of mGluR6 protein with advancing rod photoreceptor degeneration. In addition to down-regulation, the residual mGluR6 is mislocalised and forms clusters on the cell somata and axons. Similar changes occur in rat models such as the P23H (Cuenca et al. 2004), S334ter (Seiler et al. 2008) and RCS rat (Cuenca et al. 2005; Pinilla et al. 2007), however, the somatic mislocalisation of mGluR6 is more uniform and marked than in mouse models. Interestingly, the patterns of mGluR6 mislocalisation in the degenerating rat and mouse retinas appear to mimic the immature patterns of localisation seen in the respective species during early retinal development (Nomura et al. 1994; Ueda et al. 1997).

Whilst deafferentation clearly results in the down regulation of mGluR6 receptors in ON bipolar cells, the expression of ionotropic glutamate receptors in the outer plexiform layers seems to be maintained, or possibly even up-regulated, in response to photoreceptor death. Our laboratory evaluated the expression of the AMPA receptor subunits GluR1, GluR2 and GluR4 at varying stages of degeneration in the rd10 mouse. Using immunohistochemistry, we showed robust expression of all three receptor subunits, even after complete degeneration of cone photoreceptors (Puthussery et al. 2009). These data are further supported by gene expression studies that have shown the up-regulation of GluR1, GluR3 and GluR4 during and after the peak period of photoreceptor degeneration in the rd1 mouse (Namekata et al. 2006). Moreover, changes in flip-to-flop alternative splicing of the GluR1 receptor have been reported (Namekata et al. 2006), and given the expression of this receptor by OFF bipolar cells (Hack et al. 2001), it is feasible that this could lead to altered AMPA receptor kinetics in these cells. Increased protein expression of GluR1 has also been described in the rdta mouse (Liu et al. 2001).

60.2.2 Evidence for Bipolar Cell Dysfunction

60.2.2.1 Rod Bipolar Cells

To date, few studies have used single-cell electrophysiology to assay bipolar cell function during photoreceptor degeneration. In the rd1 retina, Varela et al. (2003) recorded glutamate responses from enzymatically dissociated rod bipolar cells at 4–8 weeks of age. They showed that only one of 13 cells responded to glutamate puffed onto the dendrites. Since it has been shown that the rod-to-rod bipolar cell synapse does not develop normally in the rd1 mouse (Blanks et al. 1974), it is unclear whether the observed functional deficits are due to developmental anomalies or are a secondary consequence of photoreceptor degeneration. More recently, Barhoum et al. (2008) evaluated rod bipolar cell responses to exogenously applied glutamate in a slice preparation of the rd10 retina at postnatal day 60, a time after complete degeneration of rod photoreceptors. They demonstrated that wild-type and rd10 retinas were similar in that ˜50% of RBCs responded to puff application of glutamate, with no significant difference in the response amplitudes between the two groups. This is a surprising finding, given the very marked down-regulation of the mGluR6 at P60 in the rd10 retina.

Recently, our laboratory has taken advantage of an approach originally described by Nawy (2004) to pharmacologically simulate light stimulation in ON bipolar cells in slice preparations of the degenerating retina. The group III metabotropic glutamate receptor agonist L-AP4 is applied to retinal slices to activate the mGluR6 receptors and simulate the high receptor occupancy that occurs due to glutamate release in the dark-adapted retina. We then simulate a light flash by pressure ejection of high concentrations of the mGluR6 antagonist CPPG onto the bipolar cell dendrites. The CPPG rapidly displaces the AP4 and generates an inward current that shows remarkably similar magnitude and kinetics to a true light response. Using this approach, we have found that rod bipolar cells in the rd10 retina show progressive alterations in mGluR6 currents, with decreased response amplitudes and alterations in kinetics in the early stages of degeneration (postnatal day 20), and near complete loss of rod bipolar cell responses by postnatal day 45 (Puthussery et al. 2009). While these results conflict with those of Barhoum et al. (2008) who found that mGluR6 responses were unchanged in the degenerated retina, they accord more closely with the dramatic loss in mGluR6 expression evident from the immunohistochemical findings.

The dysfunction of rod bipolar cells during retinal degeneration has also been suggested from electroretinography studies. The b-wave of the electroretinogram (ERG) is known to reflect the activity of ON-bipolar cells (Stockton and Slaughter 1989; Green and Kapousta-Bruneau 1999) and the rod contribution to the b-wave can be isolated using stimulus parameters that exploit the temporal or chromatic properties of the rods. Gargini et al. (2007) recorded ERGs in the rd10 mouse using light stimuli in which the mean luminance was sinusoidally modulated over a range of temporal frequencies in an effort to better isolate changes in b-wave kinetics. They showed that deficits in the b-wave were detectable as early as P18, and suggested that changes in bipolar cell function occur even in the early stages of photoreceptor degeneration. However, since the ERG is produced by a chain of events, it is not possible to determine where the deficit arises, be it by reduced efficiency of presynaptic signalling in the rods, or by post-synaptic alterations in rod bipolar cells, such as a reduced expression of mGluR6 receptors.

Another approach that has been employed to assess bipolar cell function during progression of retinal degeneration is the use of the organic cation 1-amino-4- guanidobutane (agmatine) to map the activity of discrete cell populations in vivo and in vitro (Marc et al. 2005). Agmatine is believed to enter cells through open cation channels, including ionotropic glutamate receptors that are activated by AMPA, kainate or NMDA as well as the unidentified cation channel downstream of the mGluR6 receptor. Immunohistochemical analysis with agmatine selective antibodies is used to assay cell activity under a variety of conditions.

Marc et al. (2007) showed that intravitreal injection of agmatine, followed by a 45 min period in mesopic light, results in the uptake of agmatine into a large proportion of rod bipolar cells in the wild-type retina. However, when the rodless coneless (rdcl) mutant mouse was examined under the same conditions, virtually no bipolar cells had any agmatine signal, suggesting permanent closure of the mGluR6gated ion channel (Marc et al. 2003). This result is surprising, since mGluR6