9.5.2  Establishing a Retinal Prosthesis/Synaptic Interface

9.5.2.1  The Proximity Requirement

Prior to the fabrication of microfluidic devices for retinal prosthesis, the general requirements for retinal stimulation via neurotransmitters must the considered. It should be noted that inter-neuronal communication occurs primarily at the synapse. Thus, neurotransmitter-based retinal prosthesis devices must localize their delivery to retinal layers that contain synapses for the target cells of interest. Proximity between target dendrites and sites of neurotransmitter delivery is critical for two primary reasons. First, diffusion is a relatively slow process that will increase the latency between stimulation and response, significantly reducing the effective stimulus update rate. Taking into account the tissue tortuosity factor, the coefficient for diffusion of L-glutamate, the primary excitatory retinal neurotransmitter, at 37°C is approximately 10 × 10−6  cm2 s−1 [37, 74]. This translates to a linear diffusion rate of approximately 33 mm/s. Thus, if the site of neurotransmitter release is 33 mm away from the target dendrites, the response latency will be 1 s. Limited to diffusional delivery, neurotransmitter-based retinal prostheses would be constrained to very low frame rates. Proximity is also critical for efficient delivery of neurotransmitter to target synapses. The concentrations required to elicit neuronal responses to the exogenous application of L-glutamate are relatively high (see discussion below). Thus, diffusional dilution over longer distances would necessitate higher total doses of L-glutamate. In addition, excitatory amino acid transmitter pumps actively remove L-glutamate from the extracellular space. This is desirable in that these pumps rapidly dampen neuronal responses to the exogenous application of L-glutamate, improving the dynamic range, spatial and temporal resolution of response. However, if there is poor proximity between stimulation sites and target dendrite populations, these pumps may increase the threshold quantity of L-glutamate release required to achieve neuronal stimulation.

The proximity requirement for neurotransmitter-based retinal prostheses may necessitate that these devices penetrate into dendritic retinal sublaminae of the inner or outer plexiform layers. The concept of chemically inducing neurons to extend synaptic contacts to a retinal prosthesis has been proposed [51, 52, 66]. Epiretinal or sub-retinal neurotransmitter-based retinal prostheses or versions of these devices that penetrate into the retina could, incorporate drug-delivery methods to release chemo-attractant molecules that induce the migration of dendrites toward stimulation sites. The loss of afferent input to bipolar cells due to photoreceptor cell loss in retinal degeneration does induce bipolar cells to re-direct their dendrites toward the inner retina where they have been reported to create self-stimulation loop circuits [60–62]. Thus, there may be a period of time during which these deafferented bipolar cells may be induced to synapse upon a sensory substitution implant. This may occur as a consequence of the sensory substitution, itself. Or, perhaps the controlled release of growth factors from a retinal prosthetic device could provide a signal to dendrites that would promote the extension and maintenance of synapses to the device. Retinal ganglion cells maintain their synaptic

contacts with their afferent bipolar and amacrine cells and do not become de-afferented as a consequence of the retinal degeneration. Thus, it may be more difficult to induce these cells to alter their well established dendritic organization.

9.5.2.2  Convective Delivery of Neurotransmitters Via Microfluidics

To overcome the temporal constraints of neurotransmitter diffusion some retinal prosthesis designs employ microfluidic technology capable of convective delivery. Two groups have worked on the development of microfluidic devices, capable of the controlling the release of neurotransmitter in space and time. Iezzi and colleagues at Wayne State University first introduced the concept of a microfluidic neurotransmitter-based retinal prosthetic device [41]. Devoid of valves, the design employs the use of phototriggered neurotransmitters. These neurotransmitters do not activate ligand-gated ion channels prior to their flash photolysis. The “uncage and release” device employs microfluidic channels that incorporate an optical sub- ­system for the spatially and temporally controlled activation of phototriggered neurotransmitters. An electrical current is then used to iontophoretically and/or electro-osmotically eject the charged, uncaged neurotransmitter from a microfluidic aperture or microneedle into close proximity to the target dendrites. This design involves storing a reservoir of caged L-glutamate prodrug and involves optical and electrical means for controlled release. This potentially minimizes the possibility of a dose-related L-glutamate induced excitoxicity. Finlayson and Iezzi [80] have shown that the localized convective delivery of L-glutamate via pneumatic ejection results in linear RGC dose-response firing with response latencies of 200 ms. These preliminary results validate the utility of convective neurotransmitter delivery for retinal prosthesis.

Another group at Stanford University has also developed microfluidic circuits that employ electroosmotic flow for the controlled delivery of neurotransmitters in space and time. They have demonstrated that electric field-driven fluid ejection of bradykinin was effective in stimulating PC-12 cells cultured on the stimulation system [81–84].

9.5.2.3  Functionalized Surfaces for Neurotransmitter Stimulation

Pepperberg and associates have been developing functionalized surfaces coated with tethered neurotransmitters for neuronal stimulation [73, 89, 105, 108]. According to the design concept, an electrical or other control signal will modulate the capacity of tethered molecules to bind to synaptic or extra-synaptic neurotransmitter receptors. Neurotransmitter analogs such as the muscimol, bound to biotin for the future purpose of adsorption to surfaces, rendering them “functionalized” have been shown to activate GABA receptors in an oocyte model. Since the neurotransmitter–biotin conjugates will ultimately be adsorbed to the surface of the implant, solid posts could be used to assure that stimulation occurs within the desired retinal layers.

9.5.2.4  Synaptic Requirements for L-Glutamate Mediated Neuronal Stimulation

Any system for delivering neurotransmitters to the retina for the purpose of retinal prosthesis will be required to match doses of L-glutamate required by target neurons. Consequently, an analysis of the anatomy and physiology of the synapse may be useful in establishing operating parameters for neurotransmitter-based retinal stimulators.

The requirements for neurotransmitter stimulation of the retina differ according to the target cells for stimulation. ON and OFF pathways are first established at the bipolar cell level. Thus, stimulation at this level may permit selective ON and OFF stimulation selectivity. Depending upon whether the retinal prosthesis is placed epiretinal or subretinal, microneedles may be necessary to deliver neurotransmitter to target neuronal cell dendrites. In degenerating retina, bipolar cells that have lost their photoreceptor input redirect their afferent dendrites toward the inner plexiform layer (IPL). Within the IPL RGC afferents synapse. Neurotransmitter stimulation directed toward ganglion cells must reach this region. Within the IPL, it may be possible to stimulate bipolar cell dendrites and/ or RGCs directly.

The rate of quantal excitation to RGCs in response to visual stimulation has been examined. Any neurotransmitter-based retinal prosthesis will need to mimic patterns of quantal excitation induced by visual stimulation. Freed determined that the just-maximal sustained RGC response to visual stimulation was induced by 3,700 quanta of L-glutamate per second, among all synapses [25, 26]. Studies of the number of L-glutamate molecules per synaptic vesicle report a range between 500 and 10,000 [87]. Thus, between 1.85 and 37 × 106 L-glutamate molecules per second would be required to induce a sustained RCG response. Freed and Sterling reported that there are approximately 550 bipolar synapses upon an ON alpha-RGC in the area centralis [27]. At 10° eccentricity, the larger membrane surface area of ON alpha-RGCs causes them to have approximately 2,200 bipolar cell synapses, since the density of bipolar cell synapses on the membrane is constant [25, 26]. Based upon a synapse diameter of 200 nm2 and a synaptic cleft of 20 nm, the volume of each synapse is approximately 2.5 al [103]. Thus, the total synaptic volume for a single ON alpha-RGC ranges between 1.38 fl near the area centralis and 5.5 fl at 10° eccentricity. Using the lowest molar quantity of L-glutamate needed for sustained RGC stimulation, combined with the largest total synaptic volume for an ON alpha RGC we arrive at a predicted minimum molar concentration of L-glutamate necessary for stimulation by a neurotransmitter-based retinal prosthesis of 0.55 mM L-glutamate. By taking the higher molar quantity of L-glutamate from the above computations, divided by the smallest total synaptic volume for an ON alpha-RGC, we predict that the upper concentration for L-glutamate required for sustained stimulation is 11.1 mM. This range is consistent with our unpublished experimental findings for RGC stimulation via exogenous application of L-glutamate in normal Sprague–Dawley, RCS and S334-ter-4 rats.