Electron microscopic immunohistochemistry, although generally providing good localization, has often failed to produce satisfying ultrastructural preservation. Techniques that result in well-preserved tissue ultrastructure often hinder penetration of immunological reagents or render antigens non-immunoreactive. These are particularly serious limitations in studies of central nervous system and retina. We have evaluated several fixatives, including picric acid, high pH paraformaldehyde, and glutaraldehyde with subsequent sodium borohydride treatment, and penetration enhancement techniques, including buffered-ethanolic treatment and freeze--thaw, for their applicability in the retina. Our best fixation was achieved with 1 hr in 4% paraformaldehyde and 0.2% glutaraldehyde (pH 7.4) followed by overnight fixation in 4% paraformaldehyde (pH 10.4). Treatment with sodium borohydride after glutaraldehyde fixation restores much of the immunoreactivity that would otherwise be undetectable. The penetration of immunological reagents can be greatly increased by using either a buffered-ethanolic series or by freezing the tissue after careful cryoprotection. Using these methods we have been able to achieve specific immunological staining throughout the full thickness of retinal slices, up to 500 microns across, while preserving good ultrastructure. The methods should prove useful in the immunocytochemical localization of many different antigens in a variety of tissues.
The mechanisms in the retina that generate light responses selective for the direction of image motion remain unresolved. Recent evidence indicates that directionally selective light responses occur first in the retina in the dendrites of an interneuron, i.e., the starburst amacrine cell, and that these responses are highly sensitive to the activity of Na-K-2Cl (NKCC) and K-Cl (KCC), two types of chloride cotransporter that determine whether the neurotransmitter GABA depolarizes or hyperpolarizes neurons, respectively. We show here that selective blockade of the NKCC2 and KCC2 cotransporters located on starburst dendrites consistently hyperpolarized and depolarized the starburst cells, respectively, and greatly reduced or eliminated their directionally selective light responses. By mapping NKCC2 and KCC2 antibody staining on these dendrites, we further show that NKCC2 and KCC2 are preferentially located in the proximal and distal dendritic compartments, respectively. Finally, measurements of the GABA reversal potential in different starburst dendritic compartments indicate that the GABA reversal potential at the distal dendrite is more hyperpolarized than at the proximal dendrite due to KCC2 activity. These results thus demonstrate that the differential distribution of NKCC2 on the proximal dendrites and KCC2 on the distal dendrites of starburst cells results in a GABA-evoked depolarization and hyperpolarization at the NKCC2 and KCC2 compartments, respectively, and underlies the directionally selective light responses of the dendrites. The functional compartmentalization of interneuron dendrites may be an important means by which the nervous system encodes complex information at the subcellular level.direction-selective ͉ GABAergic excitation ͉ interneuron
In teleost fish, centrifugal fibres originating in the olfactory bulb and containing FMRFamide-like and luteinizing hormone releasing hormone (LHRH)-like peptides project to the retina and terminate along the border of the inner nuclear and inner plexiform layers. Using a novel simultaneous two-colour immunolabelling technique, we have found that these centrifugal fibres are often closely apposed to the dopaminergic interplexiform cells. Contacts between centrifugal fibres and dopaminergic interplexiform cells were observed by electron microscopy to be conventional type synaptic junctions. Since the dopaminergic interplexiform cells make synapses on horizontal and bipolar cells, providing an intraretinal centrifugal pathway for information flow from the inner to the outer plexiform layers, we conclude that every neuron in the teleost retina is potentially susceptible to central influences via these centrifugal fibres and dopaminergic interplexiform cells.
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