PTEN Expression Regulates Gap Junction Connectivity in the Retina

Manipulation of the phosphatase and tensin homolog (PTEN) pathway has been suggested as a therapeutic approach to treat or prevent vision loss due to retinal disease. In this study, we investigated the effects of deleting one copy of Pten in a well-characterized class of retinal ganglion cells called α-ganglion cells in the mouse retina. In Pten+/– retinas, α-ganglion cells did not exhibit major changes in their dendritic structure, although most cells developed a few, unusual loop-forming dendrites. By contrast, α-ganglion cells exhibited a significant decrease in heterologous and homologous gap junction mediated cell coupling with other retinal ganglion and amacrine cells. Additionally, the majority of OFF α-ganglion cells (12/18 cells) formed novel coupling to displaced amacrine cells. The number of connexin36 puncta, the predominant connexin that mediates gap junction communication at electrical synapses, was decreased by at least 50% on OFF α-ganglion cells. Reduced and incorrect gap junction connectivity of α-ganglion cells will affect their functional properties and alter visual image processing in the retina. The anomalous connectivity of retinal ganglion cells would potentially limit future therapeutic approaches involving manipulation of the Pten pathway for treating ganglion cell degeneration in diseases like glaucoma, traumatic brain injury, Parkinson’s, and Alzheimer’s diseases.

Gap junctions are intercellular channels formed by connexins between retinal neurons that influence the propagation and integration of visual signals . Gap junctions are reported to participate in neuronal spike synchrony to enhance the saliency of visual signals, and mediate changes in light adaptation and circadian rhythms (Söhl et al., 2005;Hartveit and Veruki, 2012;Völgyi et al., 2013a;O'Brien, 2014). Gap junctions have also been linked to a number of neurological pathologies (Nakase and Nasus, 2004) as they might allow the passing of toxic molecules from dying cells to neighboring healthy cells (Krysko et al., 2005;Rodríguez-Sinovas et al., 2007). For example, blockade of Cx36 gap junctions provides RGC protection in glaucoma models (Akopian et al., 2014(Akopian et al., , 2016Chen et al., 2015).
With downregulation of Pten as a potential target to increase axonal growth and enhance RGC survival in retinal diseases (Park et al., 2008;Leibinger et al., 2012;de Lima et al., 2012;Duan et al., 2015), understanding the fundamental roles that Pten plays in forming and maintaining RGC architecture and connectivity is of high importance, which will impact future clinical therapies that manipulate Pten signaling.
To selectively study Pten signaling on RGCs, we used loxP-mediated recombination to generate mice in which parvalbumin (PV) cells lacked one copy of Pten (Baohan et al., 2016). In the present study, we evaluate the effect of Pten loss on PV-RGCs, focusing on α-RGC architecture and gap junction connectivity. Our data revealed that the lack of one copy of Pten does not alter the morphology of α-RGCs. However, ON and OFF α-RGCs exhibited a significant decrease of coupled cells or were uncoupled. Additionally, we observed that the majority of OFF α-RGCs lost their normal coupling patterns but showed novel coupling to displaced amacrine cells. The number of connexin36 puncta in OFF α-RGCs was decreased by at least 50% when compared to control OFF α-RGCs.

MATERIALS AND METHODS
These studies were conducted under protocols approved by the University of California at Los Angeles (UCLA) Animal Research Committee. All experiments were carried out in accordance with guidelines for the welfare of experimental animals issued by the U.S. Public Health Service Policy on Human Care and Use of Laboratory Animals, and the UCLA Animal Research Committee.
Both male and female mice were used for these studies. Animals were 3-5 months old at the time of experimentation. Following deep anesthesia with 1%-3% isoflurane (Abbott Laboratories, North Chicago, IL, USA), animals were euthanized by cervical dislocation. The eyes were enucleated and dissected in Hibernate A (Invitrogen, Carlsbad, CA) or Hank's Balanced Salt Solution (HBSS) (ThermoScientific, Waltham, MA) on ice for fluorescence and immunohistochemical studies, and in bicarbonate-buffered Ames medium (pH 7.4) at room temperature (RT) for the intracellular dye injection studies.

Immunohistochemistry
Immunohistochemical labeling was performed using our published protocols (Pérez de Sevilla Müller et al., 2007, 2010a,b, 2013. Whole-mounted retinas were fixed for 15 min by immersion in 4% paraformaldehyde (PFA) in 0.1 M PB (pH 7.4) at RT. They were subsequently washed in phosphate buffer (PB) three times for a total of 90 min and incubated in 10% normal goat serum (NGS) with 0.3%-0.5% Triton X-100 at 4 • C overnight. Retinas were incubated in primary antibodies (Table 1) for 7 days at 4 • C. They were then rinsed three times for 30 min each with 0.1 M PB and incubated with the corresponding secondary antibodies overnight at 4 • C. The following day, the retinas were washed three times in 0.1 M PB and mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA), Citifluor (Citifluor, London, UK) or Aqua Poly-Mount (Polysciences, Warrington, PA, USA). Control experiments for nonspecific binding of the secondary antibodies were also performed.
To prepare retinal sections, eyecups were submerged in 4% PFA in 0.1 M PB, pH 7.4, for 15, 30, 45, or 60 min at RT. They were then placed in 30% sucrose in PB overnight at 4 • C. The eyecups were embedded in optimal cutting temperature medium (Sakura Finetek, Torrance, CA, USA) and sectioned at 12-14 µm with a Leica CM3050S (Leica Microsystems, Buffalo Grove, IL, USA).
Sections were washed three times in 0.1 M PB and incubated in a solution of 10% NGS or donkey serum (DS), 1% bovine serum albumin (BSA), and 0.3-0.5% Triton X-100 in 0.1 M PB for 1-2 h at RT. Following removal of the blocking solution, the slides were immediately incubated in the primary antibodies The dilutions of the primary antibodies are given in Table 1. Secondary antibodies used in this study were Alexa-488 goat antiguinea pig IgG, Alexa-488 goat anti-mouse IgG, Alexa-488 goat anti-rat IgG, Alexa-488 or −568 goat anti-rabbit IgG, and Alexa-488 donkey anti-goat IgG. As a negative control, the omission of the primary antibodies in the single or double labeling studies confirmed the elimination of nonspecific labeling.
All antibodies employed in this study have been used previously with PFA-fixed tissue; our immunostaining patterns were identical to those previously reported in studies using mouse retina (Haverkamp and Wässle, 2000;Pérez de Sevilla Müller et al., 2013;Rodriguez et al., 2014).

Intracellular Dye Injections Studies
Intracellular injections were carried out from 1 pm to 5 pm. The retina was flattened with four radial cuts and mounted with the photoreceptor side down on black filter article. The tissue was then transferred to a bicarbonate-buffered Ames medium (pH 7.4) that was bubbled continuously with carbogen (95% O 2 /5% CO 2 ) and mounted on a Zeiss Axioskop 2.
Intracellular injections of Lucifer Yellow and Neurobiotin were performed as described earlier (Pérez de Sevilla Müller et al., 2007, 2010aVuong et al., 2015). Borosilicate glass electrodes (#60200; A-M Systems; Sequim, WA, USA) were pulled and filled at their tips with 0.5% Lucifer Yellow (Sigma-Aldrich) and 4% N-(2-aminoethyl)-biotinamide hydrochloride (Neurobiotin; Vector Laboratories, Burlingame, CA, USA), and back-filled with 0.1 M Tris buffer, pH 7.4. TdTomato fluorescent cell bodies in the GCL were visualized with a Zeiss long-working distance 40× water immersion objective and conventional epifluorescence for Cy3. Lucifer Yellow was iontophoresed with negative current of −1 nA. When the morphology of the ganglion cell was revealed, the polarity of the current was reversed (+1 nA) and Neurobiotin was injected for 3 min. Multiple cells were injected in each retina, with 1-3 injected α-RGCs in each quadrant. After the final injection, the retina was left in the chamber for at least 30 min to allow for the tracer to diffuse into the cells. Retinas were then fixed in 4% PFA for 15 min and washed for 30 min in 0.1 M PB. To visualize Neurobiotin, retinas were incubated overnight with streptavidin-fluorescein (FITC; dilution 1:500; Jackson Immunoresearch, West Grove, PA), in 0.1 M PB containing 0.3% Triton X-100 at 4 • C. On some occasions, streptavidin-fluorescein immunolabeled mis-injected retinal Müller cells close to the Neurobiotin-injected ganglion cell body. Müller cells were not included in the coupling analysis. Retinas were mounted with the GCL facing upward and cover slipped with Aqua Poly/Mount.

Analysis of Whole-Mounted Retinas
Retinal whole-mounts were imaged in their entirety as tiled mosaics and stitched together with a 5-10% overlap at the edge of each optical section, using the Zeiss Zen 2011 Black software (version 3.2) package. Individual tiles were collected as 12-bit 3D z-stacks from the nerve fiber layer to the INL using a 40× /1.20NA C-Apochromat objective and a zoom factor of 1. The confocal pinhole diameter was set to 1 Airy unit and pixel acquisition set to 1024 × 1024. Cells in retinal fields (500 × 500 µm or 1000 × 1000 µm) at 100 µm intervals from the optic nerve head to peripheral retina were manually counted. Three retinal fields per quadrant of each retina were analyzed for PV-cells and Cx36 counts.

Analysis of Retinal Sections
Retinal sections were imaged in their entirety as tiled mosaics and stitched together with a 5-10% overlap at the edge of each optical section, using the Zeiss Zen 2011 Black software (version 3.2) package using a Zeiss C-Apochromat 40 × /1.2 NA corrected water objective and a zoom factor of 0.6. The confocal pinhole diameter was set to 1 Airy unit and pixel acquisition set to 512 × 512. Cells in areas of 500 µm were manually counted. At least four retinal fields of each section were analyzed.

Analysis of Injected Ganglion Cells
Confocal images were analyzed using the Zeiss proprietary software (version 3.2), Image Browser v4 or Imaris 9.5.0 (Bitplane AG, Concord, MA, USA) software.
RGCs were reconstructed using the Imaris Filament Tracer option. The Filament Tracer operates on 3D images, which provides sufficient resolution to resolve the Filaments to be studied in all three spatial directions. The Filament Tracer option automatically computes all the paths from a user-defined starting point (RGC body) to the end of the structure. After all possible paths are calculated by the algorithm, the filaments are traced by the user by moving the mouse over the structure of interest. Imaris provided the following morphological analysis: Dendrite Length: the sum of all edges between two branch points or between a branch point and a terminal point, respectively.
Filament Volume (sum): the sum of all segment's volumes within the entire filament graph.
Filament-Dendrite Area: defined as the sum of the areas of all the segment edges. The area of an edge is defined as a surface area of a frustum (truncated cone).
Sholl Analysis: the number of dendrite intersections on concentric spheres, defining dendrite spatial distribution as a function of distance from the beginning point.
Number of Dendrite Branch Points: the number of dendrite branching points in the entire filament graph.
Filament-Dendrite Straightness: the ratio between dendrite length and radial distance between two branch points. If the Dendrite Straightness is 1 that means that the dendrite is completely straight.
Filaments-Dendrite Branching Angle B: the angle between the extending lines connecting the branch point with the neighboring branch points and the terminal points, respectively.

Analysis of Cx36 in Injected Ganglion Cells
For quantitative analysis of RGC morphology and Cx36 synaptic puncta associated with their dendrites, individual optical slices from Z-stacks were analyzed using the Imaris software. Injected RGCs were reconstructed using the Imaris Filament Tracer option to create a 3D cell-surface rendering using a combination of surface and filament objects as described above. RGCs were masked using an automated threshold determined by the software.
Cx36 puncta were first reconstructed as 3D structures using ''surface objects'' (to outline puncta borders) created using an estimated 0.5 µm diameter. Spots were created (using softwaredetermined automatic threshold) for all synaptic puncta. Puncta ''objects'' were then converted into puncta ''spots'' (with automatic intensity max spot detection thresholds and a 0.5 µm estimated diameter) using surface object centroids in Imaris. All spots located less than 0.3 µm from the surface of the RGC mask were quantified. The intensity levels and contrast of the final images presented in the figures were adjusted in Adobe Photoshop CS2 v.9.02 (Adobe Systems, San Jose, CA).

Statistical Analysis
All values are given as mean and standard error of the mean (SEM). Single statistical comparisons of a group vs. its control group were performed using a two-tailed Student's t-test in GraphPad Prism 4.0 (GraphPad Software, Inc, La Jolla, CA, USA). If data were not normally distributed, non-parametric tests (Mann-Whitney U test) were used. A p value ≤ 0.05 was considered statistically significant.
TdTomato fluorescence was also observed in somata in the inner nuclear layer (INL) adjacent to the inner plexiform layer (IPL) in PV-Pten +/+ and PV-Pten +/− retinas. Somal sizes ranged from 4.4-11.2 µm (n = 340 cells from three retinas). These neurons are likely amacrine cells due to their small somal diameter and location close to the INL/IPL border (Figure 2). TdTomato fluorescence was strong enough to visualize primary dendrites located in the IPL. The PV-Pten amacrine cell population is comprised of at least two different types (Figures 2A-C); a small-field amacrine cell with numerous varicosities (Figure 2B), and a second medium-or wide-field amacrine cell with longer and thin primary dendrites ( Figure 2C). The morphological features of the small-field amacrine cell type are similar to previous descriptions of AII amacrine cells (Casini et al., 1995;Wässle et al., 1995Wässle et al., , 2009Massey and Mills, 1999;Pang et al., 2012).
To characterize the tdTomato fluorescent cells in the GCL in the PV-Pten mouse line, whole mounts (Figures 3A-C) were immunostained with antibodies to retinal binding protein with multiple splicing (RBPMS), a pan-ganglion cell marker (Rodriguez et al., 2014). In these experiments, most tdTomato fluorescent cells expressed RBPMS, indicating that the majority (98% of cells, n = 3 retinas) are RGCs. In addition to the RGCs, 2% of the tdTomato fluorescent cells were not stained. They had smaller cell bodies, ranging from 6 to 10 µm with an average cell diameter of 8 ± 1 µm (n = 19 cells from three retinas). The small soma size and the lack of RBPMS immunostaining indicates the presence of a small number of PV-displaced amacrine cells that express tdTomato. This is consistent with a previous report that has also shown the presence of PV-positive displaced amacrine cells in the mouse retina (Kim and Jeon, 2006).

Morphology and Tracer Coupling of α-RGCs
To analyze if a single-copy loss affects RGC morphology and tracer-coupling circuitry, we injected PV-tdTomato somata in the GCL with Neurobiotin and Lucifer Yellow. Neurobiotin was confirmed to have filled the cells when tapering dendritic endings were observed.
From the 40 or more morphological, transcriptional, and function subtypes of RGCs (Sanes and Masland, 2015;Baden et al., 2016;Tran et al., 2019), we focused on α-RGCs, a well-characterized ganglion cell type with large somata, dendritic stratifications in the ON and OFF layers of the IPL, and dendritic trees that form a circular to elliptical field Völgyi et al., 2009;Sanes and Masland, 2015). In addition, the gap junction patterns of these cells are established in the mouse retina (Schubert et al., 2005;Völgyi et al., 2005). OFF α-RGCs are coupled to OFF α-RGCs in the GCL and to amacrine cells in the INL (Schubert et al., 2005;Völgyi et al., 2005). ON α-RGCs are only coupled to displaced amacrine cells and never exhibit coupling to other α-RGCs nor are they coupled to any cells in the INL (Schubert et al., 2005;Völgyi et al., 2005).
OFF α-RGC dendrites in the PV-Pten +/+ mice (n = 10 cells, Figures 5A-C) usually had an elliptical dendritic field, although some RGCs with a circular field were also observed. The dendrites rarely overlapped in PV-Pten +/+ retinas (Figures 5B,C). Loop-forming dendrites were observed in about a third of the PV-Pten +/+ OFF α-RGCs (n = 3/10 cells). In contrast, more than half of the injected cells in the PV-Pten +/− mice exhibited loop-forming dendrites (n = 14/23 cells, Figures 5D-F arrows), similar to the ON α-RGCs. Consistent with the lack of morphological differences in the ON α-RGCs, OFF α-RGCs in the PV-Pten +/− mice also did not show significant differences in their morphology compared to OFF α-RGCs in the PV-Pten +/+ mice (Figures 5G-P, P > 0.05, Mann-Whitney test).
OFF α-RGCs in the PV-Pten +/+ retinas were coupled to other OFF α-RGCs in the GCL (n = 17 cells, Figures 7A,B)  and to amacrine cells in the INL (Figures 7A,C), as reported previously (Schubert et al., 2005;Völgyi et al., 2005). The number of coupled RGCs to OFF α-RGCs in the GCL was 1.9 ± 0.2 cells, with an average of somal diameter of 16.3 ± 0.4 µm (n = 19 cells) in PV-Pten +/+ retinas. The number of coupled amacrine cells in the INL was 2.8 ± 0.6 cells, with  an average somal diameter of 7.7 ± 0.3 µm (n = 28 cells). In contrast, OFF α-RGCs in the PV-Pten +/− retinas (n = 18 cells) were coupled to either two different cell types, or a single cell type based on their somal size in the GCL (Figures 7D,F).
One cell type was the OFF α-RGC. The other cells were displaced amacrine cells. There was a significant reduction in cell coupling to other OFF α-RGCs (P < 0.0001; Mann-Whitney test, Figures 7E,G) compared to OFF α-RGCs in PV-Pten +/+  retinas. The average number of coupled RGCs to OFF α-RGCs was 0.5 ± 0.2 cells in the GCL and their average somal diameter was 16.8 ± 0.9 µm (n = 4 cells). The average number of coupled cells to displaced amacrine cells (P < 0.0001; Mann-Whitney test, Figures 7E,G) was 2.1 ± 0.5 cells in the GCL with an average somal diameter of 8.5 ± 0.3 µm (n = 12 cells). There was also a significant reduction in cell coupling to amacrine cells in the INL compared to PV-Pten +/+ retinas (P < 0.05; Mann-Whitney test, Figures 7F,G). The average number coupled amacrine cells in the INL was 1 ± 0.4 cells with an average somal diameter of 7.3 ± 0.4 µm (n = 12 cells).

Cx36 Expression in OFF α-RGCs
Next, we examined gap junction connexin (Cx) expression in OFF α-RGCs. Cx36 is the most abundant Cx in the retina and mediates coupling among the majority of RGC types, including OFF α-RGCs (Schubert et al., 2005;Völgyi et al., 2005;Pan et al., 2010).
We also used a Cx36 antibody to investigate the overall pattern of Cx36 puncta in the IPL of the PV-Pten mouse retinas. In vertical sections (Figures 9A,B), there was weak Cx36 immunoreactivity in the outer plexiform layer (OPL), and small immunoreactive puncta were observed in the IPL. The brightest immunostained puncta were found in the ON sublamina of the IPL of the PV-Pten +/+ (Figure 9A; n = 3 retinas) and PV-Pten +/− retinas (Figure 9B; n = 3 retinas), consistent with previous studies (Güldenagel et al., 2000(Güldenagel et al., , 2001Feigenspan et al., 2001;Mills et al., 2001;Deans et al., 2002).

Amacrine and Bipolar Cell Populations in the PV-Pten Mouse Line
Pten is a positive regulator of amacrine cell genesis (Tachibana et al., 2016), and suppression of PI3K/Akt signaling by Pten is crucial for proper neuronal differentiation and forming normal neuronal networks (Sakagami et al., 2012;Tachibana et al., 2016). Since PV-amacrine cells are expressed in the PV-Pten +/− retinas, we studied well-characterized amacrine and bipolar cell populations in the INL to determine if amacrine cell production is affected when a copy of Pten is deleted.
Bipolar cell populations: To study the bipolar cell population, we performed labeling experiments for Goα, a marker for ON-cone bipolar cells, PKCα, a marker for rod bipolar cells and the vesicular glutamate transporter 1 (VGluT-1), a marker for synaptic terminals of all bipolar cells as well as rod spherules and cone pedicles (Haverkamp and Wässle, 2000;Haverkamp et al., 2003;Johnson et al., 2003). We found no significant differences in the number of Goα-expressing bipolar cells and PKCα-expressing bipolar cells in PV-Pten +/− mice compared to PV-Pten +/+ mice ( Immunostaining studies with the antibodies to VGluT-1 showed no differences in the immunostaining levels and intensity in the IPL and OPL of the PV-Pten +/− retina (Supplementary  Figures 3G,H; n = 2 retinas) compared to the PV-Pten +/+ retina.
In summary, these findings indicate that the lack of one Pten copy in PV-RGCs and PV-amacrine cells does not appear to alter the number of cells in several representative bipolar and amacrine cell types, or the pattern of photoreceptor and bipolar cell terminals in the plexiform layers.

DISCUSSION
Manipulation of the Pten pathway provides insight into its potential therapeutic use in eye diseases. Pten signaling promotes RGC axon regeneration and enhances RGC survival following ocular injury (Leibinger et al., 2012;de Lima et al., 2012;Duan et al., 2015); however, to date, there is no systematic study evaluating RGC morphology, connectivity, gap junction expression, and the impact on RGC connectivity in Pten deletion lines.
The present study examines the effects of a single-copy loss of Pten in specific PV retinal types, with a focus on α-RGCs. Suppression of PI3K/Akt signaling by Pten is crucial for proper retinal neuronal differentiation and normal circuitry formation (Sakagami et al., 2012;Tachibana et al., 2016), consistent with other studies that report cortical dendritic and synaptic changes with Pten deletion (Kwon et al., 2006;Chow et al., 2009;Xiong et al., 2012). Although α-RGCs did not exhibit changes in somal size and showed modest changes in dendritic morphology, the α-RGCs did show a significant decrease in the number of Cx36 immunoreactive puncta and a reduction in cell coupling compared to α-RGCs in littermate, PV-Pten +/+ retinas. The numerical reduction of RGC tracer coupling is likely due to the reduced expression of Cx36 in OFF α-RGCs. In addition, most OFF α-RGCs showed altered changes in their connectivity, with aberrant gap junction connectivity to displaced amacrine cells. This altered connectivity (Figures 7E,G) is likely to result in Reduced Coupling in PV-RGCs Preiss et al. (2007) suggested that classical PKC isoforms may be involved in signaling to Akt phosphorylation. In addition, protein kinases are responsible for the phosphorylation of the connexins (Xia and Mills, 2004;Urschel et al., 2006;Pérez de Sevilla Müller et al., 2010a). Therefore, we hypothesize that Pten signaling modulates gap junction coupling by affecting protein kinase phosphorylation. Moreover, confocal microscopy and immunoprecipitation assays have shown that Cx43 binds to PTEN (González-Sánchez et al., 2016) and that the antiproliferative effect of Cx43, the major protein forming gap junctions in astrocytes, is reduced in glioma cells and astrocytes when Pten levels are reduced using NT-siRNA approaches (González-Sánchez et al., 2016). These experimental findings are consistent with our observations that Cx36 neuronal expression and gap junctional connectivity measured by tracer coupling is also affected by reduction of Pten signaling in retinal cells with a single copy of Pten.
In the mouse retina, Cx36 mediates coupling of the majority of RGCs (Pan et al., 2010), which underlies the synchronization of activity of neighboring RGCs. Homologous coupling between RGC neighbors is believed to underlie short-latency synchrony of impulse activity, whereas the heterologous coupling between RGCs and amacrine cells results in broader and correlated activity (Mastronarde, 1983a,b,c;Meister et al., 1995;Brivanlou et al., 1998;DeVries, 1999;Meister and Berry, 1999;Hu and Bloomfield, 2003;Völgyi et al., 2013b). A reduction in coupling due to the lack of one Pten gene could impact intracellular communication and have a deleterious influence on visual information processing. For instance, the loss of spike correlations and synchrony from gap junctions will likely decrease the propagation of visual signals (Alonso et al., 1996;Stevens and Zador, 1998;Singer, 1999;Usrey and Reid, 1999) as well as the short-latency spike synchrony in RGCs (Arnett and Spraker, 1981;Brivanlou et al., 1998;DeVries, 1999;Hu and Bloomfield, 2003).
Although OFF α-RGCs showed a decrease in the number of Cx36 immunoreactive puncta, the overall pattern of Cx36 puncta in the IPL remained the same. A possible explanation could be the formation of the aberrant Cx36 gap junctions with displaced amacrine cells, since their formation between OFF α-RGCs with a single-copy loss of Pten and displaced amacrine cells requires these amacrine cells to express Cx36. An increase in Cx gene expression has also been demonstrated in a model of neuroinflammation in the rat hippocampus (Abbasian et al., 2012). Additionally, Cx43 is upregulated following central nervous system injury (Danesh-Meyer et al., 2012). Based on these other pathological observations, changes in PTEN signaling in OFF α-RGCs lacking a single copy of Pten could also impact on the regulation of Cx36 gene expression in these ganglion cells as well as altering connectivity and influencing Cx36 expression in other retinal cell types. In addition to the altered cellular connectivity of the α-RGCs, the connectivity of other RGCs is also likely to be changed with reduction of PTEN gene expression. The altered connectivity and presumably altered functional properties as shown in cortical pyramidal neurons (Garcia-Junco-Clemente et al., 2013) would potentially impact the efficacy of future therapeutic approaches that manipulate the Pten signaling pathway for treating ophthalmic diseases.
It is important to note that four αRGC types have been described in the mouse retina based on responses to light steps: ON-sustained, ON-transient, OFF-sustained, and OFF-transient (Pang et al., 2003;Van Wyk et al., 2009;Krieger et al., 2017;Sawant et al., 2021). Morphologically, they differ in the level of dendritic stratification within the IPL with the ON types that ramify closer to the GCL and the OFF types closer to the INL (Pang et al., 2003;Van Wyk et al., 2009;Krieger et al., 2017;Sawant et al., 2021). The variety of α-RGC types could explain some of the differences we observed in the PV-Pten +/− retinas. Twelve OFF α-RGCs formed novel coupling to displaced amacrine cells while six other OFF α-RGCs were either uncoupled or with a significant reduction of their normal coupling patterns. The fact that some OFF α-RGCs had an elliptical dendritic field, or a circular field could be also due to different types of OFF α-RGCs.
While our data for the ON α-RGCs is quite consistent, another aspect to consider is that ON-sustained α-RGCs display a nasal-to-temporal gradient in cell density, size, and receptive fields (Bleckert et al., 2014). These changes in the retina might also impact in their gap junction patterns and the overall number of Cx36 expression in ON α-RGCs depending on the nasal-totemporal gradient.
In addition to alterations of gap junction connectivity of the PV-positive neurons in the PV-Pten +/− retinas, other signaling and cellular changes might occur in PV-positive neurons. Pten haploinsufficiency in cortical pyramidal neurons increases the expression of small conductance calcium-activated potassium (SK) channels, resulting in an increase in the amplitude of the after-spike hyperpolarization and a decrease in intrinsic excitability (Garcia-Junco-Clemente et al., 2013). The change in intrinsic excitability reduces the evoked firing rates of cortical pyramidal neurons (Garcia-Junco-Clemente et al., 2013). With many known calcium-activated potassium channels expressed in RGCs (Wang et al., 1998) and amacrine cells (Grimes et al., 2009;Tanimoto et al., 2012), we speculate that PV-positive retinal neurons in the PV-Pten +/− mice could also have this channelopathy and a decrease in RGC intrinsic excitability. Furthermore, calcium-activated potassium channels often co-localize with Ca 2+ channels to regulate Ca 2+ levels (Lee and Cui, 2010;Van Hook et al., 2019), suggesting the possibility that this channelopathy might also affect Ca 2+ channel function, and alter both intrinsic and extrinsic cellular signaling.

CONCLUSIONS
Precise electrical and chemical synaptic organization between retinal neurons is important for proper neural network function and visual transmission to the brain (Varadarajan and Huberman, 2018). Changes in neural wiring from disease or trauma are thus likely to alter visual information processing (Strettoi and Pignatelli, 2000;Cuenca et al., 2005;Gargini et al., 2007;Puthussery et al., 2009;Phillips et al., 2010). Altered gap junctional connectivity in the inner retina, together with functional changes in cortical cell responsivity reported for pyramidal neurons with one copy of Pten deleted (Garcia-Junco- Clemente et al., 2013;Baohan et al., 2016) presents a potential barrier for implementing Pten-related therapeutic interventions in eye diseases. These findings suggest caution in evaluating the therapeutic potential of findings that manipulation of the PTEN pathway to enhance RGC survival and promote axon regeneration (Park et al., 2008;Sun et al., 2011;de Lima et al., 2012;Duan et al., 2015;Norsworthy et al., 2017;Li et al., 2018;Wang et al., 2018). A possible approach for manipulation of the PTEN pathway would be to identify possible windows of intervention during early stages of retinal remodeling (Jones and Marc, 2005;Cuenca et al., 2014) and careful implementation of therapeutic protocols to modulate the PTEN pathway to prevent or treat visual-related abnormalities in neurodegenerative diseases.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

ETHICS STATEMENT
The animal study was reviewed and approved and these studies were conducted under protocols approved by the University of California at Los Angeles (UCLA) Animal Research Committee. All experiments were carried out in accordance with guidelines for the welfare of experimental animals issued by the U.S. Public Health Service Policy on Human Care and Use of Laboratory Animals, and the UCLA Animal Research Committee.

AUTHOR CONTRIBUTIONS
LPS conceived the project, designed the experiments, and supervised the project. LPS, AC, and SA performed the experiments. LPS, AC, SA, and AH analyzed the data. LPS, SA, and NB wrote the article. All authors contributed to the article and approved the submitted version.
FUNDING Support for these studies is from VA Merit Review (5I01BX000764; NB), NIH R01 EY04067 (NB), and NIDDDK P30 DK41301 (UCLA Cure Center Core). This work was supported in part by Career Scientist Award (14F-RCS-004) from the United States Department of Veterans Affairs. The contents do not represent the views of the U.S. Department of Veterans Affairs or the United States Government. NB is a VA Senior Career Research Scientist.