Michael Ariel, Ph.D. - US grants

Directionally sensitive (DS) retinal ganglion cells exist in most, if not all, vertebrate retinas. Yet little direct information is known about the retinal mechanism which generate directional sensitivity. This project proposes to study the DS cell and its amacrine cell inputs by use of intracellular and extracellular recording techniques and pharmacological manipulations. Using a superfused retinal eyecup of turtle and rabbit, the effects of synaptic transmitters/modulators and their antagonists can be tested on specific response properties of the DS cell. These results, coupled with the effects of the same agents in low Ca++/high Mg++ perfusate which blocks synaptic transmission, will determine whether certain synaptic interactions occur on the DS ganglion cell membrane. Information based on extracellular recordings and synaptic pharmacology indirectly suggests that several amacrine cell inputs to specialized regions of the ganglion cell membrane are responsible for directional sensitivity. In order to confirm the underlying wiring pattern of these DS cells, they will be stained by injection of intracellular dyes. Subsequent staining with histochemical markers for various amacrine cell naurotransmitters will visualize the interactions between these cells. Knowledge of the neural circuitry for DS cells in the mammalian retina may further the diagnostic usefulness of human optokinetic nystagmus since DS cells provide a major input to the accessory optic system which is responsible for such eye movements.

The vestibuloocular reflex (VOR) normally moves the eyes opposite that of head rotation in order to stabilize the visual image on the retina. VOR gain (response-stimulus) has been known to adapt to a new value alter a long-term modification of the sensory input and thus stabilize the retinal image to facilitate visual processing performed elsewhere in the brain. This proposal will study the origins of this neural plasticity in the cerebellum and brainstem during visual and vestibular mismatch produced in vitro. Using a novel in vitro turtle brain preparation with the eyes and temporal bone attached, natural sensory stimuli will provide inputs for long-term VOR adaptation. Visual patterns of whole field visual motion will be imaged onto the entire retinal surface, while the entire preparation is rotated on a rate table. Preliminary results show that under these conditions, normal visual and vestibular responses are elicited by the first order neurons in the brainstem. The velocity of visual pattern motion is encoded in neurons of the accessory optic system (Basal Optic Nucleus) and pretectum (Mesencephalic Lentiform Nucleus), while volcity of the head rotation of head rotation is encoded in the vestibular nucleus. Responses to either of these sensory signals have also been recorded in the cerebellar cortex. This turtle brain preparation remains responsive to these natural sensory stimuli after days in vitro. There are three aims of this project. First, neurons will be identified in pathways involved in adaptive changes of the VOR; the cerebellar cortex and vestibular nucleus. Second, the normal visual and vestibular responses of these neurons will be characterized using sinusoidal stimuli. Finally, the brain will be exposed to both sensory stimuli simultaneously and continuously, such that visual motion will signal an instability in the retinal image along the same axis as the rotation of the head that drives the oculomotor reflex. Within hours of exposure to these paired sensory signals, it is expected that the response to vestibular stimulation alone will change, in an attempt to reduce the instability in the retinal image. This study will identify the adaptive cells, their response properties, and the time course of adaptation. This project also develops an experimental preparation in which future intracellular studies can determine the synaptic mechanisms by which neural plasticity is generated and can identify its presynaptic and post-synaptic components. A full understanding of this neural process may lead to better treatments for visual and balance disorders.

DESCRIPTION: A neuron processes information by integrating many synaptic inputs onto its dendritic tree, leading to its spike output. This project will investigate this neural processing by stimulating individual sensory afferents to a neuron in the vertebrate brainstem, using a sensory pathway which has the unique advantage of lacking topography. As a consequence, its afferent cell bodies are widely distributed in the sensory periphery. Specifically, intracellular recordings in the accessory optic system will be performed in a unique in vitro turtle brain preparation in which the eyes remain attached and the brain remains visually responsive. Neurons in the accessory optic system presumably receive their synaptic inputs from a distinct class of retinal cell, the direction sensitive ganglion cell. The interaction between the presynaptic terminals and a postsynaptic cell performs an essential conversion from local retinal directional information into a measure of global image motion, called retinal slip, that has a well defined role in vestibular and oculomotor reflexes. The turtle's accessory optic system is called the basal optic nucleus, a surface brainstem structure whose neurons are easy to locate in vitro. Postsynaptic events of cells in the basal optic nucleus are readily recorded with high fidelity in the whole cell configuration using patch pipettes. Visual and electrical stimulation of individual retinal afferents will be used to study the size and shape of unitary postsynaptic events as well as their direction tuning. Stimulating two afferents will determine how two synaptic potentials interact on the postsynaptic membrane. This interaction is complicated by the finding that synaptic responses to individual afferents are quite variable in amplitude and that the postsynaptic membrane has voltage sensitive channels that can be modulated by synaptic potentials. To further understand this interaction, we will also study the passive membrane properties of each cell, the voltage sensitive channels in its membrane, the ionic and pharmacological nature of the synaptic currents and the cell's morphology. This full analysis will ultimately clarify the neural transformations from the retinal synaptic input to the accessory optic system output. The results of these studies will provide insights into the functioning of neurons that encode the direction of visual field motion in order to maintain our balance and stabilize our gaze. Such visual processing will also provide an excellent model to understand general mechanisms of synaptic integration and the sensory processing of the brainstem.

When moving around in the world, we see what is called 'optic flow' of the visual world, giving information about our direction and speed of movement. Signals from the neurons (nerve cells) in the retina of the eye are carried by the optic nerve to the brain. One pathway called the accessory optic system (AOS) carries information about motion over the whole visual field to nerve centers called nuclei in the brainstem. Within the retina there are many neurons that are directionally sensitive, being excited by a visual stimulus that moves in one direction, and inhibited by motion in the opposite direction. It is not clear how these excitatory and inhibitory inputs are combined by the neurons in the central nuclei, except that the interactions are more complex than simply linear addition. This project uses electrophysiological recordings from inside single cells in an unusual preparation of an entirely isolated turtle brain, to study interactions of directionally selective inputs responding to the same part of the visual world. Pharmacological manipulations are used to clarify the separate functions of the excitatory and inhibitory inputs, and better understand the membrane properties underlying the sensory integration.
Results will be critical for understanding the fundamental importance of visual signals in controlling stable eye position, posture, and locomotion in the real world, and the impact will extend beyond visual neuroscience to understanding functional integrative mechanisms at the cellular level in the brain. The project provides excellent training for a graduate student.

DESCRIPTION: (Adapted from the Investigator's Abstract) This is a revised application (R01 03894-01A) that describes novel experiments using an in vitro brain preparation with attached temporal bones to study the initial vestibular processing of the vertebrate brainstem. The Principal Investigator initially developed these innovating techniques to study visual inputs to reflex paths that stabilize the retinal image. In this grant application, only head rotation responses will be studied in the vestibular nuclei. Whole-cell patch recordings of these responses will be examined before and after unilateral reversible lidocaine inactivation of the eighth nerve. Ipsilateral monosynaptic excitatory canal afferents converge onto neurons in the vestibular nucleus. A polysynaptic input is also thought to reach the same neurons from a contralateral canal with the corresponding axis of rotation. Vestibular nucleus neurons also display several response types during natural horizontal head rotation. Cells respond to motion to or away from the side of the recording, and their spike discharges can encode the head's velocity or acceleration. To elucidate the underlying neural circuitry of the vestibular nuclei, from the eighth nerve afferents and between the two nuclei, high-resolution patch recordings of individual synaptic events will be made as head rotation and electrical nerve stimulation evoke the excitatory and/or inhibitory pathways. Effects of synaptic drug applications will help identify the neurotransmitters involved in the monosynaptic and polysynaptic circuits. The membrane properties of the vestibular nucleus cells will be analyzed to see how they modify those synaptic inputs to yield the spike output of different vestibular response types. Redundant sensory input from both labyrinths is useful to improve vestibular sensitivity and to compensate for a unilateral loss. This project analyzes these bilateral inputs to gain an understanding of their role in the natural processing that results in a normal sense of balance or produces the feeling of vertigo during a pathological condition.

[unreadable] DESCRIPTION (provided by applicant): The cerebellar cortex (Cb) is a continuous neural layer of gray matter made up of identical repeating processing modules. Although the circuitry of these modules is similar for all vertebrates, the cortical surface is highly foliated in birds and mammals with complex regional specializations. Therefore, an analysis of Cb topography and its function is difficult because only a small fraction of cortical area is observed on the exposed folial surface. [unreadable] [unreadable] We posit that a radial system of inputs onto Purkinje cells from segregated Cb afferents and [unreadable] ascending granule cell axons is the primary processing unit of the Cb module. This primary system is acted on by a secondary, orthogonal system of parallel fibers and inhibitory interneurons. The proposed study will test this hypothesis with a unique in vitro turtle Cb preparation with the brainstem and sensory nerves attached. The primary advantage of the flat turtle Cb is that it is thin enough to permit optical recordings of the entire Cb surface during trans-illumination. Using voltage-sensitive absorbance dyes, Cb topography can be characterized from large responses to individual sensory stimuli. The small cortical size also permits a topographic anatomical analysis of afferent inputs based on a complete reconstruction of serial sections. [unreadable] [unreadable] Optical recording and pathway tracing experiments will be performed to reveal how sensory afferents map onto the Cb and how these different maps overlap. This project will focus on the effects of visual and vestibular afferents by measuring optical responses in vitro to natural stimulation or electrical microstimulation within the brainstem. Additional experiments will examine the basic cerebellar module: mossy fiber inputs, the radial and orthogonal axonal outputs of the granule cell and the convergence of excitatory and inhibitory inputs onto the Purkinje cell. Finally, climbing fiber inputs to Cb will be measured anatomically and physiologically to understand their role in plasticity and modulation of information flow through the basic cerebellar module. This in vitro preparation will thereby provide a unique model system for the study of learning and adaptation of motor behaviors. [unreadable] [unreadable]