Most SCGN-positive bipolar cells are G13-negative; arrow marks one G13-positive bipolar cell. our understanding of nervous system function is obtaining a complete description of the various neural pathways, including elaborating the specific synaptic connections between identified neurons, and establishing the transmitter systems that mediate those connections. The retina has proved particularly amenable to such efforts, due in large part to the discovery of numerous cell-type-specific immunohistochemical markers, in combination with antibodies against neurotransmitter receptors. A great deal of comparative data shows that neural pathways are often well conserved across mammalian and other vertebrate species. At the first synapse in the retina, glutamate released from cone photoreceptors activates receptors in the dendrites of bipolar cells, the feedforward excitatory neurons, and the horizontal cells, which provide lateral inhibitory connections. Two types of bipolar cell, ON and OFF, encode increments and decrements in light intensity, respectively. All OFF bipolar cells are depolarized by glutamate because they express ionotropic glutamate receptors that are preferentially gated by either -amino-3-hydroxy-S-methylisoxazole-4-propionic acid (AMPA, GluA1-4, formerly GluR1-4), or kainate Rupatadine Fumarate (KA, GluK1-5, formerly GluR5-7, KA1-2). Previous studies in the ground squirrel retina suggest that the temporal response properties of OFF bipolar cells may be influenced by the type of glutamate receptor expressed in their dendrites (DeVries, 2000). In the macaque retina, the OFF bipolar cells comprise four distinct types that can be identified based on the stratification pattern of their axon terminals in the outer part of the inner plexiform layer (IPL;Boycott and Wssle, 1991). Of these, three types of diffuse bipolar (DB) cells, DB1, DB2, and DB3, make multiple connections to cone photoreceptors, while a fourth type, the flat midget bipolar (FMB) cell, makes one-to-one connections with cone photoreceptors in the central retina. Early Golgi studies combined with electron microscopy provided initial clues about the connectivity of individual bipolar cell types in the primate retina (Dowling and Boycott, 1966;Kolb, 1970;Boycott Rupatadine Fumarate and Hopkins, 1993). However, the discovery of immunohistochemical markers to stain specific bipolar cell types significantly accelerated progress. In the macaque retina, DB2 cells are labeled with antibodies against GLT-1, DB3 cells are labeled with calbindin antibodies, and FMB cells are labeled with recoverin antibodies (Grnert et al., 1994;Wssle et al., 1994;Jacoby et al., 2000). Until now, a marker of the DB1 cell has remained elusive. Previously, we demonstrated that the calcium-binding protein, secretagogin (SCGN), labels numerous subtypes of cone bipolar cell in the mouse, rat, and rabbit retina (Puthussery et al., 2010). Secretagogin is a recently identified member of the EF-hand family of calcium-binding proteins that is expressed in various brain regions and peripheral tissues (Wagner et al., 2000). Here we demonstrate that secretagogin is a marker of the DB1 bipolar cell in the macaque retina. We have utilized this marker Rupatadine Fumarate to investigate the cone connectivity, glutamate receptor expression, and synaptic inputs to the DB1 bipolar cell. == MATERIALS AND METHODS == == Animals and tissue preparation == Four retinas were collected from three adult cynomolgus macaque monkeys (Macaca fascicularis) that were euthanized after electrophysiological experiments unrelated to those described here. All procedures were approved by the local Animal Care Committee and were in accordance with the law for animal experiments issued by the German government (Tierschutzgesetz). In addition, three retinas from three adult rhesus macaques (Macaca Rabbit Polyclonal to SF3B4 mulatta) were collected postmortem as part of the Tissue Distribution Program at the Oregon National Primate Research Center. No differences in staining were observed between the two species. The anterior segment and vitreous were removed from the eye and the posterior eyecup was immersion-fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) for 560 minutes at 25 C. Although the secretagogin antibody worked well across this range of fixation times, lighter fixation was necessary to obtain good staining with the glutamate and glycine receptor antibodies. Following fixation, retinas were dissected from the eyecups, cryoprotected in graded sucrose solutions (10%, 20%, 30%), and stored at 20 C for later use. For light microscopy, samples of retina were used as a wholemount or sectioned vertically (1216 m) or horizontally (2030 m) with a.