Rod bipolar cells and horizontal cells form displaced synaptic contacts with rods in the outer nuclear layer of the nob2 retina

L-type voltage-dependent calcium channels, including Cav1.4 and the closely related Cav1.3, regulate calcium entry into the synaptic terminals of retinal photoreceptors (Yagi and Macleish, 1994; Schmitz and Witkovsky, 1997; Taylor and Morgans, 1998; Cia et al., 2005). These calcium channels are distinct from the N- and P/Q-type calcium channels that support fast neurotransmitter release in spiking neurons. Instead, at the photoreceptor dark resting potential of −40 mV (Schneeweis and Schnapf, 1999), the L-type calcium channels are tonically activated, supporting continuous glutamate release from photoreceptors. On exposure to light, photoreceptors progressively hyperpolarize, closing calcium channels and reducing glutamate release. The properties of the photoreceptor L-type channels—low-voltage activation, with little voltage dependent inactivation and rapid activation and deactivation kinetics (Barnes and Hille, 1989; Taylor and Morgans, 1998; Koschak et al., 2003; McRory et al., 2004)—allow continuous modulation of the rate of glutamate release to track changing light conditions. Furthermore, the high voltage sensitivity of the channels means that even the absorption of a single photon by a rod photoreceptor, which hyperpolarizes the membrane by ~1 mV, causes a signal that is reliably transmitted to the postsynaptic rod bipolar cell (Field and Rieke, 2002; Berntson et al., 2004). Immunolabeling has localized Cav1.4 to the synaptic layers of the retina—the outer and inner plexiform layers (OPL and IPL)—and more specifically to the ribbon synapses of rod and cone photoreceptors and bipolar cells (Morgans, 2001; Morgans et al., 2001, 2005; Berntson et al., 2003). Immunolabeling for Cav1.4, with either pan-α1-or α1F-specific antibodies, colocalizes with the synaptic ribbon proteins Bassoon and RIBEYE (Brandstatter et al., 1999; Morgans et al., 2005; tom Dieck et al., 2005) and is nested within the crescent-shape of the ribbon, suggesting a localization of Cav1.4 to the plasma membrane at the ribbon’s base. Cav1.4 forms part of the presynaptic cytomatrix at the photoreceptor active zone, where it colocalizes with the proteins RIM2, Munc13-1, and CAST1 (tom Dieck et al., 2005). Cav1.4 in the photoreceptor synapse is therefore in a position, both structurally and functionally, to coordinate presynaptic calcium entry with synaptic vesicle fusion. Mutations in the CACNA1F gene, which encodes the α1F pore-forming subunit of Cav1.4, cause the X-linked heritable disease incomplete congenital stationary night blindness (CSNB2) in humans (Bech-Hansen et al., 1998; Boycott et al., 2001). The same gene in mouse, Cacna1f, is mutated in the nob2 (no b-wave 2) strain, which is a model for CSNB2. In the nob2 mouse, the Cacna1f gene is disrupted by a naturally occurring transposable element insertion in exon 2 (Chang et al., 2006), resulting in a nonfunctional, truncated protein product. The nob2 mouse is therefore null for Cacna1f (Chang et al., 2006). A characteristic of CSNB2 is a normal a-wave but a severely reduced b-wave in the dark-adapted electroretinogram (ERG; Miyake et al., 1986, 1987; Nakamura et al., 2001). The normal a-wave indicates intact phototransduction in the photoreceptor outer segments of CSNB2 patients, but the reduced b-wave indicates disrupted synaptic transmission between photoreceptors and ON-bipolar cells (Miyake et al., 1986; Tremblay et al., 1995). Similarly, the nob2 mouse has a no-b-wave ERG phenotype much like that of CSNB2, displaying a normal a-wave but severely reduced b-wave in both the dark-adapted and the light-adapted ERG (Chang et al., 2006). Effects on both the dark- and the light-adapted ERG suggest that neurotransmission from both rod and cone photoreceptors is affected. This is supported by data from a Cav1.4 knockout mouse, which shows a dramatic decrease in depolarization-induced calcium influx into photoreceptor terminals (Mansergh et al., 2005). Preliminary characterization of the nob2 mouse also indicates that the loss of Cav1.4 results in altered retinal ganglion cell ON-responses and also in abnormal bipolar and horizontal cell morphology (Chang et al., 2006). The elimination of both electrophysiological and structural functions of Cav1.4 in CSNB2 patients and in the nob2 mice is likely to have developmental consequences, particularly for ribbon synapse formation. The formation of the invaginating photoreceptor ribbon synapse in mouse begins early in postnatal development, at about postnatal day 3 or 4 (P3/P4; Blanks et al., 1974; Rich et al., 1997), when cone photoreceptor terminals first contact horizontal cell processes in the future OPL. Contact of the cone by a second horizontal cell process coincides with invagination of the postsynaptic processes into the photo-receptor terminal, and the recruitment of the formed synaptic ribbon to the future active zone at about P6. Finally, at P7–P12 for cones, ON-bipolar cell dendrites invade the invagination to assume their position under the presynaptic ribbon (Blanks et al., 1974). Rods follow a very similar maturational sequence but are delayed with respect to cones by about 3– 4 days, forming mature synapses only by P10 –P14 (Rich et al., 1997). It is presently unknown whether normal, light-regulated glutamate release is necessary for the development of the photoreceptor-bipolar cell or photoreceptor-horizontal cell synapse. Synapse formation in the inner retina requires neuronal activity and neurotransmitter release (for review see Mumm et al., 2005), but little is known about this process in the outer retina. Study of the nob2 mouse will help to elucidate the role of the Cav1.4 calcium channel in the formation of OPL ribbon synapses. Here we examine in detail the reported aberrant outgrowth of bipolar and horizontal cells in the nob2 retina (Chang et al., 2006), focusing on the constituents of the ectopic contacts that form between rods and rod bipolar cells at the tips of aberrant bipolar cell dendrites. Furthermore, we characterize the developmental time course of the outgrowth of rod bipolar and horizontal cell processes in the nob2 retina and present data suggesting that it is rod bipolar cells, rather than horizontal cells, that are involved in the formation of ectopic synaptic contacts with rod photoreceptors in the nob2 retina in the days following eye opening.