion cells with passive or active dendritic trees coupled with each other via dendrodendritic gap junctions. Our results suggest that electrical coupling between active dendritic trees enhances the dynamic range of the ganglion cell array in comparison with both the uncoupled case and the coupled case with cells with passive dendrites. The results from our detailed computational modeling studies suggest that the key properties of the ganglion cells that endow them with a large dynamic range are large and active dendritic trees and electrical coupling via gap junctions.

An anatomical investigation of the cervicothoracic ganglion

Anatomical variability within the autonomic nervous system has long been accepted. This study evaluated the anatomical variability of the cervicothoracic ganglion (CTG) according to its form and, in addition, provided precise measurements between the CTG and the anterior tubercle of the transverse process of the sixth cervical vertebra (C6TP), the first costovertebral articulation, and the vertebral artery. Forty-two adult cadavers were dissected, 22 male and 20 females. Five main forms of CTG were documented; spindle (31.9%), dumbbell (23.2%), truncated (21.7%), perforated (14.5%), and inverted-L (8.7%). The means for length, width, and thickness of the CTG were 18.5 mm, 8.2 mm, and 4.5 mm, respectively. The dimensions were found to be slightly larger in the males than females and on the left sides as compared to the right. The mean shortest distance between the CTGs and the vertebral artery was found to be 2.8 mm, whilst the mean shortest distances to C6TP was 25.7 mm and to the first costovertebral articulation was 1.7 mm. There is great variability in the morphology of the CTG with five common forms consistently seen. The relation to the vertebral artery may influence the form of the ganglion. Two previously undocumented forms are recorded; the truncated which describes the important juxtaposition of the CTG and the vertebral artery and the perforated which describes the piercing of the ganglion itself by the artery. The findings are considered to be of clinical importance to anesthetists, surgeons, neurosurgeons, and anatomists.

Self-assembly of sensory neurons into ganglia-like microtissues

Unraveling intra- and inter-cellular signaling networks managing cell-fate control, coordinating complex differentiation regulatory circuits and shaping tissues and organs in living systems remain major challenges in the post-genomic era. Resting on the laurels of past-century monolayer culture technologies, the cell culture community has only recently begun to appreciate the potential of three-dimensional mammalian cell culture systems to reveal the full scope of mechanisms orchestrating the tissue-like cell quorum in space and time. Capitalizing on gravity-enforced self-assembly of monodispersed primary embryonic mouse cells in hanging drops, we designed and characterized a three-dimensional cell culture model for ganglion-like structures. Within 24h, a mixture of mouse embryonic fibroblasts (MEF) and cells, derived from the dorsal root ganglion (DRG) (sensory neurons and Schwann cells) grown in hanging drops, assembled to coherent spherical microtissues characterized by a MEF feeder core and a peripheral layer of DRG-derived cells. In a time-dependent manner, sensory neurons formed a polar ganglion-like cap structure, which coordinated guided axonal outgrowth and innervation of the distal pole of the MEF feeder spheroid. Schwann cells, present in embryonic DRG isolates, tended to align along axonal structures and myelinate them in an in vivo-like manner. Whenever cultivation exceeded 10 days, DRG:MEF-based microtissues disintegrated due to an as yet unknown mechanism. Using a transgenic MEF feeder spheroid, engineered for gaseous acetaldehyde-inducible interferon-beta (ifn-beta) production by cotransduction of retro-/ lenti-viral particles, a short 6-h ifn-beta induction was sufficient to rescue the integrity of DRG:MEF spheroids and enable long-term cultivation of these microtissues. In hanging drops, such microtissues fused to higher-order macrotissue-like structures, which may pave the way for sophisticated bottom-up tissue engineering strategies. DRG:MEF-based artificial micro- and macrotissue design demonstrated accurate key morphological aspects of ganglions and exemplified the potential of self-assembled scaffold-free multicellular micro-/macrotissues to provide new insight into organogenesis.

TGF-beta1 in Aplysia: role in long-term changes in the excitability of sensory neurons and distribution of TbetaR-II-like immunoreactivity.

Exogenous recombinant human transforming growth factor beta-1 (TGF-beta1) induced long-term facilitation of Aplysia sensory-motor synapses. In addition, 5-HT-induced facilitation was blocked by application of a soluble fragment of the extracellular portion of the TGF-beta1 type II receptor (TbetaR-II), which presumably acted by scavenging an endogenous TGF-beta1-like molecule. Because TbetaR-II is essential for transmembrane signaling by TGF-beta, we sought to determine whether Aplysia tissues contained TbetaR-II and specifically, whether neurons expressed the receptor. Western blot analysis of Aplysia tissue extracts demonstrated the presence of a TbetaR-II-immunoreactive protein in several tissue types. The expression and distribution of TbetaR-II-immunoreactive proteins in the central nervous system was examined by immunohistochemistry to elucidate sites that may be responsive to TGF-beta1 and thus may play a role in synaptic plasticity. Sensory neurons in the ventral-caudal cluster of the pleural ganglion were immunoreactive for TbetaR-II, as well as many neurons in the pedal, abdominal, buccal, and cerebral ganglia. Sensory neurons cultured in isolation and cocultured sensory and motor neurons were also immunoreactive. TGF-beta1 affected the biophysical properties of cultured sensory neurons, inducing an increase of excitability that persisted for at least 48 hr. Furthermore, exposure to TGF-beta1 resulted in a reduction in the firing threshold of sensory neurons. These results provide further support for the hypothesis that TGF-beta1 plays a role in long-term synaptic plasticity in Aplysia.

Eomesodermin, a target gene of Pou4f2, is required for retinal ganglion cell and optic nerve development in the mouse.

The mechanisms regulating retinal ganglion cell (RGC) development are crucial for retinogenesis and for the establishment of normal vision. However, these mechanisms are only vaguely understood. RGCs are the first neuronal lineage to segregate from pluripotent progenitors in the developing retina. As output neurons, RGCs display developmental features very distinct from those of the other retinal cell types. To better understand RGC development, we have previously constructed a gene regulatory network featuring a hierarchical cascade of transcription factors that ultimately controls the expression of downstream effector genes. This has revealed the existence of a Pou domain transcription factor, Pou4f2, that occupies a key node in the RGC gene regulatory network and that is essential for RGC differentiation. However, little is known about the genes that connect upstream regulatory genes, such as Pou4f2 with downstream effector genes responsible for RGC differentiation. The purpose of this study was to characterize the retinal function of eomesodermin (Eomes), a T-box transcription factor with previously unsuspected roles in retinogenesis. We show that Eomes is expressed in developing RGCs and is a mediator of Pou4f2 function. Pou4f2 directly regulates Eomes expression through a cis-regulatory element within a conserved retinal enhancer. Deleting Eomes in the developing retina causes defects reminiscent of those in Pou4f2(-/-) retinas. Moreover, myelin ensheathment in the optic nerves of Eomes(-/-) embryos is severely impaired, suggesting that Eomes regulates this process. We conclude that Eomes is a crucial regulator positioned immediately downstream of Pou4f2 and is required for RGC differentiation and optic nerve development.

Polysensory interneuronal projections to foot contractile pedal neurons in Hermissenda.

A Pavlovian-conditioning procedure may produce modifications in multiple behavioral responses. As an example, conditioning may result in the elicitation of a specific somatomotor conditioned response (CR) and, in addition, other motor and visceral CRs. In the mollusk Hermissenda conditioning produces two conditioned responses: foot-shortening and decreased locomotion. The neural circuitry supporting ciliary locomotion is well characterized, although the neural circuit underlying foot-shortening is poorly understood. Here we describe efferent neurons in the pedal ganglion that produce contraction or extension of specific regions of the foot in semi-intact preparations. Synaptic connections between polysensory type Ib and type Is interneurons and identified foot contractile efferent neurons were examined. Type Ib and type Is interneurons receive synaptic input from the visual, graviceptive, and somatosensory systems. Depolarization of type Ib interneurons evoked spikes in identified tail and lateral foot contractile efferent neurons. Mechanical displacement of the statocyst evoked complex excitatory postsynaptic potentials (EPSPs) and spikes recorded from type Ib and type Is interneurons and complex EPSPs and spikes in identified foot contractile efferent neurons. Depolarization of type Ib interneurons in semi-intact preparations produced contraction and shortening along the rostrocaudal axis of the foot. Depolarization of Is interneurons in semi-intact preparations produced contraction of the anterior region of the foot. Taken collectively, the results suggest that type Ib and type Is polysensory interneurons may contribute to the neural circuit underlying the foot-shortening CR in Hermissenda.

Histamine reduces flash sensitivity of on ganglion cells in the primate retina.

PURPOSE. In Old World primates, the retina receives input from histaminergic neurons in the posterior hypothalamus. They are a subset of the neurons that project throughout the central nervous system and fire maximally during the day. The contribution of these neurons to vision, was examined by applying histamine to a dark-adapted, superfused baboon eye cup preparation while making extracellular recordings from peripheral retinal ganglion cells. METHODS. The stimuli were 5-ms, 560-nm, weak, full-field flashes in the low scotopic range. Ganglion cells with sustained and transient ON responses and two cell types with OFF responses were distinguished; their responses were recorded with a 16-channel microelectrode array. RESULTS. Low micromolar doses of histamine decreased the rate of maintained firing and the light sensitivity of ON ganglion cells. Both sustained and transient ON cells responded similarly to histamine. There were no statistically significant effects of histamine in a more limited study of OFF ganglion cells. The response latencies of ON cells were approximately 5 ms slower, on average, when histamine was present. Histamine also reduced the signal-to-noise ratio of ON cells, particularly in those cells with a histamine-induced increase in maintained activity. CONCLUSIONS. A major action of histamine released from retinopetal axons under dark-adapted conditions, when rod signals dominate the response, is to reduce the sensitivity of ON ganglion cells to light flashes. These findings may relate to reports that humans are less sensitive to light stimuli in the scotopic range during the day, when histamine release in the retina is expected to be at its maximum.

Cyan fluorescent protein expression in ganglion and amacrine cells in a thy1-CFP transgenic mouse retina.

PURPOSE: To characterize cyan fluorescent protein (CFP) expression in the retina of the thy1-CFP (B6.Cg-Tg(Thy1-CFP)23Jrs/J) transgenic mouse line. METHODS: CFP expression was characterized using morphometric methods and immunohistochemistry with antibodies to neurofilament light (NF-L), neuronal nuclei (NeuN), POU-domain protein (Brn3a) and calretinin, which immunolabel ganglion cells, and syntaxin 1 (HPC-1), glutamate decarboxylase 67 (GAD(67)), GABA plasma membrane transporter-1 (GAT-1), and choline acetyltransferase (ChAT), which immunolabel amacrine cells. RESULTS: CFP was extensively expressed in the inner retina, primarily in the inner plexiform layer (IPL), ganglion cell layer (GCL), nerve fiber layer, and optic nerve. CFP fluorescent cell bodies were in all retinal regions and their processes ramified in all laminae of the IPL. Some small, weakly CFP fluorescent somata were in the inner nuclear layer (INL). CFP-containing somata in the GCL ranged from 6 to 20 microm in diameter, and they had a density of 2636+/-347 cells/mm2 at 1.5 mm from the optic nerve head. Immunohistochemical studies demonstrated colocalization of CFP with the ganglion cell markers NF-L, NeuN, Brn3a, and calretinin. Immunohistochemistry with antibodies to HPC-1, GAD(67), GAT-1, and ChAT indicated that the small, weakly fluorescent CFP cells in the INL and GCL were cholinergic amacrine cells. CONCLUSIONS: The total number and density of CFP-fluorescent cells in the GCL were within the range of previous estimates of the total number of ganglion cells in the C57BL/6J line. Together these findings suggest that most ganglion cells in the thy1-CFP mouse line 23 express CFP. In conclusion, the thy1-CFP mouse line is highly useful for studies requiring the identification of ganglion cells.