Learning Induces Transient Upregulation of Brevican in the Auditory Cortex during Consolidation of Long-Term Memories

Abstract

It is a daily challenge for our brains to establish new memories via learning while providing stable storage of remote memories. In the adult vertebrate brain, bimodal regulation of the extracellular matrix (ECM) may regulate the delicate balance of learning-dependent plasticity and stable memory formation. Here, we trained adult male mice in a cortex-dependent auditory discrimination task and measured the abundance of ECM proteins brevican (BCN) and tenascin-R over the course of acquisition learning, consolidation, and long-term recall in two learning-relevant brain regions; the auditory cortex and hippocampus. Although early training led to a general downregulation of total ECM proteins, successful retrieval correlated with a region-specific and transient upregulation of BCN levels in the auditory cortex. No other parameter such as arousal or stress could account for the transient and region-specific BCN upregulation. This performance-dependent biphasic regulation of the ECM may assist transient plasticity to facilitate initial learning and subsequently promote the long-term consolidation of memory.SIGNIFICANCE STATEMENT The capacity to learn throughout life and at the same time guarantee lifelong storage and remote recall of established memories is a daily challenge. Emerging evidence suggests an important function of the extracellular matrix (ECM), a conglomerate of secreted proteins and polysaccharides in the adult vertebrate brain. We trained mice in an auditory long-term memory task and measured learning-related dynamic changes of the ECM protein brevican. Specifically, in the auditory cortex brevican is downregulated during initial learning and subsequently upregulated in exclusively those animals that have learned the task, suggesting a performance-dependent regulation in the service of memory consolidation and storage. Our data may provide novel therapeutic implications for several neuropsychiatric diseases involving dysregulation of the ECM.

Keywords: auditory cortex; behavior; extracellular matrix; hippocampus; learning; long-term memory.

Figure 1.

Cortex-dependent auditory learning in mice trained in a shuttle-box paradigm. A, Left, Animals are trained in a two-compartmental shuttle-box. Right, Mean CRs are measured for Go (red) and NoGo trials (blue) and termed hits and false alarms, respectively (Happel et al., 2015). Dashed black line indicates mean ITS. Subjects pass through consecutive learning stages of early AV, AQ, and RT of the learned contingencies. After 4 weeks of retention interval, we tested their LTR performance. All groups contain a number of n = 8. Of all trained animals, seven subjects did not yield a significant discrimination (LPs). *Asterisks mark significant differences between hits and false alarms revealed by paired Student's t test (p < 0.05). B, Left, Based on signal detection theory, discrimination performance was measured as d′ per training day. Right, Corresponding d′ learning curve based on CR rates shown in A. C, Response latencies during CS+ trials are plotted for individual trials over all training sessions. Black crosses represent median (horizontal) and SD of escape latencies >6 s within a session. Escape latencies decreased below a range of 1 s after US onset after individual increase of the foot shock. Response latencies <6 s correspond to successful hit responses. Histograms (right) of response latencies show a bimodal distribution corresponding to hit responses (<6 s) and escape responses (6–8 s).

Figure 2.

Learning curves (LCs) of animals trained to reach distinct learning stages for subsequent Western blot analysis. A, Top, CR curves (mean ± SEM) of adult male C57BL/6N mice (n = 39; n = 8 in each group; LP = 7) in a Go/NoGo shuttle-box-paradigm during FM discrimination (4–8 vs 8–4 kHz). Relative hit rate (red) is plotted against relative false alarm rate (blue) across training days (1 session per day/60 trials). Middle, Sensitivity index d′ (mean ± SEM) was used to define distinct learning stages from avoidance, acquisition, retrieval, and a group of LP animals. Another set that reached the retrieval-stage was trained for another 5 d after a retention interval of 4 weeks to test their LTR performance. Bottom, Averaged number of sessions of each group to reach the respective learning stages (mean ± SEM) did not show significant differences across groups (Table 1A). Corresponding dotted lines in top indicate learning stage transitions. *Paired Student's t test, p < 0.05. Abbreviations for training stages will be used in following figures. B, Group comparison of mean d′ of last sessions across trained animal groups revealed significantly different performance levels between the AV and LP groups compared against AQ, RT, and LTR (Kruskal–Wallis; p < 0.001). C, Mean reaction times across all groups revealed significantly longest reaction times in low performing animals (Kruskal–Wallis; p = 0.012). D, Absolute number of shocks received over the training paradigm significantly increased with number of trained sessions and low performance (Kruskal–Wallis; p < 0.001). All indicated bars are based on Bonferroni-corrected significance levels. Box plots represent median (bar) and interquartile range, and whiskers represent full range of data.

Figure 3.

Semiquantitative Western blot analysis of learning-induced changes of BCN levels in auditory cortex and hippocampus. A, B, Top, Representative Western blot example treated with mouse anti-BCN antibody directed against the N-terminus detected the full-length BCN of 145 kDa and the 55 kDa N-terminal fragment within the cellular and the ECM fraction in auditory cortex (A) and hippocampus (B). The two lanes for each group correspond to the same sample, as probes were loaded twice. Quantification of signal intensity of BCN (55 kDa, 145 kDa) in the extracellular fraction (top) and cellular fraction (bottom). C, Across training groups, full-length BCN showed an extracellular increase exclusively in the RT group (bottom left). The 55 kDa proteolytic fragment otherwise showed a general decrease, with significant difference in low-performers. In the cellular fraction, full-length proteins showed no significant changes across all training groups, whereas the 55 kDa BCN showed consistently significantly lower levels in all training groups except of the LTR group. D, In hippocampus, significant reduction was only found in the pellet fraction of the 55 kDa BCN fragment in all training groups. Other protein levels in hippocampus did not show significant changes. *Indicates significant one-way ANOVA and post hoc Tukey tests of multiple comparisons between groups (Table 1B–D). Box plots represent median (bar) and interquartile range, and bars represent full range of data. Plus within each bar represents mean value.

Figure 4.

Semiquantitative Western blot analysis of learning-induced changes of TNR levels in auditory cortex and hippocampus. A, B, Top, Representative Western blot example treated with antibody against TNR in auditory cortex (A) and hippocampus (B). The two lanes for each group correspond to the same sample, as probes were loaded twice. Quantification of signal intensity of TNR in the extracellular fraction (top) and cellular fraction (bottom). C, Consistently with BCN, TNR also showed an extracellular increase exclusively in the RT group, but without significant difference to the naive group (left). In the cellular fraction of auditory cortex (C, right) and hippocampus (D, right) TNR showed no significant changes across all training groups. The supernatant fraction of TNR in hippocampus did also showed nonsignificant trends toward a reduction across training groups (D, left). A one-way ANOVA with multiple comparisons between groups revealed no significant differences (Table 1C–E). Box plots represent median (bar) and interquartile range, and bars represent full range of data. Plus within each bar represents mean value.

Figure 5.

Unpaired tone and foot shock presentation did not change BCN levels in auditory cortex. A, In the control group (n = 4), we presented the same amount of foot shocks and frequency modulated sounds as in the retrieval group (average number of 180 foot shocks/animal). To mimic the relief of arousal in the later phase of training due to increase in performance, we presented foot shocks in the control group only within the first six sessions. Correspondingly, spontaneous compartment changes (escape shuttlings are subtracted), as a measure of arousal (Nienhuis and Olds, 1978), were high during the first six sessions, where animals received foot shocks (>1.0/min). Dropped shuttling rates in the last four sessions indicate relief of the stress induced by the unpredictable foot shock presentation. B, Quantification of Western blot signal intensity of 145 kDa BCN, 55 kDa BCN, and TNR in the cellular (top) and extracellular fraction (bottom) of control animals. We did not observe any significant regulation of BCN or TNR measures in the pseudo-trained control group. Box plots represent median (bar) and interquartile range, and bars represent full range of data. Plus within each bar represents mean value.

Figure 6.

Correlation analysis of behavioral parameters and biochemical modulation of the 145 kDa BCN fragment in the supernatant fraction. Data were taken from training groups AV, AQ, RT, and LP. A, Relative abundance of 145 kDa BCN in the supernatant fraction was positively correlated with the d′ value in the last training session indicating its performance dependent upregulation. B, The corresponding correlation between 145 kDa BCN and reaction times was significantly negative. In contrast, no significant correlation was found between supernatant 145 kDa BCN and absolute number of shocks (C) or the ratio of relative intertrial shuttles (D). E, Relative abundance of TNR in the supernatant fraction showed a trend of a positive correlation with the d′ value in the last training session (p = 0.054). Hence, this trend is in line with the performance-dependent upregulation of 145 BCN in A. F, Furthermore, Pearson correlation of the both full-length proteins TNR and 145 kDa BCN in the supernatant fraction were highly correlated. This implies a close interaction of the two ECM full-length proteins in the supernatant fraction and their differential regulation in the loose and cell-bound/PNN-associated ECM.

Figure 7.

Changes of total levels of BCN and TNR in the auditory cortex and hippocampus along learning stages. A, Total amount of the 55 kDa BCN (left), full-length BCN (middle), and TNR (right) in auditory cortex measured as the sum of both supernatant and pellet fractions. Whereas total TNR showed no significant reduction in the auditory cortex, cleaved BCN showed a significant reduction across all training stages and recovered during LTR. Full-length BCN was also significantly reduced during early avoidance learning and also within the group of LP animals. B, Total amount of the 55 kDa BCN (left), full-length BCN (middle) and TNR (right) in hippocampus showed only generally reduced levels of the 55 kDa BCN fragment independent of learning stage. For details, see Materials and Methods. *Indicates significant one-way ANOVA and post hoc Tukey tests of multiple comparisons between groups (Table 1F,G). Box plots represent median (bar) and interquartile range, and bars represent full range of data. Plus within each bar represents mean value.

Figure 8.

Learning-dependent regulation of BCN in auditory cortex. During acquisition of learning an initial downregulation of BCN in both, the cellular and loose ECM fraction (bottom) is permissive for learning-dependent synaptic remodeling (top). The cellular ECM here refers to the cell-bound and PNN-associated forms of the ECM, as extracted in the cellular fraction (pellet) of auditory cortex samples (Deepa et al., 2006). Here, new synaptic contacts are established during acquisition learning. Note that such downregulation was not present in the non-associative control group (Fig. 5), and hence, the downregulation is specific for early associative training. During consolidation of recent memories, a transient upregulation of the supernatant 145 kDa full-length BCN (bottom) is obstructive for further structural plasticity. During this RT phase, recent memories are resistant against remodeling (Happel et al., 2014). This transient protection of recently acquired memories might be fundamental for their remote recall, as this was not found in LP animals. During LTR, levels of both BCN fragments returned to baseline, as no new (re-)learning is initiated.