Synaptic transmission between rat superior cervical ganglion neurons in dissociated cell cultures.

The principal neurons of the rat superior cervical ganglion (SCGN) when established as dissociated cells in tissue culture form synapses among themselves. In the present study we have examined this synaptic interaction when these neurons are co-cultured with several other types of tissues. Dissociated SCGN were prepared from perinatal rats and studied, after 3-4 weeks maturation, with intracellular recording techniques. Synaptic interactions between sympathetic neurons were demonstrated when these cells were: (a) grown with explants from newborn rat thoracic spinal cord, (b) when the SCGN had survived for several weeks subsequent to removal of the spinal cord explants, and (c) when the SCGN were grown in the presence of an adrenergic target (interscapular brown fat cells). Unidirectional, reciprocal, recurrent and complex chemical synaptic networks, consisting of convergence and divergence, characterized connections between SCGN. All synaptic responses were cholinergic since they were reversibly blocked by hexamethonium or mecamylamine but were not sensitive to 10(-5) M phenoxybenzamine. Removal of the spinal cord explants did not significantly alter the proportion of chemical synaptic interactions between SCGN (more than 25%) from matched cultures. Anatomical observations established that in cultures with brown fat, innervating neurites appeared on the fat cells; these neurites frequently expanded to form varicosities that resembled the adrenergic terminals normally seen on brown fat in the animal. Synaptic profiles also occurred on the neurons in these cultures and some of these were shown to be cholinergic. The proportion of neuronal interactions in the combined SCGN + fat cultures was low, however, suggesting that co-culture with target tissue might influence the frequency of interconnections developed between SCGN in culture. Other factors, such as the presence of non-neuronal cells, degree of dissociation, cellular density, culture age and the survival of certain types of SCGN in culture are discussed as variables related to the formation of synapses between SCGN. Non-rectified electrical coupling between SCGN was also observed in 17 out of 679 pairs (2.5%) of neurons. Attenuation factor for electrically coupled action potentials ranged between 1 and 43.5.     

Growth of adult rat retinal ganglion cell neurites on astrocytes

Astrocytes, as well as Schwann cells (SC), can provide suitable substrata for embryonic neurites during development, but their abilities to support adult regenerating neurites have not been directly compared. The aim of the present study was to determine the ability of astrocytes to promote adult rat retinal ganglion cell (RGC) regeneration in vitro and to compare this to previously determined growth on the surface of Schwann cells. We prepared Type I astrocytes (Raff et al: J. Neurosci. 3:1289-1300, 1983) from perinatal rats. These were subcultured and maintained in either a serum-free medium for at least 2 weeks (stellate astrocytes with little immunoreactivity for laminin) or in serum containing medium for 7 to 10 days (flat and polygonal astrocytes with immunoreactivity for laminin). Stellate astrocytes might therefore represent mature astrocytes in vivo (Ard and Bunge: J. Neurosci. 8:2844-2858, 1988), while flat astrocytes might resemble immature brain astrocytes (Liesi et al: J. Cell Biol. 96:920-924, 1983). Adult RGC survival and axonal regrowth on these glia populations was compared to that observed on different SC populations, as previously reported (Baehr and Bunge: Exp. Neurol. 106:27-40, 1989). Both astrocyte populations (either flat or stellate astrocytes) did not enhance RGC survival. Stellate astrocytes were less effective in supporting RGC axon regeneration than flat astrocytes. When these date were compared to RGC survival and axon growth on SC (Baehr and Bunge: Exp. Neurol. 106:27-40, 1989) only "activated" mature SC populations were superior to astrocytes in enhancing RGC survival and neurite regrowth.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regeneration and proliferation of embryonic and adult rat hippocampal neurons in culture.

Adult mammalian CNS neurons appear to be terminally differentiated and postmitotic. However, this conclusion may be due to nonpermissive conditions in the brain or in culture media. If embryonic rat hippocampal neurons are cultured in Neurobasal/B27 with FGF2, nearly all neurons proliferated until a maximum density was reached. Similarly, adult neurons can be cultured that fire action potentials and display immunoreactivity for neurofilament, MAP2, tau, and glutamate. Seventy percent of the 3000 isolated adult cells per milligram of brain tissue began to proliferate after 3 days in culture and incorporated BrdU. By 4 days of regeneration in culture, virtually all neuron-like cells with asymmetric processes were glutamate positive and immunoreactive for neurofilament. Immunoreactivity of the intermediate filament stem cell marker nestin increased in adult cells to levels present in freshly isolated embryonic neurons. These are the first studies to demonstrate that over 50% of adult CNS cells with neuron-like characteristics retain regenerative and proliferative potential.     

Caspase-independent retinal ganglion cell death after target ablation in the neonatal rat

In neonatal rats, superior colliculus (SC) ablation results in a massive and rapid increase in retinal ganglion cell (RGC) death that peaks about 24 h post-lesion (PL). Naturally occurring cell death during normal development, and RGC death after axonal injury in neonatal and adult rats, has primarily been ascribed to apoptosis. Given that normal developmental cell death is reported to involve caspase 3 activation, and blocking caspase activity in adults reduces axotomy-induced death, we examined whether blocking caspases in vivo reduces RGC death after neonatal SC lesions. Neither general nor specific caspase inhibitors increased neonatal RGC survival 6 and 24 h PL. These inhibitors were, however, effective in blocking caspases in another well-defined in vitro apoptosis model, the corpus luteum. Caspase 3 protein and mRNA levels in retinas from normal and SC-lesioned neonatal rats were assessed 3, 6 and 24 h after SC removal using immunohistochemistry, western and northern blots and quantitative real-time polymerase chain reaction. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) was used to independently monitor retinal cell death. The polymerase chain reaction data showed a small but insignificant increase in caspase 3 mRNA in retinas 24 h PL. Western blot analysis did not reveal a significant shift to cleaved (activated) caspase 3 protein. There was a small increase in the number of cleaved caspase 3 immunolabelled cells in the ganglion cell layer 24 h PL but this represented only a fraction of the death revealed by TUNEL. Together, these data indicate that, unlike the situation in adults, most lesion-induced RGC death in neonatal rats occurs independently of caspase activation.     

Spontaneous regeneration of adult rat retinal ganglion cell axons in vivo.

The superior brachial region of the left optic tract was lesioned in adult rats and foetal tectal tissue was implanted into the lesion site. The retinal projection from the contralateral (right) eye was examined 2 to 8 months later. In the majority of animals, retinal ganglion cell (rgc) axons were found to regenerate through cellular membranes which formed over the lesion. Axon growth could extend for up to 5 or 6 mm. Surviving tectal grafts were identified in all host rats. In animals in which regrowing rgc axons contacted tectal grafts, axons were found to innervate selectively their appropriate target regions within the graft neuropil.     

Phenotypic differentiation of neonatal rat cochlear spiral ganglion neurons following trypsin dissociation and culture*☆

BACKGROUND： Under laboratory conditions, cochlear spiral ganglion neurons are commonly isolated and cultured by mechanical dissociation. However, these neurons are extremely fragile and survive for only a short time. OBJECTIVE： To establish a trypsin dissociation and culture method for studying neonatal rat cochlear spiral ganglion neurons. DESIGN： A single sample study. SETTING： Department of Otolaryngology, Head and Neck Surgery, Xijing Hospital, Fourth Military Medical University of Chinese PLA. MATERIALS： This study was performed at the central laboratory for Department of Otolaryngology, Head and Neck Surgery, Xijing Hospital, Fourth Military Medical University of Chinese PLA from February to May 2006. A total of 40 neonatal Sprague Dawley rats of either gender, aged 2-5 days, were provided by the Laboratory Animal Center of the Fourth Military Medical University of Chinese PLA. Trypsin and neuronal-specific nuclear protein （NeuN） monoclonal antibodies were purchased from Sigma Company, USA. Culture medium was synthesized using Dulbecco＇s modified Eagle＇s medium （DMEM）/F12 （Gibco Company, USA） supplemented with 10% fetal bovine serum （Sigma Company, USA）, 100 000 U/L penicillin, and 1 mol/L NaOH. The following protocol was performed in accordance with ethical guidelines for the use and care of animals. METHODS： After anesthesia, rats were sacrificed by neck dislocation. A complete cochlear axis with spiral ganglion tissue was removed. The cochlear axis was rinsed three times in a culture dish with a diameter of 35 mm using Hank＇s balanced solution. After washings, the tissue was cut into pieces, digested with 0.25% trypsin for about 20 minutes, and incubated in a 37 ℃ water bath. The tissue was centrifuged, then mixed with serum-containing culture medium. Using a transfer pipette, the cell suspension was transferred to polylysine （0.1%）-treated culture dishes with a diameter of 35 mm. The culture dish was incubated at 37 ℃, with a 5% CO2-air environment. Once the cel     

Photoreceptor-horizontal cell reaggregation in monolayer cultures of neonatal rabbit retina

It has been widely reported that trypsin-dissociated embryonic or neonatal retinal neurons have the ability to reaggregate into histotypic structures when grown in suspension. We now demonstrate that suspension culture is not required and that cell-specific aggregates can also be generated in papain-dissociated cell cultures of neonatal rabbit retina grown on laminin-coated glass coverslips. This culture preparation permitted the direct observation of a rapid and specific reaggregation of retinal cell types into two discrete types of clusters characterized by different morphologies as well as distinct labeling patterns of cell-specific antibodies and lectins. The first cluster type was composed of cells of the outer retina (photoreceptors in specific arrangements with horizontal cells); the second was composed of cells of the inner retina (amacrines and perhaps ganglion cells). The two cluster types were not intermingled, but rather they were segregated from each other, creating a spatial separation of inner from outer retinal cells across the coverslip. Video microscopy revealed that the separation was achieved rapidly (<1 hour) and that neither cell migration nor cell division was required for construction and segregation of cell-specific reaggregates. This novel observation suggests that repulsive as well as attractive factors exist that result in an initial sorting of cells from the outer and inner retina during early development.     

Morphology and distribution of neurons in the retinal ganglion cell layer of the adult tammar wallaby--Macropus eugenii

The morphology of the ganglion cell layer of the adult tammar wallaby has been examined from Nissl-stained retinal flatmounts. From this material, neurons have been classed as ganglion cells or displaced amacrine cells according to the disposition of Nissl substance. A further subdivision of ganglion cells into a separate group of alphalike cells was assisted by determining the range of soma sizes in neurofibrillar-stained flatmounts, a method which, in the cat, has revealed the presence of alpha cells. Isodensity contour maps prepared from the Nissl-stained flatmounts show a well-developed visual streak and an area centralis in the total neuronal population. A similar pattern was also found in the ganglion cells, thus confirming Tancred's (J. Comp. Neurol. 196:585-603, '81) finding, and, as well, in the alphalike ganglion cells and the displaced amacrine cells. The relative proportions of ganglion cells to displaced amacrines (GC:DA) were evaluated from isodensity profiles drawn along and vertical to the visual streak for the two cell types and also from maps showing the variation in the GC:DA ratio throughout the retina. A comparison with results published for other species shows that the visual streak development in the tammar wallaby is consistent with the expectations of the "terrain" theory and that, in its relative proportion of displaced amacrines, the tammar closely resembles the rabbit but contrasts sharply with the cat, which has half as many ganglion cells and three times as many displaced amacrines as the other two species.     

Calbindin-immunoreactive sensory neurons in dissociated dorsal root ganglion cell cultures of chick embryo: role of culture conditions

Immunoreactivity to calbindin D-28k, a vitamin D-dependent calcium-binding protein, is expressed by neuronal subpopulations of dorsal root ganglia (DRG) in the chick embryo. To determine whether the expression of this phenotypic characteristic is maintained in vitro and controlled by environmental factors, dissociated DRG cell cultures were performed under various conditions. Subpopulations of DRG cells cultured at embryonic day 10 displayed calbindin-immunoreactive cell bodies and neurites in both neuron-enriched or mixed DRG cell cultures. The number of calbindin-immunoreactive ganglion cells increased up to 7-10 days of culture independently of the changes occurring in the whole neuronal population. The presence of non-neuronal cells, which promotes the maturation of the sensory neurons, tended to reduce the percentage of calbindin-immunoreactive cell bodies. Addition of horse serum enhanced both the number of calbindin-positive neurons and the intensity of the immunostaining, but does not prevent the decline of the subpopulation of calbindin-immunoreactive neurons during the second week of culture; on the contrary, the addition of muscular extract to cultures at 10 days maintained the number of calbindin-expressing neurons. While calbindin-immunoreactive cell bodies grown in culture were small- or medium-sized, no correlation was found between cell size and immunostaining density. At the ultrastructural level, the calbindin immunoreaction was distributed throughout the neuroplasm. These results indicate that the expression of calbindin by sensory neurons grown in vitro may be modulated by horse serum-contained factors or interaction with non-neuronal cells. As distinct from horse serum, muscular extract is able to maintain the expression of calbindin by a subpopulation of DRG cells.     

Regeneration of ganglion cell axons in the adult mouse retina

The hypothesis that regenerative failure of axons in the adult mammalian CNS is due to release of a growth inhibitor from injured oligodendrocytes and/or myelin², predicts that regeneration of injured fibers would proceed unchecked in unmyelinated CNS regions. This prediction was borne out by observations on the stratum opticarum of the mouse retina. Axonal sprouts, first seen 14–16 h post-lesion (pl), continued growing until at least 100 days pl, well beyond the time at which regeneration fails in myelinated CNS regions.     

Astrocytes from adult rat optic nerves are nonpermissive for regenerating retinal ganglion cell axons

We have directly compared the abilities of astrocytes from newborn and adult rats to support or inhibit the growth of regenerating axons in vitro. Astrocytes prepared from newborn rats were able to promote retinal ganglion cell (RGC) axon growth from embryonic and adult rat and from adult fish retinal explants. Retinal axons from E16 rat retinae grew significantly faster on astrocytes from neonatal rats than those from E18 or adult rat retinae with growth rates comparable to RGC axons from adult fish retinae. RGC regeneration from adult rat retinae was almost completely inhibited on adult rat optic nerve astrocytes. Only axons from adult fish retinae were able to extend onto monolayers from these reactive astrocytes, although their growth rates were significantly reduced. We conclude that the failure of mammalian RGC axons to regrow within the lesioned optic nerve environment is, at least in part, due to nonpermissive aspects of adult “reactive” optic nerve astrocytes. However, the cell intrinsic growth potential of RGCs also appears to influence their ability to extend axons on cellular substrates.     

Primary culture of neonatal rat olfactory neurons.

We have prepared primary cultures of purified neonatal rat olfactory neurons. Dissociated olfactory epithelial cells are maintained in modified Eagle's medium with D-valine, cytosine arabinoside, and NGF. NGF is required for neuronal survival. Immunohistochemical staining is positive for the neuronal markers vimentin, olfactory marker protein, and neuron-specific enolase, but negative for the glial markers, glial fibrillary acidic protein, and S-100 protein. Physiologic concentrations of odorants stimulate cAMP accumulation in the cells. Because of their morphology, biochemical composition, and responsiveness to odorants, these cells should enhance olfactory investigations.     

Single cell RT-PCR analysis of tyrosine kinase receptor expression in adult rat retinal ganglion cells isolated by retinal sandwiching

We describe a protocol for analysis of gene expression in single, acutely dissociated adult rat retinal ganglion cells using RT-PCR. Retrograde tracing of retinal ganglion cells from the superior colliculi was conducted using Fluorogold. Retinas were dissected and ganglion cells isolated using retinal layer separation (sandwiching). Single, fluorescently labelled retinal ganglion cells were aspirated using a micropipette and used for PCR. Two PCR protocols are described where single cell cDNA was analysed for TrkB and GAPDH or TrkB, TrkC, Ret, Met, ErbB2 and Beta-actin by multiplex-PCR. All five tyrosine kinase receptors were amplified from single retinal ganglion cells. The method will prove useful for the molecular characterization of adult retinal ganglion cells.     

Development of cholinergic retinal neurons from embryonic chicken in monolayer cultures: stimulation by glial cell-derived factors

In recent years evidence has indicated that, like the PNS, the development of the CNS is influenced by neuronotrophic polypeptide factors. In the present study, cultures of dissociated retinal neurons from 8-d-old chicken embryos were used to investigate the role of neuronotrophic factors (NTF) in the development of the neural retina. CAT, which in vivo is located in amacrine cells of the retina, served as a marker for studying the in vitro development of cholinergic retinal neurons. Differentiation of cholinergic cells under control conditions was indicated by a 10-fold increase of enzyme activity during a 7-d culture period. Addition of media conditioned by high-density retinal cultures resulted in a further stimulation of CAT activity by 100-400%. The CAT-stimulating activity was associated with a high-molecular-weight component of the retina conditioned medium (RCM) and was sensitive to protease treatment, but was not affected by other hydrolytic enzymes. The putative cholinergic factor was secreted by retinal cultures virtually free of neurons, suggesting that it is mainly produced by Müller cells. CAT-stimulating activity was also present in extracts from embryonic chicken retinae and medium conditioned by rat retinal cultures. NGF, anti-NGF antiserum, extracts from chicken brain tissues, and a number of other extracts and conditioned media, all known to contain neuronotrophic activities, were found to have no influence on cholinergic development in chicken retinal cultures. An extract from non-retinal eye tissue containing ciliary neuronotrophic factor (CNTF) stimulated CAT activity to the same extent as did RCM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphological and cytochemical studies of synapses formed in culture between isolated rat superior cervical ganglion neurons

Neurons from the superior cervical ganglia (SCG) of perinatal rats were mechanically dissociated and established in culture by a method modified from that of Bray ('70). The neurons attached to the collagen substrate with some degree of reaggregation, and their rapidly growing processes soon formed an interconnecting network. Light and electron microscope examination showed that the morphology of the cultured cells resembled that of the SCG principal cell; neither satellite cells nor the small intensely fluorescent interneurons were observed. After six to seven days in culture, synapses were seen on both the principal cell somata and on their processes. After aldehyde fixation, the synapses contained clear, pleiomorphic vesicles; after KMnO₄ fixation a variable number of the vesicles contained dense cores. After reserpine treatment and KMnO₄ fixation the synaptic vesicles of the cultured networks contained few dense cores. Similarly treated cultured networks showed an increase in the number of synaptic vesicles with dense cores if incubated with dihydroxyphenylalanine prior to fixation, but a suppression of this increase in the presence of a specific inhibitor of dopamine B-hydroxylase, FLA 63. The morphology of synapses in the cultured networks was compared to that of endings in the intact rat SCG and iris in vivo, both before and after treatment with norepinephrine and 5-hydroxydopamine. The vesicles in the synaptic endings in cultures were found to resemble those of the adrenergic endings of the rat iris, and to be similar to those seen within neuronal processes in the rat SCG. Our findings indicate that adrenergic synapses are formed between isolated SCG principal neurons in culture, and thus support recent evidence that such synapses occur in the intact rat SCG. The absence of other tissues which could receive innervation, and of ensheathing satellite cells, may provide a possible explanation for their frequency in the culture situation.     

In vitro myelination of regenerating adult rat retinal ganglion cell axons by Schwann cells.

Schwann cell cultures provide a highly favorable substrate for retinal ganglion cell (RGC) survival and axon growth in vitro (B?hr and Bunge, Exp Neurol 106:27, 1989; Hopkins and Bunge, Glia 4:46, 1991). In this report we have extended former studies to obtain axon regeneration, long-term survival, and myelination of adult rat RGC axons in co-cultures of retinal explants with purified Schwann cells. By using modified co-culture conditions, we observed myelination of regenerating adult RGC axons by Schwann cells after 3-4 weeks in vitro. Myelination was associated with a one-to-one Schwann cell-axon relationship, characteristic of the formation of peripheral myelin. Under culture conditions that supported myelination, long-term survival (more than 12 weeks) of a small population of RGCs was observed. These findings highlight the remarkable ability of Schwann cells to support long-term survival of adult rat RGCs in the absence of either central nervous system (CNS) target tissue or other peripheral nervous system (PNS) components. This tissue culture system may serve as a model for the systematic study of the molecular mechanisms which are involved in axon regeneration and myelination of adult CNS neurons.     

Catecholaminergic properties of cholinergic neurons and synapses in adult rat ciliary ganglion

Parasympathetic neurons of the ciliary ganglion are innervated by preganglionic cholinergic neurons whose cell bodies lie in the brain stem; the ganglion cells in turn provide cholinergic innervation to the intrinsic muscles of the eye. Noradrenergic innervation of the iris is supplied by sympathetic neurons of the superior cervical ganglion. Using immunocytochemical and histochemical techniques, we have examined the ciliary ganglion of adult rats for the expression of cholinergic and noradrenergic properties. As expected, the postganglionic ciliary neurons possessed detectable levels of choline acetyltransferase immunoreactivity (ChAT-IR). Unexpectedly, many ciliary neurons also exhibited immunoreactivity for tyrosine hydroxylase (TH-IR). Some had dopamine beta-hydroxylase-like (DBH-IR) immunoreactivity, but none contained detectable catecholamines, even after treatment with nialamide and L-DOPA. A sparse plexus of fibers exhibiting faint TH-IR was present in the irises of acutely sympathectomized rats. The terminals of preganglionic axons in the ciliary ganglion exhibited not only immunoreactivity for ChAT, but also for TH and contained stores of endogenous catecholamine. Neither ciliary neurons nor their preganglionic innervation accumulated detectable stores of exogenous catecholamines. Rats sympathectomized as neonates by treatment with 6-hydroxydopamine subsequently had a greater proportion of neurons possessing detectable TH-IR in the ciliary ganglion; both the TH-IR perikarya and their axons in the iris were more intensely immunofluorescent. TH-IR was present in the ciliary neuron cell bodies of mouse, guinea pig, and ferret. These species, however, lacked detectable TH-IR or catecholamine stores in preganglionic terminals. These observations indicate that mature, functionally cholinergic neurons from 2 different embryonic origins, postganglionic ciliary neurons derived from the neural crest and preganglionic neurons derived from the neural tube, display several catecholaminergic properties.     

Hypoxia-induced apoptosis in adult rat dorsal root ganglion neurons in vitro

Following spinal root injury, dorsal root ganglia suffer mechanical trauma and compromised blood supply. Little is known about the consequences for neuronal survival. Here we used cyanide treatment in vitro to examine effects of moderate hypoxia on adult rat dorsal root ganglion cells identified by GAP-43 immunostaining. 400 microM-4 mM cyanide caused sustained increases in intracellular Ca2+. Cyanide at 2 mM led to a significant increase in apoptosis, detected using TUNEL labelling and confirmed by ultrastructural analysis, and a further increase when cultures were left overnight in fresh medium. Our study shows that dorsal root ganglion neurons die by apoptosis following hypoxia and that cell death increases over time. Cyanide response provides a simple assay for testing neuroprotective agents and examining underlying mechanisms.     

Birthdates of neurons in the retinal ganglion cell layer of the ferret

The present study determined the temporal and spatial patterns of genesis for neurons of different sizes in the retinal ganglion cell layer of the ferret. Fetal ferrets were exposed to tritiated thymidine on embryonic days E-22 through E-36. One to 3 months after birth, they were perfused and their retinae dissected, and autoradiographs were prepared from resinembedded sections throughout the entire flattened retinal ganglion cell layer. Soma size differences in conjunction with separate retrograde labeling and calbindin immunocytochemical studies were used as criteria for identifying different retinal ganglion cell subtypes in juvenile and adult ferrets. Neurons of different sizes in the ganglion cell layer were generated at different stages during development. Medium sized cells were generated primarily between E-22 and E-26; the largest cells were generated between E-24 and E-29; small cells were generated between E-26 and E-32; and very small cells were generated between E-29 and E-36. The former three groups were interpreted to be three subtypes of retinal ganglion cells, while the latter group was interpreted to be displaced amacrine cells. This temporal order of the genesis of ganglion cell classes is consistent with the spatial ordering of their fibers in the mature optic chiasm and tract, and it is consistent with the developmental change in decussation pattern recently shown in the optic pathway of embryonic ferrets. The spatial pattern of genesis suggests that ganglion cells of a particular class are added to the ganglion cell layer in a centroperipheral fashion initiated in the dorsocentral retina nasal to the area centralis. No evidence was found for a wave of ganglion cell addition that proceeded in a spiralling pattern around the area centralis, as has been reported in the cat.