The Dorsal Raphe Nucleus Receives Afferents From Alpha-Like Retinal Ganglion Cells and Intrinsically Photosensitive Retinal Ganglion Cells in the Rat

Abstract

Purpose: A retinal projection into the dorsal raphe nucleus (DRN), namely, the retino-raphe projection, exists in many species. The rat is one of several species in which a retino-raphe projection has been described; however, the retinal ganglion cell (RGC) types that contribute to this pathway are unknown.

Methods: Retrograde tracing via cholera toxin subunit B (CTB) was used to reveal DRN-projecting RGCs in rats, combined with intracellular injection in vitro, melanopsin immunostaining in whole-mounted retinas, and serotonin immunostaining to define the DRN. We modified methods of CTB injection into DRN used previously in order to avoid possible contamination with other retinorecipient regions, particularly the superior colliculus (SC).

Results: The majority of DRN-projecting RGCs showed alpha-like morphology, and some CTB-positive RGCs were colabeled with melanopsin. Approximately 80% of the total population of CTB-labeled DRN-projecting RGCs was alpha-like cells including ON alpha cells and OFF alpha cells; these alpha-like cells were melanopsin immunonegative. Approximately 10% of the remaining DRN-projecting RGCs were melanopsin immunopositive, in which the M1 subtype of intrinsically photosensitive retinal ganglion cell (ipRGC) provided the dominant projection of ipRGCs into DRN, with only few non-M1 ipRGCs involved. The DRN-projecting ipRGCs could be retrogradely labeled following tracer injection into all rostrocaudal aspects of the DRN.

Conclusions: Both conventional RGCs with alpha-like morphology and melanopsin-expressing ipRGCs project into the rat DRN. Approximately 10% of DRN-projecting RGCs were colabeled with melanopsin, and the majority of these were the M1 subtype of ipRGCs. An ipRGC component of the retino-raphe projection may contribute to a sustained light-mediated modulation of DRN serotonin release.

The dorsal raphe nucleus (DRN) is a key source of the neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) into the forebrain.^1,2 The DRN is located in the ventral part of the periaqueductal gray (PAG) of the midbrain^2,3 and contains the largest number of 5-HT neurons of the mammalian raphe system.^1,4 Due to its widely spread serotonergic projections, the DRN has been associated with a broad assortment of behavioral and physiological functions including the modulation of mood.^2,4 

In addition to many afferent projections from the forebrain including the orbitofrontal cortex, hypothalamus, and amygdala,^5–8 the DRN also receives afferents from the retina. The retino-raphe projection has been described in several species, including the cat,⁹ rat,^10,11 Mongolian gerbil,^11,12 tree shrew,¹³ and Octodon degus,¹⁴ as well as the monkey Cebus apella,¹⁵ and light stimulation induces c-Fos expression in DRN.^16,17 However, a retino-raphe projection has not been observed in a few species, such as the mouse,^18,19 ground squirrel,²⁰ and Nile grass rat,²¹ although some of these negative findings may be technique related.²² Dorsal raphe nucleus-projecting retinal ganglion cells (RGCs) have recently been described in detail in the gerbil, and the vast majority of these cells have alpha-like morphology and Y-cell physiological responses to light.¹² These alpha Y-cell DRN-projecting cells showed no intrinsic responses to light using extracellular recordings and using conventional staining techniques were not immunopositive for melanopsin,¹² a novel opsin conferring light sensitivity to a subset of RGCs.²² Although the visual system of the rat has been extensively investigated, the retino-raphe projection has received only limited attention,^10,11 and the details on the classification and function of DRN-projecting RGCs are still unknown. 

In our study of the retino-raphe projection in the rat, we conducted anterograde and retrograde tracing using cholera toxin subunit B (CTB) combined with melanopsin immunostaining in retinal whole mounts. In addition to a large population of alpha-like RGCs, a small percentage of DRN-projecting RGCs was observed to express melanopsin, supporting the interpretation that these cells are probably intrinsically photosensitive retinal ganglion cells (ipRGCs). 

Materials and Methods

Animals

Adult male Sprague-Dawley rats (Rattus norvegicus, 280–350 g, obtained from the Laboratory Animal Unit at the University of Hong Kong and originating from Charles River Lab, Hollister, CA, USA) were the subjects of investigation in this study. All animals were kept on a 12:12 light:dark cycle (light = 30–40 lux; dark = ∼0 lux, light onset at 7:00 AM) with food and water provided ad libitum. All animal experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and policies on the utilization of animals and humans in neuroscience research, and were approved by the Faculty Committee on the Use of Live Animals in Teaching and Research in The University of Hong Kong. 

Intraocular Injection of CTB

Rats were anesthetized with an intraperitoneal injection of 100 mg/kg ketamine and 10 mg/kg xylazine. Following topical use of corneal anesthetic (0.5% proparacaine hydrochloride), a 35-G needle attached to a 5.0-μL Hamilton syringe was inserted into the vitreous chamber of the eye at the temporal cornea–conjunctival margin. Then 5.0 μL 2% (wt/vol) Alexa Fluor 488-conjugated CTB (C-22841; Molecular Probes, Eugene, OR, USA) dissolved in 2% dimethyl sulfoxide (156914; Sigma-Aldrich Corp. St. Louis, MO, USA) was slowly injected into the eye, and the needle was held in place for a period of 5 minutes. After the withdrawal of the needle, the injected site was immediately washed with saline, and antibiotic (bacitracin) was applied topically.¹¹ 

DRN Injection of CTB

Animals were anesthetized with an intraperitoneal injection of 100 mg/kg ketamine and 10 mg/kg xylazine. After inducing deep anesthesia, the animal was placed in a small-animal stereotaxic apparatus (68001; RWD Life Science, San Diego, CA, USA), and the incisor bar was adjusted so that the dorsal surface of the hindbrain skull was level. Using a dental drill, a small burr hole was made in the skull, which was 5.2 mm posterior to lambda. A 5.0-μL Hamilton syringe inclined at a 28° angle from vertical (see Fig. 2A) was positioned with the 27-G needle tip just touching the surface of the dura. The needle was then advanced slowly 7.2 mm. Cholera toxin subunit B (0.4–0.8 μL) was gradually pressure injected using a microsyringe pump controller (100 nL/min; World Precision Instruments, Sarasota, FL, USA), followed by 0.2 μL oil (sesame oil; Sigma-Aldrich Corp.) to limit the diffusion of CTB tracer.²³ The needle was left in place for 10 minutes and then was slowly withdrawn. The skull was sealed with bone wax and the skin incision closed with wound clips. After a survival period of 7 days, animals were killed and retinas and brains were collected.^11,23 

Figure 1

 

Labeling of axons of retinal ganglion cells projecting into the DRN. (A) Labeled RGC axons are shown in a sagittal section contralateral to the eye injected with CTB with the DRN outlined. (B–E) CTB-labeled fibers in the rostral (B), mid (C, E), and caudal portions (D) of the DRN. More CTB-positive fibers were observed in the mid-DRN (C, E). Some retinal fibers were in close apposition to the somas or dendrites of 5-HT-positive cells (arrows in [B–E]). 4V, fourth ventricle; DRN, dorsal raphe nucleus; SC, superior colliculus; 5-HT, 5-hydroxytryptamine or serotonin; CTB, cholera toxin subunit B conjugated to Alexa 488. Scale bars: 500 μm (A); 20 μm (B–D); 2 μm (E).

Figure 2

 

Retrograde labeling of DRN-projecting retinal ganglion cells. (A) The approach used for CTB injection into the DRN (sky-blue arrow) to avoid the superior colliculus (SC). Examples are provided of CTB injection sites in the rostral portion of DRN (B1), mid portion of DRN (C1), and caudal portion of DRN (D1); and examples of CTB-labeled RGCs (green) are shown in (B2–D2) while melanopsin immunostaining of the same retinas is provided in (B3–D3). (B4–D4) CTB-positive RGCs colabeling with melanopsin (Opn4, magenta; arrows). DRN, dorsal raphe nucleus; SC, superior colliculus; Aq, cerebral aqueduct; 5-HT, 5-hydroxytryptamine or serotonin; CTB, cholera toxin subunit B conjugated to Alexa 488. Scale bars: 1000 μm (A); 50 μm (B1–D1); 10 μm (B2–D4).

Intracellular Injection to Fill CTB-Positive RGCs

Seven days after CTB injection into the DRN, rats were anesthetized as above; the eyeballs were enucleated, and the fresh retinas were carefully but rapidly dissected from the eyecup. Flat-mounted retinas were placed on filter paper (GS, 0.22 mm; Millipore Corp., Bedford, MA, USA) with the ganglion cell layer facing up, and then set on the injection chamber and superfused with oxygenated Ames' medium (Sigma-Aldrich Corp.) continually at a fixed rate (3 mL/min). Retrogradely CTB-labeled cells in the retina were filled using a microelectrode containing 4% neurobiotin (SP-1120; Vector Laboratories, Burlingame, CA, USA) with 1% Lucifer yellow (L3510; Sigma-Aldrich Corp.). A small amount of current (1–2 nA for 1–2 minutes) was applied to the electrode until the soma and process were completely stained. After completion of the intracellular injection, the retinas were incubated in 4% paraformaldehyde (PFA) for 2 hours, followed by PBS washing for 10 minutes × 3 and blocking incubation of 5% goat serum for 1 hour. Retinas were then incubated in Alexa Fluor 488-conjugated streptavidin (1:200, S32354; Molecular Probes) for 2 hours at room temperature. Retinas were washed in PBS and coverslipped in antifade aqueous mounting medium. Details of the process have been described previously.^12,24 

Tissue Collection and Preparation

Following deep anesthesia, animals were perfused with saline (pH 7.4) followed by 4% PFA in 0.01 M phosphate buffer (PB, pH 7.4). Retinas were collected followed by postfixation with 4% PFA for 45 minutes at room temperature. Brains were removed from the skulls and stored in 4% PFA solution at 4°C overnight. The next day, brains were rinsed in 0.01 M PB and then stored in 30% sucrose in 0.01 M PB until they had sunk. The hindbrain including the midbrain raphe complex was then serially sectioned at 30 μm using a microtome (Model 860; American Optical, Buffalo, NY, USA). Brain sections were collected as six alternate sets of slices and stored at −20°C in a cryoprotectant storage buffer (30% ethylene glycol, 30% sucrose in 0.01 M PB; pH 7.4) until they were used for immunocytochemical analysis.^25,26 

Immunocytochemistry

Details of the staining techniques for retinal whole mounts have been described previously.^24,26 Briefly, whole retinas were washed in 0.1% PBS-Tween 20 (PBST, 3 × 10 minutes) and then placed into blocking solution with 5% donkey serum in 0.1% Triton X-100 in PBS for 1 hour, followed by incubation in anti-melanopsin polyclonal antibody (1:1000, rabbit, PA1-780, generated against an N-terminal peptide of melanopsin; Thermo Fisher Scientific, Waltham, MA, USA) and anti-choline acetyltransferase (ChAT) polyclonal antibody (1:500, goat, AB144P; Millipore Corp.) for 48 hours at 4°C. After washing with PBS (3 × 10 minutes), the retinas were placed in donkey anti-rabbit Alexa 647-conjugated IgG (1:500; Thermo Fisher Scientific) and donkey anti-goat Alexa 568 (1:500; Molecular Probes) for 2 hours at room temperature. After washing with PBS (3 × 10 minutes), retinas were coverslipped with the ganglion cell side up in an aqueous mounting medium (Dako Corp., Carpinteria, CA, USA). Immunocytochemistry performed on brain sections was similar except that brain sections were stained with rabbit anti-serotonin antibody (1:1000, S5545; Sigma-Aldrich Corp.). 

Tyramide signal amplification (TSA kit, T20925; Molecular Probes) to immunostain melanopsin was performed according to the manufacturer's instructions and as modified by Berson and colleagues.^27,28 Briefly, endogenous peroxidase activity was quenched by incubating the retinas in 0.1% H₂O₂ in PBS for 30 minutes, followed by washing in PBST, 3 × 10 minutes. Then the retinas were placed in TSA-12 blocking solution for 1 hour and incubated with melanopsin antibody PA1-780 (1:1,000, rabbit; Thermo Fisher Scientific) in TSA-12 blocking solution for 48 hours at 4°C. The retinas were washed in PBST (6 × 10 minutes) and then incubated with a goat anti-rabbit horseradish peroxidase secondary antibody at 1:100 in the TSA-12 blocking solution, for 2 hours at room temperature. After washing in PBST (6 × 10 minutes), retinas were then incubated for exactly 7 minutes with tyramide-488 at 1:100 in 1× Plus Amplification Diluent (provided by the TSA kit) and quickly transferred to PBST for washing (6 × 10 minutes). Finally retinas were mounted on glass slides with the ganglion cell layers up and coverslipped with aqueous mounting medium (Dako Corp.). 

Data Collection and Analysis

The retinas were scanned under a confocal microscope (LSM 700; Carl Zeiss, Oberkochen, Germany). The z-axis interval was 0.3 to 0.4 μm. Each stack of optical sections covered a retinal area of 325.75 × 325.75 μm (1024 × 1024 pixels). The thickness of the stack was kept at ∼65 μm. With use of the confocal software ZEN 2012 (gained from Zeiss Group Official Web site), each stack of optical sections was montaged and projected to a 0° x–y plane and a 90° y–z plane to obtain a three-dimensional reconstruction of the cells. Details of three-dimensional reconstruction and confocal calibration procedures are described elsewhere.²⁹ Quantification of CTB-positive cells and CTB-melanopsin colabeled cells was conducted using a ×40 objective in the fluorescent microscope (Nikon, Kawasaki, Japan) with Neurolucida software (MBF Bioscience, Williston, VT, USA). Analysis of soma size and dendritic field (DF) size was performed with the software of ImageJ (National Institutes of Health, Bethesda, MD, USA) and Photoshop (Adobe Corp., San Jose, CA, USA). 

Results

Anterograde Labeling of Retinal Axons in the DRN

Unilateral intraocular CTB injection in the rat produced labeled retinal axons throughout the visual system including the DRN as illustrated in a sagittal section through the superior colliculus (SC) and midbrain DRN (Fig. 1A). The CTB-labeled fibers in the DRN were beaded in appearance (Figs. 1B–E), and were distributed throughout the rostrocaudal regions of the DRN (Figs. 1B–E). Some CTB fibers were very close to the somas (Figs. 1B, 1C, 1E) or dendrites (Fig. 1D) of 5-HT cells. Compared with the rostral portion (Fig. 1B) and caudal portion (Fig. 1D), the greatest number of labeled fibers were present in the midregion of the DRN, namely mid-DRN (Figs. 1C, 1E), and the relatively long fibers were more frequently observed in the ventrolateral parts of mid-DRN (DRVL) (Fig. 1E). 

Retrograde Labeling of DRN-Projecting RGCs

As shown in Figure 2A, to perform CTB injections into the DRN, we modified methods used previously,^11,12 to limit possible contamination with other retinorecipient regions, particularly the SC. Seven days following the injection of CTB into the DRN, CTB-positive RGCs were detected in the retinal whole mounts (Fig. 2). Following injection of CTB (0.4 μL) and 0.2 μL oil into the subregions of DRN, including rostral (Fig. 2B1), mid (Fig. 2C1), and caudal portions (Fig. 2D1), respectively, CTB-positive RGCs were observed in the flat-mounted retinas (Figs. 2B2, 2C2, 2D2). These results suggest that RGCs may project throughout the rostrocaudal extent of the DRN. Compared with the CTB injection sites located in the rostral and caudal portions of the DRN (Figs. 2B1, 2D1), CTB injections located in the mid-DRN (Fig. 2C1) resulted in a greater number of CTB-positive RGCs (Tables 1, 2), consistent with the results of anterograde labeling of retinal axons in the DRN (Fig. 1). Interestingly, after staining with melanopsin antibody (Figs. 2B3, 2C3, 2D3), a few CTB-labeled DRN-projecting RGCs were melanopsin immunopositive (Figs. 2B4, 2C4, 2D4, arrows). Similar to the non-melanopsin expressing DRN-projecting RGCs, melanopsin-immunopositive DRN-projecting RGCs were observed following CTB injections into the rostral, mid, and caudal DRN (Fig. 2; Tables 1, 2). 

Table 1

 

The Number of CTB-Labeled DRN-Projecting Retinal Ganglion Cells Following CTB (0.8 μL) Injection Into the Mid-DRN

 

Table 2

 

The Number of CTB-Labeled DRN-Projecting Retinal Ganglion Cells Following CTB (0.8 μL) Injection Into the Rostral or Caudal DRN

 

Morphology of DRN-Projecting RGCs

The dendritic stratification pattern of DRN-projecting RGCs was determined by intracellular injection of neurobiotin into CTB-labeled RGCs. A DRN-projecting RGC with dendrites arborizing in the proximal ON region of the inner plexiform layer (IPL) is illustrated in Figure 3A, whereas a DRN-projecting RGC with dendrites terminating in the distal OFF IPL is shown in Figure 3B. Similar to the DRN-projecting RGCs in the gerbil previously reported from our group,¹² most of the DRN-projecting RGCs in the rat retina had large somas and large dendritic fields (Figs. 3A, 3B, Fig. 4A), conforming to those of alpha RGCs.^30,31 The DRN-projecting alpha-like cells were not melanopsin immunopositive even after tyramide signal amplification (Figs. 3A, 3B, inserted panel at lower left corner), despite intensive melanopsin immunostaining in the rat retina. 

Figure 3

 

Morphology of DRN-projecting retinal ganglion cells. A typical ON alpha-like cell (A) and an OFF alpha-like cell (B) retrogradely labeled from the DRN and after intracellular filling with neurobiotin are shown. DRN-projecting ipRGCs include M1 cells (C) and non-M1 cells (D) while the DRN-projecting RGC in (E) is a brush-like CTB-labeled ganglion cell. Arrows indicate the axons. ChAT staining (blue) indicates two lines of cholinergic amacrine cells: a, inner (ON) sublayer of inner plexiform layer; b, outer (OFF) sublayer of inner plexiform layer. NB, neurobiotin; Opn4, melanopsin; ChAT, choline acetyltransferase; CTB, cholera toxin subunit B conjugated to Alexa 488. Scale bars: 20 μm.

Figure 4

 

Types of DRN-projecting retinal ganglion cells. (A) Plot of cell soma diameter versus dendritic field sizes of DRN-projecting RGCs (n = 44: 5 ipRGCs and 39 conventional RGCs). Open circles are melanopsin-positive DRN-projecting RGCs, and filled circles are melanopsin-negative DRN-projecting RGCs. (B) Percentage of different types of DRN-projecting RGCs. The percentage of alpha cells is based on the analysis after intracellular filling, while the ipRGC percentage was quantified from melanopsin immuostaining of CTB-labeled whole-mounted retinas.

At least five types of ipRGCs have been described based on their dendritic stratification pattern, melanopsin expression level, soma size, and other characteristics.^28,32 The M1 ipRGC is the only type that has dendrites that arborize exclusively in the OFF layer of the IPL adjacent to the border of the inner nuclear layer (INL).²⁸ The DRN-projecting melanopsin-positive RGCs typically resembled the M1 ipRGC subtype based on the dendritic stratification pattern in the IPL revealed by the intracellular injection of neurobiotin; filled dendrites extended to the border of the INL (Fig. 3C). Non-M1 ipRGCs were also observed (Fig. 3D), but much less frequently. In addition to alpha cells, other melanopsin-negative DRN-projecting RGCs were also observed, with small and brush-like dendritic morphology (Fig. 3E). However, these RGCs represented only approximately 10% of the total population of DRN-projecting RGCs (Fig. 4B). 

Quantification of DRN-Projecting RGCs

Since CTB tracer injected at mid-DRN could potentially involve more of the DRN (i.e., tracer could defuse rostrally and caudally in the DRN from the injection site), we used the labeled retinas from animals (n = 6) with CTB injection sites at the mid-DRN for quantification. The mean number of CTB-labeled DRN-projecting RGCs observed was 685 ± 55 cells per retina (Table 1), and of these, 67 ± 6 RGCs were CTB-melanopsin colabeled or 9.8% of DRN-projecting RGCs (Table 1). A similar number of labeled RGCs was observed in the contralateral and ipsilateral retinas if CTB was injected into the DRN midline (first three samples in Table 1). When CTB was injected into a DRVL part of mid-DRN, the number of labeled RGCs was higher in the contralateral retina (last three samples in Table 1). The number of CTB-labeled RGCs ranged from approximately 1000 to 1400 in each animal (both retinas together) based on CTB (0.8 μL) injections into the mid-DRN. When the injection sites were located at rostral or caudal DRN portions, the number of CTB-labeled cells was often no more than 50 in each retina (Table 2). 

The majority of DRN-projecting RGCs were classified as large cells based on the analysis of soma sizes (≥15 μm) and DF sizes (≥290 μm) (Fig. 4A, n = 44: 5 ipRGCs and 39 conventional RGCs). As shown in Figure 4B, the DRN-projecting alpha cells accounted for approximately 80% while DRN-projecting ipRGCs made up approximately 10%. Among the DRN-projecting ipRGCs, the majority belonged to M1 subtype (Fig. 3C), with a small percentage of non-M1 ipRGCs (Fig. 3D), based on observations from all available samples (n = 20 retinas, only 7 non-M1 cells of 64 CTB-melanopsin colabeled cells with three-dimensional reconstruction). And the average soma size of DRN-projecting ipRGCs was 14.03 ± 0.43 μm (Table 1), consistent with the soma sizes of M1 ipRGCs reported previously.^28,32,33 

Discussion

In this study a retino-raphe projection was confirmed in the rat using both anterograde and retrograde tracing of connections between the retina and DRN, similar to the first observation of a retino-raphe projection described in the cat.⁹ The majority (≈80%) of DRN-projecting RGCs had large somas and alpha-like dendritic morphology that arborized in either the ON or OFF layers of the IPL, and these cells did not stain for melanopsin protein even after signal amplification techniques were employed. In addition, it was observed for the first time that of the DRN-projecting RGCs, approximately 10% were ipRGCs based on the expression of melanopsin in CTB-labeled cells. The vast majority of DRN-projecting ipRGCs appeared to belong to the M1 subtype of ipRGCs. 

In the mammalian retina, ipRGCs are a conserved type of RGC exhibiting the expression of melanopsin, rendering these cells intrinsically photosensitive.³⁴ The population of ipRGCs represents approximately 2% to 4% of all ganglion cells in rodents and approximately 0.2% in primates.^35,36 Since ipRGCs mainly innervate the suprachiasmatic nucleus and intergeniculate leaflet as well as olivary pretectal nucleus,¹⁸ they primarily play a role in non–image-forming visual functions, in particular, controlling circadian photoentrainment, pupillary light reflex, and melatonin suppression by light.³⁷ However, ipRGCs are also involved in visual functions,^26,38,39 particularly in primates.³⁶ The peak photosensitivity of ipRGCs is located at the spectrum of blue light (∼480 nm), and blue light stimulation also significantly activated neural activity in the human brainstem detected by fMRI.^40,41 These findings might suggest the possibility that ipRGCs project to the human DRN. 

Pituitary adenylate cyclase-activating polypeptide (PACAP) coexists in ipRGCs in the rat and primate retinas and immunostaining in brain sections has been used as a surrogate marker for melanopsin axons.^42,43 However, no PACAP-labeled fibers have been described in either the DRN or PAG,⁴² while a very sparse ipRGC projection into PAG had been observed in mice.¹⁸ We attempted to determine if DRN-projecting RGC axons colabeled with PACAP, but the colabeling of CTB-filled fibers in DRN was extremely rare in this study (data not shown), perhaps due to the relatively small number of DRN-projecting ipRGCs. 

The M1 subtype of ipRGC was found to be the primary type of ipRGC projecting to the DRN. The morphological features of M1 cells in rats are similar to those in mice,³² including dendritic stratification close to the border of the INL, often robust expression of melanopsin in the few primary dendrites, and relatively smaller soma size. Based on these morphological characteristics we conclude that most DRN-projecting ipRGCs were M1 cells. Many fewer non-M1 DRN-projecting ipRGCs were observed compared to M1 ipRGCs. However, since dendrites of CTB-labeled melanopsin cells could not always be clearly observed using immunostaining, some cells classified as non-M1 may have been M1 ipRGCs. That is also in agreement with a previous study showing that the majority of ipRGCs found in rats belong to the M1 ipRGCs.^32,33,44 

Dorsal raphe nucleus-projecting RGCs have been analyzed in rat and gerbil retinas. Similar to the report by Fite et al.,¹¹ DRN-projecting RGCs in the rat were not distributed in the retina as regularly as those in gerbils.¹² However, DRN-projecting RGCs in rats were found distributed throughout the entire retina, rather than restricted to the inferior retina as reported by Fite et al.¹¹ The soma sizes of DRN-projecting RGCs in rats were also more variable compared with those in gerbils.¹¹ It also appears that there is a greater diversity in the types of DRN-projecting RGCs in rats compared with gerbils.¹² At least three types of DRN-projecting RGCs were observed in rats in the current study, including alpha-like cells, ipRGCs, and brush-like RGCs. 

The DRN is a major source of 5-HT afferents into the forebrain.¹ It is thought that 5-HT plays important roles in many neural activities.^4,6,45 However, the retinal projection into the DRN has not attracted sufficient attention so far, and the functions of this retinal pathway are still unknown. Our previous study in gerbils revealed that light signals could directly transmit into the DRN through the retino-raphe projection; and in the rod/cone photoreceptor degeneration model used, modulated serotonergic tone as well as subsequent affective behavior.^12,24 These findings open the possibility that the retino-raphe projection may play a role in light therapy for depression, especially for seasonal affective disorder,⁴⁶ although to date, the retino-raphe pathway has not been identified in humans. Different RGCs provide different types of information to their central targets, and it remains to be determined if different types of RGCs innervate different targets in the DRN (e.g. 5-HT or gamma-aminobutyric acid [GABA] neurons). The role of alpha-like DRN-projecting RGCs is unknown²²; the ipRGC component of the retino-raphe projection in the rat might contribute to a sustained light-mediated modulation of serotonin release from the DRN. 

In conclusion, this study reports for the first time that a small population ipRGCs project into the rat DRN in addition to the alpha-like RGCs. Approximately 10% of DRN-projecting RGCs were colabeled with melanopsin, and the majority were the M1 subtype of ipRGC. The function of the retino-raphe projection remains ill defined. 

Acknowledgments

We thank Phillis W. F. Kau for her technical support for immunostaining and Bo Peng for his techniques for intracellular injection. 

Supported by funding from the Jessie Ho Professorship in Neuroscience (the University of Hong Kong Foundation for Educational Development and Research Ltd.), Guangdong Natural Science Foundation (2014A030313387), funds of Leading Talents of Guangdong (2013), Programme of Introducing Talents of Discipline to Universities (B14036), National Natural Science Foundation of China (31400942), and The National Program on Key Basic Research Project of China (K-FS, 973 Program: 2011CB707501 and 2014CB542205). 

Disclosure: X. Li, None; C. Ren, None; L. Huang, None; B. Lin, None; M. Pu, None; G.E. Pickard, None; K.-F. So, None