Justin Elstrott

Genetic Identification of an On-Off Direction- Selective Retinal Ganglion Cell Subtype Reveals a Layer-Specific Subcortical Map of Posterior Motion

Motion detection is an essential component of visual processing. On-Off direction-selective retinal ganglion cells (On-Off DSGCs) detect objects moving along specific axes of the visual field due to their precise retinal circuitry. The brain circuitry of On-Off DSGCs, however, is largely unknown. We report a mouse with GFP expressed selectively by the On-Off DSGCs that detect posterior motion (On-Off pDSGCs), allowing two-photon targeted recordings of their light responses and delineation of their complete map of central connections. On-Off pDSGCs project exclusively to the dorsal lateral geniculate nucleus and superior colliculus and in both targets form synaptic lamina that are separate from a lamina corresponding to non-DSGCs. Thus, individual On-Off DSGC subtypes are molecularly distinct and establish circuits that map specific qualities of directional motion to dedicated subcortical areas. This suggests that each RGC subtype represents a unique parallel pathway whose synaptic specificity in the retina is recapitulated in central targets.

DSCAM and DSCAML1 function in self-avoidance in multiple cell types in the developing mouse retina.

DSCAM and DSCAM-LIKE1 (DSCAML1) serve diverse neurodevelopmental functions, including axon guidance, synaptic adhesion, and self-avoidance, depending on the species, cell type, and gene family member studied. We examined the function of DSCAM and DSCAML1 in the developing mouse retina. In addition to a subset of amacrine cells, Dscam was expressed in most retinal ganglion cells (RGCs). RGCs had fasciculated dendrites and clumped cell bodies in Dscam(-/-) mice, suggesting a role in self-avoidance. Dscaml1 was expressed in the rod circuit, and mice lacking Dscaml1 had fasciculated rod bipolar cell dendrites and clumped AII amacrine cell bodies, also indicating a role in self-avoidance. Neurons in Dscam or Dscaml1 mutant retinas stratified their processes appropriately in synaptic laminae in the inner plexiform layer, and functional synapses formed in the rod circuit in mice lacking Dscaml1. Therefore, DSCAM and DSCAML1 function similarly in self-avoidance, and are not essential for synaptic specificity in the mouse retina.

Transgenic Mice Reveal Unexpected Diversity of On-Off Direction-Selective Retinal Ganglion Cell Subtypes and Brain Structures Involved in Motion Processing

On-Off direction-selective retinal ganglion cells (DSGCs) encode the axis of visual motion. They respond strongly to an object moving in a preferred direction and weakly to an object moving in the opposite, “null,” direction. Historically, On-Off DSGCs were classified into four subtypes according to their directional preference (anterior, posterior, superior, or inferior). Here, we compare two genetically identified populations of On-Off DSGCs: dopamine receptor 4 (DRD4)-DSGCs and thyrotropin-releasing hormone receptor (TRHR)-DSGCs. We find that although both populations are tuned for posterior motion, they can be distinguished by a variety of physiological and anatomical criteria. First, the directional tuning of TRHR-DSGCs is broader than that of DRD4-DSGCs. Second, whereas both populations project similarly to the dorsal lateral geniculate nucleus, they project differently to the ventral lateral geniculate nucleus and the superior colliculus. Moreover, TRHR-DSGCs, but not DRD4-DSGCs, also project to the zona incerta, a thalamic area not previously known to receive direction-tuned visual information. Our findings reveal unexpected diversity among mouse On-Off DSGC subtypes that uniquely process and convey image motion to the brain.

Direction Selectivity in the Retina Is Established Independent of Visual Experience and Cholinergic Retinal Waves

Direction selectivity in the retina requires the asymmetric wiring of inhibitory inputs onto four subtypes of On-Off direction-selective ganglion cells (DSGCs), each preferring motion in one of four cardinal directions. The primary model for the development of direction selectivity is that patterned activity plays an instructive role. Here, we use a unique, large-scale multielectrode array to demonstrate that DSGCs are present at eye opening, in mice that have been reared in darkness and in mice that lack cholinergic retinal waves. These data suggest that direction selectivity in the retina is established largely independent of patterned activity and is therefore likely to emerge as a result of complex molecular interactions.

Parallel Regulation of Feedforward Inhibition and Excitation during Whisker Map Plasticity

Sensory experience drives robust plasticity of sensory maps in cerebral cortex, but the role of inhibitory circuits in this process is not fully understood. We show that classical deprivation-induced whisker map plasticity in layer 2/3 (L2/3) of rat somatosensory (S1) cortex involves robust weakening of L4-L2/3 feedforward inhibition. This weakening was caused by reduced L4 excitation onto L2/3 fast-spiking (FS) interneurons, which mediate sensitive feedforward inhibition and was partially offset by strengthening of unitary FS to L2/3 pyramidal cell synapses. Weakening of feedforward inhibition paralleled the known weakening of feedforward excitation. As a result, mean excitation-inhibition balance and timing onto L2/3 pyramidal cells were preserved. Thus, reduced feedforward inhibition is a covert compensatory process that can maintain excitatory-inhibitory balance during classical deprivation-induced Hebbian map plasticity.

Two-photon targeted recording of GFP-expressing neurons for light responses and live-cell imaging in the mouse retina.

Cell type–specific green fluorescent protein (GFP) expression in the retina has been achieved in an expanding repertoire of transgenic mouse lines, which are valuable tools for dissecting the retinal circuitry. However, measuring light responses from GFP-labeled cells is challenging because single-photon excitation of GFP easily bleaches photoreceptors. To circumvent this problem, we use two-photon excitation at 920 nm to target GFP-expressing cells, followed by electrophysiological recording of light responses using conventional infrared optics. This protocol offers fast and sensitive detection of GFP while preserving the light sensitivity of the retina, and can be used to obtain light responses and the detailed morphology of a GFP-expressing cell. Targeting of a GFP-expressing neuron takes less than 3 min, and the retina preparation remains light sensitive and suitable for recording for at least 8 h. This protocol can also be applied to study retinal neurons labeled with other two photon–excitable fluorophores. It is assumed that potential users of this protocol will have a basic understanding of retinal physiology and patch-clamp recording, which are not described in detail here.

GABA-A receptor-mediated signaling alters the structure of spontaneous activity in the developing retina

Ambient GABA modulates firing patterns in adult neural circuits by tonically activating extrasynaptic GABAA receptors. Here, we demonstrate that during a developmental period when activation of GABAA receptors causes membrane depolarization, tonic activation of GABAA receptors blocks all spontaneous activity recorded in retinal ganglion cells (RGCs) and starburst amacrine cells (SACs). Bath application of the GABAA receptor agonist muscimol blocked spontaneous correlated increases in intracellular calcium concentration and compound postsynaptic currents in RGCs associated with retinal waves. In addition, GABAA receptor agonists activated a tonic current in RGCs that significantly reduced their excitability. Using a transgenic mouse in which green fluorescent protein is expressed under the metabotropic glutamate receptor subtype 2 promoter to target recordings from SACs, we found that GABAA receptor agonists blocked compound postsynaptic currents and also activated a tonic current. GABAA receptor antagonists reduced the holding current in SACs but not RGCs, indicating that ambient levels of GABA tonically activate GABAA receptors in SACs. GABAA receptor antagonists did not block retinal waves but did alter the frequency and correlation structure of spontaneous RGC firing. Interestingly, the drug aminophylline, a general adenosine receptor antagonist used to block retinal waves, induced a tonic GABAA receptor antagonist-sensitive current in outside-out patches excised from RGCs, indicating that aminophylline exerts its action on retinal waves by direct activation of GABAA receptors. These findings have implications for how various neuroactive drugs and neurohormones known to modulate extrasynaptic GABAA receptors may influence spontaneous firing patterns that are critical for the establishment of adult neural circuits.

Receptive field mosaics of retinal ganglion cells are established without visual experience.

A characteristic feature of adult retina is mosaic organization: a spatial arrangement of cells of each morphological and functional type that produces uniform sampling of visual space. How the mosaics of visual receptive fields emerge in the retina during development is not fully understood. Here we use a large-scale multielectrode array to determine the mosaic organization of retinal ganglion cells (RGCs) in rats around the time of eye opening and in the adult. At the time of eye opening, we were able to reliably distinguish two types of ON RGCs and two types of OFF RGCs in rat retina based on their light response and intrinsic firing properties. Although the light responses of individual cells were not yet mature at this age, each of the identified functional RGC types formed a receptive field mosaic, where the spacing of the receptive field centers and the overlap of the receptive field extents were similar to those observed in the retinas of adult rats. These findings suggest that, although the light response properties of RGCs may need vision to reach full maturity, extensive visual experience is not required for individual RGC types to form a regular sensory map of visual space.

Expression and function of the neuronal gap junction protein connexin 36 in developing mammalian retina

With the advent of transgenic mice, much has been learned about the expression and function of gap junctions. Previously, we reported that retinal ganglion cells in mice lacking the neuronal gap junction protein connexin 36 (Cx36) have nearly normal firing patterns at postnatal day 4 (P4) but many more asynchronous action potentials than wild‐type mice at P10 (Torborg et al. [2005] Nat. Neurosci. 8:72–78). With the goal of understanding the origin of this increased activity in Cx36–/– mice, we used a transgenic mouse (Deans et al. [2001] Neuron 31:477–485) to characterize the developmental expression of a Cx36 reporter in the retina. We found that Cx36 was first detected weakly at P2 and gradually increased in expression until it reached an adult pattern at P14. Although the onset of expression varied by cell type, we identified Cx36 in the glycinergic AII amacrine cell, glutamatergic cone bipolar cell, and retinal ganglion cells (RGCs). In addition, we used calcium imaging and multielectrode array recording to characterize further the firing patterns in Cx36–/– mice. Both correlated and asynchronous action potentials in P10 Cx36–/– RGCs were significantly inhibited by bath application of an ionotropic glutamate receptor antagonist, indicating that the increase in activity was synaptically mediated. Hence, both the expression patterns and the physiology suggest an increasing role for Cx36‐containing gap junctions in suppressing RGC firing between waves during postnatal retinal development. J. Comp. Neurol. 493:309–320, 2005.

Role of adenylate cyclase 1 in retinofugal map development

The development of topographic maps of the sensory periphery is sensitive to the disruption of adenylate cyclase 1 (AC1) signaling. AC1 catalyzes the production of cAMP in a Ca2+/calmodulin‐dependent manner, and AC1 mutant mice (AC1−/−) have disordered visual and somatotopic maps. However, the broad expression of AC1 in the brain and the promiscuous nature of cAMP signaling have frustrated attempts to determine the underlying mechanism of AC1‐dependent map development. In the mammalian visual system, the initial coarse targeting of retinal ganglion cell (RGC) projections to the superior colliculus (SC) and lateral geniculate nucleus (LGN) is guided by molecular cues, and the subsequent refinement of these crude projections occurs via an activity‐dependent process that depends on spontaneous retinal waves. Here, we show that AC1−/− mice have normal retinal waves but disrupted map refinement. We demonstrate that AC1 is required for the emergence of dense and focused termination zones and elimination of inaccurately targeted collaterals at the level of individual retinofugal arbors. Conditional deletion of AC1 in the retina recapitulates map defects, indicating that the locus of map disruptions in the SC and dorsal LGN of AC1−/− mice is presynaptic. Finally, map defects in mice without AC1 and disrupted retinal waves (AC1−/−;β2−/− double KO mice) are no worse than those in mice lacking only β2−/−, but loss of AC1 occludes map recovery in β2−/− mice during the second postnatal week. These results suggest that AC1 in RGC axons mediates the development of retinotopy and eye‐specific segregation in the SC and dorsal LGN. J. Comp. Neurol. 520:1562–1583, 2012.