Visual Neuroscience



Robust syntaxin-4 immunoreactivity in mammalian horizontal cell processes


ARLENE A.  HIRANO  a1 a4 c1 , JOHANN HELMUT  BRANDSTÄTTER  a5 a6 , ALEJANDRO  VILA  a1 and NICHOLAS C.  BRECHA  a1 a2 a3 a4
a1 Departments of Neurobiology & Medicine, Geffen School of Medicine at UCLA, Los Angeles, California
a2 Jules Stein Eye Institute, Geffen School of Medicine at UCLA, Los Angeles, California
a3 CURE Digestive Diseases Research Center, Geffen School of Medicine at UCLA, Los Angeles, California
a4 VAGLAHS, Los Angeles, California
a5 Institute for Biology, Department of Animal Physiology, University of Erlangen-Nuernberg, Erlangen, Germany
a6 Department of Neuroanatomy, Max Planck Institute for Brain Research, Frankfurt am Main, Germany

Article author query
hirano aa   [Google Scholar] 
brandstatter jh   [Google Scholar] 
vila a   [Google Scholar] 
brecha nc   [Google Scholar] 
 

Abstract

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.

(Received January 19 2007)
(Accepted March 12 2007)


Key Words: SNARE complex; Exocytosis; Vesicular release.

Correspondence:
c1 Address correspondence and reprint requests to: Arlene A. Hirano, Ph.D., Geffen School of Medicine at UCLA, Dept. of Neurobiology, 10833 Le Conte Ave., Box 951763, Los Angeles, CA 90095. E-mail: ahirano@mednet.ucla.edu