Mudpuppy retinal ganglion cell morphology revealed by an HRP impregnation technique which provides Golgi-like staining.

A new technique of retrograde labeling of ganglion cells with horseradish peroxidase (HRP) has been developed, based on orbital injections of HRP combined with a detergent (lysolecithin). When injections are followed by an appropriate survival time, dense staining of a small number of widely scattered cells results in Golgi-like filling of each neuron. This technique, as well as a variation which causes mass staining of ganglion cell somas, has been used to analyze the morphology of mudpuppy retinal ganglion cells. Morphological analysis has relied on computer reconstruction techniques for display, analysis of dendritic sublamination pattern, and morphometric analysis of the dendrites and soma. Based on morphological criteria, the mudpuppy retina contains a rich variety of ganglion cell types which vary according to soma placement, dendritic field size, polar vs. non-polar dendritic fields, dendritic branching pattern, and dendritic sublamination. The mudpuppy retina contains both conventional and displaced ganglion cells: the latter constitute about 15% of the total ganglion cell population. Both conventional and displaced ganglion cells show morphological diversity of dendritic sublamination branching pattern; cells from each group have a dendritic branching pattern confined to either distal or proximal divisions of the inner plexiform layer, whereas other cells have dendrites which branch in both sublaminae. Using morphological criteria, two subtypes of ganglion cells were identified, which have a distinctive branching pattern and dendritic tree size. The size and distribution of ganglion cell somas were analyzed from retinas in which mass staining of ganglion cells was present. The total number of ganglion cells was estimated at approximately 14,500 cells per retina. There was a tendency for soma size and density to decrease near the optic disk. The somas of displaced ganglion cells are smaller than their conventional counterparts, at the same retinal eccentricity. The somas of all HRP filled cells swell when compared to those of unstained fixed and freshly dissected retinas. The degree of swelling is proportional to the length of exposure to HRP. Cell swelling was evident for both retrograde labeling and intracellularly injected HRP. This artifact of HRP staining could influence the interpretation of studies in which quantitative differences in soma sizes are based on the use of HRP labeling.

Subtle actions of 2-amino-4-phosphonobutyrate (APB) on the Off pathway in the mudpuppy retina.

The principal action of 2-amino-4-phosphonobutyrate (APB) applied to the retina is the elimination of the On-bipolar cell light response. In this study, we report that APB also has more subtle effects; it sometimes enhances Off responses of Off bipolar cells and On, Off, and On-Off inner retina neurons. Possible mechanisms for this enhancement are considered.

Preservation of physical dimensions in a model of reactive synaptogenesis in the red nucleus.

Collateral sprouting of cerebral cortical fibers in the red nucleus following destruction of the interpositus nucleus may be effective in restoring activity of rubral neurons. Shrinkage of the deafferented red nucleus was measured to estimate its effect on recording neural activity and its contribution as a stimulus for sprouting. The results suggest that rubral morphology is preserved during the early time course of collateral sprouting when electrophysiological changes are evident.

Anterior chiasmal optic nerve avulsion.

In a case of traumatic avulsion of the optic nerve at the anterior chiasm, MR imaging provided highly specific images of the injury site, including the absence of the optic nerve within the optic canal and the point of transection at the anterior portion of the chiasm. This was confirmed clinically and histopathologically. MR imaging should be considered in cases of suspected chiasmal injury.

Bipolar origin of synaptic inputs to sustained OFF-ganglion cells in the mudpuppy retina.

1. The synaptic inputs to sustained OFF-center ganglion cells of the mudpuppy retina were studied using a superfused retina-eye-cup preparation. Intra- and extracellular electrophysiological recording techniques were carried out during bath application of 2-amino-4-phosphonobutyrate (APB), a glutamate analog that selectively blocks the light responses of ON-bipolars but has minor effects on OFF-bipolar or horizontal cells. 2. The use of APB reduced ganglion cell inputs to those arising from the OFF-bipolar channel. In this way, the existence and polarity (depolarizing vs. hyperpolarizing) of direct or indirect bipolar connections to ganglion cells was determined. 3. Cobalt application was used to block synaptic transmission and demonstrate that APB does not have a direct excitatory action on ganglion cells. 4. Intracellular recording experiments included the use of pulsatile and sustained current injection to evaluate the input resistance changes associated with light, the action of APB, and the excitatory, inhibitory, or disafacilitory nature of the postsynaptic potentials. 5. Some intracellularly recorded cells were stained with horseradish peroxidase (HRP) to verify the ganglion cell origin of the recordings. 6. The OFF-ganglion cell population of the mudpuppy appears to be a heterogeneous group of cells. Sustained OFF-ganglion cells can receive dominant inputs through either the ON- or OFF-bipolar cell pathway or through a mixture of the two. 7. Based on the analysis of this study, we divided sustained OFF-ganglion cells into three subclasses. For one class, light causes the removal of a sustained excitatory input which originates from the OFF-bipolar channel (i.e., a light-evoked disfacilitation); a second class of cells is almost entirely driven by the ON-bipolar channel through a sustained light-evoked inhibitory input; and a third class receives both a light-evoked sustained disfacilitory input from the OFF-bipolar channel and a sustained inhibitory input through the ON-bipolar pathway. Thus the retina appears to use a variety of mechanisms that result in a common response to flashing light stimuli. 8. The results of this study show that APB can be a powerful tool for pharmacologically deciphering the functional connections that exist between outer and inner retinal neurons.

Synaptic inputs and morphology of sustained ON-ganglion cells in the mudpuppy retina.

1. Sustained ON-ganglion cells from the mudpuppy retina were studied with a combined approach, including intracellular and extracellular recording from the superfused retina-eyecup preparation, pharmacology with bath-applied 2-amino-4-phosphonobutyrate (APB), and retrograde and intracellular staining using horse radish peroxidase (HRP). 2. Bath application of micromolar levels of APB selectively blocks the light response of ON-bipolar cells; APB was used to separate synaptic inputs into those which originate from ON- vs. OFF-bipolar cells. This approach clearly demonstrates that the light response of the vast majority of sustained ON-ganglion cells is primarily the result of sustained excitatory inputs that arise (directly or indirectly) from ON-bipolar cells. 3. APB revealed the presence of transient excitatory OFF-inputs in many sustained ON-ganglion cells that are normally not evident. 4. Five sustained ON-ganglion cells were intracellularly stained with HRP and their morphology was analyzed with the aid of a computer-assisted neuron reconstruction system. The stained cells are anatomically similar, based on quantitative analysis of a number of morphological parameters. The dendritic trees of all five cells are primarily confined to sublamina b of the inner plexiform layer, although some cells have a small number of processes that ramify in sublamina a. These latter processes may relate to the transient excitatory OFF-inputs revealed with APB application. 5. Ganglion cells which are morphologically similar to the stained, intracellularly sustained ON-ganglion cells were found in a collection of Golgi-like cells that were labeled by retrograde HRP transport. This raises the possibility that sustained ON-ganglion cells in the mudpuppy may constitute a morphologically identifiable class of retinal ganglion cells in this species. There is also some suggestion that a morphologically similar class of OFF-cells may be present.