Upregulation of cAMP response element-mediated gene expression during experience-dependent plasticity in adult neocortex

Abstract

Gene transcription is thought to be essential for memory consolidation and long-lasting changes in synaptic function. In particular, the signal transduction pathways that activate the transcription factor cAMP response element binding protein (CREB) have been implicated in the process of synaptic potentiation. To study the involvement of this pathway in neocortical plasticity within the barrel cortex, we have used a strain of mice carrying a LacZ reporter gene with six cAMP response elements (CREs) upstream of a minimal promoter. Removal of all but one facial whisker results in the expansion of the spared whisker's functional representation within somatosensory cortex. Under the same conditions of whisker deprivation, we observed a strong (eightfold compared with baseline) and highly place-specific upregulation of CRE-mediated gene transcription in layer IV of the spared whisker barrel. Reporter gene upregulation occurred rapidly after deprivation (16 hr) and was only observed under experimental conditions capable of inducing whisker response potentiation. LacZ expression in layer IV was accompanied by an increase in responsiveness of a subpopulation of layers II/III cells to spared whisker stimulation as determined by in vivo single-unit recording. Given that CREB is involved in the expression of plasticity in superficial layers (Glazewski et al., 1999), and yet CRE-mediated gene expression occurs in layer IV, it is likely that the molecular events initiating plasticity occur presynaptically to the cells that exhibit changes in their receptive field properties.

Fig. 1.

Basal levels of transgene expression in mouse brain. A, Dorsal view of cortical areas showing blue β-galactosidase reaction product reveals marked staining in visual cortex (arrowhead) and superior and inferior colliculi (arrow). B, Lateral view of the same brain shows staining in piriform (arrowheads), entorhinal cortex (arrow), and frontal cortex.C, Medial view through a bisected brain shows labeling of retrosplenial (arrowhead) and cingulate cortex. The pontine nuclei can also be seen to stain strongly (arrow). Scale bar, 2 mm.

Fig. 2.

A period of single-whisker experience leads to upregulation of CRE-mediated gene expression in the spared barrel. Four examples are shown of CRE–LacZ expression in the spared D1 barrel. A, The D1 barrel is visible macroscopically as a blue dot on the cortical surface contralateral to the spared whisker (arrow). The arrowheadindicates the location of an arc of staining between the anterior and posterior barrels. There are no equivalent areas of staining in the barrel cortex of the contralateral hemisphere. Scale bar, 1 mm.B–D, Despite some variability in the degree of CRE–LacZ expression, there are several common features including the D1 barrel, the visual cortex staining, and a faintly visible arc of staining at the border of the PMBSF and the ALBSF (A, arrowhead) (also see Fig. 8). After X-gal staining, brains were sunk in 30% sucrose, which cleared the tissue and enabled visualization of expression levels below the pial surface of the brain. The right hemisphere receiving normal input from the vibrissae shows no expression over baseline levels from undeprived control animals (Fig. 1).

Fig. 3.

CRE-mediated gene expression after 16 hr of single-whisker experience. A, Fluorescence image of layer IV of the hemisphere corresponding to the deprived barrel field showing barrels outlined by nuclei stained with propidium iodide. Theasterisk indicates the D1 barrel; the E1 and C1 barrels are above and below D1, respectively. B, Bright-field view of the same area as A. Tissue has been reacted with X-gal and reveals strong β-gal activity in the spared D1 barrel but not the surrounding barrels. C, Fluorescence image of layer IV barrel field from the undeprived hemisphere of the same animal. The D1 barrel is marked by an asterisk, and the orientation is the same as in A. D, Bright-field view of C reacted in X-gal, showing little β-gal activity. Scale bar, 150 μm.

Fig. 4.

Frequency of X-gal-positive cells in spared versus deprived barrels, by layer. Flattened brains were sectioned tangential to the pial surface to allow identification of the individual barrels, and blood vessels were used to orient adjacent sections to this barrel map. The number of blue cells in an area within an identified barrel was counted and divided by the total number of cells within that area (n = 4 animals). CRE-mediated LacZexpression was greatest within layer IV of the spared D1 barrel (black bars), where labeled cells were scattered throughout the barrel. Neighboring barrels in adjacent rows were identified, and expression within these barrels was also quantified (C1, gray bars; E1 barrels, hatched bars).

Fig. 5.

In vivo recording from 16 hr single-whisker spared versus control undeprived animals shows potentiation of spared-whisker responses. A, Responses (number of spikes per stimulus) of single units within layer IV and layers II/III of the D1 barrel to D1 whisker deflection from both control and single-whisker spared were recorded and averaged to generate a response magnitude average ± SEM (control undeprived, n = 3 animals; single-whisker spared, n = 4 animals). No statistically significant difference between the two experimental groups was demonstrated using this comparison. B, The distribution of cells within the D1 barrel is shown for deprived and undeprived cases and is indistinguishable. C, E, Responses from single units were sorted according to the magnitude of their response to D1 whisker deflection in a cumulative distribution function (CDF). This analysis revealed a shift in the distribution of responses of layers II/III cells (n = 89 cells in deprived animals and 83 cells in controls) but not layer IV cells (deprived animals,n = 50 cells; control animals,n = 64 cells). D, F, CDFs from control and single-whisker spared animals were subtracted to determine the number of cells that potentiated their responses after single-whisker experience. The difference in CDFs occurs among cells responding between 1.35 and 2.5 spikes per stimulus within layers II/III and suggests that ∼20% are potentiated. No difference was observed in the subtracted CDFs for layer IV cells (F).

Fig. 6.

CRE-mediated gene expression can be sustained for long periods of single-whisker experience. An example of reporter gene expression within the spared D1 barrel at 16 hr (A) and 7 d (B) of single-whisker experience is shown. In both cases, the margin of the D1 barrel as determined by fluorescent staining of cell nuclei (data not shown) can be identified by a boundary of X-gal-positive cells. The plane of section in B does not include the entire D1 barrel in layer IV; adjacent sections reveal that the pattern ofLacZ expression is roughly uniform throughout the barrel in this layer. See Results for quantification.

Fig. 7.

Upregulation of reporter gene expression is a specific response to single-whisker experience. The fraction ofLacZ-expressing cells within layer IV was determined for (A) control, undeprived animals (n = 4), (B) unilateral all-whisker-deprived animals, spared side (n = 4), (C) unilateral all-whisker-deprived animals, deprived side (n = 4), (D) single-whisker spared animals, deprived barrels (n= 4), and (E) single-whisker spared animals, spared D1 barrel (n = 4). Data are for the 16 hr time point where deprivations are involved. A schematicof the deprivation conditions and whisker representation area that was quantified (gray patch) is shown below each bar. Values for the fraction of X-gal-positive cells did not significantly differ between control undeprived animals and sensory input-deprived areas under various deprivation conditions, suggesting that the upregulation of CRE-mediated gene expression observed with the “single-whisker spared” deprivation pattern is not a response to changes in the general level of evoked activity but is highly correlated with events known to induce potentiation of neuronal responses.

Fig. 8.

The spared anterior barrels show upregulation of CRE–LacZ after unilateral deprivation.A, Propidium iodide-labeled section through the barrel field showing the location of the barrels. B, The same section as in A under normal illumination showing the location of LacZ-positive cells that occur in the spared barrels at the border of the deprived posterior medial barrel subfield (PMBSF) and the anterior barrels (which were not deprived) in the anterior lateral barrel subfield (ALBSF). The location of the barrels has been superimposed in outline. Note that the number of positive cells is very low in the deprived whisker region, highest at the border, and fades back toward low levels in the spared barrels farther away from the deprived barrels. Scale bar, 100 μm.C, The number of cells showing CRE-mediated gene expression is quantified by counting LacZ-positive nuclei on either side of the spared/deprived border. The approximate locations of the four, five, six, and seven arcs of barrels are indicated by dashed lines. Expression is highest in the first row of spared barrels and decreases in both directions away from the spared/deprived barrel border. The contour of expression can be explained by the pattern of deprivation acting on barrels linked by reciprocal inhibitory pathways.