The central nervous system (CNS) in adult mammals loses the ability to regenerate after injury. Although electrical stimulation (ES) has been shown to promote neural regeneration, the underlying mechanisms by which ES enhances CNS regeneration remain elusive. The aim of the present study was to investigate the effect of ES on neurite outgrowth of goldfish retinal explants. The optic nerve of adult goldfish was intraorbitally crushed 7-14 days before retinal explants preparation. The explants were cultured in a laminin-coated indium tin oxide (ITO, transparent conducting oxide) device for ES application. Various strengths and waveforms of ES were applied to examine their effect on neural outgrowth. When the retinal explants were stimulated for 1h with intermittent pulses on day 1 (20 Hz; 10 pulses every 2s), the regenerated neurite length was significantly increased compared to explants that were stimulated with continuous square waves or continuous pulses over the same time course. It was also found that increased ES strength and repeated ES each day can enhance neurite outgrowth significantly. These results suggest that intermittent pulse ES is able to promote neurite regeneration most effectively in goldfish retinal explants, and that this electrically stimulated neurite outgrowth using ITO conductive electrodes may provide a useful platform for investigating cellular mechanisms of CNS regeneration.

Chondroitin sulfate proteoglycans produced in glial scar tissue are a major inhibitory factor for axonal regeneration after central nervous system injury in mammals. The inhibition is largely due to chondroitin sulfates, whose effects differ according to the sulfation pattern. In contrast to mammals, fish nerves spontaneously regenerate beyond the scar tissue after spinal cord injury, although the mechanisms that allow for axons to pass through the scar are unclear. Here, we used immunohistochemistry to examine the expression of two chondroitin sulfates with different sulfation variants at the lesion site in goldfish spinal cord. The intact spinal cord was immunoreactive for both chondroitin sulfate-A (CS-A) and chondroitin sulfate-C (CS-C), and CS-A immunoreactivity overlapped extensively with glial processes positive for glial fibrillary acidic protein. At 1week after inducing the spinal lesion, CS-A immunoreactivity was observed in the cell bodies and extracellular matrix, as well as in glial processes surrounding the lesion center. At 2weeks after the spinal lesion, regenerating axons entering the lesion center overtook the CS-A abundant area. In contrast, at 1week after lesion induction, CS-C immunoreactivity was significantly decreased, and at 2weeks after lesion induction, CS-C immunoreactivity was observed along the regenerating axons entering the lesion center. The present findings suggest that after spinal cord injury in goldfish, chondroitin sulfate proteoglycans are deposited in the extracellular matrix at the lesion site but do not form an impenetrable barrier to the growth of regenerating axons.

Although it has been reported that dendritic neurotransmitter releases from amacrine cells are regulated by the intracellular Ca(2+) concentration ([Ca(2+)](i)), their spatiotemporal patterns are not well explained. Fast Ca(2+) imagings of amacrine cells in the horizontal slice preparation of goldfish retinas under whole-cell patch-clamp recordings were undertaken to better investigate the spatiotemporal patterns of dendritic [Ca(2+)](i). We found that amacrine cell dendrites showed inhomogeneous [Ca(2+)](i) increases in both Na(+) spiking cells and cells without Na(+) spikes. The spatiotemporal properties of inhomogeneous [Ca(2+)](i) increases were classified into three patterns: local, regional and global. Local [Ca(2+)](i) increases were observed in very discrete regions and appeared as discontinuous patches, presumably evoked by local excitatory postsynaptic potentials. Regional [Ca(2+)](i) increases were observed in either a single or a small number of dendrites, presumably reflecting the result of dendritic action potentials. Global [Ca(2+)](i) increases were observed in the entire dendrites of a cell and were mediated by Na(+) action potentials or multiple Na(+) action potentials riding on slow depolarization. Ca(2+)-mediated potentials also evoked global [Ca(2+)](i) increase in cells without Na(+) spikes. These spatiotemporal dynamics of dendritic Ca(2+) signals may reflect multiple modes of synaptic integration on the dendrites of amacrine cells.

Retinal amacrine cells of the same class in cyprinid fish are homotypically connected by gap junctions. The permeability of their gap junctions examined by the diffusion of Neurobiotin into neighboring amacrine cells under application of dopamine or cyclic nucleotides to elucidate whether electrical synapses between the cells are regulated by internal messengers. Neurobiotin injected intracellularly into amacrine cells in isolated retinas of goldfish, and passage currents through the electrical synapses investigated by dual whole-patch clamp recordings under similar application of their ligands. Control conditions led us to observe large passage currents between connected cells and adequate transjunctional conductance between the cells (2.02±0.82nS). Experimental results show that high level of intracellular cyclic AMP within examined cells block transfer of Neurobiotin and suppress electrical synapses between the neighboring cells. Transjunctional conductance between examined cells reduced to 0.23nS. However, dopamine, 8-bromo-cyclic AMP or high elevation of intracellular cyclic GMP leaves gap junction channels of the cells permeable to Neurobiotin as in the control level. Under application of dopamine (1.25±0.06nS), 8-bromo-cyclic AMP (1.79±0.51nS) or intracellular cyclic GMP (0.98±0.23nS), the transjunctional conductance also remains as in the control level. These results demonstrate that channel opening of gap junctions between cyprinid retinal amacrine cells is regulated by high level of intracellular cyclic AMP.

A strategy based upon a comparative decrease in bilateral symmetry of cytochrome oxidase (COX) histochemistry following unilateral eye enucleation was used to identify the primary visual area in the area dorsalis of the telencephalon of the goldfish, Carassius auratus. The lateral zone of area dorsalis (Dl) at about the level of the anterior commissure exhibits such a bilateral difference. A parallel decline in the symmetry COX reactivity was observed in the associated part of the central zone (Dc). Electrophysiological activity using extracellular techniques confirmed the visually-driven activity of neurons in these areas. Lesions confirmed the loci in the lateral zone of area dorsalis, including both its dorsal and ventral parts. Single- and multi-unit recordings exhibited a variety of responses to different light stimuli. Single unit latency measures proved not to be a reliable measure of target areas. Responses habituated to stimuli repeated within 5 s and were only reliably evoked with intervals greater than several seconds.

GAD65 and GAD67 are the two major isoforms of the enzyme that converts glutamate into GABA in a single step reaction. Despite studies describing GAD65 and GAD67 mRNA expression in the mammalian brain, both GAD65 and GAD67 mRNA expression has not yet been fully described for a non-mammalian vertebrate model. Similarly, the expression patterns of GABA-T mRNA, the major enzyme involved in metabolizing GABA, have not been described for any vertebrate. In the present study, we utilized non-radioactive in situ hybridization to localize GAD65, GAD67, and GABA-T in the adult goldfish brain and complimented this with an in vitro assessment of total GAD and GABA-T enzyme activities. A partial fragment of goldfish GABA-T was cloned for a riboprobe that showed approximately 92% deduced amino acid identity to zebrafish GABA-T and 78% identity to human GABA-T. Transcripts for GAD65, GAD67, and GABA-T were detected throughout the brain and were detected largely in the medial and ventral regions of the telencephalon, nucleus preopticus, nucleus recessus lateralis of the hypothalamus, and Purkinje cell layer of the cerebellum. GAD65 mRNA was significantly more abundant in the nucleus recessus posterioris of the hypothalamus than GAD67 and GABA-T mRNA. Total GAD and GABA-T specific enzyme activity was highest in the hypothalamus and optic tectum and GABA-T activity was significantly higher than total GAD enzyme activity. Our results show that GAD65, GAD67, and GABA-T mRNAs are generally correlated with total GAD and GABA-T activity and all three transcripts have a largely overlapping mRNA distribution in the goldfish forebrain.

The transcription factor Pax2 actively participates in the development of the vertebrate visual system. In adults, Pax2 expression persists in a subpopulation of Müller cells and/or astrocytes in the retina and optic nerve head (ONH), although its function remains elusive. In a previous work we showed that the pax2 gene expression is modified and the Pax2(+) astrocyte population in the ONH strongly reacted during the regeneration of the retina after a lesion in goldfish. In the present work we have analyzed Pax2 expression in the goldfish ONH after optic nerve (ON) crush. At one week post-injury, when the regenerating axons arrive at the ONH, the pax2 gene expression level increases as well as the number of Pax2(+) astrocytes in this region. These Pax2(+) astrocytes show a higher number of Cytokeratin (Ck)(+)/GFAP(+) processes compared with control animals. In contrast, a different S100(+) astrocyte population is not modified and persists similar to that of controls. Furthermore, we find a ring that surrounds the posterior ONH that is formed by highly reactive astrocytes, positive to Pax2, GFAP, Ck, S100, GS and ZO1. In this region we also find a source of new astrocytes Pax2(+)/PCNA(+) that is activated after the injury. We conclude that Pax2(+) astrocytes constitute a subpopulation of ONH astrocytes that strongly reacts after ON crush and supports our previous results obtained after retina regeneration. Altogether, this suggests that pax2 gene expression and Pax2(+) astrocytes are probably directly involved in the process of axonal regeneration.

A great deal of effort has been invested in using trophic factors and other bioactive molecules to promote cell survival and axonal regeneration in the adult central nervous system. Far less attention has been paid to investigating potential effects that trophic factors may have that might interfere with recovery. In the visual system, BDNF has been previously reported to prevent regeneration. To test if BDNF is inherently incompatible with regeneration, BDNF was given intraocularly during optic nerve regeneration in the adult goldfish. In vivo imaging and anatomical analysis of selectively labeled axons were used as a sensitive assay for effects on regeneration within the tectum. BDNF had no detectable inhibitory effect on the ability of axons to regenerate. Normal numbers of axons regenerated into the tectum, exhibited dynamic growth and retractions similar to controls, and were able to navigate to their correct target zone in the tectum. However, BDNF was found to have additional effects that adversely affected the quality of regeneration. It promoted premature branching at ectopic locations, diminished the growth rate of axons through the tectum, and resulted in the formation of ectopic collaterals. Thus, although BDNF has robust effects on axonal behavior, it is, nevertheless, compatible with axonal regeneration, axon navigation and the formation of terminal arbors.