Plasticity-Related Gene 1 Affects Mouse Barrel Cortex Function via Strengthening of Glutamatergic Thalamocortical Transmission

Abstract

Plasticity-related gene-1 (PRG-1) is a brain-specific protein that modulates glutamatergic synaptic transmission. Here we investigated the functional role of PRG-1 in adolescent and adult mouse barrel cortex both in vitro and in vivo. Compared with wild-type (WT) animals, PRG-1-deficient (KO) mice showed specific behavioral deficits in tests assessing sensorimotor integration and whisker-based sensory discrimination as shown in the beam balance/walking test and sandpaper tactile discrimination test, respectively. At P25-31, spontaneous network activity in the barrel cortex in vivo was higher in KO mice compared with WT littermates, but not at P16-19. At P16-19, sensory evoked cortical responses in vivo elicited by single whisker stimulation were comparable in KO and WT mice. In contrast, at P25-31 evoked responses were smaller in amplitude and longer in duration in WT animals, whereas KO mice revealed no such developmental changes. In thalamocortical slices from KO mice, spontaneous activity was increased already at P16-19, and glutamatergic thalamocortical inputs to Layer 4 spiny stellate neurons were potentiated. We conclude that genetic ablation of PRG-1 modulates already at P16-19 spontaneous and evoked excitability of the barrel cortex, including enhancement of thalamocortical glutamatergic inputs to Layer 4, which distorts sensory processing in adulthood.

Keywords: behavior; in vitro; in vivo; network activity; patch-clamp recordings.

Figure 1.

Behavioral assessment of sensorimotor integration and whisker-mediated sensory perception. (A–F) Bar diagrams demonstrating quantitative differences in the beam-walking test performance between PRG-1-KO (KO, gray columns) mice (n = 18) and wild-type (WT, open columns) littermates (n = 18). The following parameters have been assessed: number of slides (A), mean number of slides per 100 cm (B), number of turns (C), running speed (D), running time (E), and path length (F). Results show mice performance on 20, 10, and 5 mm wide beams during 180 s and summary of all beams (all). (G) Bar diagrams demonstrating quantitative differences in the Rota Rod test performance between PRG-1-KO (KO, grey columns) mice (n = 18) and wild-type (WT, open columns) littermates (n = 18) in terms of speed at the time of fall. Data were obtained from 3 trials per mouse. (H) Mouse performance in a sandpaper maze designed to assess whisker-mediated sensory perception. While WT animals improved over time (n = 7 WT mice), PRG-1 KO mice did not improve above chance levels (n = 12 PRG-1 KO mice; performance over time and between groups was calculated using a 2-way ANOVA). Values represent mean ± SEM. Asterisks mark statistically significant differences, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Figure 2.

PRG-1 genetic ablation influences spontaneous neuronal activity in the somatosensory cortex in vivo. (A1) Representative traces showing 10 s in vivo recordings of spontaneous extracellular local field potentials (LFP) in P16–19 wild-type (WT, black) and PRG-1-KO (KO, gray) mouse. Corresponding multi-unit activity (MUA) is shown below the LFP traces. Horizontal lines below MUA traces display MUA bursts. (A2) Averaged power spectra of 30 min continuous LFP recordings from 12 barrels in 5 WT mice and 15 barrels in 7 PRG-1-KO mice. (A3) Box plots illustrating the power spectra in different frequency bands. (B1) Representative traces showing 10 s in vivo recordings of spontaneous LFPs in P25–31 WT (black) and PRG-1-KO (KO, gray) mouse and corresponding MUA with MUA bursts. (B2) Average power spectra of 30 min continuous LFP recordings from 10 barrels in 5 WT mice and 8 barrels in 4 KO mice. (B3) Box plot illustrating the power spectra in different frequency bands. Asterisks mark significant differences, *P < 0.05, **P < 0.01, and ***P < 0.001, Mann–Whitney–Wilcoxon test.

Figure 3.

Genetic ablation of PRG-1 affects multiunit activity (MUA) in the somatosensory cortex in vivo. (A–C) Box plots showing spontaneous MUA firing rate (A), number of MUA bursts (B), and MUA burst duration (C) in WT and PRG-1-KO mice of the P16–19 and P25–31 age group. Statistical analyses were performed using 1-way ANOVA test followed by post hoc Tukey's multiple comparison test. Asterisks mark significant differences, *P < 0.05, **P < 0.01, ***P < 0.001, and ns, not significant.

Figure 4.

PRG-1 deletion prevents functional maturation of sensory-evoked responses in the somatosensory cortex in vivo. (A) Average of evoked LFP responses from P16–19 WT (12 barrels, 5 mice), P16–19 PRG-1-KO (15 barrels, mice, 7 mice), P25–31 WT (10 barrels, 5 mice), and P25–31 PRG-1-KO (8 barrels, 4 mice). The black arrowheads indicate the time point of mechanical single whisker stimulation. (B–D) Box plots showing the peak amplitude (B), slope (C), and duration (D) of the evoked responses in the 4 groups. (E) Average of evoked sink responses from P16–19 WT (12 barrels, 5 mice), P16–19 PRG-1-KO (15 barrels, 7 mice), P25–31 WT (10 barrels, 5 mice), and P25–31 PRG-1-KO (8 barrels, 4 mice). (F–H) Box plots showing the peak amplitude (F), slope (G), and duration (H) of the evoked sink responses in the 4 groups. Statistical analyses were performed using 1-way ANOVA test followed by Tukey's multiple comparison test. Asterisks mark significant differences, *P < 0.05, **P < 0.01, ***P < 0.001, and ns, not significant.

Figure 5.

PRG-1-KO does not affect paired-pulse plasticity in vivo. (A1) Average of paired-pulse evoked responses at 100 ms inter-stimulus interval in 4 groups. (A2) Box plots showing corresponding S2/S1 ratios from A1. (B1) Average of paired-pulse evoked responses at 250 ms inter-stimulus interval. (B2) Box plots illustrating corresponding S2/S1 ratios from B1. Statistical analyses were performed using 1-way ANOVA test followed by Tukey's multiple comparison test. Asterisks mark significant differences, *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 6.

PRG-1-KO mice show increased evoked (e)EPSCs in somatosensory cortex in vitro. (A) Representative eEPSCs recorded from Layer 4 spiny stellate neurons in WT (left) and KO (right panel) mice. eEPSCs were elicited by paired-pulse electrical stimulation in the ventrobasal thalamus. Upper traces demonstrate original eEPSCs (20 in both cases); lower traces show the averaged responses. (B–D) Significantly larger eEPSC amplitudes (B), smaller failure rates (C), and smaller paired-pulse ratios (at 50, 100, and 200 ms ISIs; D) were observed in PRG-1-KO mice compared with WT littermates. Numbers indicate number of cells. *, **, and ***, P < 0.05, P < 0.01, and P < 0.001, respectively, unpaired Student’s t-test.

Figure 7.

Layer 4 spiny stellate cells in PRG-1-KO mice demonstrate increased mEPSC frequency. (A) Representative mEPSCs recorded in WT (left) and KO (right panel) mice. (B) Significantly higher frequency of mEPSCs observed in KO mice compared with WT littermates. Left panel shows mean values. Right panel shows cumulative distribution of interevent intervals. (C) Mean mEPSC amplitude did not vary between WT and PRG-1-KO groups. Numbers in (B) indicate number of cells. **P < 0.01, ns, not significant, unpaired Student’s t-test.

Figure 8.

Re-expression of PRG-1 in a subset of cells in constitutive PRG-1-KO mice influenced the frequency of mEPSCs. (A) Representative image of in utero electroporated Layer 4 spiny stellate cell. (B) Representative recordings of mEPSCs from PRG-1-deficient (KO) and PRG-1-expressing Layer 4 spiny stellate cells. (C,D) Re-constitution of PRG-1 expression reduced the frequency of mEPSCs (C) and did not alter the mean amplitude of mEPSCs (D). Numbers in (B) indicate number of cells. ***P < 0.001; ns, not significant, unpaired Student’s t-test.