KCC2-dependent subcellular E(Cl) difference of ON-OFF retinal ganglion cells in larval zebrafish

Abstract

Subcellular difference in the reversal potential of Cl(-) (ECl) has been found in many types of neurons. As local ECl largely determines the action of nearby GABAergic/glycinergic synapses, subcellular ECl difference can effectively regulate neuronal computation. The ON-OFF retinal ganglion cell (RGC) processes both ON and OFF visual signals via its ON and OFF dendrites, respectively. It is thus interesting to investigate whether the ON and OFF dendrites of single RGCs exhibit different local ECl. Here, using in vivo gramicidin-perforated patch recording in larval zebrafish ON-OFF RGCs, we examine local ECl at the ON and OFF dendrites, and soma through measuring light-evoked ON and OFF inhibitory responses, and GABA-induced response at the soma, respectively. We find there are subcellular ECl differences between the soma and dendrite, as well as between the ON and OFF dendrites of single RGCs. These somato-dendritic and inter-dendritic ECl differences are dependent on the Cl(-) extruder, K(+)/Cl(-) co-transporter (KCC2), because they are largely diminished by down-regulating kcc2 expression with morpholino oligonucleotides (MOs) or by blocking KCC2 function with furosemide. Thus, our findings indicate that there exists KCC2-dependent ECl difference between the ON and OFF dendrites of individual ON-OFF RGCs that may differentially affect visual processing in the ON and OFF pathways.

Keywords: Cl− reversal potential; GABA; KCC2; in vivo whole-cell recording; retinal ganglion cells; subcellular; zebrafish.

Figure 1

Measurement of dendritic E_Cl in zebrafish RGCs. (A) An example showing light-evoked OFF synaptic responses recorded at different holding potentials from −80 to 0 mV when the ON-OFF RGC was loaded with 14 mM Cl⁻ internal solution under conventional whole-cell recording mode. (B) I–V curves of early (black square) and late (green triangle) components of light-evoked responses. Dendritic E_Cl, calculated by fitting the I–V curve of the late-phase component with linear regression, is about −60 mV, close to the Cl⁻ equilibrium potential (−59.8 mV). (C) Theoretical (square) and measured (circle) dendritic E_Cl when the RGCs was loaded with internal solution with different Cl⁻ concentration (4, 8, 14, or 24 mM). The data were obtained from 10 cells at each data point. The values are represented as mean ± SEM.

Figure 2

Somato-dendritic E_Cl difference of zebrafish ON-OFF RGCs. (A) A schematic showing the experimental procedure. (B) Light-evoked ON (red circle) and OFF (green triangle) synaptic responses and GABA-induced currents (black square) were simultaneously recorded at different holding potentials from an ON-OFF RGC under gramicidin-perforated patch recording mode. (C) I–V curves of GABA-induced current at the RGC soma (black square), and the late-phase components of ON (red circle) and OFF (green triangle) light-evoked responses. The data were obtained from the same cell with (B). (D) Plots of dendritic E_Cl against somatic E_Cl. The dotted line represents the orthogonal, and the blue circle and yellow triangle represent the mean E_Cl of ON and OFF responses, respectively. The data were obtained from 98 ON-OFF RGCs. (E) Distribution of somato-dendritic E_Cl difference. (F) Mean E_Cl differences between soma and ON or OFF dendrites of ON-OFF RGCs from 2.5 to 6 dpf. At each data points, the cell number is more than 13. The values are represented as mean ± SEM.

Figure 3

Inter-dendritic E_Cl difference between ON and OFF dendrites of zebrafish ON-OFF RGCs. (A,B) Left, light-evoked ON and OFF synaptic responses recorded at different holding potentials from two ON-OFF RGCs under gramicidin-perforated patch recording mode. Right, I–V curves of the late-phase components of light-evoked ON (red circle) and OFF (green triangle) responses. (C) Distribution of the inter-dendritic E_Cl difference from 152 cells. (D) Mean absolute values of inter-dendritic E_Cl difference from 2.5 to 6 dpf. At each data points, the cell number is more than 15. ^*P < 0.05, unpaired Student's t-test. The values are represented as mean ± SEM.

Figure 4

Knockdown of zebrafish kcc2 diminishes subcellular E_Cl difference. (A,B) Mean dendritic (A) and somatic (B) E_Cl of control (black square) and kcc2 morphants (blue circle). At each data points, the cell number is more than 8. (C,D) Left, Light-evoked ON and OFF responses of RGCs from a control (C) and kcc2 morphants (D) under gramicidin-perforated patch recording mode. Right, I–V curves of the late-phase components of light responses. (E,F) Cumulative probability of inter-dendritic (E) and somato-dendritic (F) E_Cl differences in control (n = 35 cells) and kcc2 morphants (n = 71 cells). The symbols near the X-axis represent the mean E_Cl differences. ^*P < 0.01, Kolmogorov–Smirnov test. The values are represented as mean ± SEM.

Figure 5

Blockade of KCC2 reduces subcellular E_Cl difference. (A,B) Cumulative probability of inter-dendritic (A) and somato-dendritic (B) E_Cl difference in control (n = 44 cells) and furosemide-treated (n = 29 cells) animals. The symbols near the X-axis represent the mean E_Cl differences. ^*P < 0.01, Kolmogorov–Smirnov test. The values are represented as mean ± SEM.

Figure 6

Schematic of subcellular E_Cl differences within single zebrafish RGCs. (A,B) Cartoon showing subcellular E_Cl differences between soma, ON and OFF dendrites in zebrafish ON-OFF RGCs. The [Cl⁻]_i is color-coded.