Arlene A. Hirano

Immunocytochemical Description of Five Bipolar Cell Types of the Mouse Retina

With the ever‐growing number of transgenic mice being used in vision research, a precise knowledge of the cellular organization of the mouse retina is required. As with the cat, rabbit, rat, and primate retinae, as many as 10 cone bipolar types and one rod bipolar type can be expected to exist in the mouse retina; however, they still have to be defined. In the current study, several immunocytochemical markers were applied to sections of mouse retina, and the labeling of bipolar cells was studied using confocal microscopy and electron microscopy. By using antibodies against the neurokinin‐3 receptor NK3R; the plasma membrane calcium ATPase1 (PMCA1); and the calcium (Ca)‐binding proteins CaB1, CaB5, caldendrin, and recoverin, three different OFF‐cone bipolar cells could be identified. One type of ON‐cone bipolar cell was identified through its immunoreactivity for CaB5 and PMCA1. Rod bipolar cells, comparable in morphology to those of other mammalian retinae, expressed protein kinase Cα and CaB5. It was also shown that putative OFF‐cone bipolar cells receive light signals through flat contacts at the cone pedicle base, whereas ON‐cone bipolar signaling involves invaginating contacts. The distribution of the kainate receptor subunit GluR5 was studied by confocal and electron microscopy. GluR5 was expressed at flat bipolar cell contacts; however, it appears to be involved with only certain types of OFF‐cone bipolar cells. This suggests that different bipolar cell types receive their light signals through different sets of glutamate receptors. J. Comp. Neurol. 455:463–476, 2003.

The nob2 mouse, a null mutation in Cacna1f : Anatomical and functional abnormalities in the outer retina and their consequences on ganglion cell visual responses

Glutamate release from photoreceptor terminals is controlled by voltage-dependent calcium channels (VDCCs). In humans, mutations in the Cacna1f gene, encoding the alpha1F subunit of VDCCs, underlie the incomplete form of X-linked congenital stationary night blindness (CSNB2). These mutations impair synaptic transmission from rod and cone photoreceptors to bipolar cells. Here, we report anatomical and functional characterizations of the retina in the nob2 (no b-wave 2) mouse, a naturally occurring mutant caused by a null mutation in Cacna1f. Not surprisingly, the b-waves of both the light- and dark-adapted electroretinogram are abnormal in nob2 mice. The outer plexiform layer (OPL) is disorganized, with extension of ectopic neurites through the outer nuclear layer that originate from rod bipolar and horizontal cells, but not from hyperpolarizing bipolar cells. These ectopic neurites continue to express mGluR6, which is frequently associated with profiles that label with the presynaptic marker Ribeye, indicating potential points of ectopic synapse formation. However, the morphology of the presynaptic Ribeye-positive profiles is abnormal. While cone pedicles are present their morphology also appears compromised. Characterizations of visual responses in retinal ganglion cells in vivo, under photopic conditions, demonstrate that ON-center cells have a reduced dynamic range, although their basic center-surround organization is retained; no alteration in the responses of OFF-center cells was evident. These results indicate that nob2 mice are a valuable model in which to explore the pathophysiological mechanisms associated with Cacna1f mutations causing CSNB2, and the subsequent effects on visual information processing. Further, the nob2 mouse represents a model system in which to define the signals that guide synapse formation and/or maintenance in the OPL.

Protein Tyrosine Kinase Activity and Its Endogenous Substrates in Rat Brain: A Subcellular and Regional Survey

Abstract: The rat CNS contains high levels of tyrosine‐specific protein kinases that specifically phosphorylate the tyrosine‐containing synthetic peptide poly(Glu80,Tyr20). The phosphorylation of this peptide is rapid and occurs with normal Michaelis‐Menten kinetics. Using this peptide to assay for enzyme activity, we have measured the protein tyrosine kinase activity in homogenates from various regions of rat CNS. A marked regional distribution pattern vas observed, with high activity present in cerebellum, hippocampus, olfactory bulb, and pyriform cortex, and low activity in the pons/medulla and spinal cord. The distribution of protein tyrosine kinase activity was examined in various subcellular fractions of rat forebrain. The majority of the activity was associated with the particulate fractions, with enrichment in the crude microsomal (P3) and crude synaptic vesicle (LP2) fractions. Moreover, the subcellular distribution of pp60csrc, a well‐characterized protein tyrosine kinase, was examined by immunoblot analysis using an affinity‐purified antibody specific for pp60csrc. The subcellular distribution of pp60csrc paralleled the overall protein tyrosine kinase activity. In addition, using an antibody specific for phosphotyrosine, endogenous substrates for protein tyrosine kinases were demonstrated on immunoblots of homogenates from the various regions and the subcellular fractions. The immunoblots revealed numerous phosphotyrosine‐containing proteins that were present in many of the CNS regions examined and were associated with specific subcellular fractions. The differences in tyrosine‐specific protein kinase activity, and in phosphotyrosine‐containing proteins, observed in various regional areas and subcellular fractions may reflect specific functional roles for protein tyrosine kinase activity in mammalian brain.

Cellular distribution and subcellular localization of molecular components of vesicular transmitter release in horizontal cells of rabbit retina.

The mechanism underlying transmitter release from retinal horizontal cells is poorly understood. We investigated the possibility of vesicular transmitter release from mammalian horizontal cells by examining the expression of synaptic proteins that participate in vesicular transmitter release at chemical synapses. Using immunocytochemistry, we evaluated the cellular and subcellular distribution of complexin I/II, syntaxin‐1, and synapsin I in rabbit retina. Strong labeling for complexin I/II, proteins that regulate a late step in vesicular transmitter release, was found in both synaptic layers of the retina, and in somata of A‐ and B‐type horizontal cells, of γ‐aminobutyric acid (GABA)‐ and glycinergic amacrine cells, and of ganglion cells. Immunoelectron microscopy demonstrated the presence of complexin I/II in horizontal cell processes postsynaptic to rod and cone ribbon synapses. Syntaxin‐1, a core protein of the soluble N‐ethylmaleimide‐sensitive‐factor attachment protein receptor (SNARE) complex known to bind to complexin, and synapsin I, a synaptic vesicle‐associated protein involved in the Ca2+‐dependent recruitment of synaptic vesicles for transmitter release, were also present in the horizontal cells and their processes at photoreceptor synapses. Photoreceptors and bipolar cells did not express any of these proteins at their axon terminals. The presence of complexin I/II, syntaxin‐1, and synapsin I in rabbit horizontal cell processes and tips suggests that a vesicular mechanism may underlie transmitter release from mammalian horizontal cells. J. Comp. Neurol. 488:70–81, 2005. Published 2005 Wiley‐Liss, Inc.

Calcium channels in rat horizontal cells regulate feedback inhibition of photoreceptors through an unconventional GABA‐ and pH‐sensitive mechanism

•  Inhibitory feedback from horizontal cells to photoreceptors regulates synaptic gain and contributes to centre–surround receptive field formation via mechanisms that are not fully understood. •  We show that horizontal cell calcium channels and ionotropic GABA receptors mediate the inhibitory feedback, and that the results of their actions are blocked by strong pH buffering with Hepes. •  GABA appears to act not upon the photoreceptor but instead upon the horizontal cell itself. The horizontal cell GABA receptors are permeable to chloride and bicarbonate, meaning their activation can produce changes in synaptic cleft pH. •  These results suggest that activation of calcium channels in a depolarized horizontal cell releases GABA, which acts in an autaptic manner to increase bicarbonate permeability. The resulting influx of bicarbonate contributes to acidification of the synaptic cleft, inhibiting photoreceptor calcium channels, the hallmark of inhibitory feedback at this synapse.

Plasmalemmal and Vesicular γ-Aminobutyric Acid Transporter Expression in the Developing Mouse Retina

Plasmalemmal and vesicular γ‐aminobutyric acid (GABA) transporters influence neurotransmission by regulating high‐affinity GABA uptake and GABA release into the synaptic cleft and extracellular space. Postnatal expression of the plasmalemmal GABA transporter‐1 (GAT‐1), GAT‐3, and the vesicular GABA/glycine transporter (VGAT) were evaluated in the developing mouse retina by using immunohistochemistry with affinity‐purified antibodies. Weak transporter immunoreactivity was observed in the inner retina at postnatal day 0 (P0). GAT‐1 immunostaining at P0 and at older ages was in amacrine and displaced amacrine cells in the inner nuclear layer (INL) and ganglion cell layer (GCL), respectively, and in their processes in the inner plexiform layer (IPL). At P10, weak GAT‐1 immunostaining was in Müller cell processes. GAT‐3 immunostaining at P0 and older ages was in amacrine cells and their processes, as well as in Müller cells and their processes that extended radially across the retina. At P10, Müller cell somata were observed in the middle of the INL. VGAT immunostaining was present at P0 and older ages in amacrine cells in the INL as well as processes in the IPL. At P5, weak VGAT immunostaining was also observed in horizontal cell somata and processes. By P15, the GAT and VGAT immunostaining patterns appear similar to the adult immunostaining patterns; they reached adult levels by about P20. These findings demonstrate that GABA uptake and release are initially established in the inner retina during the first postnatal week and that these systems subsequently mature in the outer retina during the second postnatal week. J. Comp. Neurol. 512:6–26, 2009. Published 2008 Wiley‐Liss, Inc.

Galanin receptors in the rat gastrointestinal tract

Galanin functions are mediated by three distinct G-protein-coupled receptors, galanin receptor 1 (GalR1), GalR2 and GalR3, which activate different intracellular signaling pathways. Here, we quantified mRNA levels of GalR1, GalR2 and GalR3 in the gastrointestinal tract using real time RT-PCR. GalR1 and GalR2 mRNAs were detected in all segments with the highest levels in the large intestine and stomach, respectively. GalR3 mRNA levels were quite low and mostly confined to the colon. We also investigated the effect of galanin 1-16, which has high affinity for GalR1 and GalR2 and low affinity for GalR3 on depolarization-evoked Ca2+ increases in rat cultured myenteric neurons using Ca2+-imaging. Intracellular Ca2+ changes in myenteric neurons were monitored using the Ca2+ sensitive dye, fluo-4, and confocal microscopy. Galanin 1-16 (1 microM) markedly inhibited the K+-evoked Ca2+ increases in myenteric neurons. In summary, the differential distribution of GalRs supports the hypothesis that the complex effects of galanin in the gastrointestinal tract result from the activation of multiple receptor subtypes. Furthermore, this study confirms the presence of functional GalRs and suggests that galanin modulates transmitter release from myenteric neurons through inhibition of voltage-dependent calcium channels involving a G(i/o)-coupled GalR.

Guinea Pig Horizontal Cells Express GABA, the GABA-Synthesizing Enzyme GAD65, and the GABA Vesicular Transporter

γ‐Aminobutyric acid (GABA) is likely expressed in horizontal cells of all species, although conflicting physiological findings have led to considerable controversy regarding its role as a transmitter in the outer retina. This study has evaluated key components of the GABA system in the outer retina of guinea pig, an emerging retinal model system. The presence of GABA, its rate‐limiting synthetic enzyme glutamic acid decarboxylase (GAD65 and GAD67 isoforms), the plasma membrane GABA transporters (GAT‐1 and GAT‐3), and the vesicular GABA transporter (VGAT) was evaluated by using immunohistochemistry with well‐characterized antibodies. The presence of GAD65 mRNA was also evaluated by using laser capture microdissection and reverse transcriptase‐polymerase chain reaction. Specific GABA, GAD65, and VGAT immunostaining was localized to horizontal cell bodies, as well as to their processes and tips in the outer plexiform layer. Furthermore, immunostaining of retinal whole mounts and acutely dissociated retinas showed GAD65 and VGAT immunoreactivity in both A‐type and B‐type horizontal cells. However, these cells did not contain GAD67, GAT‐1, or GAT‐3 immunoreactivity. GAD65 mRNA was detected in horizontal cells, and sequencing of the amplified GAD65 fragment showed approximately 85% identity with other mammalian GAD65 mRNAs. These studies demonstrate the presence of GABA, GAD65, and VGAT in horizontal cells of the guinea pig retina, and support the idea that GABA is synthesized from GAD65, taken up into synaptic vesicles by VGAT, and likely released by a vesicular mechanism from horizontal cells. J. Comp. Neurol. 518:1647–1669, 2010.

SNAP25 expression in mammalian retinal horizontal cells

Horizontal cells mediate inhibitory feedforward and feedback lateral interactions in the outer retina at photoreceptor terminals and bipolar cell dendrites; however, the mechanisms that underlie synaptic transmission from mammalian horizontal cells are poorly understood. The localization of a vesicular γ‐aminobutyric acid (GABA) transporter (VGAT) to horizontal cell processes in primate and rodent retinae suggested that mammalian horizontal cells release transmitter in a vesicular manner. Toward determining whether the molecular machinery for vesicular transmitter release is present in horizontal cells, we investigated the expression of SNAP25 (synaptosomal‐associated protein of 25 kDa), a key SNARE protein, by immunocytochemistry with cell type‐specific markers in the retinae of mouse, rat, rabbit, and monkey. Different commercial antibodies to SNAP25 were tested on vertical sections of retina. We report the robust expression of SNAP25 in both plexiform layers. Double labeling with SNAP25 and calbindin antibodies demonstrated that horizontal cell processes and their endings in photoreceptor triad synapses were strongly labeled for both proteins in mouse, rat, rabbit, and monkey retinae. Double labeling with parvalbumin antibodies in monkey retina verified SNAP25 immunoreactivity in all horizontal cells. Pre‐embedding immunoelectron microscopy in rabbit retina confirmed expression of SNAP25 in lateral elements within photoreceptor triad synapses. The SNAP25 immunoreactivity in the plexiform layers and outer nuclear layer fell into at least three patterns depending on the antibody, suggesting a differential distribution of SNAP25 isoforms. The presence of SNAP25a and SNAP25b isoforms in mouse retina was established by reverse transcriptase‐polymerase chain reaction. SNAP25 expression in mammalian horizontal cells along with other SNARE proteins is consistent with vesicular exocytosis. J. Comp. Neurol. 519:972–988, 2011.

Robust syntaxin-4 immunoreactivity in mammalian horizontal cell processes.

Horizontal cells mediate inhibitory feed-forward and feedback communication in the outer retina; however, mechanisms that underlie transmitter release from mammalian horizontal cells are poorly understood. Toward determining whether the molecular machinery for exocytosis is present in horizontal cells, we investigated the localization of syntaxin-4, a SNARE protein involved in targeting vesicles to the plasma membrane, in mouse, rat, and rabbit retinae using immunocytochemistry. We report robust expression of syntaxin-4 in the outer plexiform layer of all three species. Syntaxin-4 occurred in processes and tips of horizontal cells, with regularly spaced, thicker sandwich-like structures along the processes. Double labeling with syntaxin-4 and calbindin antibodies, a horizontal cell marker, demonstrated syntaxin-4 localization to horizontal cell processes; whereas, double labeling with PKC antibodies, a rod bipolar cell (RBC) marker, showed a lack of co-localization, with syntaxin-4 immunolabeling occurring just distal to RBC dendritic tips. Syntaxin-4 immunolabeling occurred within VGLUT-1-immunoreactive photoreceptor terminals and underneath synaptic ribbons, labeled by CtBP2/RIBEYE antibodies, consistent with localization in invaginating horizontal cell tips at photoreceptor triad synapses. Vertical sections of retina immunostained for syntaxin-4 and peanut agglutinin (PNA) established that the prominent patches of syntaxin-4 immunoreactivity were adjacent to the base of cone pedicles. Horizontal sections through the OPL indicate a one-to-one co-localization of syntaxin-4 densities at likely all cone pedicles, with syntaxin-4 immunoreactivity interdigitating with PNA labeling. Pre-embedding immuno-electron microscopy confirmed the subcellular localization of syntaxin-4 labeling to lateral elements at both rod and cone triad synapses. Finally, co-localization with SNAP-25, a possible binding partner of syntaxin-4, indicated co-expression of these SNARE proteins in the same subcellular compartment of the horizontal cell. Taken together, the strong expression of these two SNARE proteins in the processes and endings of horizontal cells at rod and cone terminals suggests that horizontal cell axons and dendrites are likely sites of exocytotic activity.