ObjectiveMelanopsin retinal ganglion cells (mRGCs) are photoreceptors driving circadian photoentrainment, and circadian dysfunction characterizes Alzheimer disease (AD). We investigated mRGCs in AD, hypothesizing that they contribute to circadian dysfunction.MethodsWe assessed retinal nerve fiber layer (RNFL) thickness by optical coherence tomography (OCT) in 21 mild‐moderate AD patients, and in a subgroup of 16 we evaluated rest–activity circadian rhythm by actigraphy. We studied postmortem mRGCs by immunohistochemistry in retinas, and axons in optic nerve cross‐sections of 14 neuropathologically confirmed AD patients. We coimmunostained for retinal amyloid β (Aβ) deposition and melanopsin to locate mRGCs. All AD cohorts were compared with age‐matched controls.ResultsWe demonstrated an age‐related optic neuropathy in AD by OCT, with a significant reduction of RNFL thickness (p = 0.038), more evident in the superior quadrant (p = 0.006). Axonal loss was confirmed in postmortem AD optic nerves. Abnormal circadian function characterized only a subgroup of AD patients. Sleep efficiency was significantly reduced in AD patients (p = 0.001). We also found a significant loss of mRGCs in postmortem AD retinal specimens (p = 0.003) across all ages and abnormal mRGC dendritic morphology and size (p = 0.003). In flat‐mounted AD retinas, Aβ accumulation was remarkably evident inside and around mRGCs.InterpretationWe show variable degrees of rest–activity circadian dysfunction in AD patients. We also demonstrate age‐related loss of optic nerve axons and specifically mRGC loss and pathology in postmortem AD retinal specimens, associated with Aβ deposition. These results all support the concept that mRGC degeneration is a contributor to circadian rhythm dysfunction in AD. ANN NEUROL 2016;79:90–109

In the present study the localization of pituitary adenylate cyclase-activating peptide (PACAP)-expressing cell bodies and PACAP projections were mapped in the adult rat brain and spinal cord by using immunohistochemistry and in situ hybridization histochemistry. A widespread occurrence of PACAP-containing cell bodies was found, with the greatest accumulation in several hypothalamic nuclei and in several brainstem nuclei, especially the habenular nuclei, the pontine nucleus, the lateral parabrachial nucleus (LPB), and the vagal complex. PACAP was also present in cell bodies in the olfactory areas, in neocortical areas, in the hippocampus, in the vestibulo- and cochlear nuclei, in cell bodies of the intermediolateral cell column of the spinal cord and in Purkinje cells of the cerebellum, in the subfornical organ, and in the organum vasculosum of the lamina terminalis. An intense accumulation of PACAP-immunoreactive (-IR) nerve fibers was observed throughout the hypothalamus, in the amydaloid and extended amygdaloid complex, in the anterior and paraventricular thalamic nuclei, in the intergeniculate leaflet, in the pretectum, and in several brainstem nuclei, such as the parabrachial nucleus, the sensory trigeminal nucleus, and the nucleus of the solitary tract. PACAP-IR nerve fibers were also found in the area postrema, the posterior pituitary and the choroid plexus, and the dorsal and ventral horn of the spinal cord. The widespread distribution of PACAP in the brain and spinal cord suggests that PACAP is involved in the control of many autonomic and sensory functions as well as higher cortical processes.

Mammalian circadian rhythms generated in the hypothalamic suprachiasmatic nuclei are entrained to the environmental light/dark cycle via a monosynaptic pathway, the retinohypothalamic tract (RHT). We have shown previously that retinal ganglion cells containing pituitary adenylate cyclase-activating polypeptide (PACAP) constitute the RHT. Light activates the RHT via unknown photoreceptors different from the classical photoreceptors located in the outer retina. Two types of photopigments, melanopsin and the cryptochromes (CRY1 and CRY2), both of which are located in the inner retina, have been suggested as "circadian photopigments." In the present study, we cloned rat melanopsin photopigment cDNA and produced a specific melanopsin antibody. Using in situ hybridization histochemistry combined with immunohistochemistry, we demonstrate that the distribution of melanopsin was identical to that of the PACAP-containing retinal ganglion cells. Colocalization studies using the specific melanopsin antibody and/or cRNA probes in combination with PACAP immunostaining revealed that melanopsin was found exclusively in the PACAP-containing retinal ganglion cells located at the surface of somata and dendrites. These data, in conjunction with published action spectra analyses and work in retinally degenerated (rd/rd/cl) mutant mice, suggest that melanopsin is a circadian photopigment located in retinal ganglion cells projecting to the biological clock.

The retinohypothalamic tract (RHT) relays photic information from the eyes to the suprachiasmatic nucleus (SCN). Activation of this pathway by light plays a role in adjusting circadian timing via a glutamatergic pathway at night. Here we report a new signaling pathway by which the RHT may regulate circadian timing in the daytime as well. We used dual immunocytochemistry for pituitary adenylate cyclase-activating peptide (PACAP) and the in vivo tracer cholera toxin subunit B and observed intense PACAP-immunoreactivity (PACAP-IR) in retinal afferents in the rat SCN as well as in the intergeniculate leaflet (IGL) of the thalamus. This PACAP-IR in the SCN as well as in the IGL was nearly lost after bilateral eye enucleation. PACAP afferents originated from small ganglion cells distributed throughout the retina. The phase of circadian rhythm measured as SCN neuronal activity in vitro was significantly advanced (3.5 +/- 0.4 hr) by application of 1 x 10(-6) M PACAP-38 during the subjective day [circadian time (CT)-6] but not at night (CT14 and CT19). The phase-shifting effect is channeled to the clock via a PACAP-R1 receptor, because mRNA from this receptor was demonstrated in the ventral SCN by in situ hybridization. Furthermore, vasoactive intestinal peptide was nearly 1000-fold less potent in stimulating a phase advance at CT6. The signaling mechanism was through a cAMP-dependent pathway, which could be blocked by a specific cAMP antagonist, Rp-cAMPS. Thus, in addition to its role in nocturnal regulation by glutamatergic neurotransmission, the RHT may adjust the biological clock by a PACAP/cAMP-dependent mechanism during the daytime.

Melanopsin is a photopigment expressed in retinal ganglion cells, which are intrinsically photosensitive and are also involved in retinal circuits arising from rod and cone photoreceptors. This circuitry, however, is poorly understood. Here, we studied the morphology, distribution and synaptic input to melanopsin-containing ganglion cells in a New World monkey, the common marmoset (Callithrix jacchus). The dendrites of melanopsin-containing cells in marmoset stratify either close to the inner nuclear layer (outer stratifying), or close to the ganglion cell layer (inner stratifying). The dendritic fields of outer-stratifying cells tile the retina, with little overlap. However, the dendritic fields of outer-stratifying cells largely overlap with the dendritic fields of inner-stratifying cells. Thus, inner-stratifying and outer-stratifying cells may form functionally independent populations. The synaptic input to melanopsin-containing cells was determined using synaptic markers (antibodies to C-terminal binding protein 2, CtBP2, for presumed bipolar synapses, and antibodies to gephyrin for presumed amacrine synapses). Both outer-stratifying and inner-stratifying cells show colocalized immunoreactive puncta across their entire dendritic tree for both markers. The density of CtBP2 puncta on inner dendrites was about 50% higher than that on outer dendrites. The density of gephyrin puncta was comparable for outer and inner dendrites but higher than the density of CtBP2 puncta. The inner-stratifying cells may receive their input from a type of diffuse bipolar cell (DB6). Our results are consistent with the idea that both outer and inner melanopsin cells receive bipolar and amacrine input across their dendritic tree.

Analyses in mice deficient for the blue-light-sensitive photopigment melanopsin show that direct effects of light on behavior and EEG depend on the time of day. The data further suggest an unexpected role for melanopsin in sleep homeostasis.

Given the expression of melanopsin in PACAP-containing RGCs of the human RHT, this photoreceptor is a likely first base in the chain of events leading to photoentrainment of both normal and blind people.

Mitochondrial optic neuropathies, that is, Leber hereditary optic neuropathy and dominant optic atrophy, selectively affect retinal ganglion cells, causing visual loss with relatively preserved pupillary light reflex. The mammalian eye contains a light detection system based on a subset of retinal ganglion cells containing the photopigment melanopsin. These cells give origin to the retinohypothalamic tract and support the non-image-forming visual functions of the eye, which include the photoentrainment of circadian rhythms, light-induced suppression of melatonin secretion and pupillary light reflex. We studied the integrity of the retinohypothalamic tract in five patients with Leber hereditary optic neuropathy, in four with dominant optic atrophy and in nine controls by testing the light-induced suppression of nocturnal melatonin secretion. This response was maintained in optic neuropathy subjects as in controls, indicating that the retinohypothalamic tract is sufficiently preserved to drive light information detected by melanopsin retinal ganglion cells. We then investigated the histology of post-mortem eyes from two patients with Leber hereditary optic neuropathy and one case with dominant optic atrophy, compared with three age-matched controls. On these retinas, melanopsin retinal ganglion cells were characterized by immunohistochemistry and their number and distribution evaluated by a new protocol. In control retinas, we show that melanopsin retinal ganglion cells are lost with age and are more represented in the parafoveal region. In patients, we demonstrate a relative sparing of these cells compared with the massive loss of total retinal ganglion cells, even in the most affected areas of the retina. Our results demonstrate that melanopsin retinal ganglion cells resist neurodegeneration due to mitochondrial dysfunction and maintain non-image-forming functions of the eye in these visually impaired patients. We also show that in normal human retinas, these cells are more concentrated around the fovea and are lost with ageing. The current results provide a plausible explanation for the preservation of pupillary light reaction despite profound visual loss in patients with mitochondrial optic neuropathy, revealing the robustness of melanopsin retinal ganglion cells to a metabolic insult and opening the question of mechanisms that might protect these cells.