Fig. 2. Mild focal cooling does not impact basal dendritic plasticity and excitability.
A) Schematic depicting low-frequency stimulation-induced plasticity in basal dendrites under mild focal cooling (left). An exemplar layer 5 pyramidal neuron loaded with Alexa 488 (100 μM) (scale bars, 50 μm). (Inset) dendritic segments that underwent the plasticity protocol without (top, red) and under the influence of focal cooling (bottom, blue) (scale bars, 5 μm).
B) Field-evoked EPSP amplitude as a function of low-frequency plasticity induction in the presence and absence of mild focal temperature modulation (data not shown for the case without cooling). The initial readout at 1 Hz (gray) is followed by potentiation at 0.1 Hz and subsequent readout again at 1 Hz (blue). While the initial (grey) and final readouts (blue) are performed at 35°C, the plasticity induction is carried out at two different temperatures for comparison − 35°C (data not shown) and 30°C (grey). For clarity, the initial and final readouts at 1Hz, as well as the potentiation (0.1 Hz) are only shown for the case in which plasticity was induced at 30°C. Note the induced plasticity is not significantly changed under the influence of mild focal cooling.
C) Comparison between the percentage of increase (post vs. control) in the field-evoked EPSP amplitude while low-frequency potentiation is performed at different temperatures. Results of field stimulation show an insignificant change in the EPSP amplitude after LTP (Wilcoxon Mann Whitney test, n=7 neurons, p=0.4136). Error bars represent the standard error of the mean (SEM).
D) (i) Uncaging-evoked responses from spines located on basal dendritic branches plasticized in the absence of cooling (top, red) and under the influence of cooling (bottom, blue) (scale bars, 50 μm). (Inset) Dendritic segments where the spines are uncaged before and after plasticity induction without (top, red) and under the influence of focal cooling (bottom, blue) (scale bars, 5 μm). (ii) Uncaging-evoked responses across spines located on target dendrites (left), depicting the difference between baseline (gray) and post-LTP induction (red: 35°C; blue: 30°C; signifies the temperatures at which plasticity was carried out), and percentage of increase (post-LTP vs. baseline control) for uncaging-evoked response (right) (Wilcoxon Mann Whitney test, n=6 neurons, p=0.6282). Error bars represent the standard error of the mean (SEM).
E) Schematic of the experimental protocol for probing the temperature dependence of branch-specific potentiation via a readout of nonlinear input-output transformations across the basal dendrite. Low-frequency field stimulation is used to evoke plasticity, while two-photon glutamate uncaging probes nonlinear input-output characteristics before (control, grey) and after (post, either red or blue) plasticity induction. Arrows indicate increasing number of spines stimulated. Focal cooling is only applied during the induction phase, while control and post-induction readout occur at 35°C. Note the absence of amplification in the dendritic nonlinear response between the baseline (grey) and post-plasticity period (red, blue). Arrows indicate an increasing number of spines uncaged.
F) (i) Normalized dendritic input-output response measured at the soma before and after plasticity induction at 35°C (Wilcoxon signed rank test, n=7 neurons, seven spines: p=0.3750, six spines: p=0.98125, five spines: p=0.3750, four spines: p=0.0313, three spines: p=0.2969, two spines: p=0.2969, one spine: p=0.1094). (ii) Similar to (i), but here plasticity induction was performed alongside moderate cooling (Wilcoxon signed rank test, n=6 neurons, seven spines: p=0.0303, six spines: p=0.4375, five spines: p=0.0313, four spines p=0.3125, three spines p=1.0, two spines p=0.3125, one spine p=0.3125). (iii) Comparison showing the percentage of amplitude increase for plasticity induced with focal cooling and without focal cooling (Wilcoxon Mann Whitney test, n=7 neurons, seven spines: p=0.5338, six spines: p=0.3660, five spines: p=0.7308, four spines: p=0.0734, three spines: p=0.5338, two spines: p=1.0, one spine: p=0.1014). Error bars represent the standard error of the mean (SEM).
G) (left) As an additional control, a spike timing-dependent plasticity (STDP) protocol was used instead of low-frequency stimulation to evoke potentiation across basal dendrites with and without the application of mild focal cooling. (middle) Baseline (black) and post-induction readout without cooling (red) or with cooling (blue) applied. (right) Comparison of evoked amplitudes as a percentage of control for the two cases (Mann Whitney test, n=6 neurons, p = 0.002). A mild reduction in temperature during plasticity induction does not amplify excitability.