CREB controls cortical circuit plasticity and functional recovery after stroke

Treatments that stimulate neuronal excitability enhance motor performance after stroke. cAMP-response-element binding protein (CREB) is a transcription factor that plays a key role in neuronal excitability. Increasing the levels of CREB with a viral vector in a small pool of motor neurons enhances motor recovery after stroke, while blocking CREB signaling prevents stroke recovery. Silencing CREB-transfected neurons in the peri-infarct region with the hM4Di-DREADD blocks motor recovery. Reversing this inhibition allows recovery to continue, demonstrating that by manipulating the activity of CREB-transfected neurons it is possible to turn off and on stroke recovery. CREB transfection enhances remapping of injured somatosensory and motor circuits, and induces the formation of new connections within these circuits. CREB is a central molecular node in the circuit responses after stroke that lead to recovery from motor deficits.

. Lentiviral CREB transfection 1 week after stroke. All images are from one section with multi-fluorescent confocal imaging of neurons (NeuN, purple), astrocytes (GFAP, yellow), and blood vessels (Glut-1, red). CREB is localized as a GFP fusion protein in the nucleus. CREB co-localizes with neurons but not astrocytes or blood vessels.
Supplementary Figure 4. Lentiviral CREB transfection 1 week after stroke. Low magnification confocal images of same multi-fluorescent immunohistochemical staining as in Supplementary  Figure 3. In each panel the top is the pial surface of cortex. The bottom is the subcortical white matter. These images are taken from coronal sections and anterior is to the left and ventral to the bottom. The stroke site is not visible but is to the right of the panels.
Supplementary Figure 5. Increased excitability with lentivirus CREB transduction. (a-c). CREB expression leads to increased excitability in in cortical pyramidal neurons. CREB-tdTOM POS = lentivirus with CAMKIIa promoter CREB and tdTomato genes. tdTOM POS = control lentivirus, with CAMKIIA promoter and tdTomato. CREB-tdTOM NEG = non-fluorescent (virus negative) neurons adjacent to CREB transfected neurons in the same slice. (a) Rheobase measurements, with all cells held at a membrane potential of -80 mV. , Rheobase measurements were significantly smaller in CREB-containing cells (546.9+/-70.5 pA, n=8) when compared to non-CREB-containing cells (840.8+/-99.2 pA, n=6) in the same animal [t(12)=2.491, p=0.0284]. No differences were seen in rheobase measurements between adjacent cells no virus and control virus cells (660.0+/-88.5 pA, n=6)(t(10)=1. 374, p=0.199) Figure 12. (a) Effect of effect of inhibitory DREADD in motor cortex in control (non-stroke) mice on gait. The data from the two behavioral studies, lentivirus control and lentivirus-inhibitory DREADD control, were separately compared to determine if the presence of inhibitory DREADD activation by itself impairs motor control. The data on gridwalking from Fig.  2b and Fig. 3f was isolated to look at motor performance in the conditions of control lentivirus and lentivirus-DREADD+CNO (i.e. with the inhibitory DREADD effect). There is some variance in motor performance over the testing periods, but no significant difference between the two groups (f(1,90)=1.27, p=0.2634). (b) Effect of inhibitory DREADD in motor cortex in stroke mice on gait. The same data as in (a) were separately compared for stroke+control lentivirus vs. stroke+lentivirus-DREADD+CNO. Both groups have worsening motor control after stroke, as seen in the increase in footfaults. There is a non-significant difference between control-lenti in stroke and control-lenti-DREADD+CNO in stroke at 4 weeks, but these two groups are otherwise overlapping in their behavioral performance (f(1,80) = 1.44, p=0.2293). (c). Compare the effect of activation of the inhibitory DREADD in control (a) and stroke (b) to the effect of activation of the inhibitory DREADD after CREB induction in stroke. Note that the scale is higher for Y in this graph than for (a,b), because the effect on motor control is much greater after after CREB induction than the non-significant effect of just inhibitory DREADD induction without CREB.
Supplementary Figure 13. Remapping of forepaw somatosensory cortex after stroke. Top row shows location of center of forepaw S1 cortex in lentivirus control with the fluorescent reporter tdTomato, and with CREB. Over time there is no significant shift in the location of the S1 forepaw location. Middle row shows the location of the forepaw S1 cortex center after stroke. In TOMATO+STROKE (middle row, left panel) there is no activation in cortex from forepaw stimulation in weeks 1, 2, and 4 (second row of images from top in Fig. 5c). In stroke with control (non-CREB) induction there is a significant long distance shift of the center of the forepaw location. In CREB induction after stroke there is non-significant shift in location. The statistical testing of this data is in Fig. 5d.
Supplementary Figure 14. Hindpaw somatosensory cortex does not remap after stroke. Same conventions as in Supplementary Figure 11. Compare the bottom panel with Fig. 5d. There is an early shift of the hindpaw somatosensory cortex at week 1 that is not sustained.
Supplementary Figure 15. Laser speckle contrast imaging of cerebral blood flow 1 week after stroke: Laser speckle contrast imaging was performed through the cranial window at different intervals before and after stroke. The cortical surface was illuminated with an expanded laser diode beam (785 nm, 80mW) coupled to a 600 μm diameter fiber optic cable. Blue color represent regular blood flow while green-yellow show the reduced blood flow in the stroke area. Top row shows laser speckle imaging of control (left) and stroke control virus (right) one week after stroke. Center row shows laser speckle imaging of CREB alone (left) and stroke CREB (right) one week after stroke. Bottom row show the quantification of the cortical blood flow between stroke and relative control. Relative cortical blood flow values were obtained as the ratio K02/Kt2.