Masao Tachibana

A Key Role of Starburst Amacrine Cells in Originating Retinal Directional Selectivity and Optokinetic Eye Movement

The directional selectivity of retinal ganglion cell responses represents a primitive pattern recognition that operates within a retinal neural circuit. The cellular origin and mechanism of directional selectivity were investigated by selectively eliminating retinal starburst amacrine cells, using immunotoxin-mediated cell targeting techniques. Starburst cell ablation in the adult retina abolished not only directional selectivity of ganglion cell responses but also an optokinetic eye reflex derived by stimulus movement. Starburst cells therefore serve as the key element that discriminates the direction of stimulus movement through integrative synaptic transmission and play a pivotal role in information processing that stabilizes image motion.

TRPM1 is a component of the retinal ON bipolar cell transduction channel in the mGluR6 cascade

An essential step in intricate visual processing is the segregation of visual signals into ON and OFF pathways by retinal bipolar cells (BCs). Glutamate released from photoreceptors modulates the photoresponse of ON BCs via metabotropic glutamate receptor 6 (mGluR6) and G protein (Go) that regulates a cation channel. However, the cation channel has not yet been unequivocally identified. Here, we report a mouse TRPM1 long form (TRPM1-L) as the cation channel. We found that TRPM1-L localization is developmentally restricted to the dendritic tips of ON BCs in colocalization with mGluR6. TRPM1 null mutant mice completely lose the photoresponse of ON BCs but not that of OFF BCs. In the TRPM1-L-expressing cells, TRPM1-L functions as a constitutively active nonselective cation channel and its activity is negatively regulated by Go in the mGluR6 cascade. These results demonstrate that TRPM1-L is a component of the ON BC transduction channel downstream of mGluR6 in ON BCs.

Submillisecond Kinetics of Glutamate Release from a Sensory Synapse

Exocytosis-mediated glutamate release from ribbon-type synaptic terminals of retinal bipolar cells was studied using AMPA receptors and simultaneous membrane capacitance measurements. Release onset (delay <0.8 ms) and offset were closely tied to Ca2+ channel opening and closing. Asynchronous release was not copious and we estimate that there are approximately 5 Ca2+ channels per docked synaptic vesicle. Depending on Ca2+ current amplitude, release occurred in a single fast bout or in two successive bouts with fast and slow onset kinetics. The second, slower bout may reflect a mobilization rate of reserve vesicles toward fusion sites that is accelerated by increasing Ca2+ influx. Bipolar cell synaptic ribbons thus are remarkably versatile signal transducers, capable of transmitting rapidly changing sensory input, as well as sustained stimuli, due to their large pool of releasable vesicles.

Effects of glycine and GABA on isolated bipolar cells of the mouse retina.

1. Bipolar cells were enzymatically (papain) dissociated from the mouse retina. Responses to exogenously applied glycine and GABA were recorded using the whole‐cell voltage clamp method (pipette solution contained 121 mM‐Cl‐). Both glycine and GABA evoked inward currents in cells voltage clamped at negative membrane voltages (e.g. ‐60 mV) and superfused with the control solution containing 146 mM‐Cl‐. 2. Polarities of both glycine‐ and GABA‐induced currents reversed near 0 mV under our control conditions. The reversal potential depended on both external [( Cl‐]o) and internal (intrapipette; [Cl‐]p) Cl‐ concentrations, but on neither Na+ nor K+ concentration. The reversal potentials were very close to the calculated equilibrium potential for Cl‐ estimated by using the Nernst equation with various external and internal Cl‐ activities. 3. The sensitivity to both glycine and GABA was highest at the axon terminal bulb. 4. Glycine‐induced responses were antagonized by 10 nM‐strychnine (competitively and non‐competitively), but by neither bicuculline nor picrotoxin. GABA‐induced responses were antagonized by 30 microM‐bicuculline (competitively) and 30 microM‐picrotoxin (non‐competitively), but not by 100 nM‐strychnine. Muscimol was as effective as GABA. Baclofen evoked no response even at 100 microM and did not modulate voltage‐dependent Ca2+ current. Pentobarbitone (10 microM) increased the sensitivity to GABA. These observations suggest that glycine and GABA worked on separate receptor molecules and that the receptors for GABA were GABAA type. 5. The present study suggests that glycine and GABA, both putative neurotransmitters of amacrine cells, mediate inhibition of bipolar cells in the mouse retina.

Transient calcium current of retinal bipolar cells of the mouse.

1. Isolated bipolar cells were obtained by enzymic (papain) dissociation of the adult mouse retina. The membrane voltage was clamped and the membrane currents were measured by the whole‐cell version of the patch‐clamp technique. Isolated bipolar cells and horizontal cells of the goldfish retina were also studied for comparison. 2. Hyperpolarization from the holding voltage, Vh, of ‐46 mV evoked a slowly activating, Cs+‐sensitive, inward current (probably an h‐current), and depolarization evoked a TEA‐ and Cs+‐sensitive outward current (probably a combination of K+ currents). 3. Depolarization from a more negative Vh (e.g. ‐96 mV) evoked a transient inward current that had maximal amplitude between ‐40 and ‐20 mV. This current was identified as a Ca2+ current (ICa): its amplitude was increased with elevated [Ca2+]o and was decreased with reduced [Ca2+]o, and it was blocked by 4 mM‐Co2+, but not by 5 microM‐TTX. 4. Both the perikaryon and the axon terminal generated ICa with similar properties. 5. The plot of Ca2+ conductance (gCa) against membrane voltage (activation curve) was sigmoidal: in 10 mM [Ca2+]o, gCa increased for membrane voltages more positive than ‐65 mV, was half‐maximal at about ‐25 mV, and reached saturation at about +30 mV. The plot of inactivation of gCa against membrane voltage was also sigmoidal: with 1 s conditioning depolarization in 10 mM [Ca2+]o, gCa decreased for membrane voltages more positive than ‐80 mV, was half‐maximal at about ‐50 mV, and was fully suppressed for voltages greater than ‐30 mV. 6. ICa in the mouse bipolar cells was insensitive to 50 microM‐Cd2+, 10 microM‐nifedipine and 10 microM‐Bay K 8644. In contrast, the calcium currents of bipolar and horizontal cells of the goldfish retina were markedly suppressed by 50 microM‐Cd2+ and 10 microM‐nifedipine, and were augmented several fold by 10 microM‐Bay K 8644. The calcium currents of goldfish bipolar and horizontal cells were sustained, and were activated in a more positive range of potentials than the ICa of mouse bipolar cells. 7. The voltage range at which the ICa of mouse bipolar cells is activated includes the presumed range of membrane potentials spanned during light‐evoked responses; thus, this current may participate in synaptic transmission. The transient character of ICa may also help to shape transient responses of ganglion cells.

Synchronized retinal oscillations encode essential information for escape behavior in frogs

Synchronized oscillatory activity is generated among visual neurons in a manner that depends on certain key features of visual stimulation. Although this activity may be important for perceptual integration, its functional significance has yet to be explained. Here we find a very strong correlation between synchronized oscillatory activity in a class of frog retinal ganglion cells (dimming detectors) and a well-known escape response, as shown by behavioral tests and multi-electrode recordings from isolated retinas. Escape behavior elicited by an expanding dark spot was suppressed and potentiated by intraocular injection of GABAA receptor and GABAC receptor antagonists, respectively. Changes in escape behavior correlated with antagonist-evoked changes in synchronized oscillatory activity but not with changes in the discharge rate of dimming detectors. These antagonists did not affect the expanding dark spot–induced responses in retinal ganglion cells other than dimming detectors. Thus, synchronized oscillations in the retina are likely to encode escape-related information in frogs.

Retinal bipolar cells receive negative feedback input from GABAergic amacrine cells

Bipolar cells make reciprocal synapses with amacrine cells in the inner plexiform layer; both feedforward connections and feedback connections are present. The physiological properties of the feedback synapse have not been well described. Since some amacrine cells are thought to be GABAergic, we examined bipolar cells for feedback input from gamma-aminobtyric acid (GABA)ergic amacrine cells. Solitary bipolar cells were dissociated enzymatically from the goldfish retina. Cells were voltage clamped with a patch pipette and their GABA sensitivity was examined. GABA evoked responses in all bipolar cells with a large axon terminal, which were identified to be the rod dominant ON type, and in some bipolar cells with a small axon terminal. The highest GABA sensitivity was located at the axon terminal. The least effective dose was as low as 100 nM. A small insignificant response of high threshold was evoked when GABA was applied to the dendrite and soma. GABA increased the Cl conductance and caused membrane hyperpolarization. The bipolar cells had the GABAA receptor coupled with a benzodiazepine receptor. The GABA-evoked response was not susceptible to Co ions, which suppressed the GABA-induced responses in turtle cones by 50% at 5 microM concentration. Incomplete desensitization was observed, suggesting that the GABAergic pathway seems capable of transmitting signals tonically. The present results strongly indicate that the rod-dominant ON-type bipolar cells and some bipolar cells with a small axon terminal receive negative feedback inputs from GABAergic amacrine cells.

High-Density Presynaptic Transporters Are Required for Glutamate Removal from the First Visual Synapse

Reliable synaptic transmission depends not only on the release machinery and the postsynaptic response mechanism but also on removal or degradation of transmitter from the synaptic cleft. Accumulating evidence indicates that postsynaptic and glial excitatory amino acid transporters (EAATs) contribute to glutamate removal. However, the role of presynaptic EAATs is unclear. Here, we show in the mouse retina that glutamate is removed from the synaptic cleft at the rod to rod bipolar cell (RBC) synapse by presynaptic EAATs rather than by postsynaptic or glial EAATs. The RBC currents evoked by electrical stimulation of rods decayed slowly after pharmacological blockade of EAATs. Recordings of the evoked RBC currents from EAAT subtype-deficient mice and the EAAT-coupled anion current reveal that functional EAATs are localized to rod terminals. Model simulations suggest that rod EAATs are densely packed near the release site and that rods are equipped with an almost self-sufficient glutamate recollecting system.

Two components of transmitter release in retinal bipolar cells : exocytosis and mobilization of synaptic vesicles

Ca2+-transmitter release coupling was examined using bipolar cells with large presynaptic terminals dissociated from the goldfish retina. Presynaptic Ca2+ current (I(Ca)) was recorded under the whole-cell voltage clamp. Release of excitatory amino acid transmitter was simultaneously monitored as the current through N-methyl-D-asperate (NMDA) receptors of reporter cells or as the membrane capacitance (C(m)) change associated with exocytosis. When I(Ca) was activated by a long depolarizing pulse, a double-peaked transmitter-induced current (I(tr)) was elicited in reporter cells. The rapid component of I(tr) was evoked immediately after the onset of depolarization, and was affected only slightly by intracellularly applied Ca2+ chelators. The delayed slow component of I(tr) was elicited during depolarization once a fixed amount of Ca2+ was accumulated in presynaptic terminals, and its appearance was suppressed or retarded by Ca2+ chelators. Two components of transmitter release were also recognized by monitoring C(m) changes elicited by the activation of I(Ca). These results suggest that bipolar cells have at least two pools of synaptic vesicles; a small, immediately releasable pool and a large releasable pool. The rapid and the delayed slow components of transmitter release may reflect exocytosis and mobilization of synaptic vesicles, respectively.

Group III Metabotropic Glutamate Receptors and Exocytosed Protons Inhibit L-Type Calcium Currents in Cones But Not in Rods

Light responses of photoreceptors (rods and cones) are transmitted to the second-order neurons (bipolar cells and horizontal cells) via glutamatergic synapses located in the outer plexiform layer of the retina. Although it has been well established that postsynaptic group III metabotropic glutamate receptors (mGluRs) of ON bipolar cells contribute to generating the ON signal, presynaptic roles of group III mGluRs remain to be elucidated at this synaptic connection. We addressed this issue by applying the slice patch-clamp technique to the newt retina. OFF bipolar cells and horizontal cells generate a steady inward current in the dark and a transient inward current at light offset, both of which are mediated via postsynaptic non-NMDA receptors. A group III mGluR-specific agonist, l-2-amino-4-phosphonobutyric acid (l-AP-4), inhibited both the steady and off-transient inward currents but did not affect the glutamate-induced current in these postsynaptic neurons. l-AP-4 inhibited the presynaptic L-type calcium current (ICa) in cones by shifting the voltage dependence of activation to more positive membrane potentials. The inhibition of ICa was most prominent around the physiological range of cone membrane potentials. In contrast, l-AP-4 did not affect L-type ICa in rods. Paired recordings from photoreceptors and the synaptically connected second-order neurons confirmed that l-AP-4 inhibited both ICa and glutamate release in cones but not in rods. Furthermore, we found that exocytosed protons also inhibited ICa in cones but not in rods. Selective modulation of ICa in cones may help broaden the dynamic range of synaptic transfer by controlling the amount of transmitter release from cones.