Christianne E. Strang

Central projections of intrinsically photosensitive retinal ganglion cells in the macaque monkey

Circadian rhythms generated by the suprachiasmatic nucleus (SCN) are entrained to the environmental light/dark cycle via intrinsically photosensitive retinal ganglion cells (ipRGCs) expressing the photopigment melanopsin and the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP). The ipRGCs regulate other nonimage-forming visual functions such as the pupillary light reflex, masking behavior, and light-induced melatonin suppression. To evaluate whether PACAP-immunoreactive retinal projections are useful as a marker for central projection of ipRGCs in the monkey brain, we characterized the occurrence of PACAP in melanopsin-expressing ipRGCs and in the retinal target areas in the brain visualized by the anterograde tracer cholera toxin subunit B (CtB) in combination with PACAP staining. In the retina, PACAP and melanopsin were found to be costored in 99% of melanopsin-expressing cells characterized as inner and outer stratifying melanopsin RGCs. Two macaque monkeys were anesthetized and received a unilateral intravitreal injection of CtB. Bilateral retinal projections containing colocalized CtB and PACAP immunostaining were identified in the SCN, the lateral geniculate complex including the pregeniculate nucleus, the pretectal olivary nucleus, the nucleus of the optic tract, the brachium of the superior colliculus, and the superior colliculus. In conclusion, PACAP-immunoreactive projections with colocalized CtB represent retinal projections of ipRGCs in the macaque monkey, supporting previous retrograde tracer studies demonstrating that melanopsin-containing retinal projections reach areas in the primate brain involved in both image- and nonimage-forming visual processing.

Central projections of intrinsically photosensitive retinal ganglion cells in the macaque monkey: Central projections of intrinsically photosensitive RGCs in macaque

Circadian rhythms generated by the suprachiasmatic nucleus (SCN) are entrained to the environmental light/dark cycle via intrinsically photosensitive retinal ganglion cells (ipRGCs) expressing the photopigment melanopsin and the neuropeptide pituitary adenylate cyclase‐activating polypeptide (PACAP). The ipRGCs regulate other nonimage‐forming visual functions such as the pupillary light reflex, masking behavior, and light‐induced melatonin suppression. To evaluate whether PACAP‐immunoreactive retinal projections are useful as a marker for central projection of ipRGCs in the monkey brain, we characterized the occurrence of PACAP in melanopsin‐expressing ipRGCs and in the retinal target areas in the brain visualized by the anterograde tracer cholera toxin subunit B (CtB) in combination with PACAP staining. In the retina, PACAP and melanopsin were found to be costored in 99% of melanopsin‐expressing cells characterized as inner and outer stratifying melanopsin RGCs. Two macaque monkeys were anesthetized and received a unilateral intravitreal injection of CtB. Bilateral retinal projections containing colocalized CtB and PACAP immunostaining were identified in the SCN, the lateral geniculate complex including the pregeniculate nucleus, the pretectal olivary nucleus, the nucleus of the optic tract, the brachium of the superior colliculus, and the superior colliculus. In conclusion, PACAP‐immunoreactive projections with colocalized CtB represent retinal projections of ipRGCs in the macaque monkey, supporting previous retrograde tracer studies demonstrating that melanopsin‐containing retinal projections reach areas in the primate brain involved in both image‐ and nonimage‐forming visual processing. J. Comp. Neurol. 522:2231–2248, 2014.

Expression of Alpha 7 Nicotinic Acetylcholine Receptors by Bipolar, Amacrine, and Ganglion Cells of the Rabbit Retina

Cholinergic agents affect the light responses of many ganglion cells (GCs) in the mammalian retina by activating nicotinic acetylcholine receptors (nAChRs). Whereas retinal neurons that express β2 subunit-containing nAChRs have been characterized in the rabbit retina, expression patterns of other nAChR subtypes remain unclear. Therefore, we evaluated the expression of α7 nAChRs in retinal neurons by means of single-, double-, and triple-label immunohistochemistry. Our data demonstrate that, in the rabbit retina, several types of bipolar cells, amacrine cells, and cells in the GC layer express α7 nAChRs. At least three different populations of cone bipolar cells exhibited α7 labeling, whereas glycine-immunoreactive amacrine cells comprised the majority of α7-positive amacrine cells. Some GABAergic amacrine cells also displayed α7 immunoreactivity; α7 labeling was never detected in rod bipolar cells or rod amacrine cells (All amacrine cells). Our data suggest that activation of α7 nAChRs by acetylcholine (ACh) or choline may affect glutamate release from several types of cone bipolar cells, modulating GC responses. ACh-induced excitation of inhibitory amacrine cells might cause either inhibition or disinhibition of other amacrine and GC circuits. Finally, ACh may act on α7 nAChRs expressed by GCs themselves.

Parallel optical monitoring of visual signal propagation from the photoreceptors to the inner retina layers

Understanding of visual signal processing can benefit from simultaneous measurement of different types of retinal neurons working together. In this Letter, we demonstrate that intrinsic optical signal (IOS) imaging of frog retina slices allows simultaneous observation of stimulus-evoked responses propagating from the photoreceptors to the inner neurons. High-resolution imaging revealed robust IOSs at the photoreceptor, the inner plexiform, and the ganglion cell layers. While IOSs of the photoreceptor layer were mainly confined to the area directly stimulated by the visible light, IOSs of the inner retinal layers spread from the stimulus site into relatively large areas with a characteristic near-to-far time course.

Nicotinic Acetylcholine Receptor Subunits in Rhesus Monkey Retina

PURPOSE The purpose of this study was to detect and establish the cellular localizations of nicotinic acetylcholine receptor (nAChR) subunits in Rhesus monkey retina. METHODS Retinas were dissected from the eyes of monkeys killed after unrelated experiments. RNA was extracted and analyzed by RT-PCR, using primers designed against human sequences of alpha3-alpha7, alpha9, and beta2-beta4 nAChR subunits. The RT-PCR products were separated by gel electrophoresis and sequenced. Frozen sections of postmortem fixed monkey eyes were immunolabeled with well-characterized and specific monoclonal antibodies against the alpha3, alpha4, alpha6, alpha7, beta2, or beta4 nAChR subunits and visualized with fluorescence labeling. RESULTS Products of the predicted size for the alpha3-alpha7, alpha9, and beta2-beta4 nAChR subunits were detected by RT-PCR in Rhesus monkey retina. Homology between transcripts from monkey retina and human nucleotide sequences ranged from 93 to 99%. Immunohistochemical studies demonstrated that neurons in various cell layers of monkey retina expressed alpha3, alpha4, alpha7, or beta2 nAChR subunits and cells with the morphology of microglia were immunoreactive for the alpha6 or beta4 nAChR subunits. CONCLUSIONS nAChR subunits are expressed in the monkey retina and localize to diverse retinal neurons as well as putative microglia. Besides mediating visual processing, retinal nAChRs may influence refractive development and ocular pathologies such as neovascularization.

Activation of the cGMP/nitric oxide signal transduction system by nicotine in the retina.

Acetylcholine is one of the primary excitatory neurotransmitters/neuromodulators in the retina, but little is known about the downstream signaling pathways it can activate. The present study immunocytochemically examines the potential sources of acetylcholine and the location of the nicotinic cholinergic receptors in the turtle retina. It also examines how activation of these receptors can influence the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signal-transduction pathways. Photoreceptors, amacrine cells, and potentially ganglion cells contain choline acetyltransferase-like immunoreactivity (LI). Nicotinic acetylcholine receptors are immunocytochemically localized on photoreceptors, horizontal, bipolar, and ganglion cells. Nitric oxide imaging indicates that stimulation with nicotine increases NO production primarily in photoreceptors, horizontal, Muller, bipolar, and ganglion cells. In turn, very select populations of amacrine cells respond to this NO with increased levels of cGMP-LI. Selective inhibitors reveal that nitric oxide synthase is involved in most, but not all, of these increases in cGMP-LI. These results show that acetylcholine can activate the NO/cGMP signal-transduction pathways in both the inner and outer retina. This indicates that both of the major excitatory retinal transmitters, glutamate and acetylcholine, can stimulate NO production that increases levels of cGMP-LI in overlapping populations of retinal cells.

Rabbit retinal ganglion cell responses to nicotine can be mediated by β2-containing nicotinic acetylcholine receptors

Acetylcholine (ACh) affects the response properties of many retinal ganglion cells (GCs) through the activation of nicotinic acetylcholine receptors (nAChRs). To date there have been few studies directly correlating the expression of specific nAChR subtypes with the physiological and morphological characteristics of specific retinal GCs. This study was designed to correlate responses to nicotine application with immunohistochemical evidence of nAChR expression in physiologically and morphologically identified ganglion cells. Extracellular recordings were used to physiologically identify rabbit retinal GCs, based on responses to light stimulation. Cells were then tested for responses to nicotine application and/or for expression of nAChRs, as judged by immunoreactivity to mAb210, an nAChR antibody. The morphologies of many physiologically identified cells were also determined by dye injection. More than three-fourths of ganglion cells tested responded to nicotine application under cobalt-induced synaptic blockade. The nicotine sensitivity was consistent with nAChR immunoreactivity and was also correlated with specific morphological subgroups of GCs. Overall, approximately two-thirds of all physiologically identified GCs that were processed for immunohistochemistry displayed immunoreactivity. In total, 18 of 22 physiologically identified cells demonstrated both sensitivity to nicotine application under synaptic blockade and mAb210 immunoreactivity (mAb210-IR). Thus, mAb210-IR is likely to represent functional nAChRs that can modulate retinal information processing and visual functioning via direct excitation of a number of GC classes.

Role of acetylcholine in nitric oxide production in the salamander retina

Although acetylcholine is one of the most widely studied neurotransmitters in the retina, many questions remain about its downstream signaling mechanisms. In this study we initially characterized the cholinergic neurotransmitter system in the salamander retina by localizing a variety of cholinergic markers. We then examined the link between both muscarinic and nicotinic receptor activation and nitric oxide production by using immunocytochemistry for cyclic guanosine monophosphate (cGMP) as an indicator. We found a large increase in cGMP‐like immunoreactivity (cGMP‐LI) in the inner retina in response to muscarinic (but not nicotinic) receptor activation. Based on the amplification of mRNA transcripts, receptor immunocytochemistry, and the use of selective antagonists, we identified these receptors as M2 muscarinic receptors. Using double‐labeling techniques, we established that these increases in cGMP‐LI were seen in GABAergic but not cholinergic amacrine cells, and that the increases were blocked by inhibitors of nitric oxide production. The creation of nitric oxide in response to cholinergic receptor activation may provide a mechanism for modulating the well‐known mutual interactions of acetylcholine‐glycine‐GABA in the inner retina. As GABA and glycine are the primary inhibitory neurotransmitters in the retina, signaling pathways that modulate their levels or release will have major implications for the processing of complex stimuli by the retina. J. Comp. Neurol. 507:1952–1963, 2008.

Nicotinic acetylcholine receptor expression by directionally selective ganglion cells

Acetylcholine (ACh) enhances the preferred direction responses of directionally selective ganglion cells (DS GCs; Ariel & Daw, 1982; Ariel & Adolph, 1985) through the activation of nicotinic acetylcholine receptors (nAChRs; Ariel & Daw, 1982; Massey et al., 1997; Kittila & Massey, 1997). DS GCs appear to express at least two types of nAChRs, those that are sensitive to the partially subtype-specific antagonist methyllycaconitine (MLA), and those that are MLA-insensitive (Reed et al., 2002). Our purpose was to confirm the expression of alpha7 nAChRs by DS GCs and to assess the contributions of other nAChR subtypes to DS GC responses. Using choline as a nAChR partially subtype-specific agonist, we found that the majority of DS GCs demonstrated responses to choline while under synaptic blockade. The blockade or reduction of choline-induced responses by bath application of nanomolar (nM) concentrations of MLA provided direct evidence that the choline responses were mediated by alpha7 nAChRs. Because choline is a partial agonist for alpha3beta4 nAChRs (Alkondon et al., 1997), the residual choline responses are consistent with mediation by alpha3beta4 nAChRs. Additionally, a subset of DS GCs responded to nicotine but not to choline, indicating the expression of a third nAChR subtype. The pharmacological results were supported by single cell reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry experiments. The expression of alpha7 and specific non-alpha7 nAChR subtypes was correlated with the preferred direction. This indicates the possibility of differential responses to ACh depending on the direction of movement. This is the first description of differential expression of multiple nAChR subtypes by DS GCs.

Two-photon excited autofluorescence imaging of freshly isolated frog retinas

The purpose of this study was to investigate cellular sources of autofluorescence signals in freshly isolated frog (Rana pipiens) retinas. Equipped with an ultrafast laser, a laser scanning two-photon excitation fluorescence microscope was employed for sub-cellular resolution examination of both sliced and flat-mounted retinas. Two-photon imaging of retinal slices revealed autofluorescence signals over multiple functional layers, including the photoreceptor layer (PRL), outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), and ganglion cell layer (GCL). Using flat-mounted retinas, depth-resolved imaging of individual retinal layers further confirmed multiple sources of autofluorescence signals. Cellular structures were clearly observed at the PRL, ONL, INL, and GCL. At the PRL, the autofluorescence was dominantly recorded from the intracellular compartment of the photoreceptors; while mixed intracellular and extracellular autofluorescence signals were observed at the ONL, INL, and GCL. High resolution autofluorescence imaging clearly revealed mosaic organization of rod and cone photoreceptors; and sub-cellular bright autofluorescence spots, which might relate to connecting cilium, was observed in the cone photoreceptors only. Moreover, single-cone and double-cone outer segments could be directly differentiated.