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. 2023 Sep 15:17:1258773.
doi: 10.3389/fncel.2023.1258773. eCollection 2023.

Electrophysiological properties of layer 2/3 pyramidal neurons in the primary visual cortex of a retinitis pigmentosa mouse model (rd10)

Claas Halfmann  1 , Thomas Rüland  1   2   3 , Frank Müller  2   3   4 , Kevin Jehasse  1 , Björn M Kampa  1   3   4   5
Affiliations

Electrophysiological properties of layer 2/3 pyramidal neurons in the primary visual cortex of a retinitis pigmentosa mouse model (rd10)

Claas Halfmann et al. Front Cell Neurosci. .

Abstract

Retinal degeneration is one of the main causes of visual impairment and blindness. One group of retinal degenerative diseases, leading to the loss of photoreceptors, is collectively termed retinitis pigmentosa. In this group of diseases, the remaining retina is largely spared from initial cell death making retinal ganglion cells an interesting target for vision restoration methods. However, it is unknown how downstream brain areas, in particular the visual cortex, are affected by the progression of blindness. Visual deprivation studies have shown dramatic changes in the electrophysiological properties of visual cortex neurons, but changes on a cellular level in retinitis pigmentosa have not been investigated yet. Therefore, we used the rd10 mouse model to perform patch-clamp recordings of pyramidal neurons in layer 2/3 of the primary visual cortex to screen for potential changes in electrophysiological properties resulting from retinal degeneration. Compared to wild-type C57BL/6 mice, we only found an increase in intrinsic excitability around the time point of maximal retinal degeneration. In addition, we saw an increase in the current amplitude of spontaneous putative inhibitory events after a longer progression of retinal degeneration. However, we did not observe a long-lasting shift in excitability after prolonged retinal degeneration. Together, our results provide evidence of an intact visual cortex with promising potential for future therapeutic strategies to restore vision.

Keywords: electrophysiology; patch-clamp; rd10 mouse model; retinitis pigmentosa; visual cortex.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Figures

Figure 1
Figure 1
Acute but non-long-lasting shift of subthreshold membrane properties of rd10 L2/3 PNs. (A) Examples of voltage responses to hyperpolarizing current steps for each of the three age groups (I, II, and III) and genotypes (gray traces B6, blue traces rd10). Bottom traces show injected current steps (−50 and −100 pA). (B) No change in resting membrane potential between B6 and rd10 in all age groups. (C) Voltage sag is not changed between B6 and rd10 in all age groups. (D) Time constant is not different between B6 and rd10 in all age groups. (E) Input resistance of age groups I and II showed a significant difference between B6 and rd10. Sample size of recorded cells for (B–E) I: n = 12 B6; 15 rd10, II: n = 35 B6; 65 rd10, III: n = 60 B6; 66 rd10.
Figure 2
Figure 2
Acute but non-long-lasting changes in action potential properties in rd10 L2/3 PNs. (A) Example traces of action potential firing elicited by depolarizing current steps. Traces in the background show the first few action potentials elicited by current step injections. Traces in front are examples of the first action potential of these trains for each of the three age groups (I, II, and III) and genotypes (gray traces B6, blue traces rd10). Vertical and horizontal lines indicate action potential amplitude and half-width, respectively, bottom line indicates action potential threshold. (B) Action potential threshold and (C) action potential amplitude did not show significant differences between B6 and rd10. (D) Action potential rise time and (E) Action potential half-width were significantly increased in rd10 from age group II. Sample size of recorded cells for (B–E) I: n = 12 B6; 15 rd10, II: n = 35 B6; 65 rd10, III: n = 60 B6; 66 rd10.
Figure 3
Figure 3
Acute but non-long-lasting shift of suprathreshold properties in rd10 L2/3 PNs. (A) Example traces of action potential firing. For each of the three age groups (I, II, and III) and two genotypes (B6 gray traces and rd10 blue traces), one exemplary cell is presented. Each of these cells is shown at three different depolarizing current steps, from rheobase to the maximal current step. (B) AP firing frequency at each depolarizing current step (FI curve). Insets show the area under the FI curve. Only rd10 neurons from age group II had significantly higher evoked AP firing rates. (C) Rheobase of rd10 L2/3 PNs from age group II is significantly decreased. (D) AP amount in 100 ms time bins of the stimulation period, dashed lines indicate the different time bins for analysis of AP firing in the respective time bin. Age group II had a significantly higher number of APs in each time bin. (E) Intensity plot of the adaptation ratio (ISI-n/ISI-9) did not reveal any differences between rd10 and B6. (F) Example traces of ongoing AP firing while membrane potential is raised to −40 mV. (G) Quantification of spontaneous AP firing did not reveal significant differences between B6 and rd10 across all age groups. Sample size of recorded cells for (B–D) I: n = 12 B6; 15 rd10, II: n = 35 B6; 65 rd10, III: n = 60 B6; 66 rd10, (E) I: n = 11 B6; 15 rd10, II: n = 22 B6; 48 rd10, III: n = 47 B6; 54 rd10, and (G) I: n = 8 B6; 12 rd10, II: n = 12 B6; 23 rd10, III: n = 14 B6; 33 rd10.
Figure 4
Figure 4
Consistent pre- and postsynaptic network properties in rd10 and B6 L2/3 PNs. (A) Schematic representation of the experimental setup. V1 in the brain slice is highlighted in green. Layer 2/3 PNs were recorded in current-clamp mode (green highlighted cell), while a second pipette is used for the extracellular stimulation of the surrounding L2/3 neurons (yellow area of stimulation and red highlighted cells affected by stimulation). (B) Example traces of voltage responses evoked by the extracellular stimulation of the surrounding network (five stimuli at 20 Hz and two stimuli at 40 Hz). (C) Paired pulse ratios {PPR [event(n)/event(1)]} at 20 Hz did not reveal any difference between B6 and rd10. (D) Paired pulse ratios {PPR [event(2)/event(1)]} at 40 Hz show a significant difference between B6 and rd10 in age group II. Sample size of recorded cells for (C) I: n = 8 B6; 13 rd10, II: n = 13 B6; 16 rd10, III: n = 21 B6; 29 rd10, (D) I: n = 8 B6; 13 rd10, II: n = 14 B6; 19 rd10, III: n = 24 B6; 37 rd10.
Figure 5
Figure 5
Spontaneous synaptic inputs are stable in rd10 L2/3 PNs. (A) Example traces of spontaneous depolarizing events in the current clamp at membrane potential raised to −50 mV (for hyperpolarizing events, see Supplementary Figure 2). (B) Mean events per second for depolarizing and hyperpolarizing events measured at −50 mV membrane potential and (C) Mean median amplitudes of depolarizing and hyperpolarizing events remain similar between B6 and rd10 across all age groups. (D) Example traces of spontaneous inhibitory and excitatory currents in voltage clamp at +10 mV (two upper sweeps) and −45 mV (two lower sweeps), respectively. (E) Mean events per second of spontaneous postsynaptic currents at +10 mV and −45 mV were not different between B6 and rd10. (F) Mean median amplitudes of spontaneous membrane currents at +10 mV and −45 mV. Age group III currents at +10 mV show a significant difference between B6 and rd10. Sample size of recorded cells for (B, C) I: n = 13 B6; 15 rd10, II: n = 14 B6; 44 rd10, III: n = 31 B6; 41 rd10 cells, (E, F) (+10 mV): II: n = 16 B6; 19 rd10, III: n = 18 B6; 11 rd10 and (E, F) (−45 mV): II: n = 18 B6; 24 rd10, III: n = 19 B6; 17 rd10.
Figure 6
Figure 6
No change in synaptic properties from L4 in rd10 L2/3 PNs. (A) Schematic representation of the experimental setup. V1 in the brain slice is highlighted in green. L2/3 PNs were recorded in voltage clamp mode (green highlighted cell), while a second pipette was used for the extracellular stimulation of L4 neurons (yellow area of stimulation and red highlighted cells affected by stimulation). (B) Example traces of postsynaptic current responses to stimulation in L4 recorded in L2/3 PNs at different holding membrane potentials (stimulation protocol was five stimuli at 20 Hz and two stimuli at 40 Hz). (C) Paired pulse ratio {PPR [event(n)/event(1)]} with 5 stimuli at 20 Hz at +10 mV. (D) Paired pulse ratio with two stimuli at 40 Hz at +10 mV. (E) Paired pulse ratio with five stimuli at 20 Hz at −45 mV. (F) Paired pulse ratio with two stimuli at 40 Hz at −45 mV. None of the paired pulse ratio values (C–F) was significantly different between B6 and rd10. Sample size of recorded cells for (C) II: n = 10 B6; 13 rd10, III: n = 12 B6; 8 rd10, (D) II: n = 11 B6; 17 rd10, III: n = 16 B6; 10 rd10, (E) II: n = 17 B6; 14 rd10, III: n = 19 B6; 7 rd10, (F) II: n = 18 B6; 20 rd10, III: n = 19 B6; 7 rd10.
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Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This study was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)−368482240/GRK2416 and GRK2610: Innoretvision.
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