µ-Opioid Receptor Activation Directly Modulates Intrinsically Photosensitive Retinal Ganglion Cells.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) encode light intensity and trigger reflexive responses to changes in environmental illumination. In addition to functioning as photoreceptors, ipRGCs are post-synaptic neurons in the inner retina, and there is increasing evidence that their output can be influenced by retinal neuromodulators. Here we show that opioids can modulate light-evoked ipRGC signaling, and we demonstrate that the M1, M2 and M3 types of ipRGCs are immunoreactive for µ-opioid receptors (MORs) in both mouse and rat. In the rat retina, application of the MOR-selective agonist DAMGO attenuated light-evoked firing ipRGCs in a dose-dependent manner (IC50 < 40 nM), and this effect was reversed or prevented by co-application of the MOR-selective antagonists CTOP or CTAP. Recordings from solitary ipRGCs, enzymatically dissociated from retinas obtained from melanopsin-driven fluorescent reporter mice, confirmed that DAMGO exerts its effect directly through MORs expressed by ipRGCs. Reduced ipRGC excitability occurred via modulation of voltage-gated potassium and calcium currents. These findings suggest a potential new role for endogenous opioids in the mammalian retina and identify a novel site of action-MORs on ipRGCs-through which opioids might exert effects on reflexive responses to environmental light.

Light-evoked S-nitrosylation in the retina.

Nitric oxide (NO) synthesis in the retina is triggered by light stimulation. NO has been shown to modulate visual signal processing at multiple sites in the vertebrate retina, via activation of the most sensitive target of NO signaling, soluble guanylate cyclase. NO can also alter protein structure and function and exert biological effects directly by binding to free thiol groups of cysteine residues in a chemical reaction called S-nitrosylation. However, in the central nervous system, including the retina, this reaction has not been considered to be significant under physiological conditions. Here we provide immunohistochemical evidence for extensive S-nitrosylation that takes place in the goldfish and mouse retinas under physiologically relevant light intensities, in an intensity-dependent manner, with a strikingly similar pattern in both species. Pretreatment with N-ethylmaleimide (NEM), which occludes S-nitrosylation, or with 1-(2-trifluromethylphenyl)imidazole (TRIM), an inhibitor of neuronal NO synthase, eliminated the light-evoked increase in S-nitrosylated protein immunofluorescence (SNI) in the retinas of both species. Similarly, light did not increase SNI, above basal levels, in retinas of transgenic mice lacking neuronal NO synthase. Qualitative analysis of the light-adapted mouse retina with mass spectrometry revealed more than 300 proteins that were S-nitrosylated upon illumination, many of which are known to participate directly in retinal signal processing. Our data strongly suggest that in the retina light-evoked NO production leads to extensive S-nitrosylation and that this process is a significant posttranslational modification affecting a wide range of proteins under physiological conditions.

Nitric oxide mediates activity-dependent plasticity of retinal bipolar cell output via S-nitrosylation.

Coding a wide range of light intensities in natural scenes poses a challenge for the retina: adaptation to bright light should not compromise sensitivity to dim light. Here we report a novel form of activity-dependent synaptic plasticity, specifically, a "weighted potentiation" that selectively increases output of Mb-type bipolar cells in the goldfish retina in response to weak inputs but leaves the input-output ratio for strong stimuli unaffected. In retinal slice preparation, strong depolarization of bipolar terminals significantly lowered the threshold for calcium spike initiation, which originated from a shift in activation of voltage-gated calcium currents (ICa) to more negative potentials. The process depended upon glutamate-evoked retrograde nitric oxide (NO) signaling as it was eliminated by pretreatment with an NO synthase blocker, TRIM. The NO-dependent ICa modulation was cGMP independent but could be blocked by N-ethylmaleimide (NEM), indicating that NO acted via an S-nitrosylation mechanism. Importantly, the NO action resulted in a weighted potentiation of Mb output in response to small (≤-30 mV) depolarizations. Coincidentally, light flashes with intensity ≥ 2.4 × 10(8) photons/cm(2)/s lowered the latency of scotopic (≤ 2.4 × 10(8) photons/cm(2)/s) light-evoked calcium spikes in Mb axon terminals in an NEM-sensitive manner, but light responses above cone threshold (≥ 3.5 × 10(9) photons/cm(2)/s) were unaltered. Under bright scotopic/mesopic conditions, this novel form of Mb output potentiation selectively amplifies dim retinal inputs at Mb → ganglion cell synapses. We propose that this process might counteract decreases in retinal sensitivity during light adaptation by preventing the loss of visual information carried by dim scotopic signals.

Regulation of GABA and glutamate release from proopiomelanocortin neuron terminals in intact hypothalamic networks.

Hypothalamic proopiomelanocortin (POMC) neurons and their peptide products mediate important aspects of energy balance, analgesia, and reward. In addition to peptide products, there is evidence that POMC neurons can also express the amino acid transmitters GABA and glutamate, suggesting these neurons may acutely inhibit or activate downstream neurons. However, the release of amino acid transmitters from POMC neurons has not been thoroughly investigated in an intact system. In the present study, the light-activated cation channel channelrhodopsin-2 (ChR2) was used to selectively evoke transmitter release from POMC neurons. Whole-cell electrophysiologic recordings were made in brain slices taken from POMC-Cre transgenic mice that had been injected with a viral vector containing a floxed ChR2 sequence. Brief pulses of blue light depolarized POMC-ChR2 neurons and induced the release of GABA and glutamate onto unidentified neurons within the arcuate nucleus, as well as onto other POMC neurons. To determine whether the release of GABA and glutamate from POMC terminals can be readily modulated, opioid and GABA(B) receptor agonists were applied. Agonists for µ- and κ-, but not δ-opioid receptors inhibited transmitter release from POMC neurons, as did the GABA(B) receptor agonist baclofen. This regulation indicates that opioids and GABA released from POMC neurons may act at presynaptic receptors on POMC terminals in an autoregulatory manner to limit continued transmission. The results show that, in addition to the relatively slow and long-lasting actions of peptides, POMC neurons can rapidly affect the activity of downstream neurons via GABA and glutamate release.