Cortical Feedback Regulates Feedforward Retinogeniculate Refinement

Abstract

According to the prevailing view of neural development, sensory pathways develop sequentially in a feedforward manner, whereby each local microcircuit refines and stabilizes before directing the wiring of its downstream target. In the visual system, retinal circuits are thought to mature first and direct refinement in the thalamus, after which cortical circuits refine with experience-dependent plasticity. In contrast, we now show that feedback from cortex to thalamus critically regulates refinement of the retinogeniculate projection during a discrete window in development, beginning at postnatal day 20 in mice. Disrupting cortical activity during this window, pharmacologically or chemogenetically, increases the number of retinal ganglion cells innervating each thalamic relay neuron. These results suggest that primary sensory structures develop through the concurrent and interdependent remodeling of subcortical and cortical circuits in response to sensory experience, rather than through a simple feedforward process. Our findings also highlight an unexpected function for the corticothalamic projection.

Figure 1. Disrupting activity in V1 with muscimol between P20-P27 alters connectivity of the retinogeniculate synapse

A) Schematic of the primary visual pathway. The feedforward pathway projections are colored in black, the feedback pathway in blue. (+) indicates excitatory synapses, (−) indicates inhibitory synapses. TRN = thalamic reticular nucleus, LIN = local inhibitory neuron B) Immunohistochemistry reveals expression of the activity dependent marker c-fos is reduced unilaterally in V1 both 24 and 48 hours after muscimol injection (injection tract marked by CTB, n = 5 sections, 3 mice for each time point, scale bar = 200μm) C) Schematic depicting experimental design. D) Example recordings from relay neurons of vehicle- or muscimol-injected mice. Each graph shows overlaid AMPAR- (inward currents, recorded at −70mV) and AMPA and NMDAR- (outward currents, recorded at +40mV) mediated EPSCs evoked by incrementally increasing optic tract stimulation. To the right of overlaid traces, EPSC amplitudes are plotted by stimulus intensity. E) Cumulative probability plot of AMPAR-mediated single fiber EPSCs shows a significant shift toward weaker retinal inputs in muscimol-treated mice. F) AMPAR- and NMDAR-mediated single-fiber strengths were significantly reduced after muscimol injections, as compared with younger mice (P20, grey) as well as vehicle-injected age-matched littermates (white). G) Maximal EPSCs were not significantly altered. H) The number of retinal inputs innervating each relay neuron increased after one week of muscimol injections, indicated by the decreased fiber fraction. (For E-H, n = P20: 30 cells from 6 mice; vehicle-injected: 25 cells from 4 mice; muscimol-injected: 20 cells from 4 mice) * P < 0.05, ** P < 0.01.

Figure 2. The inhibitory DREADD HM4Di can suppress L6 corticothalamic cells

A) mCherry expression labels L6 somas in a P20 NTSR1-Cre mouse injected neonatally with AAV2/8-hSyn-DIO-HM4Di mCherry. B) Diffuse mCherry fluorescence from corticothalamic axons is visible in the dLGN. scale bar = 200μm C) Current-clamp recordings of L6 cells expressing HM4Di show a robust hyperpolarization and reduction in firing rates upon application of CNO (n = 8 cells from 8 slices). D) Schematic depicting experimental design for chronic manipulation of L6 activity during the critical period. E) Schematic of in vivo recording approach. F) Pyramidal cells recorded from L6 cells of NTSR1-Cre mice expressing HM4Di shows decreased firing within 30 minutes after IP injection of CNO (n=25 cells from 6 mice, Friedman test with Dunn's multiple comparison test to assess changes longitudinally). G) Activity of L4 pyramidal cells in NTSR1-Cre mice expressing HM4Di shows significantly increased firing 30 minutes after intraperitoneal injection of CNO, consistent with a suppressed output from L6 cells (n=21 cells). H) Pyramidal cells in superficial layers also increased firing after CNO injection (n=10 cells).

Figure 3. HM4Di-mediated suppression of L6 induces the recruitment of additional, strong retinal inputs to relay neurons

A) Schematic depicting experimental design. B) Example recordings from relay neurons of NTSR1-Cre mice expressing HM4Di, injected with vehicle or CNO from P20 to P27-33. EPSC amplitudes plotted by stimulus intensity again show few discrete steps in the control example, and more steps in the cell from a CNO-injected mouse. C) Cumulative probability plot of AMPAR-mediated single fibers shows no shift in the strength of retinal inputs in CNO-injected mice. D) Neither AMPAR- nor NMDAR-mediated single fiber strength was altered by suppressing L6 with HM4Di. E) Both AMPAR- and NMDAR-mediated maximum EPSCs were increased after CNO injections. F) The number of retinal inputs converging on each relay neuron was significantly increased in mice injected with CNO. (For C-F, n = vehicle-injected: 34 cells from 9 mice; CNO-injected: 50 cells from 11 mice) ** P < 0.01, *** P < 0.001.

Figure 4. Chronic HM4Di–mediated suppression of L6 cells in NTSR1-Cre mice leads to increased activity in dLGN relay neurons

A) Recording site in dLGN labeled by DiO painted on the electrode. scale bar = 200μm B) Spontaneous activity of relay neurons was not significantly affected by L6 suppression (n= 105 vs. 133 cells from vehicle (white) and CNO (red) treated mice). C) The distribution of relay neuron response profiles was significantly affected by chronic HM4DI-mediated L6 suppression. D) Evoked activity of visually responsive dLGN relay neurons was increased over 2-fold after 10 days of HM4DI-mediated suppression of L6 cells. (n=41 vs. 75 cells). ** P < 0.01.

Figure 5. Enhancing L6 neuronal activity also triggers remodeling of the retinogeniculate synapse

A) Schematic depicting experimental design. B) Example recordings from relay neurons of NTSR1-Cre mice expressing HM3Dq, injected with vehicle or CNO from P20 to P27-33. C) Cumulative probability plot of AMPAR-mediated single fibers shows a significant shift toward weaker retinal inputs in CNO-injected mice. D) AMPA- and NMDA-mediated single fiber strengths after increased L6 activity. E) AMPAR-and NMDAR-mediated maximum EPSCs were not significantly altered in CNO-injected mice. F) The number of RGCs innervating each relay neuron was significantly larger after HM3Dq activation, as shown by the decreased fiber fraction. (For C-F, n = vehicle-injected: 51 cells from 13 mice; CNO-injected: 46 cells from 12 mice) * P < 0.05, ** P < 0.01.

Figure 6. Suppressing cortical feedback does not trigger significant retinogeniculate remodeling in older mice

A) Schematic of experimental design. B) Example recordings from relay neurons of vehicle- or CNO-injected mice. Single fiber strengths (C, D) and maximum EPSCs (E) did not significantly change with L6 inhibited from P30-P37. F) The number of retinal inputs converging onto each relay neuron was not significantly affected by altering cortical feedback after P30. (For C-F, n = vehicle-injected: 21 cells from 9 mice; CNO-injected: 35 cells from 11 mice).

Figure 7. Revised model for retinogeniculate refinement during development

Thickness of lines and size of circles indicate the relative strength of RGC inputs to relay neurons. At P10, retinal inputs to relay neurons are weak and numerous, and the cortical feedback circuit is not fully connected. By P20, many retinal inputs have been functionally eliminated (axon arbors remain present, indicated by gray contacts, but represent latent or extremely weak synapses), while others have strengthened. By this point, cortical feedback has begun to influence relay neuron activity, and thus drastic changes in cortical activity during the subsequent critical period (indicated by dashed lines in the bottom row at P30) disrupts the final stage of refinement. This leads to an increase rather than decrease in the number of functional inputs at P30, similar to the effect of visual deprivation during this stage of development. This phenotype is seen with all three perturbations of cortical activity reported here. In the case of HM4Di-manipulated L6 activity, relay neuron activity is elevated, and there is an additional effect of new retinal inputs strengthening.