## 3 Role of PACAP and Its Receptors in the Eye  
The presence of PACAP and its receptors has been largely shown in the eye [33, 56]. PACAP positive expression was found in mammalian, teleost, turtle, and chicken retina [57]. In particular, in situ hybridization and immunohistochemical analysis have revealed the presence of PACAP in specific cell populations of retinal tissue samples. PACAP was positively expressed in the nerve fiber layer (NFL), the ganglion cell layer (GCL), the inner plexiform layer (IPL), and the pigment epithelium (PE). The immunoreactivity of PACAP appeared in the early phase of retinal development [58], as demonstrated by its presence in the chick inner nuclear layer (INL) from embryonic day 8 [59]. PACAP mRNA expression was detectable in the rat GCL at embryonic day 20 [60], whereas, in the zebrafish, PACAP immune-positive signal was found in the retina at 24 h post-fertilization [61]. PAC1R was strongly expressed in the GCL, in neuronal cell bodies of amacrine and horizontal cells localized in the INL and in the PE. On the contrary, PAC1R was weakly expressed in the IPL, outer plexiform layer (OPL), outer nuclear layer (ONL), and photoreceptor layer [62, 63, 64, 65]. The expression profile of PAC1R splice variants (Null, Hip, Hop1, Hop2, Hiphop1, and Hiphop2) was described during retina development [11]. The expression of PAC1 Rs at the subcellular level was identified at the plasma membrane, in the rough endoplasmic reticulum, in the cytoplasmic matrix of retinal ganglion cells (RGCs) and amacrine cells in the INL [66]. PAC1R immunoreactivity was also detected in retinal tissue and in rat primary cultures of Muller cells [67, 68]. In the rat retina, the expression of VPAC1R and VPAC2R was demonstrated [69]. Moreover, [PERSON] et al. [11] showed the potential involvement of VPACRs at all stages of retinal development in the rat. The protective effects of PACAP in the visual system have been widely studied in the neural and non-neuronal parts of the eye, including the cornea (Figure 2). Figure 2: Schematic diagram showing the main pro-survival/antiapoptotic/protective intracellular pathways activated by PACAP in different ocular structures. Several studies showed that PACAP has protective effects in the retina against toxic or ischemic insults, inflammation, hypoxia, oxygen-induced retinopathy, traumatic injuries, glaucoma, and diabetic retinopathy [70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81]. The protective role of PACAP was observed in different cell types, including bipolar neurons, amacrine, and pigment epithelial cells [82; 83]. In particular, in the retinal pigment epithelial cells, PACAP counteracted oxidative stress and hypoxic insult by exerting protective and pro-survival effects [83; 84; 85; 86]. PACAP is also expressed in a subpopulation of retinal ganglion cells participating in the modulation of the circadian rhythm through a photoneuroendocrine circuit. In fact, it transfers light signals from the retina to the suprachiasmatic nucleus via the retinoblastoma tract [87]. In accord, it has been shown that the intracerebral injection of PACAP induced a phase-delay of the circadian rhythm similarly to light and the treatment with PACAP receptor antagonist reduced light changes in vitro [88]. This evidence let hypothesized that exogenous administration of PACAP as a repair agent for damage to the structure of the eye might alter the wake-sleep cycle. PACAP is not only expressed in the retina, but it has also been demonstrated in other ocular tissues. A rich plexus of PACAP-immunoreactive nerve fibers were found in the cat lacrimal gland, arising mainly from the splenopalatine ganglion, where 10% of the neuronal cell bodies show PACAP immunoreactivity [89]. Moreover, PAC1R immunoreactivity was observed in mouse infraorbital lacrimal gland acinar cells [90]. Here, PACAP eye drops promoted tear secretion via the PAC1R/AC/cAMP/PKA/AQP5 cascade [91]. PACAP receptors were found in both the anterior uvea and choroid [92].