Retinal input to efferent target amacrine cells in the avian retina

Abstract

The bird visual system includes a substantial projection, of unknown function, from a midbrain nucleus to the contralateral retina. Every centrifugal, or efferent, neuron originating in the midbrain nucleus makes synaptic contact with the soma of a single unique amacrine cell, the target cell (TC). By labeling efferent neurons in the midbrain, we have been able to identify their terminals in retinal slices and make patch-clamp recordings from TCs. TCs generate Na+-based action potentials (APs) triggered by spontaneous EPSPs originating from multiple classes of presynaptic neurons. Exogenously applied glutamate elicited inward currents having the mixed pharmacology of NMDA, kainate, and inward rectifying AMPA receptors. Exogenously applied GABA elicited currents entirely suppressed by GABAzine and therefore mediated by GABAA receptors. Immunohistochemistry showed the vesicular glutamate transporter, vGluT2, to be present in the characteristic synaptic boutons of efferent terminals, whereas the GABA synthetic enzyme, GAD, was present in much smaller processes of intrinsic retinal neurons. Extracellular recording showed that exogenously applied GABA was directly excitatory to TCs and, consistent with this, NKCC, the Cl- transporter often associated with excitatory GABAergic synapses, was identified in TCs by antibody staining. The presence of excitatory retinal input to TCs implies that TCs are not merely slaves to their midbrain input; instead, their output reflects local retinal activity and descending input from the midbrain.

Figure 1. Methods used to Label rEFs and Record from TCs

A: Schematic illustration of the labeling method. Under stereotaxic control 0.5 μL of the anatomical tracer, Fluoro-Ruby, was injected unilaterally into the ION. IO-neurons took up Fluoro-Ruby and transported it to their axon terminals (red arrow). After a minimum survival of 3 days, sufficient Fluoro-Ruby accumulated in the rEF terminals to allow their visualization in retinal slices. B: A retinal slice imaged in IR-DIC and fluorescence optics. A single rEF is shown (red) crossing the IPL and terminating in a pericellular nest in the INL. Identifying the postsynaptic TC from its DIC outline and contact with the rEF terminal, a patch clamp pipet (shown in cartoon) was used to fill the cell with Lucifer Yellow (LY). This cell could also be stimulated with exogenous neurotransmitters from a puff-pipet placed as illustrated. C: A typical LY-filled TC with attached pipet (yellow), showing the TC axon (arrowheads), and close apposition between the stubby TC dendrites and rEF pericellular nest (red); features that were used to confirm identity of the recorded TC. The boundary between the INL and IPL is indicated with a dashed line.

Figure 2. TCs Produce APs that can be Triggered by Spontaneous PSPs

A: Sample traces from a current clamp recording of a TC showing many spontaneous PSPs, 5 are ridden by APs (arrows). Inset shows detail of a single AP (black) superimposed on a single PSP (grey). Recorded using KCl intracellular solution. B: Extracellular recording of APs obtained during loose patch recording from a TC. C: A voltage-step from −79.5 mV to −59.5 mV (top trace) elicited a rapid and transient current in a voltage-clamped TC (lower black trace). Application of 0.1 μM TTX completely suppressed this current (grey trace), implying that TCs have functional voltage-gated Na⁺-channels. Recorded using CsMs intracellular solution. D: Voltage-clamp recordings from a TC held at −20 mV show that High Mg²⁺external blocks most of the spontaneous PSCs recorded in the TC. In Normal external (upper black traces) this TC received a steady stream of spontaneous synaptic input. The vast majority of this input was blocked by High Mg²⁺/0 Ca²⁺ external (grey traces). This effect was fully reversible by washing with Normal external (lower black traces). Recorded using CsMs intracellular solution.

Figure 3. Pharmacological Characterization of Glutamate Receptors on TCs

A: Voltage-clamp recordings from a TC showing the typical response (black trace) to a 20 ms puff of glutamate (V_H = −20 mV). 100 μM glutamate (Glu, black bar) elicited a large inward current in Normal external (black trace). The glutamate puff response was significantly decreased by DNQX (red trace) and completely eliminated by DNQX & APV (green trace), but GYKI-53655 (blue trace) had little effect at this voltage. Full recovery was achieved in Wash (grey trace). B: Comparison of the amplitude of the glutamate puff response in Normal and High Mg²⁺/0 Ca²⁺ external. Prior to grouping, the mean amplitude of the glutamate-puff response in each cell was normalized to the mean amplitude of the response in High Mg²⁺. No significant difference was detected (n=5 cells). C: Summary of the effects of glutamate antagonists on the glutamate puff responses of 8 TCs. Data have been pooled from cells recorded in Normal and High Mg²⁺/0 Ca²⁺ external (see text), thus the term “Initial” has been used to signify the response prior to application of any glutamate antagonist. D: I-V relations of the glutamate puff response. The protocol used to determine the I-V relation of the puff response is illustrated in the inset; grey trace shows the timing of the puff of agonist (glutamate), black trace shows the timing of the voltage ramps. In Normal external (black), the I-V relation of the glutamate puff response showed strong outward rectification and reversed at −8.4 mV. Evidence for the presence of Ca²⁺-permeable AMPA receptors is provided by the ability of GYKI-53655 (blue) to reduce current at more negative voltages but not at positive voltages. The isolated AMPA-mediated component of the I-V relation (i.e. the difference between Normal and GYKI-53655), is shown in E. The negative slope between −80 and −30 mV is indicative of a significant NMDA mediated component. The combined application of DNQX & APV blocked the glutamate puff response at all voltages (bright green trace). The Wash trace is omitted.

Figure 4. Pharmacological Characterization of GABA Receptors on TCs

A: Voltage clamp recording from a TC during puff application of 100 μM epibatidine (Epibat, black bar). Trace shown is the average of 4 trials, in none of which was a response elicited. The two small events visible in this trace are the result of spontaneous PSCs that occurred during the third trial. B: 150 μM GABA (black bar) elicited a large outward current in Normal external (black trace), which was completely eliminated by 20 μM GABAzine (light grey trace) and partially recovered after washing (dark grey trace). C: Comparison of the amplitude of the GABA-puff response in Normal and High Mg²⁺/0 Ca²⁺ external, as in Figure 3B. No significant difference was detected (n=5 cells). D: Summary of the effect of GABAzine (with or without TPMPA) on the GABA-puff response in 10 TCs. Data have been pooled as for the glutamate experiments (Figure 3C). The mean amplitude of the GABA-puff response was reduced to practically zero by GABAzine and further addition of TPMPA (n=3 cells) did not further reduce the amplitude. Partial reversal was seen in Wash. E: I-V relations of the GABA puff response using the same protocol as illustrated in Figure 3D. In Normal external (black), the I-V relation of the GABA-puff response was weakly outward rectifying, reversing at −59 mV. GABAzine blocked the response at all voltages (light grey). This effect was only partially reversed in Wash (dark grey).

Figure 5. GABA input to the TC is Excitatory

A: Extracellularly recorded spontaneous APs from a TC. In this cell, APs were observed at a frequency of 3.1 Hz in Normal external (upper trace). GABAzine (middle trace) reduced AP frequency to 1.3 Hz; no significant recovery was observed in Wash (lower trace). B: Summary of the reduction in spontaneous AP frequency following the application of GABAzine (Gz; n=3). GABAzine significantly decreased AP frequency; however, reversal of this effect was not seen. N=Normal; W=Wash. C: Sample traces from a TC showing the burst of APs elicited by short (20 ms) puffs of GABA (bars). In Normal external, High Mg²⁺, and Wash each puff evoked 1-2 APs. Spontaneous APs, common in Normal external, were eliminated by High Mg²⁺, while GABA-puff evoked APs were unchanged by High Mg²⁺/0 Ca²⁺ but eliminated by GABAzine. D: Summary of the effect of High Mg²⁺/0 Ca²⁺ (Mg) or GABAzine (Gz) on GABA-puff evoked APs for the cell illustrated in C and two others. Neither the number of APs (left) nor the latency (right) of the GABA-puff evoked burst were significantly altered by High Mg²⁺. However, GABAzine significantly reduced the number of APs and increased the latency. E: Spontaneous PSC frequency is reduced, but not eliminated, by GABA and glutamate antagonists. GABA antagonists alone (Gb) suppress approximately half of the spontaneous PSCs recorded in TCs (n=11), while Glutamate antagonists alone (Gl) only suppress about 25% (n=9). Combined application of GABA and glutamate antagonists (Gb&Gl) reduced spontaneous PSC by 80% (n=4), roughly the value expected from additive effects of the individual antagonists.

Figure 6. Exogenous Glutamate has Direct and Indirect Excitatory Effects on the TC

A: Intracellular recording of the glutamate puff response revealed that glutamate increased the frequency of the outward PSCs. Given the V_H of −20 mV, current through glutamate receptors is expected to be inward, while current through GABA and glycine receptors should be outward. Top, an example of a TCs response to a single glutamate-puff illustrates the relationship between the single, large inward current (associated with activation of glutamate receptors on the TC) and the multiple, small outward PSCs (associated with GABA or glycinergic input to the TC). The glutamate-evoked (GE)-PSCs observed during the decay of the large inward glutamate puff response (black trace) are likely mediated by synaptic transmission as they are eliminated by High Mg²⁺/0 Ca²⁺ (grey trace) whereas, the inward current is not eliminated and thus is mediated by glutamate receptors on the TC itself. Bottom, PSTHs from 2 different TCs showing the dramatic increase in outward PSC frequency following a glutamate puff, and the significant reduction of spont- and GE-PSCs caused by application of High Mg²⁺/0 Ca²⁺ (left) or GABAzine (right). Bars above the current traces and PSTHs show the timing of the glutamate puff (thick bar) and the TCs inward glutamate puff response (thin bar). B: Summary of the effects of High Mg²⁺/0 Ca²⁺ and GABAzine on spontaneous- and GE- PSCs. Spontaneous (spont) frequency was measured during a period preceding each glutamate puff; GE frequency was measured during the 50-100 msec following each puff. In Normal external solution, short puffs of glutamate cause a 5-fold increase in the frequency of PSC (n=10). High Mg²⁺/0 Ca²⁺ external solution was able to virtually eliminate both spont- and GE-PSCs (n=7). GABAzine was able to suppress most of the spont- and GE PSCs, but the remaining PSCs suggest that some of these events might be glycinergic(n=3). C: Sample traces from a TC showing the burst of APs elicited by short (20 ms) puffs of 100 μM glutamate (thick bar). In Normal external (upper black traces) each glutamate puff evoked 3-5 APs, in addition spontaneous APs were observed (arrows). In High Mg²⁺/0 Ca²⁺ external (grey traces) spontaneous APs were eliminated and the number of APs in the glutamate puff response was decreased to 1-2. In Wash (lower black trace), spontaneous APs returned and the number of APs in the glutamate puff response increased to 10. D: Summary of the effect of High Mg²⁺/0 Ca²⁺ on the glutamate puff evoked APs. High Mg²⁺/0 Ca²⁺ significantly decreased the number of APs, but did not alter the latency. This effect was partially reversed in Wash (n=5).

Figure 7. Immunohistochemical Characterization of Neurotransmitter Receptors on TCs

Shown here are TC somata identified by an antibody to parvalbumin (red) together with antibodies to transmitter receptors (green). Images are collapsed confocal stacks showing the entire thickness of the retina with photoreceptors at the top and the ganglion cells or optic fiber layer at the bottom. Dotted lines indicate the INL-IPL border. The intensity and contrast of these images were adjusted to allow clear visualization of antibody labeling on and around the TC somata where all synapses to TCs are known to lie (Lindstrom et al., 2009); TCs make no synapses in the IPL. Scale Bars are 20 μm. A: Soma of a TC (a) stains weakly for the nAChR α7 subunit (b), apparent colocalization can be seen in (yellow, c). The faintness of the green channel signal is indicated relatively high autofluorescence shown in the outer segments at the top of the images (d), a DIC image is shown here for orientation but omitted from subsequent figures. B: GABA_A receptors colocalize with TCs. Double label immunohistochemistry for parvalbumin identifies a TC (a), that is also positive for the α1 subunit of the GABA_A receptor (b), merged in (c). C: Retina double labeled for parvalbumin (red, a) and GluR2/3 (green, b) shows strong colocalization (c).

Figure 8. Cellular Localization of Transmitter Associated Molecules

A: ChAT staining is absent from the region surrounding the rEF terminal. Double labeling for rEF terminals and choline acetyltransferase, the synthetic enzyme for ACh, revealed no colocalization between the two. (a), ChAT staining (green) shows 2 strongly stained laminae in the IPL and regular arrays of starburst amacrine cells both above and below the IPL. The rEF terminal, labeled with antibodies against TMR (red), used to increase the signal of Fluoro-Ruby in rEF terminals, lies in a region of the INL that lacks ChAT staining. Scale bar is 20 μm. (b&c) Higher magnification images of the region surrounding the rEF shown in the merged image, (a). Scale bar is 20 μm. B&C: Presynaptic markers for glutamate and GABA have distinct staining patterns. In both B and C the larger merged image (a) shows the entire thickness of the retina with photoreceptors at the top and the optic fiber layer at the bottom. Dotted lines indicate the INL-IPL border. The three images on the right (b-d) show higher magnification images of the region surrounding the TC so that colocalization between the two antibodies can be more closely examined. All images are compressed confocal stacks. Scale bars are 10 μm. B: Double labeling with antibodies against rhodamine (red), identifying the rEF terminal and vGluT2 (a presynaptic marker for glutamatergic synapses, green) shows a matching pattern. In b, c, & d, individual presynaptic boutons, approximately 2μm in diameter, can often be seen in both images (arrows). C: Double labeling with antibodies against parvalbumin (red) and GAD65/67 (green) reveals that a few small, punctate GABAergic terminals contact thin, dendritic extensions of the TC (yellow in merged images and arrows in b & c). These GABAergic terminals do not have the same staining pattern as vGluT2 in B and do not correspond to the rEF terminals. D: TCs express high levels of NKCC. A retinal slice double labeled with antibodies against parvalbumin, used to identify the TC (a) and NKCC (b). In the merged image (c) the soma and dendritic region of the TC is seen to be strongly NKCC positive. Scale bar is 20 μm.