Molecular mechanisms underlying selective synapse formation of vertebrate retinal photoreceptor cells

Abstract

In vertebrate central nervous systems (CNSs), highly diverse neurons are selectively connected via synapses, which are essential for building an intricate neural network. The vertebrate retina is part of the CNS and is comprised of a distinct laminar organization, which serves as a good model system to study developmental synapse formation mechanisms. In the retina outer plexiform layer, rods and cones, two types of photoreceptor cells, elaborate selective synaptic contacts with ON- and/or OFF-bipolar cell terminals as well as with horizontal cell terminals. In the mouse retina, three photoreceptor subtypes and at least 15 bipolar subtypes exist. Previous and recent studies have significantly progressed our understanding of how selective synapse formation, between specific subtypes of photoreceptor and bipolar cells, is designed at the molecular level. In the ON pathway, photoreceptor-derived secreted and transmembrane proteins directly interact in trans with the GRM6 (mGluR6) complex, which is localized to ON-bipolar cell dendritic terminals, leading to selective synapse formation. Here, we review our current understanding of the key factors and mechanisms underlying selective synapse formation of photoreceptor cells with bipolar and horizontal cells in the retina. In addition, we describe how defects/mutations of the molecules involved in photoreceptor synapse formation are associated with human retinal diseases and visual disorders.

Introduction

Proper neural connection, mediated by selective synapse formation, is crucial for normal development and function of the vertebrate CNS, including the brain and the retina. How the genetic program encoded on the genome establishes selective synapse formations among numerous neurons is still poorly understood. The vertebrate retina provides us with an excellent system to investigate selective synapse formation at the molecular level because of the relatively simple circuit, fixed synaptic location, and availability of various synaptic markers. The retina contains five major classes of neurons: photoreceptor cells, bipolar cells, horizontal cells, amacrine cells, and ganglion cells. Retinal synapses are formed in two distinct layers, the outer plexiform layer (OPL) and the inner plexiform layer (IPL). Photoreceptor cell axon terminals form synapses with bipolar cell dendrites and horizontal cell processes in the OPL, and bipolar cell axon terminals, connect with amacrine cell and ganglion cell dendrites in the IPL [1, 2] (Fig. 1).

Fig. 1.

Organization of the retinal structure and photoreceptor synaptic structure. a Immunostaining of a wild type mouse retina with an α-Lrit1 (red) and an PKCα antibody. Nuclei were stained with DAPI (blue). Lrit1 is localized to photoreceptor and bipolar cell axon terminals. Illustrations of photoreceptor cells (green), bipolar cells (purple), and ganglion cells (magenta) are shown. OS outer segments, ONL outer nuclear layer, INL inner nuclear layer, GCL ganglion cell layer, OPL outer plexiform layer, IPL inner plexiform layer. b A schematic retina diagram. The retina consists of Müller glial cells and five types of neurons: photoreceptor cells (gray, blue, and green), bipolar cells (yellow), horizontal cells (orange), amacrine cells (pink), and ganglion cells (purple). Photoreceptor axon terminals connect with bipolar cell dendrites and horizontal cell processes in the OPL. Bipolar cell axon terminals connect with amacrine and ganglion cell dendrites in the IPL. Bipolar cells are classified into ON- and OFF-types. ON- and OFF-bipolar axon terminals form synapses with amacrine cells and ganglion cells at the different layers in the IPL. c A schematic diagram of the photoreceptor synaptic structure. A schematic of rod (left) and cone (right) photoreceptor ribbon synapses. ON-bipolar cell dendrites (pink) and horizontal cell processes (blue) invaginate into the photoreceptor axon terminals (yellow). The synaptic ribbon (gray) is located close to the surface membrane and tethers synaptic vesicles (white circles). OFF-bipolar cells (orange) make flat contacts with the cone pedicle. d 3D-SIM images of rod (left) and cone (right) photoreceptor synapses, immunostained with anti-Pikachurin (green), anti-Lrit1 (red), and anti-Ctbp2 (white) antibodies. e An electron micrograph of rod (left) and cone (right) photoreceptor axon terminals in the adult mouse retina. Photoreceptor axonal terminals are colored in yellow, horizontal cell processes in blue, and bipolar cell dendrites in pink. R synaptic ribbon.

The photographs in a, d, and e were obtained from [58]

Vision is initiated by rod and cone photoreceptor cells that respond to light stimuli, convert light information to electrical signals, and transmit them to bipolar cells and horizontal cells, which are second-order neurons. Rod photoreceptors are highly sensitive and respond to even a single photon. In contrast, cone photoreceptors are less sensitive; however, they respond to a broad range of light intensities [3, 4]. Rod and cone photoreceptors selectively connect to different classes of bipolar cells. Rod photoreceptor terminals are connected with dendrites of rod bipolar cells which are only the ON type. Cone photoreceptors make synaptic contacts with both ON and OFF-bipolar cells which are called cone ON-bipolar cells and (cone) OFF-bipolar cells, respectively. In dark conditions, photoreceptors release more neurotransmitter glutamate than in light conditions. Glutamate depolarizes OFF-bipolar cells by activating an ionotropic glutamate receptor (iGluR) [5], whereas glutamate hyperpolarizes ON-bipolar cells by closing the TRPM1 cation channel, which follows GRM6 activation, an ON-bipolar cell-specific glutamate receptor. GRM6 activates G_o, a heterotrimeric G protein, that closes the TRPM1 cation channel [6–9]. G_o is composed of Gα_o, Gβ3, and Gγ13 in ON-bipolar cells. Acceleration of GTP hydrolysis by Gα_o stabilized by GTPase activating protein (GAP), allows for rapid onset of ON-bipolar cell depolarization during light-evoked responses. GAP stabilizes the Gα_o transition state by interacting with the switch regions of Gα_o subunits, thereby accelerating GTP hydrolysis of Gα_o [10]. Several studies have suggested that, mainly, RGS7 and RGS11 deactivate G_o by acting as a GAP of G_o [11, 12]. Horizontal cells modulate synaptic transmission from photoreceptors to ON and OFF-bipolar cells by providing inhibitory feedback to photoreceptor cells [13, 14]. Three different inhibitory feedback mechanisms by horizontal cells have been proposed [15]; GABA-mediated feedback [16–18], pH-based feedback [19–21] and hemichannel-mediated ephaptic feedback [22–25].

Photoreceptor synaptic structure and development in the vertebrate retina

A photoreceptor axon terminal forms a ribbon synapse with bipolar cell dendrites and horizontal cell processes in the OPL. The ribbon synapse elaborates a specialized structure, known as the synaptic ribbon. It is an electron-dense, plate-like structure that tethers 100 or more synaptic vesicles on the surface [26]. Vesicles tethered to the ribbon enable both acute and sustained releases [27, 28]. The photoreceptor ribbon synapse is characterized by the L-type calcium channel CACNA1F (Cav 1.4), which is activated by high voltage and enables sustained glutamate release by slow inactivation [28]. Two ON-bipolar cell dendritic terminals and two horizontal cell processes invaginate into a single rod photoreceptor axon terminal (Fig. 1). The OFF-bipolar cell dendrites make flat, non-invaginating synaptic contacts with cone photoreceptor axon terminals [29, 30]. The cone pedicles form flat contacts with OFF-bipolar dendrites, and invaginations with ON-bipolar dendrites and horizontal cell processes, while rod spherules develop a single invagination with ON-bipolar dendrites and horizontal cell processes (Fig. 1). The number of ribbon synapses per pedicle varies depending on the species. For example, the zebrafish cone pedicle contains 2–7 ribbons [31] while the mouse cone pedicle contains ~ 10 ribbons [32]. In the macaque, the number of ribbons per pedicle depends on the location in the retina. In the central retina, each pedicle contains on average ~ 21 ribbons, while a cone pedicle has on average ~ 42 ribbons in the peripheral retina [33]. In vertebrates, retinal development commonly occurs in a sequential manner [34–38]. In the mouse retina, photoreceptor synapses are formed postnatally [35].

Cone and rod photoreceptor synaptogenesis occur at different developmental stages. Cone photoreceptors initiate ribbon synapse formation around postnatal day 4 (P4) and P5 [39] in mice. Horizontal cell processes contact with cone terminals and the synaptic ribbons are attached to the terminal membrane, forming monad contacts. Horizontal cell processes begin to invaginate cone photoreceptor terminals and form dyad contacts on P6. Cone ON-bipolar cell dendrites begin to invaginate cone terminals and occupy the central position, just beneath the ribbon. These dendrites form the triad contacts between P7 and P10. Rod photoreceptor synaptogenesis proceeds in the same manner as that of cones but begins 3–4 days later. Both rod and cone synapse formations are completed by P14 to P15 [39, 40]. In this review, we describe the functions of transmembrane and secreted proteins, both of which contribute to photoreceptor synapse formation. Genes that contribute to photoreceptor synapse formation are presented in Table 1, and the proposed model of photoreceptor synapse formation is summarized in Fig. 2.

Table 1.

List of genes contributing photoreceptor synapse formation

Gene name	Protein localization	Histological phenotype of KO mouse	Electrophysiological phenotype of KO mouse	Human disease	Protein interaction	References
Bassoon	Photoreceptor cell axon terminal	Invagination defect of horizontal and ON-bipolar cells into photoreceptor synaptic terminus, floating ribbons in photoreceptor synaptic terminus	Reduction of b-wave amplitude and delay of implicit time in scotopic and photopic ERGs	N/A	RYBEYE, Piccolo, CAST	[91, 97]
Cacna1f	Photoreceptor cell axon terminal	Small and floating ribbons, invagination defect of horizontal and ON-bipolar cells into photoreceptor synaptic terminus, mislocalization of α2δ4 and Ctbp2	No b-wave in scotopic and photopic ERGs	CSNB, Cone-rod dystrophy	Cabp4, Cav1.4α2δ4, Cav1.4β2	[85–87, 89, 90, 102, 104, 106, 108]
Cabp4	Photoreceptor cell axon terminal	Thinner OPL	Reduction of a-wave and b-wave amplitudes in scotopic and photopic ERGs	CSNB, Cone-rod synaptic disorder, congenital nonprogressive	Cav1.4α1,Cav1.4α2δ4	[90, 110]
Cacnb2	Photoreceptor cell axon terminal	Thinner OPL	Reduction of b-wave amplitude in scotopic and photopic ERGs	Brugada syndrome 4	Cav1.4α1	[88, 89, 127, 129]
Cacna2d4	Photoreceptor cell axon terminal	Invagination defect, mislocalization of mGluR6,Cav1.4 and Elfn1 in rod ON-bipolar cell dendrites	No b-wave in scotopic and photopic ERGs	CSNB, cone dystrophy	Elfn1,Cabp4,Cav1.4α1	[55, 89, 114, 128]
Dystrophin	Photoreceptor cell axon terminal	Mislocalization of Gpr179 in ON-bipolar cell dendritic terminus, mislocalization of Gpr179 and dystroglycan in photoreceptor synaptic clefts	Delay of ERG b-wave implicit time and reduction of b-wave amplitude in scotopic ERG	Muscular dystrophy, abnormal retinal neurotransmission	Dystroglycan, Dystrobrevin, Syntrophin, actin	[45–48, 118, 120, 121]
Dystroglycan	Photoreceptor synaptic cleft	Invagination defect of ON-bipolar cell terminus into photoreceptor synaptic terminus, mislocalization of Gpr179 and Pikachurin in photoreceptor synaptic clefts	Delay of b-wave implicit time in scotopic and photopic ERGs and reduction of b-wave amplitude in photopic ERG	Muscular dystrophy-dystroglycanopathy (congenital with brain and eye anomalies)	Dystrophin, Pikachurin	[42, 43, 118, 119]
Elfn1	Photoreceptor synaptic cleft	Invagination defect of ON-bipolar cell terminus into rod photoreceptor synaptic terminus, mGluR6 mislocalization in rod bipolar cell dendritic terminus	No b-wave in scotopic ERG	N/A	mGluR6, Lrit1, Cacna2d4	[51, 55, 58]
Lrit1	Photoreceptor synaptic cleft	Morphological defect of cone photoreceptor synaptic terminus, Normal invagination	Photopic ERG b-wave amplitude reduction and implicit time delay in photopic ERG, Impaired OKR	N/A	Lrit1, mGluR6,Frmpd2	[58, 59]
Pikachurin	Photoreceptor synaptic cleft	Invagination defect of ON-bipolar cell terminus into photoreceptor synaptic terminus, mislocalization of Gpr179 and dystroglycan in photoreceptor synaptic clefts	Delay of b-wave implicit time in scotopic and photopic ERGs and reduction of b-wave amplitude in photopic ERG, Impaired OKR	N/A	Dystroglycan,Gpr179	[42, 43, 49, 131]
Gpr179	ON-bipolar cell dendritic tip	Normal invagination	No b-wave in scotopic and photopic ERGs	CSNB	Trpm1, Pikachurin	[49, 50, 53, 79, 100, 131, 132]
Lrit3	ON-bipolar cell dendritic tip	Invagination defect of ON-bipolar cell terminus into cone photoreceptor synaptic terminus, mislocalization of Trpm1, mGluR6 and Gpr179 in ON-bipolar cell dendritic terminus	No b-wave in scotopic and photopic ERGs	CSNB	N/A	[61, 62, 64, 109]
mGluR6	ON-bipolar cell dendritic tip	Invagination defect of ON-bipolar cell terminus into photoreceptor synaptic terminus	No b-wave in scotopic and photopic ERGs	CSNB	Elfn1, Lrit1, Nyx, Gαo	[51, 58, 59, 65, 66, 72, 75, 79, 80, 112, 130]
Nyx	ON-bipolar cell dendritic tip	Mislocalization of Trpm1 in ON-bipolar cell dendritic terminus	No b-wave in scotopic and photopic ERGs	CSNB	mGluR6, Trpm1	[68, 80, 103, 105],
Gnb3	ON-bipolar cell dendritic tip	Invagination defect of bipolar cell terminus into photoreceptor axon terminus	No b-wave in scotopic and photopic ERGs	CSNB	Trpm1	[70, 76, 107]
NGL2	Horizontal cell process	Invagination defect of bipolar cell terminus into photoreceptor axon terminus	Decreased b-wave amplitudes in scotopic and photopic ERGs	N/A	Netrin-G	[83, 84]
PlexA4	Horizontal cell process	Invagination defect of bipolar cell terminus into photoreceptor axon terminus	Normal ERG	N/A	Sema6a	[81, 82]
CAST	Photoreceptor axon terminal	Reduction of presynaptic active zone size in photoreceptor cells, Thinner OPL	Decreased b-wave amplitude in scotopic ERG	N/A	Bassoon	[97, 99]

Fig. 2.

Proposed model of the rod (left panel) and cone (right panel) photoreceptor synapse formation. a The molecules involved in rod and cone photoreceptor synapse formation with ON-bipolar cells. The α2δ4–Elfn1–mGluR6 complex and the DGC–Pikachurin–Gpr179 complex function independently in the synapse formation between rod photoreceptors and bipolar cells. Lrit1 is localized to rod photoreceptor axon terminals and interacts with Elfn1, although Lrit1 is dispensable for rod photoreceptor synaptic function and structure (left panel). The Frmpd2–Lrit1–mGluR6 complex and the DGC–Pikachurin–Gpr179 complex independently function in synapse formation between cone photoreceptors and cone ON-bipolar cells (right panel). b Molecules which are thought to contribute directly to synapse pairings between rod/cone photoreceptor cells and ON-bipolar cells

Pikachurin: a secreted protein required for selective synapse formation of photoreceptor cells

In the vertebrate retina, rod and cone photoreceptor cells selectively connect with their respective ON-bipolar cells [41]. These selective connections are assumed to be crucial for vision. The molecular mechanisms underlying selective connectivity are gradually becoming clear. Pikachurin was identified as a secretory protein-encoding gene, predominantly expressed in photoreceptor cells in the mouse retina [42]. Pikachurin encodes a 1017 amino acid protein containing fibronectin-3, Laminin-G, and EGF-like domains with N-terminal signal sequences. PIKACHURIN localizes to the synaptic cleft between the photoreceptor axon terminal and the ON-bipolar cell dendritic terminal. In Pikachurin knock out mice (Pikachurin^−/−), ON-bipolar cell dendritic tips fail to invaginate into rod and cone photoreceptor axon terminals, whereas horizontal cell processes invaginate normally into photoreceptor cell terminals. Electroretinogram (ERG) analysis showed that Pikachurin^−/− mice had a significantly delayed implicit time of ERG b-waves under both scotopic and photopic conditions [42]. In contrast, synaptic transmission, from photoreceptors to OFF-bipolar cells, were unaffected in the Pikachurin^−/− mouse retina. PIKACHURIN localization to the photoreceptor synaptic cleft depends on the function of the Dystrophin–glycoprotein complex (DGC) [43]. DYSTROGLYCAN (DG), a key DGC component, is localized to photoreceptor axon terminals. It consists of an extracellular α-subunit (α-DG) and a transmembrane β-subunit (β-DG). DG, which is encoded by a single gene, is cleaved after translation into two subunits, α and β [44]. DG interacts with DYSTROPHIN through the β-subunit and with PIKACHURIN through the α-subunit. Retinal photoreceptor-specific DG conditional knock out (CKO) mice show an invagination defect of ON-bipolar dendrites, which is similar to that of Pikachurin^−/− mice. On the other hand, DG CKO mice exhibit a more severe delay in neurotransmission from photoreceptor cells to ON-bipolar cells than Pikachurin^−/− mice [42, 43]. These observations indicate that the synaptic connection between photoreceptors and ON-bipolar cells is more severely impaired in the DG CKO retina than in the Pikachurin^−/− retina. Both PIKACHURIN and DG are predominantly expressed in photoreceptor cells. Immunohistochemical analysis in the Pikachurin^−/− retina revealed that DG staining in the photoreceptor synaptic terminals was significantly reduced, suggesting that PIKACHURIN and DG are localized to the photoreceptor axon terminals in an interdependent manner [43]. DYSTROPHIN, an ACTIN-binding cytoskeletal protein [45] and a component of the DGC, is required for proper localization of PIKACHURIN and DG to the photoreceptor synaptic cleft; however, DYSTROPHIN localizes in photoreceptor axon terminals independent of DG and PIKACHURIN [43]. DYSTROPHIN also interacts with the actin cytoskeleton and other DGC components, including DYSTROBREVIN and SYNTROPHIN [46, 47]; these factors may be important for proper DYSTROPHIN localization in the photoreceptor axon terminals. Mutations in the Dystrophin gene displayed an implicit time delay of b-waves in scotopic ERGs in mice [48].

Recently, it was reported that Pikachurin binds to GPR179, an orphan G protein-coupled receptor. Immunohistochemical analysis showed that PIKACHURIN was required for proper localization of both GPR179 and the postsynaptic GAP complex, including RGS7 and RGS11, to ON-bipolar cell dendritic terminals [49]. It should be noted that no invagination defect is observed in the Gpr179^−/− retina, indicating that GPR179 is not essential for PIKACHURIN localization to the photoreceptor synaptic cleft [50]. This suggests that the PIKACHURIN-GPR179 interaction is not essential for forming invagination [49, 50]. Other factors on ON-bipolar cell dendrites might interact with PIKACHURIN to facilitate invagination. DGC, PIKACHURIN, and GPR179 begin to localize photoreceptor synapses between P8 and P12, just before bipolar cell dendrite invagination occurs in the mouse retina [43, 49]. Taken together, during development, photoreceptor cell-secreted PIKACHURIN binds to DGC at photoreceptor axon terminals and recruits GPR179 to the ON-bipolar cell dendritic terminals. This trans-synaptic complex underlies selective synapse formation and normal synaptic transmission between photoreceptor and ON-bipolar cells.

Synapse formation of rod photoreceptor cells

Recently, it was reported that ELFN1 (extracellular leucine-rich repeat and fibronectin type III domain 1), a cell adhesion protein-containing leucine-rich repeat (LRR), fibronectin type III, and transmembrane domains with an N-terminal signal sequence, plays an important role in selective synapse formation between rod photoreceptors and rod ON-bipolar cells [51]. ELFN1 is predominantly expressed in rod photoreceptor cells and localizes to the rod photoreceptor synaptic terminals. ELFN1 interacts in trans with GRM6 in the synaptic cleft between rod photoreceptors and rod ON-bipolar cells. Both Elfn1^−/− and Grm6^−/− mice exhibit an ON-bipolar cell invagination defect, indicating the interaction between ELFN1 and GRM6 is essential for rod-rod ON-bipolar cell synaptic connection [51, 52]. ELFN1 and GRM6 begin to localize to the photoreceptor synaptic cleft between P7 and P11 [51]. Loss of Elfn1 abrogates rod synapse formation during synaptogenesis. In the Elfn1^−/− retina, rod ON-bipolar cell dendrites fail to invaginate into rod photoreceptor axon terminals, and synaptic transmission from rod photoreceptors to rod ON-bipolar cells is impaired. Cone photoreceptor synaptic structure and function are unaffected. Loss of ON-bipolar cell invagination into photoreceptor cells is also observed in Pikachurin^−/− mice and DG CKO mice [43]. However, GRM6 and ELFN1 localization are unaffected in Pikachurin^−/− mice, and GPR179 is normally localized to ON-bipolar cell dendrites in the Grm6^−/− retina [43, 49, 53]. Moreover, there is no detectable physical interaction between PIKACHURIN and GRM6 [49]. These findings suggest the ELFN1–GRM6 complex and DGC–PIKACHURIN complex function in parallel in photoreceptor synapse formation; however, both complexes are required for invagination formation of the photoreceptor terminal. In addition to its role in selective synapse formation, ELFN1 serves as an allosteric modulator of GRM6, which alters its ability to activate G proteins [54]. Similar to the ELFN1 defect, the CACNA1F auxiliary α2δ4 subunit (α2δ4) deficiency impairs ON-bipolar cell invagination, specifically in rod photoreceptors, as shown by electron microscopic analysis, while α2δ4^−/− mice show disrupted neurotransmission from both rod and cone photoreceptors to ON-bipolar cells [55, 56]. Apart from its function as a calcium channel auxiliary subunit, the α2δ4 directly binds to ELFN1 and renders ELFN1 localization to the photoreceptor axon terminal [55]. Therefore, the rod selective ON-bipolar cell invagination defect, observed in the α2δ4^−/− retina, is likely due to the absence of ELFN1 at photoreceptor synaptic terminals. α2δ4 is already localized to photoreceptor axon terminals in the P7 mouse retina. ELFN1 also contributes to synapse formation in the hippocampus with a mechanism similar to that in the retina. In the hippocampus, ELFN1 binds in trans to GRM7, which belongs to the same subfamily with GRM6, the group III mGluR [57].

Synapse formation between cone photoreceptors and cone ON-bipolar cells

Cone photoreceptors connect to two types of bipolar cells: cone ON-bipolar cells and cone OFF-bipolar cells. LRIT1, an LRR cell adhesion protein, plays an essential role in selective synapse formation between cone photoreceptor cells and cone ON-bipolar cells [58, 59]. In contrast, ELFN1 functions for rod photoreceptor-rod ON-bipolar cell selective synapse formation. LRIT1 is localized to photoreceptor synaptic terminals and, similar to ELFN1, interacts trans-synaptically with GRM6. Lrit1-deficient mice show an aberrant morphology of cone photoreceptor pedicles, reducing and delaying synaptic transmission from cone photoreceptors to cone ON-bipolar cells. Lrit1 loss prevents cone synapse formation during synaptogenesis. These findings propose LRIT1 and ELFN1 may play similar roles in the selective synapse formation of rods and cones, respectively. However, there are some differences between LRIT1 and ELFN1. First, LRIT1 and ELFN1 show different localization patterns in the retina. LRIT1 is localized to both rod and cone photoreceptor axon terminals, whereas ELFN1 is specifically localized to rod axon terminals [51, 58, 59]. Furthermore, LRIT1 and ELFN1 are localized to different regions of rod photoreceptor synaptic terminals. ELFN1 is localized to a larger area than LRIT1 and is partially co-localized with LRIT1. LRIT1 is localized to a narrow area, just outside the synaptic ribbon in rod axon terminals. Second, Lrit1^−/− mice show mild defects in neurotransmission and synaptic connections between photoreceptors and ON-bipolar cells compared to those in Elfn1^−/− mice [58]. Although Lrit1 is localized to both cone and rod photoreceptor synaptic terminals, rod photoreceptor synaptic function and structure were unaffected in Lrit1^−/− mice. Synapse formation between rod photoreceptors and rod ON-bipolar cells is probably regulated by the ELFN–GRM6 complex even in the Lrit1^−/− retina, since the localization of ELFN1 and GRM6 in the OPL is unaffected in the Lrit1^−/− retina. Although LRIT1 is localized to photoreceptor axon terminals and interacts with ELFN1, the LRIT1 function in rod axon terminals remains unknown. ON-bipolar cell dendrites and horizontal cell processes normally invaginate into rod and cone photoreceptor cells in the Lrit1^−/− retina. The DGC–PIKACHURIN complex appears to function normally for the cone photoreceptor-cone ON-bipolar cell synapse formation in the Lrit1^−/− retina, since Pikachurin is normally localized to photoreceptor axon terminals, even in the Lrit1^−/− retina [58]. PIKACHURIN and LRIT1 are localized to adjacent different sub-regions in the photoreceptor synaptic cleft. Pikachurin^−/− mice and Lrit1^−/− mice exhibit different defects in the optokinetic responses (OKRs). Lrit1^−/− mice show more severe defects in the OKR amplitudes than those observed in Pikachurin^−/− mice, whereas Lrit1^−/− mice show milder defects in the OKR spatial frequencies and temporal frequencies than the Pikachurin^−/− mice [58, 60]. These findings suggest that PIKACHURIN and LRIT1 function independently in photoreceptor synapse formation. Ablation of the mouse LRIT3, a cell adhesion molecule of the same family as LRIT1, selectively impairs the ON-bipolar cell invagination of cone photoreceptor synaptic terminals [61]; however, LRIT3 is crucial for synaptic transmission from rod and cone photoreceptors to ON-bipolar cells. LRIT3 was reported to be expressed in ON-bipolar cells [62] and rods [63]. Lrit3-deficient mice show almost complete loss of ERG b-wave under scotopic and photopic conditions [64]. In Lrit3^−/− mice, RGS7/11, GRM6, and GPR179 are selectively reduced in cone ON-bipolar cell dendritic terminals [62]; however, the interacting partner of LRIT3 and the mechanism underlying selective impairment of cone photoreceptor synapse assembly in Lrit3^−/− mice has not been clarified. It is conceivable that LRIT3 may bind in trans to proteins localized to cone photoreceptor axon terminals.

Postsynaptic components of ON-bipolar cell dendritic terminals

Glutamate released from photoreceptor cells hyperpolarizes ON-bipolar cells through the components of the metabotropic signaling cascade containing GRM6, TRPM1, GPR179, a leucine-rich proteoglycan NYCTALOPIN (NYX), and LRIT3. These components are localized to ON-bipolar cell dendritic terminals [62, 65–69]. Under light conditions, less glutamate leads to the opening of the TRPM1 channel, depolarizing ON-bipolar cells, and generation of ERG b-wave which is absent or severely reduced in congenital stationary night blindness (CSNB) patients in humans. Under dark conditions, more glutamate activates GRM6 and the G protein G_o, which is composed of Gα_o, Gβ3, and Gγ13, leading to TRPM1 channel closure [8, 67, 70, 71]. All these components are required to depolarize light response in ON-bipolar cells, and loss of each component in mice disrupts synaptic transmission from photoreceptor cells to ON-bipolar cells [9, 50, 64, 66, 68, 72–78]. Interestingly, Grm6-, Gβ3-, or Lrit3-deficient mice show a disruption of the photoreceptor synaptic structure, indicating these components are also required for photoreceptor synapse formation [51, 52, 61, 76]. Gβ3 plays an important role in synaptic maintenance. ON-bipolar cell invagination is reduced in the Gβ3^−/− retina in 1-month old mice, although normal invagination is observed in 3-week old mice [76]. GPR179 and NYX interact with TRPM1 [79, 80]. NYX also binds to GRM6. NYX is necessary for TRPM1 localization to ON-bipolar cell synaptic terminals [65], whereas GPR179 is dispensable for TRPM1 localization [53]. GPR179 localization to ON-bipolar cell dendrites is independent of GRM6, TRPM1, and NYX [53]. There is no interaction between GRM6 and TRPM1 using the yeast two-hybrid system [65]. LRIT3 is required for proper TRPM1 localization to rod and cone ON-bipolar dendritic terminals, and for the localization of GRM6 and GPR179 to cones, but not for rod ON-bipolar cell dendritic terminals [62]. However, interactions between LRIT3 and TRPM1, GRM6 or GPR179 have not been reported.

Photoreceptor-horizontal cell synapse formation

Pikachurin^−/−, ELFN1^−/−, Grm6^−/−, or Gβ3^−/− mice show an invagination defect of ON-bipolar cell dendritic terminals into photoreceptors; however, horizontal cell processes normally invaginate photoreceptor axons in these mice. In contrast, PlexinA4 (PlexA4)^−/− mice display defects in the insertion of horizontal cell processes in photoreceptor axon terminals with no apparent defect in the invagination of ON-bipolar cell dendrites [81]. PLEXA4 is localized to horizontal cell processes and interacts with SEMA6A, a transmembrane Semaphorin [82]. An ectopic outgrowth of horizontal cell processes into the ONL is observed in Sema6A^−/− and PlexA4^−/− retina [81]. Synaptic transmissions from photoreceptor cells to ON-bipolar cells are unaffected in the PlexA4^−/− retina, suggesting horizontal cell invagination is dispensable for synaptic transmission from photoreceptors to ON-bipolar cells. Since PLEXA4 localizes to horizontal cell processes already at P5 [81], it can be assumed that PLEXA4 localizes to horizontal cell processes at P4, when it begins to invaginate into rod axons. NETRIN G-LIGAND 2 (NGL-2), a leucine-rich repeat (LRR)-containing protein, is required for synapse formation between rod photoreceptor cells and horizontal cells [83]. Loss of NGL-2 causes abnormal invasion of horizontal cell processes into the ONL. NGL-2 is localized to horizontal cell processes and interacts with NETRIN-G2, which is expressed in photoreceptor cells [83, 84]. Electron microscopy revealed defects in the insertion of horizontal cell processes into rod axon terminals in the NGL-2^−/− retina [83]. ON-bipolar cell dendrite invagination is unaffected, and horizontal cell processes invaginate normally into cone photoreceptor axon terminals in the NGL-2^−/− retina. Consistent with these observations, the photopic ERGs are normal; however, the scotopic ERGs are abnormal in NGL-2^−/− mice, indicating that NGL-2 is essential for the selective wiring between rod photoreceptors and horizontal cells. It remains unclear why ERGs were affected in NGL-2^−/− mice. NGL-2 begins to localize to horizontal cell processes at around P10, which is consistent with the fact that NGL-2 regulates the synaptic connection between horizontal cells and rods but not between horizontal cells and cones. At P10, invagination of horizontal cell processes to cone axon terminals has almost completed, while invagination to rod axon terminals has only just begun. Additional proteins may regulate photoreceptor-horizontal cell synapse formation since some horizontal cell processes invaginate into photoreceptor axon terminals in NGL-2^−/− and PlexA4^−/− retinas. Moreover, since NGL-2 contributes to rod-horizontal cell-selective synapse formation, other proteins may regulate cone-horizontal cell-selective synapse formation. Further studies are awaited to identify molecules involved in synapse formation between horizontal cells and photoreceptors.

Photoreceptor presynaptic proteins required for ribbon synapse structure formation

In the photoreceptor ribbon synapse, Ca²⁺ flows through CACNA1F, a voltage-gated, L-type calcium channel. CACNA1F is vital for the assembly and function of photoreceptor ribbon synapses and localizes to photoreceptor axon terminals [85]. Loss of the Cacna1f gene, encoding the pore-forming subunit of the CACNA1F calcium channel, causes a neurotransmission defect from photoreceptors to ON-bipolar cells and an invagination failure in the photoreceptor synaptic terminals. In the Cacna1f^−/− retina, both bipolar and horizontal cell invaginations are disrupted. In addition, photoreceptor synaptic ribbons are absent, and horizontal cell processes and rod bipolar cell dendrites invade into the ONL [85–87]. Mice lacking CACNA1F beta 2 subunit, which is encoded by Cacnb2, also exhibit an impaired synaptic transmission from photoreceptors to ON-bipolar cells [88, 89]. Furthermore, CABP4, a CACNA1F regulator, is crucial for the development and/or maintenance of photoreceptor synapses. CABP4 interacts directly with the C-terminal domain of the CACNA1F α₁-subunit and shifts the CACNA1F activations to hyperpolarized voltages [90]. Cabp4^−/− mice show a defect in neurotransmission from photoreceptors to ON-bipolar cells, thinner OPL, invasion of horizontal cell processes and ON-bipolar cell dendrites into the ONL, and reduction in the number of photoreceptor synaptic terminals and synaptic ribbons. Deficiency in the regulator and components of CACNA1F in the mouse retina impairs both rod and cone photoreceptor synaptic structures. However, α2δ4-deficient mice show a defect of rod selective synapse formation, caused by ELFN1 delocalization, which selectively regulates the rod photoreceptor-rod ON-bipolar cell synaptic connection [55]. Photoreceptor glutamate release is also essential for the synaptic connection between photoreceptors and ON-bipolar cells. Inhibition of glutamate release from photoreceptor cells in the mouse retina causes dissipation of ELFN1 and GRM6 from photoreceptor synapses and an aberrant CACNA1F localization pattern [51, 55]. Several proteins that are localized intracellularly in photoreceptor axon terminals also play roles in photoreceptor synapse formation. BASSOON, a major photoreceptor ribbon component, regulates photoreceptor synapse formation [91]. BASSOON is localized to the synaptic ribbons in photoreceptor axon terminals, and this localization begins at around P4-P6 in rodents [91, 92]. BASSOON localization in photoreceptor ribbon synapses is disturbed in Cacna1f^−/− mice [85]. In the Bassoon^−/− retina, photoreceptor ribbons are “floating” since they are not anchored to the presynaptic active zones [91]. Bassoon^−/− mice show an invagination defect of bipolar and horizontal cells, an aberrant invasion of rod ON-bipolar cell dendrites and horizontal cell processes into the ONL, and impaired photoreceptor synaptic transmission. Decreased b-wave amplitude and delay of b-wave implicit time in scotopic and photopic ERGs are observed in Bassoon^−/− mice. In zebrafish cone photoreceptor axon terminals, SYNAPTOJANIN plays a similar role to BASSOON [93, 94]. BASSOON binds directly to, and functionally interacts with, RIBEYE, which serves as a central building block of the synaptic ribbon [95–97]. Furthermore, Bassoon interacts directly with CAST/Erc2 (CAZ-associated structural protein) [97, 98]. CAST, one of the CAZ (cytomatrix of the active zone) proteins, is required for proper formation of the CAZ, which is a densely interlinked network of proteins overlying the presynaptic membrane in photoreceptor synapses [99]. CAST^−/− mice exhibit a decrease in the size of photoreceptor presynaptic active zone and invasion of the horizontal and bipolar cell processes into the ONL. Decreased amplitudes of the b-wave in scotopic ERGs and a thinner OPL are observed in CAST^−/− mice, while the a-wave is normal [99]. Loss of proteins localized in photoreceptor presynaptic active zones and synaptic ribbons causes aberrant invasion of bipolar cell dendrites and horizontal cell processes into the ONL [55, 85, 87, 90, 91, 99]. These observations suggest that components of photoreceptor presynaptic active zones and synaptic ribbons are essential, not only for the formation of photoreceptor synaptic structures but also for correct photoreceptor synaptic location in the retina.

Photoreceptor synapse formation defect and human retinal diseases

Mutations in several of the genes that are related to photoreceptor synapse formation or phototransduction, such as CACNA1F, CABP4, CACNA2D4, GRM6, GPR179, LRIT3, TRPM1, and NYX in humans, cause CSNB [50, 100–112]. CSNB is a clinically and genetically heterogeneous retinal disorder characterized by impaired night vision. Scotopic ERG b-wave amplitude is severely reduced in CSNB patients. Two forms of CSNB can be clinically distinguished: incomplete CSNB (icCSNB) and complete CSNB (cCSNB). Mutations in the protein, localized to photoreceptor axon terminals are associated with icCSNB, while mutations in the protein localized to ON-bipolar cell dendrites are associated with cCSNB. icCSNB is characterized by reduced scotopic ERG b-wave amplitudes, and substantially reduced cone responses, and is associated with mutations in CABP4, CACNA1F, and CACNA2D4 [102, 106, 110–113]. These ERG phenotypes observed in icCSNB patients are less severe than those of the mouse models with mutations in Cabp4, Cacna1f, and Cacna2d4. In addition, these mouse models exhibit more severe phenotypes in synaptic structures than human patients [111]. Mutations in CACNA2D4 also cause cone dystrophy accompanied by night blindness [114], and mutations in CACNA1F or CACNA2D4 are also associated with cone-rod dystrophy [114–117]. cCSNB is characterized by a drastically reduced scotopic ERG b-wave amplitude as well as specific photopic ERG waveforms and has been associated with mutations in GRM6, GNB3, GPR179, LRIT3, NYX, or TRPM1. The mode of inheritance of cCSNB is X-linked or autosomal recessive [109, 111]. cCSNB patients have other ocular disorders, such as myopia, nystagmus, decreased visual acuity, and strabismus [111]. While the scotopic ERG phenotype of b-wave loss in cCSNB patients is similar to that of mouse models, the photopic b-wave phenotype in cCSNB model mice is more severe than that in human cCSNB patients. It is unclear whether ocular defects other than the ERG phenotype are observed in cCSNB model mice. Mutations in the DYSTROPHIN and DG genes cause various forms of muscular dystrophy [118–120]. Furthermore, it has been reported that patients with Becker and Duchenne muscular dystrophy with DMD mutations frequently show abnormal ERGs, with markedly reduced b-wave amplitudes, under scotopic conditions [121]. No association between visual disorders and mutations in the factors that contribute to photoreceptor synapse formation, including ELFN1, LRIT1, PIKACHURIN, NGL-2, PLEXA4, and BASSOON, has been reported so far; future genome studies might find these factors are also involved in the pathogenesis of human visual disorders. Taken together, various findings show that photoreceptor synapse formation is indispensable for normal human vision. Further studies on the mechanisms of photoreceptor synapse formation may also contribute to the development of regenerative medicine of human iPS/ES cell transplantation therapies for severe visual impairment patients.

Similar mechanisms underlying synapse formation between the retina and the brain

Different mechanisms and molecules play important roles in synapse formation between rod photoreceptors and rod ON-bipolar cells, cone photoreceptors and cone ON-bipolar cells, rod photoreceptors and horizontal cells, and cone photoreceptors and horizontal cells. In the retina, presynaptic proteins trans-synaptically bind to postsynaptic proteins and establish a “trans-synaptic bridge”, which contributes to synapse formation between photoreceptors and bipolar cells or horizontal cells. This synapse formation mechanism in the OPL is similar to that in the brain. Recently, it was revealed that GPC4, a secreted HSPG (heparan sulfate proteoglycan), regulates mossy fiber-CA3 pyramidal cell synapse formation in the hippocampus with a mechanism similar to PIKACHURIN [122] (Fig. 3). GPC4 binds directly to a presynaptic receptor protein tyrosine phosphatase leukocyte common antigen-related (LAR) and a postsynaptic orphan receptor, GPR158, a close homolog of GPR179. These proteins build a “trans-synaptic bridge”. GPC4 interacts with GPR158 in a synaptic, input-specific manner and selectively organizes mossy fiber-CA3 synaptic architecture. Other secreted proteins, such as CBLN1, C1QL2, and C1QL3, also form a “trans-synaptic bridge”, spanning the synaptic cleft in the cerebellum and hippocampus. The Cbln family proteins regulate synapse formation and synaptic functions (Fig. 3). CBLN1, secreted from presynaptic parallel fibers and the cerebellar granule cell axons, directly interacts with presynaptic, membrane-tethered NEUREXIN and postsynaptic GLUD2, an orphan glutamate receptor DELTA2. Thus, CBLN1 regulates the synaptic connection between parallel fibers and Purkinje cells, and facilitates bi-directional synaptic differentiation [123–125]. C1Q-like proteins, C1QL2 and C1QL3, are secreted by mossy fibers and bind in trans to postsynaptic GLUK2 and GLUK4 kainate-type glutamate receptor (KAR) subunits. These three proteins also bind to the presynaptic NEUREXIN3 and regulate mossy fiber-CA3 pyramidal cell synaptic function [126]. These findings may imply that synapse formation, regulated by secreted proteins in the brain and retina, is controlled through similar mechanisms. Hence, mechanisms underlying synapse formation in the retina are considered useful for uncovering the mechanisms of synapse and neural circuit formation in the brain.

Fig. 3.

Schematic diagram of the functional mechanisms of secreted proteins in the synaptic clefts. a A schematic representation of the functional mechanism of Pikachurin in photoreceptor synapse formation in the retina. Pikachurin binds to presynaptic Dystroglycan and postsynaptic Gpr179. b A schematic diagram of the functional mechanism of Gpc4 in mossy fiber-CA3 pyramidal cell synapse formation in the hippocampus. Gpc4, secreted from mossy fibers, interacts with presynaptic LAR and postsynaptic Gpr158, a close homolog of Gpr179. c A schematic diagram of the functional mechanism of Cbln1 in synapse formation between parallel fibers and Purkinje cells in the cerebellum

Conclusion

There have been many findings regarding the mechanisms of photoreceptor synapse formation; however, some important questions still remain unresolved. For example, the molecular mechanisms underlying synapse formation between cone photoreceptors and OFF-bipolar cells have not been clarified, even though mechanisms of photoreceptor-ON-bipolar cell connections have been reported in several studies. Some molecules in cone photoreceptor axon terminals may interact in trans with iGluR on the OFF-bipolar dendrites. Further progress in this field is essential for understanding the mechanisms of vision and neural circuit formation in the retina.