Experience-dependent expression of NPAS4 regulates plasticity in adult visual cortex

Abstract

There is evidence that developmental-like plasticity can be reactivated in the adult visual cortex. Although activity-dependent transcription factors underlying the process of plasticity reactivation are currently unknown, recent studies point towards NPAS4 as a candidate gene for the occurrence of plasticity in the adult visual system. Here, we addressed whether NPAS4 is involved in the reinstatement of plasticity by using the monocular deprivation protocol and long-term fluoxetine treatment as a pharmacological strategy that restores plasticity in adulthood. A combination of molecular assays for gene expression and epigenetic analysis, gene delivery by lentiviral infection, shRNA interference and electrophysiology as a functional read-out, revealed a previously unknown role for the transcription factor NPAS4 in the regulation of adult visual cortical plasticity. We found that NPAS4 overexpression restores ocular dominance plasticity in adult naıve animals whereas NPAS4 down-regulation prevents the plastic outcome caused by fluoxetine in adulthood.Our findings lead the way to the identification of novel therapeutic targets for pathological conditions where reorganization of neuronal networks would be beneficial in adult life.

Figure 1. Increased NPAS4 expression parallels the fluoxetine-induced reinstatement of plasticity in the adult visual system

A, experimental protocol used for long-term FLX administration (28 days). B, long-term treatment with FLX increases NPAS4 mRNA levels in the VC of adult animals (t test, P < 0.05, n= 8). Inset 1: a marked decrease of H3K27 tri-methylation status at the NPAS4 promoter region (t test, P < 0.05, n= 8) was observed in the VC after long-term FLX administration. Inset 2: no change of H3K9 acetylation at the NPAS4 promoter occurred after pharmacological treatment (t test, P= 0.1, n= 8). C, long-term FLX administration increases NPAS4 protein levels (t test, P < 0.05, n= 8) in the VC of adult animals after 2 days of either MD or binocular vision (Bin Vision; D23) during the last week of treatment.

Figure 2. NPAS4 overexpression reactivates plasticity in the adult visual system

A, schematic diagram of the lentiviral infection protocol (28 days) in naïve animals. Adult rats were infected with Syn-NPAS4 in the binocular region of the primary VC contralateral to the deprived eye (see Methods). B and C, functional analysis of plasticity. NPAS4 overexpression in naïve animals restores susceptibility to MD in adulthood as assessed by VEPs (C/I ratio 0.9 ± 0.07; one-way ANOVA, P < 0.0001; post hoc Tukey's test, P < 0.001, n= 8) and single units recordings (CBI: 0.37 ± 0.04; one-way ANOVA, P < 0.0001; post hoc Tukey's test, P < 0.001, n= 6, 112 cells). No change of binocularity was observed in any of the control experimental groups. The shift of OD caused by NPAS4 overexpression was similar to that observed during the critical period for plasticity at early stages of development (CP (P28)). D, the cumulative distribution of OD score was biased towards the ipsilateral eye in MD NPAS4-infected animals compared with controls (Kolmorogov–Smirnov test, P < 0.05). E, the shift of OD in response to 1 week of MD was due to a decrease in the response stimulation of the deprived eye (t test, P < 0.05, VEP amplitude 0.68 ± 0.12, n= 6 and 0.73 ± 0.03, n= 8 for naïve and scramble, respectively; VEP amplitude 0.34 ± 0.05, n= 8 for NPAS4). VEPs amplitudes at the recording site in the VC contralateral to occlusion were normalized to the sum of the response to stimulation of the contralateral and ipsilateral eye of control animals. F, VA for the deprived eye (0.72 ± 0.02 cycles (c) deg⁻¹) was markedly reduced as compared with the fellow eye (0.99 ± 0.01 cycles deg⁻¹) in NPAS4-overexpressing animals (t test; P < 0.001, n= 6) but not in any of the control experimental groups. Error bars represent SEM. *Statistical significance.

Figure 3. Response properties of visual cortical neurons following the lentiviral infection approach

Functional properties of visual cortical neurons were unaltered after either NPAS4 overexpression in naïve animals or NPAS4 down-regulation during FLX treatment. Data are represented as box charts. The central horizontal line represents the median value while the other two horizontal lines are the 25th and 75th percentiles; error bars denote the 5th and 95th percentiles; square symbols indicate the mean value. A, receptive field (RF) size (degrees of visual angle) was higher in NPAS4-overexpressing animals (n= 112 cells) compared with controls (one-way ANOVA, P < 0.0001; post hoc Tukey's test, P < 0.001; n= 86 cells for adult MD rats; n= 62 cells for naïve controls). B, peak discharge decreased significantly in NPAS4-overexpressing and FLX-treated animals as compared to controls (one-way ANOVA, P < 0.0001; post hoc Tukey's test, P < 0.001). C, spontaneous activity did not change in any of the experimental groups (n= 63 cells for FLX+MD-NPAS4-shRNAi; n= 62 cells for FLX+MD-Scramble). *Statistical significance.

Figure 4. NPAS4 underlies the FLX-induced reactivation of adult visual cortical plasticity

A, schematic diagram of the lentiviral delivering of NPAS4-shRNAi in rats long-term treated with FLX or vehicle. B, silencing of NPAS4 by RNA interference (NPAS4-shRNAi) during FLX treatment counteracts the process of plasticity reactivation. The shift of OD in FLX-treated rats after MD (C/I ratio, 1.0 ± 0.08; one-way ANOVA, P < 0.0001; post hoc Tukey's test, P < 0.001, n= 5) was totally prevented in NPAS4-shRNAi infected animals (C/I ratio, 2.4 ± 0.07, n= 6). Scramble interference did not alter the effects caused by FLX in VC plasticity (C/I ratio, 1.13 ± 0.06). Binocularity in naïve animals with binocular vision (C/I ratio, 2.34 ± 0.15, n= 4) or after 1 week of MD (C/I ratio, 2.24 ± 0.06, n= 6) did not change after silencing of NPAS4 basal levels. C, the cumulative distribution of OD score was biased towards the ipsilateral eye in both adult MD rats treated with FLX and scramble-infected animals after FLX treatment as compared with controls (Kolmorogov–Smirnov test, P < 0.05) but not in any other experimental group (n= 92 cells for FLX+MD-treated rats; n= 63 cells for FLX+MD-NPAS4-shRNAi; n= 42 cells for FLX+MD-Scramble; n= 55 cells for Naïve+MD-NPAS4-shRNAi; n= 70 cells for Naïve+Bin-NPAS4-shRNAi. *Statistical significance.

Figure 5. The process of plasticity reactivation caused by FLX in the adult visual system

The reinstatement of plasticity caused by FLX in adult life is associated with signal transduction pathways that involve the activation of long-distance serotonergic transmission, a down-regulation of local intracortical inhibitory circuitries and enhanced NPAS4 expression. We propose a model in which the increased serotonergic signalling shifts the inhibitory/excitatory balance in favor of excitation, thus activating intracellular mechanisms that eventually promote epigenetic modifications of chromatin structure that, in turn, allow for the expression of plasticity genes in adult life among which NPAS4 plays a key role. NPAS4 seems to turn on a transcriptional program that underlies structural and functional plasticity while facilitating, in parallel or in series, a reorganization of inhibitory circuitries that might contribute to the homeostasis of cortical excitability by driving inhibition during this phase of enhanced plasticity. The expression of NPAS4 target genes may ultimately set in motion downstream physiological mechanisms that provide a permissive environment for adult visual cortical plasticity (e.g. enhanced BDNF-trkB signalling, removal of extracellular matrix components that are inhibitory for plasticity, enhanced dendritic spines density and remodelling). Accordingly, an increased expression of BDNF and transcription factors involved in the regulation of dendritic spine density and structural plasticity was observed in the VC of NPAS4-infected rats (E.C. L.M. E.T. and J.F.M.V. unpublished data). Continuous arrows represent established interactions between molecular and cellular processes mentioned (boxes). Dashed lines represent interactions that remain to be ascertained.