Axonal stratification patterns and glutamate‐gated conductance mechanisms in zebrafish retinal bipolar cells

Whole-cell patch recording and puff pipette techniques were used to identify glutamate receptor mechanisms on bipolar cell (BC) dendrites in the zebrafish retinal slice. Recorded neurons were stained with Lucifer Yellow, to correlate glutamate responses with BC morphology. BC axon terminals (ATs) consisted of swellings or varicosities along the axon, as well as at its end. AT stratification patterns identified three regions in the inner plexiform layer (IPL): a thick sublamina a, with three bands of ATs, a narrow terminal-free zone in the mid-IPL, and a thin sublamina b, with two bands of ATs. BCs occurred with ATs restricted to sublamina a(Group a), sublamina b(Group b) or with ATs in both sublaminae (Group a/b). OFF-BCs belonged to Group aor Group a/b. These cells responded to glutamate or kainate with a CNQX-sensitive conductance increase. Reversal potential (Erev) ranged from −0.6 to +18 mV. Bipolar cells stimulated sequentially with both kainate and glutamate revealed a population of glutamate-insensitive, kainate-sensitive cells in addition to cells sensitive to both agonists. ON-BCs responded to glutamate via one of three mechanisms: (a) a conductance decrease with Erev≈ 0 mV, mimicked by L-(+)-2-amino-4-phosphonobutyric acid (APB) or trans-1-amino-1,3-cyclopentanedicarboxylic acid (trans-ACPD), (b) a glutamate-gated chloride conductance increase (IGlu-like) characterized by Erev≥ECl (where ECl is the chloride equilibrium potential) and partial blockade by extracellular Li+/Na+ substitution or (c) the activation of both APB and chloride mechanisms simultaneously to produce a response with outward currents at all holding potentials. APB-like responses were found only among BCs in Group b, with a single AT ramifying deep within sublamina b; whereas, cells expressing IGlu-like currents had one or more ATs, and occurred within Groups b or a/b. Multistratified cells (Group a/b) were common and occurred with either ON- or OFF-BC physiology. OFF-BCs typically had one or more ATs in sublamina a and only one AT in sublamina b. In contrast, multistratified ON-BCs had one or more ATs in sublamina b and a single AT ramifying deep in sublamina a. Multistratified ON-BCs expressed the IGlu-like mechanism only. Visual processing in the zebrafish retina involves at least 13 BC types. Some of these BCs have ATs in both the ON- and OFF-sublaminae, suggesting a significant role for ON- and OFF-inputs throughout the IPL. Retinal bipolar cells are physiologically separated into ON- and OFF-subtypes based on responses to light and/or glutamate, the photoreceptor neurotransmitter. OFF-bipolar cells are depolarized by glutamate release in the dark. ON-bipolar cells, in contrast, are hyperpolarized by the very same glutamate release. The division of the light signal into ON- and OFF-pathways is achieved through a variety of glutamate receptors mediating these responses. OFF-bipolar cells express ionotropic glutamate receptors (iGluRs) that are directly activated by glutamate or the glutamate agonists, kainate and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA). Glutamate responses restricted to the kainate receptor family were recently described in OFF-bipolar cells of ground squirrel (DeVries & Schwartz, 1999). Kainate receptor currents are typically characterized by slower desensitization and recovery than AMPA receptor currents. Glutamate responses of the N-methyl-D-aspartate (NMDA) family are not seen in retinal bipolar cells. Glutamate binding onto OFF-bipolar dendrites opens non-selective cation channels, increasing membrane conductance, and depolarizing the cell (Murakami et al. 1975; Sasaki & Kaneko, 1996). ON-bipolar cells express a metabotropic glutamate receptor (mGluR) that indirectly gates ion channels via a second messenger cascade (Nawy & Jahr, 1990; Nawy, 1999). This receptor is selectively agonized by L-(+)-2-amino-4-phosphonobutyric acid (L-AP4 or APB) (Slaughter & Miller, 1981). In addition, APB has been reported to directly activate a potassium conductance (Hirano & MacLeish, 1991). Recently, a third ON-type glutamate-gated conductance, in which glutamate gates a chloride channel with transporter-like pharmacology (Grant & Dowling, 1995) was identified in ON-bipolar cells of white perch. Classically, ON- and OFF-bipolar cells also differ by axon terminal ramification in the inner plexiform layer (IPL). OFF-bipolar cell axon terminals ramify in the distal IPL (sublamina a); whereas ON-bipolar cell axon terminals ramify in the proximal IPL (sublamina b) (Famiglietti et al. 1977; Stell et al. 1977). Multistratified bipolar cells have been identified in fish (Scholes, 1975; Sherry & Yazulla, 1993), turtle (Kolb, 1982), and salamander (Maple & Wu, 1997). Early anatomical studies of bipolar cell dendritic contacts identified basal contacts at synapses between photoreceptors and OFF-bipolar cells; while ON-bipolar cell dendrites terminated in invaginating ribbon synapses (Stell et al. 1977). Thus, the identification of basal contacts between photoreceptors and multistratified bipolar cells in monkey (Mariani, 1983) suggested that these cells represented a further population of OFF-bipolar cells. In accord with this, recent work in the salamander retina (Maple & Wu, 1997) physiologically identified bipolar cells with axon terminals ramifying in both sublaminae as OFF-cells. In this report, we describe glutamate-gated currents and glutamate receptor types present on zebrafish bipolar cell dendrites and correlate the observed responses with axon terminal morphology. Zebrafish is an important model in developmental/molecular studies. It is the goal of the present work to define further the organization of the visual system in the zebrafish model. Based on the observed patterns of axonal ramification we have identified 13 morphological types of bipolar cells. In addition to classical ON or OFF stratification patterns, a diverse multistratified group containing both ON- and OFF-type responses was discovered. Glutamatergic mechanisms in ON-bipolar cells exhibited one of three different conductance patterns. OFF-bipolar cells exhibited a single ionotropic conductance pattern, but activated either by kainate-selective, or glutamate/kainate-selective receptors.