Although NMDA currents were described in AII amacrine cells previously (Hartveit and Veruki, 1997
; Zhou and Dacheux, 2004
), the physiological significance of these currents was uncertain. We now describe a role for these NMDA receptors in driving increased phosphorylation of Cx36 gap junctions on AIIs, thereby increasing cell-cell coupling in the AII amacrine cell network. Indeed, driving increased phosphorylation of Cx36 may be the primary purpose of NMDA receptors on AIIs.
NMDA receptors on AII amacrine cells contribute minimally or not at all to the postsynaptic response elicited by evoked glutamate release from a presynaptic rod bipolar cell (Singer and Diamond, 2003
; Trexler et al., 2005
; Veruki et al., 2003
). Our anatomical examination confirmed that NMDA receptors on AII amacrine cells are nonsynaptic, in that they are not associated with presynaptic terminals or sites of glutamate release, but instead colocalize with Cx36 gap junctions. Furthermore, NMDA receptors on AIIs are functionally coupled to a pathway driving increased phosphorylation of Cx36 at the regulatory Ser293 site (Ouyang et al., 2005
), which promotes increased gap junctional coupling in the AII amacrine cell network (Kothmann et al., 2009
), and this pathway requires activation of CamKII. Cx36 phosphorylation is sensitive to the concentration of extracellular glutamate, and activation of all glutamatergic ON-type bipolar cells drove increased phosphorylation of Cx36 on AIIs. Together, these data describe a mechanism to potentiate Cx36-mediated electrical coupling between neurons via activation of NMDA receptors.
The precise source of glutamate leading to activation of NMDA receptors on AIIs is still unclear, as we found no relationship between the NR1 puncta on AIIs and the presynaptic ribbons in rod bipolar cells or nearby ON-type cone bipolar cells. Spillover of glutamate does occur at the rod bipolar-AII synapse (Veruki et al., 2006
), but the relatively large mean distance between NR1 puncta and presynaptic ribbons and the high variability in the measure (3.3 ± 2.2 μm) suggests that overall extracellular glutamate “tone” may be an important influence on AII NMDA receptors as well (but see below). Noise analysis has been used to demonstrate the contribution of ambient extracellular glutamate to the excitability of pyramidal neurons in hippocampal slices (Sah et al., 1989
) as well as to the basal noise in retinal ganglion cells (Gottesman and Miller, 2003
), in both cases via binding to NMDA receptors. Thus, it is conceivable that NMDA receptors on AIIs might detect changes in ambient glutamate. This could explain why addition of AMPA alone was able to drive Cx36 phosphorylation, especially as we found that this still required NMDA receptor activation. However, we found that reducing the Mg2+
block of NMDA receptors in dark-adapted retina by itself was not sufficient to drive phosphorylation of Cx36. It may be that the depolarization provided by AMPA application is also important. AIIs express L-type calcium channels, which are primarily localized to their lobular dendrites. However, calcium imaging of AIIs during a depolarizing pulse revealed small Ca2+
elevations in the arboreal dendrites of most cells as well (Habermann et al., 2003
). While our experiments show that NMDA receptors are required for AMPA-induced increases in phosphorylation of Cx36 in dark-adapted retina, we cannot exclude the possibility that Ca2+
influx through voltage-gated calcium channels is also required under these conditions.
Given that previous studies have not found an NMDA component in the AII EPSC in response to activation of a single presynaptic rod bipolar cell, it may be that multiple nearby bipolar cells must be activated within a short temporal window in order to sufficiently alter extracellular glutamate concentration for significant NMDA receptor activation. However, since depolarizing a single rod bipolar cell causes sufficient spillover to activate glutamate transporters on neighboring rod bipolar cell terminals (Veruki et al., 2006
), and that the glutamate transporter in rod bipolar cells (EAAT5 (Pow and Barnett, 2000
)) has a lower affinity for glutamate than does NMDA (64 μM vs. 1 μM (Arriza et al., 1997
; Olverman et al., 1984
)), it seems most likely that NMDA receptors on AIIs do “see” glutamate released by individual rod bipolar cells, even if it does not activate a significant NMDA current. Because spillover from rod bipolar cells seems to have a relatively large diffusion domain, when they release glutamate (particularly when two or more nearby rod bipolar cells release nearly synchronously) it is likely to cause a temporary elevation in ambient glutamate within that domain (since glutamate transport does not fully clear the extracellular glutamate). This is one potential explanation for why we see heterogeneous phosphorylation of Cx36 within individual AII amacrine cells in our light-adapted control conditions (Kothmann et al., 2009
). The expression and activity of glutamate transporters in the inner retina, as well as their potential modulation by luminance and adaptation conditions, is no doubt also a crucial component to such a mechanism of NMDA receptor activation. While NMDA currents activated by ambient glutamate would likely be relatively small, they could contribute sufficient Ca2+
influx to activate CaMKII due to the long time constant of deactivation conferred by many NMDA receptor subunits (Cull-Candy et al., 2001
Application of NMDA to well dark-adapted retina was sufficient to robustly increase Cx36 phosphorylation, even though AII amacrine cells should be hyperpolarized under this condition and the Mg2+
concentration in our Ames solution was physiological (1.2 mM Mg2+
). This may simply be a result of regenerative activation of NMDA receptors in response to a minutes-long exposure to agonist. It is also important to note that compared to many other neurons AIIs have a relatively depolarized resting potential (−46 ± 1.2 mV in dark-adapted retina (Dunn et al., 2006
)), a potential at which the conductance of NMDA receptors is ~15% of maximum in 1 mM Mg2+
(Jahr and Stevens, 1990
, their ), and thus some fraction of the receptors should be available to begin with. NR2 subunits also significantly alter the Mg2+
sensitivity of NMDA receptors (Monyer et al., 1994
), although the identity of the NR2 subunit composition of AII NMDA receptors is unknown. Our data indicate that even in very well dark-adapted retina, NMDA receptors are still responsive to exogenous agonist, potentially due to the above reasons.
It is more straightforward to imagine the circumstances that would activate NMDA receptors on AII amacrine cells in vivo
under scotopic conditions, when they are depolarized directly by glutamate released from rod bipolar cells onto postsynaptic AMPA receptors. Based on our results, we propose that ambient and/or spillover glutamate drives opening of nonsynaptic NMDA receptors on depolarized AII dendrites, thus linking modulation of Cx36-mediated coupling with the excitation of AII amacrine cells. Such a situation fits with the observed increase in AII-AII coupling adapted to scotopic background illumination (Bloomfield and Volgyi, 2004
; Bloomfield et al., 1997
), which will evoke glutamate release from some rod bipolar cells. Since rod photoreceptors diverge to two rod bipolar cells (Sterling et al., 1988
), even single rod activations have the potential to activate multiple rod bipolar cell inputs onto a single AII, which might cause a local elevation in ambient glutamate and increase local AII-AII coupling, as proposed above. Coupling then increases further as increased background illumination more strongly stimulates a larger pool of rod bipolar cells. Our data from dark-adapted retina support this scenario, as pharmacological activation of ON-type bipolar cells and activation by a background light increment both drive increased phosphorylation of Cx36 gap junctions. Thus in our model nonsynaptic NMDA receptors on AIIs provide a link between local activity in rod bipolar cells and local coupling in the AII network, while synaptic AMPA receptors provide the depolarization to overcome the threshold for NMDA receptor activation.
The NMDA-driven increase in phosphorylation of Cx36 requires activation of CaMKII. This arrangement in AII amacrine cells bears some similarities with fish Mauthner cells, where Cx35 gap junctions are encircled by postsynaptic densities that contain synaptic NMDA receptors (Flores et al., 2010
; Tuttle et al., 1986
), and potentiation of electrical coupling also requires NMDA receptor and CaMKII activation (Pereda et al., 1998
; Pereda and Faber, 1996
). A similar physical organization of synaptic NMDA receptors with Cx36 gap junctions in mammalian inferior olive neurons that show highly variable coupling (Hoge et al., 2011
) may presage a similar regulatory scheme. However, the AII amacrine cell also shows a distinct difference in that there do not appear to be excitatory chemical synapses directly associated with the NMDA/Cx36 complexes. This unique arrangement has led us to conclude that the primary purpose of NMDA receptors in AII amacrine cells is to drive increased coupling through Cx36 gap junctions.
A critical design feature of an efficient dynamic regulatory mechanism is to employ the opposing actions of two independent pathways to increase and decrease the amplitude of the regulated parameter. In the AII amacrine cell, the glutamate-driven increase in Cx36 phosphorylation and AII coupling is opposed by dopaminergic signaling via a D1-like receptor. The latter involves a cAMP/PKA signaling pathway that activates protein phosphatase 2A and reduces coupling by reducing Cx36 phosphorylation (Kothmann et al., 2009
). Retinal dopamine secretion is increased by bright light adaptation, and we have shown that it is sufficient to overwhelm the pathway favoring increased Cx36 phosphorylation (Kothmann et al., 2009
). The presence of these independent, opposed signaling pathways allows for precise, dynamic control of coupling wherein the relative strength of each pathway is continuously compared to dictate the coupling state. This push-pull organization results in a non-linear relationship between AII coupling and background illumination. Such an organization is likely a common theme in regulation of Cx36-mediated electrical coupling.
Cx36 displays a tremendous potential for regulation, which need not be limited to only two opposing pathways. Multiple kinases converge onto the regulatory residues of Cx36 (Alev et al., 2008
; Kothmann et al., 2007
; Ouyang et al., 2005
; Patel et al., 2006
; Urschel et al., 2006
), and in all likelihood multiple phosphatases do as well. Furthermore, alternate signaling pathway organization can lead to distinct properties. This is exemplified by retinal photoreceptors in which a cAMP/PKA signaling pathway that lacks an intervening phosphatase drives increased photoreceptor coupling via increased Cx35 phosphorylation (Li et al., 2009
), precisely the reverse of the cAMP signaling found in AIIs.
Electrical synapses are widespread in the central nervous system (Connors and Long, 2004
), but plasticity has thus far been detected in a relatively small number of circuits. Noradrenergic modulation of coupling in hippocampal neurogliaform cells (Zsiros and Maccaferri, 2008
) strongly resembles the dopaminergic modulation of AII amacrine cell coupling. Metabotropic glutamate receptors have also been found to drive uncoupling of interneurons in the thalamic reticular nucleus (Landisman and Connors, 2005
). Such forms of plasticity are an intrinsic property of electrical synapses containing Cx36 and are likely widespread in the central nervous system.
Plasticity of electrical synapses imparts important emergent properties to the neural networks that employ them. While strong coupling promotes spike synchrony, weaker coupling can promote antiphase and phase-locked states (Marder, 1998
; Saraga et al., 2006
). The dynamic modulation of electrical synaptic strength can efficiently regulate these complex network properties. Disruption of higher-order processes such as memory of complex objects and motor learning in Cx36 knockout animals (Frisch et al., 2005
; Van Der Giessen et al., 2008
) is likely a manifestation of the loss of this plasticity, as is impaired context-dependent fear learning when Cx36 gap junctions are blocked (Bissiere et al., 2011
). As both NMDA receptors and Cx36 gap junctions are widely expressed throughout the vertebrate brain, it seems likely that nonsynaptic NMDA/Cx36 complexes may exist in other central neurons, where NMDA receptors could modulate electrical coupling as a function of local excitation history.