Structure and functional connections of presynaptic terminals in the vertebrate retina revealed by activity-dependent dyes and confocal microscopy.

The fluorescent dyes sulforhodamine 101 (SR 101) and FM1-43 were used as activity-dependent dyes (ADDs) to label presynaptic terminals in the retinas of a broad range of animals, including amphibians, mammals, fish, and turtles. The pattern of dye uptake was studied in live retinal preparations by using brightfield, fluorescence, and confocal microscopy. When bath-applied to the retina-eyecup, these dyes were avidly sequestered by the presynaptic terminals of virtually all rods, cones, and bipolar and amacrine cells; ganglion cell dendrites and horizontal cells lacked significant dye accumulation. Other structures stained with these dyes included pigment epithelial cells, cone outer segments, and Müller cell end-feet. Studies of dye uptake in dark- and light-adapted preparations showed significant differences in the dye accumulation pattern in the inner plexiform layer (IPL), suggesting a dynamic, light-modulated control of endocytotic activity. Presynaptic terminals in the IPL could be segregated on the basis of volume: bipolar varicosities in the IPL were typically larger than those of amacrine cells. The combination of retrograde labeling of ganglion cells and presynaptic terminal labeling with ADDs served as the experimental preparation for three-dimensional reconstruction of both structures, based on dual detector, confocal microscopy. Our results demonstrate a new approach for studying synaptic interactions in retinal function. These findings provide new insights into the likely number and position of functional connections from amacrine and bipolar cell terminals onto ganglion cell dendrites.

Responses to vertical vestibular stimulation of neurons in the nucleus reticularis gigantocellularis in rabbits.

1. Because the nucleus reticularis gigantocellularis (NRGc) receives a substantial descending projection from the caudal vestibular nuclei, we used extracellular single-unit recording combined with natural vestibular stimulation to examine the possible peripheral origins of the vestibularly modulated activity of caudal NRGc neurons located within 500 microns of the midline. Chloralose-urethan anesthetized rabbits were stimulated with an exponential "step" and/or static head-tilt stimulus, as well as sinusoidal rotation about the longitudinal or interaural axes providing various combinations of roll or pitch, respectively. Recording sites were reconstructed from electrolytic lesions confirmed histologically. 2. More than 85% of the 151 neurons, in the medial aspect of the caudal NRGc, responded to vertical vestibular stimulation. Ninety-six percent of these responded to rotation onto the contralateral side (beta responses). Only a few also responded to horizontal stimulation. Seventy-eight percent of the neurons that responded to vestibular stimulation responded during static roll-tilt. One-half of these neurons also responded transiently to the change in head position during exponential "step" stimulation, suggesting input mediated by otolith and semicircular canal receptors or tonic-phasic otolith neurons. 3. Seventy-five percent of the responsive neurons had a "null plane." The planes of stimulation resulting in maximal responses, for cells that responded to static stimulation, were distributed throughout 150 degrees in both roll and pitch quadrants. Five of these cells responded only transiently during exponential "step" stimulation and responded maximally when stimulated in the plane of one of the vertical semicircular canals. 4. The phase of the response of the 25% of medial NRGc neurons that lacked "null planes" gradually shifted approximately 180 degrees during sinusoidal vestibular stimulation as the plane of stimulation was shifted about the vertical axis. These neurons likely received convergent input with differing spatial and temporal properties. 5. The activity of neurons in the medial aspect of the caudal NRGc of rabbits was modulated by both otolithic macular and vertical semicircular canal receptor stimulation. This vestibular information may be important for controlling the intensity of the muscle activity in muscles such as neck muscles where the load on the muscle is affected by the position of the head with respect to gravity. Some of these neurons may also shift muscle function from an agonist to an antagonist as the direction of head tilt changes.

Cholinergic projection to the dorsal cap of the inferior olive of the rat, rabbit, and monkey.

The inferior olive is divided into several subnuclei that receive specific sensory information. The caudal dorsal cap of the medial accessory subdivision of the inferior olive receives horizontal optokinetic information from the nucleus of the optic tract. The immediately subjacent beta-nucleus receives vertical vestibular information mediated by a GABAergic pathway originating from the ipsilateral descending and medial vestibular nuclei. None of the transmitters to the dorsal cap have been identified. Using choline acetyltransferase (ChAT) immunohistochemistry, we have identified a cholinergic pathway that terminates exclusively in the dorsal cap of rats and monkeys. No other division of the inferior olive received a significant cholinergic innervation. In the rabbit, immunostaining for ChAT reveals a weaker and more diffuse cholinergic innervation of both the dorsal cap and the subjacent beta-nucleus. In rats and rabbits we injected iontophoretically the orthograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) into the medial and descending vestibular nuclei (MVN, DVN) as well as the nucleus prepositus hypoglossi (NPH) in order to trace the possible origin of the cholinergic projection. PHA-L injections into the NPH and medial aspect of the MVN labeled terminals within the contralateral dorsal cap. PHA-L injections in the central and lateral aspects of the MVN as well as the DVN labeled the ipsilateral beta-nucleus. Pressure injections of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) in the caudal dorsal cap of the rabbit inferior olive demonstrated a predominantly contralateral projection to the dorsal cap from the lateral aspect of the NPH. However, pressure injections of HRP into the caudal dorsal cap combined with ChAT immunohistochemistry in the rabbit demonstrated that most of the neurons of the NPH that projected to the dorsal cap were not cholinergic, and that most of the ChAT-positive neurons within the NPH occupied a more ventral location than the neurons within the NPH that were retrogradely labeled from the HRP injection into the contralateral dorsal cap. In the rat, we made lesions in the MVN, DVN and NPH by injection of ibotenic acid (0.3-0.5 microliter), in an attempt to deplete the dorsal cap of the inferior olive of its cholinergic input. Lesions confined to the NPH and medial aspect of the MVN of the rat caused a loss of ChAT staining in the contralateral dorsal cap. Lesions placed more laterally within the MVN or DVN failed to deplete ChAT-positive terminals in the contralateral or ipsilateral dorsal caps. The dorsal cap of the rat and monkey receives a discrete cholinergic projection.(ABSTRACT TRUNCATED AT 400 WORDS)

Activity of neurons in the beta nucleus of the inferior olive of the rabbit evoked by natural vestibular stimulation.

The inferior olive (IO) appears to be organized functionally in discrete subnuclei that receive transmitter-specific inputs. In particular, the IO receives a GABAergic input that is most densely concentrated in the beta-nucleus. In this experiment, we examined the functional specificity of neurons in the beta-nucleus of the IO of rabbits by recording their activity during natural vestibular and optokinetic stimulation. Rabbits were anesthetized and positioned in a triaxial servo- controlled rate table with the head fixed at the center of rotation. Contour-rich visual stimuli were rear-projected onto a 70 deg tangent screen and moved at constant velocities. Recording sites in the beta-nucleus were verified by subsequent histological analysis of marking microlesions. Neurons in the beta-nucleus responded to roll vestibular stimulation about the longitudinal axis. These neurons were excited when the rabbit was rolled onto the side which was contralateral to the recording site, and inhibited when the rabbit was rolled ipsilaterally. Thirty-eight of the 75 beta-nucleus neurons that were responsive to roll vestibular stimulation also responded to static tilt, indicating an otolithic as well as a vertical semicircular canal origin of the vestibular input. The modulated activity of none of the neurons could be attributed to stimulation of the horizontal semicircular canals. All the recorded neurons were found in a region of the beta-nucleus that was retrogradely labeled following HRP injections into the cerebellar nodulus. Using a "null point" technique, we found that there was a differential projection of information from the anterior and posterior semicircular canals onto to the beta-nucleus. Stimulation of the ipsilateral anterior-contralateral posterior semicircular canals modulates activity of the neurons in the caudal 500 microns of the beta-nucleus. Stimulation of the ipsilateral posterior-contralateral anterior semicircular canals modulates activity of neurons located more rostrally. beta-nucleus neurons and the olivocerebellar circuits in which they participate may constitute an important pathway for the control and adaptive modification of postural reflexes.