Charles L. Zucker

Comparison of fixation and penetration enhancement techniques for use in ultrastructural immunocytochemistry.

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.

Dendritic compartmentalization of chloride cotransporters underlies directional responses of starburst amacrine cells in retina

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.

Ultrastructure of human retinal cell transplants with long survival times in rats

Human fetal retinas (6-12 weeks post-conception) were obtained from elective abortions, transplanted to rat retinas and examined by electron microscopy. The oldest transplants that form the basis of this report were obtained 40 and 41 total weeks post-conception. The host rats were immunosuppressed with cyclosporin A. The transplants developed according to their intrinsic, genetically determined timetable. The development was heterogeneous with some parts showing almost normal differentiation and others, little. Both rods and cones developed with inner and outer segments and synaptic terminals. In regions corresponding to the inner plexiform layer, bipolar cell processes were seen in the typical dyad arrangement. Likewise, amacrine cell processes formed typical conventional synapses. Serial synapses were seen, engaging amacrine cell synapses as well as a few reciprocal synapses at the bipolar cell dyads. Monad-type synaptic complexes, a sign of immaturity, were common in bipolar cell processes. Similarly, incompletely differentiated synapses of both the amacrine and bipolar cell types were often observed. Ganglion cell processes could not be identified with certainty. A structure with morphological characteristics similar to the inner limiting membrane was noted to form inside the transplant. Both epi-retinal and sub-retinal transplants were obtained. Transplant cells touched host photoreceptor cells or pigment epithelium without any obvious specializations. The host pigment epithelium microvilli were absent adjacent to the graft. However, graft cells did appear in the host retina, and nerve cell processes were observed to cross the membrane separating the transplant and host.

Transplantation of embryonic retina to the subretinal space in rabbits

Embryonic rabbit retina can be transplanted to the subretinal space of adult rabbit with a new method, which gives a high rate of successful short-term transplants. Embryonic (stage E 15) neural retina cells were injected through an incision just behind the sclerocorneal border with a thin (inner diameter 0.15-0.4 mm, outer diameter 0.3-0.5 mm) plastic tube attached to a specially designed instrument, by which the length of the protruding plastic tip could be controlled. The retina was penetrated from the vitreous side and the donor tissue was injected into the subretinal space. The cells survived in the host for at least 5 months, although the long-term survival rate tended to decrease. The transplanted cells matured and differentiated, forming an approximation of the layered, retinal structure with some anomalies (e.g. rosettes). The subretinal location offers an interesting and convenient way of studying the development of retinal cell transplants in rabbits. Large transplants can be produced, and the risk for failures due to erroneous vitreous placement is small.

Function and Structure in Retinal Transplants

Embryonic mammalian donor retina transplanted into the subretinal space of a mature host develops into a graft with wellorganized, but atypical retinal structure. We tested the effect of this organization on rabbitto-rabbit graft functional properties, isolating the graft to avoid contamination of graft responses by host retinal activity. Transient ON or ON-OFF spike-like responses and local electroretinograms (L-ERGs) were recorded simultaneously via a single electrode on the graft surface. These response components depended on stimulus diameter, sometimes in a way indicating antagonistic center-surround receptive field organization and spatial tuning (43%). Other times, the responses were an increasing function of stimulus diameter which saturated for large spots (57%). Response amplitudes were transplantation surgery is to be done with therapeutic aims.

Regulation of Dopamine Release from Interplexiform Cell Processes in the Outer Plexiform Layer of the Carp Retina

Abstract: The 7‐aminobutyric acid (GABA) antagonists bicuculline and picrotoxin stimulate a four‐ to fivefold increase in endogenous dopamine release from isolated intact carp retina. The release evoked by these agents is Ca2+ dependent, a finding suggesting a vesicular release. Using light microscopic autoradiography, we have localized the sites of dopamine release to the dopaminergic interplexiform cell processes of the outer plexiform layer, which synapse onto horizontal cells. Our findings support previous suggestions that the dopaminergic interplexiform cells receive GABAergic inhibitory input and that the effects of GABA antagonists on horizontal cells are mediated by dopamine release from the interplexiform cells.

Correlations between cholinergic neurons and muscarinic m2 receptors in the rat retina

ACETYLCHOLINE is well established as the neurotransmitter of starburst amacrine cells in the vertebrate retina but their function is poorly understood. We compared the distribution of muscarinic m2 receptors in the rat retina with the localization of the starburst cell processes. mAChR2 immunoreactivity appeared in a central band in the inner plexiform layer, which did not co-localize with the processes of the cholinergic amacrine cells. We found co-labelling of VAChT and ChAT making it highly unlikely that there are undetected cholinergic neurons in rat retina. Most mAChR2 receptors were located far from the cholinergic neurons, suggesting that most of them are unlikely to be associated with conventional cholinergic synapses.

Ultrastructural Circuitry in Retinal Cell Transplants to Rat Retina

The development of five transplants of fetal retinal tissue to adult rat eyes was examined with the electron microscope. The transplants were of 9 to 10 weeks total age after conception in four cases and 20 weeks in one case. They were at stage E15 when transplanted. Transplants developed in both the epiretinal and subretinal spaces. The transplants were heterogeneously developed with some parts showing almost normal differentiation and others little. Subretinal transplants examined in this study were more developed than epiretinal grafts. Photoreceptor cells developed both inner and outer segments. Their synaptic terminals possessed output ribbon synapses with postsynaptic processes similar to those seen in normal retinas. In regions corresponding to the inner plexiform layer, the adult complement of synapses was seen, including advanced features such as serial synapses as well as reciprocal synapses at bipolar cell dyads. Incompletely differentiated synapses of both the amacrine and bipolar cell types were often observed, especially in the rat epiretinal transplants. Ganglion cell processes could not be identified with certainty. Although transplant cells were adjacent to host photoreceptor cells and pigment epithelium, obvious specializations or interactions were not observed. The experiments suggest that embryonic rat retinal cell transplants develop most or perhaps all of the structural components and neuronal circuitry necessary to transduce light and process some visual information.

Gamma-aminobutyric acidA receptors on a bistratified amacrine cell type in the rabbit retina.

γ‐Aminobutyric acid (GABA) is considered to be a major inhibitory neurotransmitter in the inner plexiform layer of the retinas of all vertebrate species. It is contained in and released from nearly 40% of the amacrine cells and is known to play a major role in many aspects of visual processing. By using well‐characterized antibodies to several subunits of the GABAA receptor, we have analyzed their localization on the cell bodies and dendritic trees of two amacrine cell populations in the rabbit retina, which have been either filled intracellularly with Lucifer yellow or stained immunohistochemically. Both populations are selectively stained by intravitreal injection of the fluorescent nuclear dye 4′,6‐diaminidin‐2‐phenylindoldihydrochloride (DAPI). We have found that the most significant concentration of the α1 and β2/3 GABAA receptor subunits is localized to the DAPI‐3 type amacrine cell. The perikarya of the DAPI‐3 cells are found in the proximal inner nuclear layer and send their processes into two sublayers in sublaminae a and b of the inner plexiform layer. These processes abut but do not directly overlap those of the two mirror‐symmetric populations of starburst amacrine cells. Because the cell bodies of the DAPI‐3 cells are the only ones in the inner nuclear layer that stain strongly for either the α1 or β2/3 subunits, such staining is a diagnostic feature of these cells. Their processes also constitute the most strongly staining ones found within the inner plexiform layer. The dendritic trees of DAPI‐3 cells, which range from about 150 μm up to about 300 μm, exhibit recurvate looping processes reminiscent of those described for directionally selective ganglion cells. In contrast to the DAPI‐3 cell, we have also shown that the starburst amacrine cells exhibit no immunoreactivity for the α1 GABAA receptor subunit and very little for the β2/3 subunit. Thus, we have shown that the DAPI‐3 cells contain the highest concentrations of the α1 and β2/3 GABAA receptor subunits in the rabbit retina. These cells, which costratify near the processes of both the starburst amacrine cells and the ON‐OFF directionally selective ganglion cells, thus, are situated both anatomically and by virtue of their receptor content to potentially interact. J. Comp. Neurol. 393:309–319, 1998.

Compartmental localization of gamma-aminobutyric acid type B receptors in the cholinergic circuitry of the rabbit retina

Although many effects of γ‐aminobutyric acid (GABA) on retinal function have been attributed to GABAA and GABAC receptors, specific retinal functions have also been shown to be mediated by GABAB receptors, including facilitation of light‐evoked acetylcholine release from the rabbit retina (Neal and Cunningham [ 1995 ] J. Physiol. 482:363–372). To explain the results of a rich set of experiments, Neal and Cunningham proposed a model for this facilitation. In this model, GABAB receptor‐mediated inhibition of glycinergic cells would reduce their own inhibition of cholinergic cells. In turn, muscarinic input from the latter to the glycinergic cells would complete a negative‐feedback circuitry. In this study, we have used immunohistochemical techniques to test elements of this model. We report that glycinergic amacrine cells are GABAB receptor negative. In contrast, our data reveal the localization of GABAB receptors on cholinergic/GABAergic starburst amacrine cells. High‐resolution localization of GABAB receptors on starburst amacrine cells shows that they are discretely localized to a limited population of its varicosities, the majority of likely synaptic‐release sites being devoid of detectable levels of GABAB receptors. Finally, we identify a glycinergic cell that is a potential muscarinic receptor‐bearing target of GABAB‐modulated acetylcholine release. This target is the DAPI‐3 cell. We propose, based on these data, a modification of the Neal and Cunningham model in which GABAB receptors are on starburst, not glycinergic amacrine cells. J. Comp. Neurol. 493:448–459, 2005.