Turtle Accessory Optic System Research Articles

Analysis of quantal size of voltage responses to retinal stimulation in the accessory optic system

In the intact vertebrate central nervous system, the quantal nature of synaptic transmission is difficult to measure because the postsynaptic sites may be distributed along a tortuous dendritic tree that cannot be readily clamped spatially to a uniform potential. Titrating the intact brain's extracellular concentration of calcium ions is also challenging because of its strong buffering mechanisms. In this study, using a whole brain with eye attached preparation, quantal neurotransmission was examined in the turtle brainstem in vitro, by recording from accessory optic system neurons that receive direct input from visually responsive retinal ganglion cells. Unitary EPSPs, evoked by microstimulation of a single ganglion cell, were measured during whole cell current-clamp recordings. In this preparation, the neurons exhibit direction-selectivity, despite the hypoxic conditions. Bath application of cadmium to reduce calcium influx also reduced evoked EPSP amplitudes to that of the spontaneous synaptic events. Statistical analyses indicated that these evoked response amplitudes could be well fitted to a Poisson distribution for most brainstem neurons. Therefore, the spontaneous miniature excitatory synaptic events of approximately 1 mV, as also observed during spike blockade of the retina [Kogo, N., Ariel, M., 1997. Membrane properties and monosynaptic retinal excitation of neurons in the turtle accessory optic system. Journal of Neurophysiology 78, 614–627], are likely responses to the neurotransmitter of single vesicles release by retinal axon terminals.

Localization of GABA (γ-aminobutyric acid) markers in the turtle's basal optic nucleus

Recent physiological data have demonstrated that retinal slip, the sensory code of global visual pattern motion, results from complex interactions of excitatory and inhibitory visual inputs to neurons in the turtle's accessory optic system (the basal optic nucleus, BON) [M. Ariel, N. Kogo, Direction tuning of inhibitory inputs to the turtle accessory optic system, J. Neurophysiol. 86 (2001) 2919–2930. [6], N. Kogo, T.X. Fan, M. Ariel, Synaptic pharmacology in the turtle accessory optic system, Exp. Brain Res. 147 (2002) 464–472. [23]]. In the present study, the inhibitory neurotransmitter γ-aminobutyric acid (GABA), its synthetic enzyme, glutamic acid decarboxylase (GAD-67) and its receptor subtypes GABA A and GABA B receptors were localized within the BON. GABA antibodies revealed cell bodies and processes, whereas antibodies against GAD revealed a moderate density of immunoreactive puncta throughout the BON. GAD in situ hybridization labeled BON cell bodies, indicating a possible source of inhibition intrinsic to the nucleus. Ultrastructural analysis revealed terminals positive for GAD that exhibit symmetric synaptic specializations, mainly at neuronal processes having small diameters. Neurons exhibiting immunoreactivity for GABA A receptors were diffusely labeled throughout the BON, with neuronal processes exhibiting more labeling than cell bodies. In contrast, GABA B-receptor-immunoreactive neurons exhibited strong labeling at the cell body and proximal neuronal processes. Both these receptor subtypes are functional, as evidenced by changes of visual responses of BON neurons during application to the brainstem of selective receptor agonists and antagonists. Therefore, GABA may be synthesized by BON neurons, released by terminals within its neuropil and stimulate both receptor subtypes, supporting its role in mediating visually evoked inhibition contributing to modulation of the retinal slip signals in the turtle accessory optic system.

Morphology of the turtle accessory optic system.

Neural signals of the moving visual world are detected by a subclass of retinal ganglion cells that project to the accessory optic system in the vertebrate brainstem. We studied the dendritic morphologies and direction tuning of these brainstem neurons in turtle (Pseudemys scripta elegans) to understand their role in visual processing. Full-field checkerboard patterns were drifted on the contralateral retina while whole-cell recordings were made in the basal optic nucleus in an intact brainstem preparation in vitro. Neurobiotin diffused into the neurons during the recording and was subsequently localized in brain sections. Neuronal morphologies were traced using appropriate computer software to analyze their position in the brainstem. Most labeled neurons were fusiform in shape and had numerous varicosities along their processes. The majority of dendritic trees spread out in a transverse plane perpendicular to the rostrocaudal axis of the nucleus. Neurons near the brainstem surface were often oriented tangential to that surface, whereas more cells at the dorsal side of the nucleus were oriented radial to the brainstem surface. Further analysis of Nissl-stained neurons revealed the largest neurons are located in the rostral and medial portions of the nucleus although neurons are most densely packed in the middle of the nucleus. The preferred directions of the visual responses of the neurons in this sample did not correlate with their morphology and position in the nucleus. Therefore, the morphology of the cells in the turtle accessory optic system appears dependent on its position within the nucleus while its visual responses may depend on the synaptic inputs that contact each cell.

Connectivity of the turtle accessory optic system

Recent whole-cell recordings show that there are multiple synaptic inputs to the accessory optic system of the pond turtle Pseudemys scripta elegans (the basal optic nucleus, BON), suggesting a complex role in visual processing. The BON outputs have now been investigated using transport of diI, rhodamine-conjugated and biotinylated dextrans. Although transport was primarily anterograde, contralateral retinal ganglion cells were labeled retrogradely, confirming that the injection site was a retinal target. Other retrogradely labeled neurons were found ipsilateral to the injection site, in the pretectum, the ventral tegmentum, the dorsal nucleus of the posterior commissure and the lateral habenular nucleus. However, other data indicate that the habenular cells were labeled by spread of the tracer from the BON to the adjacent fasciculus retroflexus and interpeduncular nucleus. Anterogradely labeled fibers projected from BON following three paths, a lateral bundle to the ipsilateral dorsal midbrain, an intermediate bundle to the ipsilateral pretectal area or the posterior commissure and a ventral fiber bundle to the tegmentum bilaterally. Some of these fibers projected caudally through the tegmentum and cerebellar peduncle to terminate just below the Purkinje cell layer of the cerebellar cortex. Fibers that coursed via the intermediate bundle to the posterior commissure were also seen reaching the contralateral pretectal area and the contralateral BON. Injections of the retrograde tracer Fluorogold were also made in the BON to confirm the reciprocal connectivity of both basal optic nuclei. The pathways revealed by these experiments indicate the existence of multiple afferent and efferent connections of the BON, supporting the view that the accessory optic system is more than a simple relay of retinal signals into the brainstem for optokinetic reflexes.

Synaptic pharmacology in the turtle accessory optic system.

The accessory optic system of the turtle (the basal optic nucleus, BON) receives both excitatory and inhibitory inputs that are direction-sensitive. When the dorsal midbrain is ablated, only the monosynaptic direction-sensitive input from the retina to the BON remains. To better understand the central visual processing performed by the accessory optic system, this study identifies the neurotransmitters and their receptors that mediate the synaptic excitation and inhibition of BON cells. We used a reduced in vitro turtle brainstem preparation in which the two eyes and brain were isolated pharmacologically. Patch recordings were made on BON neurons while drugs were applied to the brain, with the eyes bathed in control media and either exposed to visual pattern motion or subjected to electrical stimulation. An antagonist of the AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) subtype of glutamate receptor applied within the brain chamber blocked the visual responses. In response to electrical stimulation both excitatory and inhibitory synaptic events were blocked in BON cells, presumably by blocking direct excitation by retinal ganglion cell axons in the BON and indirect excitation of inhibitory interneurons elsewhere in the brainstem. An NMDA receptor antagonist was ineffective, even when the response was measured in a BON cell depolarized in Mg(2+)-free media. A GABA(A) receptor on the BON cell mediates the inhibitory responses to retinal stimulation. Injection of lidocaine into the contralateral eye caused an increase in spontaneous inhibitory post-synaptic potentials (IPSPs), suggesting that a tonic retinal output exists that reduces brainstem inhibition of BON cells. Also, there may be tonic inhibition of an excitatory path to BON neurons from within the brainstem, because bicuculline increased spontaneous excitatory post-synaptic potentials (EPSPs) observed in a BON cell without retinal input. These results indicate that the BON is a site of complex visual processing of competing visual signals and provide insight into how an interaction of excitation and inhibition creates a retinal slip signal in the accessory optic system.

Direction tuning of inhibitory inputs to the turtle accessory optic system.

Neurons in turtle accessory optic system (basal optic nucleus, BON) were studied to compare excitatory and inhibitory visual inputs. Using a reduced in vitro brain stem preparation with the eyes attached, previous studies only showed a monosynaptic retinal input to the BON from direction-sensitive retinal ganglion cells that share a common preferred direction. Now using an intact brain stem preparation, not only did BON neurons display inhibitory postsynaptic potentials [IPSP(C)s] spontaneously, but IPSP(C)s were also evoked by visual pattern motion, they had their polarity reversed near the chloride equilibrium potential and they were blocked by the GABA(A) antagonist bicuculline. Because excitatory postsynaptic currents had reversal potentials >0 mV, BON cells were recorded using patch electrodes filled with QX-314 or Cs+ to measure the cell's direction tuning also at that higher reversal potential. For most of the BON neurons studied, their visual excitation and inhibition had a very similar preferred direction, indicating that both synaptic inputs were maximally active onto the same cell under the same stimulus conditions. These competing inputs may result from connections between the pretectum and accessory optic nuclei. Such synaptic interactions may serve a functional role in the visual processing necessary to create retinal slip signals for oculomotor control.

Analysis of direction-tuning curves of neurons in the turtle's accessory optic system.

Visual-movement sensitivity of neurons in the turtle's accessory optic system was investigated. Neuronal responses to stimulus direction and speed were analyzed to determine whether they reflect processing by a one-dimensional encoder of visual motion or whether they indicate directional integration of presynaptic direction-sensitive responses whose maximal-response directions are distributed. Both of these mechanisms make predictions about the functional relationship between stimulus direction and response. The responses of single units in the basal optic nucleus to visual stimulation in different directions were described by both cosine and wrapped normal fitting functions. The wrapped normal function (a Gaussian curve mapped onto a circle) performed at least as well as the cosine function and described directional tuning curves of varying widths. Unlike cosines, the addition of two wrapped normals could describe multi-lobed directional data. Next, it was demonstrated that these neurons did not encode visual motion projected onto a single, spatial axis. Responses to the projected speed along the maximal-response direction were systematically lower than responses to the actual speed along that direction. Thus, for speeds above 1 degrees/s, neuronal response varies with respect to direction but not speed. Summation of presynaptic direction-sensitive responses with distributed maximal-response directions (referred to as directional integration) is discussed as a means of accounting for these results.

Direction Tuning of Individual Retinal Inputs to the Turtle Accessory Optic System

Neurons in turtle accessory optic system [basal optic nucleus (BON)] were recorded to study convergence of retinal afferents, using whole-cell patch electrodes in a reduced in vitro brainstem preparation with the eyes attached. BON cells primarily exhibit EPSPs from a contralateral retinal ganglion cell input and generate an output of action potentials. Visual responses were evoked by different directions of either full-field or local moving patterns. Direction tuning of action potentials was compared with that of EPSPs detected by passing the membrane voltage through an AC amplifier and window discriminator. This rough measure of retinal input indicated that the direction tuning of the full-field excitatory input from the retina matched that of the spike output for the same BON cell. Using local patterns within the receptive fields of the BON cells, it was estimated that one to four adjacent retinal inputs were being stimulated. The direction tuning of these inputs had preferred directions that were similar to that of the full-field spike output of the cell, irrespective of where the small window was placed within the receptive field. Because more than one retinal input may have been stimulated by the small stimulus window, subsets of those EPSPs that may represent responses of a single retinal afferent were identified based on their amplitude and rise time. Again, the preferred direction of those putative single retinal afferents matched the direction tuning of the spike output of the BON cell. These findings are discussed in terms of the formation of the retinal slip signal by the BON.

Membrane properties and monosynaptic retinal excitation of neurons in the turtle accessory optic system.

Using an eye-attached isolated brain stem preparation of a turtle, Pseudemys scripta elegans, in conjunction with whole cell patch techniques, we recorded intracellular activity of accessory optic system neurons in the basal optic nucleus (BON). This technique offered long-lasting stable recordings of individual synaptic events. In the reduced preparation (most of the dorsal structures were removed), large spontaneous excitatory synaptic inputs [excitatory postsynaptic potentials (EPSPs)] were frequently recorded. Spontaneous inhibitory postsynaptic potentials were rarely observed except in few cases. Most EPSPs disappeared after injection of lidocaine into the retina. A few EPSPs of small size remained, suggesting that these EPSPs either were from intracranial sources or may have been miniature spontaneous synaptic potentials from retinal ganglion cell axon terminals. Population EPSPs were synchronously evoked by electrical stimulation of the contralateral optic nerve. Their constant onset latency and their ability to follow short-interval paired stimulation indicated that much of the population EPSP's response was monosynaptic. Visually evoked BON spikes and EPSP inputs to BON showed direction sensitivity when a moving pattern was projected onto the entire contralateral retina. With the use of smaller moving patterns, the receptive field of an individual BON cell was identified. A small spot of light, projected within the receptive field, guided the placement of a bipolar stimulation electrode to activate retinal ganglion cells that provided input to that BON cell. EPSPs evoked by this retinal microstimulation showed features of unitary EPSPs. Those EPSPs had distinct low current thresholds. Recruitment of other inputs was only evident when the stimulation level was increased substantially above threshold. The average size of evoked unitary EPSPs was 7.8 mV, confirming the large size of synaptic inputs of this system relative to nonsynaptic noise. EPSP shape was plotted (rise time vs. amplitude), with the use of either evoked unitary EPSPs or spontaneous EPSPs. Unlike samples of spontaneous EPSPs, data from many unitary EPSPs formed distinct clusters in these scatterplots, indicating that these EPSPs had a unique shape among the whole population of EPSPs. In most BON cells studied, hyperpolarization-activated channels caused a slow depolarization sag that reached a plateau within 0.5-1 s. This property suggests that BON cells may be more complicated than a simple site for convergence of direction-sensitive retinal ganglion cells to form a central retinal slip signal for control of oculomotor reflexes.

Electrophysiological evidence for a direct projection of direction-sensitive retinal ganglion cells to the turtle's accessory optic system

1. The direct retinal input pathway to the basal optic nucleus (BON), the primary nucleus of the turtle accessory optic system, was characterized physiologically. We tested the hypothesis that directional information encoded in retinal ganglion cells can influence the BON via a direct pathway. Using an in vitro whole-brain, eyes-attached preparation, we demonstrated the directness of this pathway by 1) antidromic activation of retinal ganglion cells from the contralateral BON and 2) orthodromic activation of the BON from the contralateral optic nerve. 2. Of 72 physiologically classified retinal ganglion cells, 9 could be antidromically activated from the contralateral BON with low current (less than 200 micro A). Eight of these cells were direction-sensitive (DS). The ninth cell did not respond to visual stimulus movement. The antidromic latencies ranged from 2.2 to 6.1 ms with a mean of 3.8 ms. These latencies were quite consistent for each cell, having an average SD of 0.08 ms. Moreover, consistent responses could always be recorded at stimulation rates up to 100 Hz. 3. With current stimulation of the contralateral optic nerve, the orthodromic conduction latency of 17 BON single units ranged from 2.5 to 6.6 ms with a mean of 4.6 ms. These latencies were more variable for an individual cell, having an average SD of 0.3 ms. Responses to individual current pulses could never be consistently evoked at stimulation rates greater than 40 Hz. 4. DS responses were recorded in BON single units after the removal of the dorsal midbrain, including the optic tectum and pretectum as well as the telencephalon. Three of these cells were activated orthodromically by current stimulation delivered to the contralateral optic nerve. Thus directional information reaches the BON via a direct projection from the contralateral retina. 5. Visual response properties of DS retinal ganglion cells were compared with those of BON cells to examine the transformations that take place in the brain stem. Applying a limaçon model to the responses of both DS retinal ganglion cells and BON cells revealed that both types of cells have very similar direction tuning. However, the distribution of maximally responsive directions in the retina may differ from that of the BON. 6. Because DS retinal ganglion cells project directly to the BON, and because BON cells lose their direction sensitivity after retinal application of GABA antagonists, we conclude that the BON receives essential directional information directly from DS retinal ganglion cells. This directional information in the BON may represent a retinal slip error signal necessary for retinal image stabilization.

Independent eye movements in the turtle

In order to evaluate the normal eye movements of the turtle, Pseudemys scripta elegans, the positions of each eye were recorded simultaneously using two search-coil contact lenses. Optokinetic nystagmus (OKN) was strikingly unyoked in this animal such that one eye's slow-phase velocity was substantially independent of that of the other eye. On the other hand, the fast-phase motions of both eyes occurred more or less in synchrony. An eye's slow-phase gain is primarily dependent on the direction and velocity of the stimulus to that eye. Using monocular stimuli, the highest mean gain (0.54 +/- 0.047; mean +/- standard error of mean) occurred using temporal-to-nasal movement at 2.5 deg/s. The mean OKN gain for nasal-to-temporal movement was only 0.13 +/- 0.015 at that velocity. Additionally, using the optimal monocular stimulus (temporal-to-nasal stimulation at 2.5 deg/s) only drove the occluded eye to move nasal-to-temporally at 0.085 deg/s, equivalent to a "gain" of only 0.034 +/- 0.011. The binocular OKN gain during rotational stimuli was higher than monocular gain, especially during nasal-to-temporal movement at high velocities. Also the difference in slow-phase eye velocity between the two eyes was smaller during binocular rotational stimuli. In contrast, when each eye simultaneously viewed its temporal-to-nasal stimulus at an equal velocity, two behaviors were observed. Often, OKN alternated between an animal's left eye and right eye. Occasionally, both eyes moved at equal but opposite velocities. These behavioral data provide a quantitative baseline to interpret the properties of the retinal slip information in the turtle's accessory optic system. Those properties are similar to the behavior of the turtle in that both are tuned to direction and velocity independently for each eye (Rosenberg & Ariel, 1990).