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

Abstract

The nob2 mouse carries a null mutation in the Cacna1f gene, which encodes the pore-forming subunit of the L-type calcium channel, Ca v 1.4. The loss of the electroretinogram b-wave in these mice suggests a severe reduction in transmission between photoreceptors and second-order neurons in the retina and supports a central role for the Ca v 1.4 calcium channel at photoreceptor ribbon synapses, to which it has been localized. Here we show that the loss of Ca v 1.4 leads to the aberrant outgrowth of rod bipolar cell dendrites and horizontal cell processes into the outer nuclear layer (ONL) of the nob2 retina and to the formation of ectopic synaptic contacts with rod photoreceptors in the ONL. Ectopic contacts are predominantly between rods and rod bipolar cells, with horizontal cell processes also present at some sites. Ectopic contacts contain apposed pre-and postsynaptic specializations, albeit with malformed synaptic ribbons. Cone photoreceptor terminals do not participate in ectopic contacts in the ONL. During retinal development, ectopic contacts appear in the days after eye opening, appearing progressively farther into the ONL at later postnatal stages. Ectopic contacts develop at the tips of rod bipolar cell dendrites and are less frequently associated with the tips of horizontal cell processes, consistent with the adult phenotype. The relative occurrence of pre-and postsynaptic markers in the ONL during development suggests a mechanism for the formation of ectopic synaptic contacts that is driven by the retraction of rod photoreceptor terminals and neurite outgrowth by rod bipolar cell dendrites. Indexing termsbipolar cell; horizontal cell; rod retraction; second-order neuron outgrowth L-type voltage-dependent calcium channels, including Ca v 1.4 and the closely related Ca v 1.3, 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) (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 Ca v 1.4 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., 2001Morgans et al., , 2005Berntson et al., 2003). Immunolabeling for Ca v 1.4, with either p...

Neurturin-Mediated Ret Activation Is Required for Retinal Function

“…We found that NRTN Ϫ/Ϫ and Ret RETfloxEGFP/RETfloxEGFP :Six3Cre mice displayed a paucity of axonal projections as well as highly aberrant bipolar and horizontal cell dendrites, suggesting that abnormal synapse organization, involving the photoreceptors, bipolar cells, and horizontal cells, is a contributing factor to retinal dysfunction in these mice. Interestingly, similar aberrant dendrites are seen in other mouse mutants with reduced photoreceptor-bipolar cell signaling, including nob2 mice, which have a mutation in the ␣ subunit of a presynaptic voltage-dependent calcium channel (Chang et al, 2006;Bayley and Morgans, 2007) and mice deficient in Ca 2ϩ -binding protein 4 (CaBP4), which interacts with these calcium channels in photoreceptor termini (Haeseleer et al, 2004; Maeda et al , 2005). Abnormal elongation of bipolar and horizontal cell processes is also seen in mice with mutations in the Bsn and Rs1h genes (Dick et al, 2003;Johnson et al, 2006), as well as in response to photoreceptor degeneration in the RCS rat model of retinitis pigmentosa (Cuenca et al, 2005).…”

“…Additional investigation of the NRTN Ϫ/Ϫ and Ret RETfloxEGFP/RETfloxEGFP : Six3Cre retinas revealed that presynaptic vesicle markers and synaptic ribbon structures are present among the photoreceptor cell bodies of the ONL in these mice. Such aberrantly located synapse structures are also seen in Bsn Ϫ/Ϫ and nob2 mice (Dick et al, 2003;Chang et al, 2006;Bayley and Morgans, 2007). Recent studies in hippocampal neurons showed that GDNF triggers adhesion between GFR␣1-expressing neurons, a critical step in appropriate synapse formation between these cells (Ledda et al, 2007).…”

“…The b-wave is driven primarily by the ON-bipolar cells, and any abnormality in ONL synapses would reduce the b-wave. In fact, the Rs1h and nob2 mutants display "electronegative" waveforms (Chang et al, 2006;Johnson et al, 2006;Bayley and Morgans, 2007), in which the b-wave amplitude is less than the a-wave amplitude. We also observed reduced a-waves in Ret DN/ϩ , Ret RETfloxEGFP/RETfloxEGFP :Six3Cre, and NRTN Ϫ/Ϫ mice.…”

“…Notably, the integrity of the synaptic triad of the OPL, which is crucial for feedforward as well as feedback signaling, depends on dendrites of horizontal cells (which express Ret and GFR␣1), bipolar cells, and rod spherules or cone pedicles. In mice with mutations that cause aberrant synaptic transmission between pho- Bayley and Morgans, 2007). To determine whether similar bipolar or horizontal dendrite abnormalities exist in NRTN Ϫ/Ϫ mice, we stained these cells with PKC and calbindin antibodies and visualized retinas using highresolution confocal microscopy.…”

Ablation of Retinal Horizontal Cells from Adult Mice Leads to Rod Degeneration and Remodeling in the Outer Retina

“…Interestingly, the loss of postsynaptic proteins does not induce remodeling: postsynaptic no-b-wave mutants (McCall and Gregg, 2008), such as mutants for proteins in the mGluR6-mediated signaling cascade of ON bipolar cells, show no morphological alterations in the OPL. Thus, it was hypothesized that the changes in presynaptic no-b-wave mutants are caused by changes in calcium signaling or homeostasis in presynaptic photoreceptor terminals (Bayley and Morgans, 2007). As the negative feedback from horizontal cells to cones was shown to regulate the calcium current in photoreceptors (Kamermans et al, 2001), loss of horizontal cell feedback in horizontal cell-ablated retinae likely impairs the normal function of presynaptic calcium channels, leading to a similar phenotype as in calcium channel mutants (Ball et al, 2002;Chang et al, 2006).…”