Supplementary Figure 7: Response of dynorphin cells to somatostatin. Whole-cell patch-clamp recordings were made from eight eGFP⁺ neurons in spinal cord slices from Pdyn^Cre mice that had received intraspinal injections of AAV.flex.eGFP.

A.An example trace showing membrane hyperpolarization in response to bath-applied somatostatin (2 μM) in a dynorphin cell. All 8 cells tested showed clear hyperpolarization within a few minutes of the start of somatostatin application. B. Changes in the membrane potential of individual cells (circle, n = 8) are plotted, and the mean values (gray bar ± SD) are shown in control conditions (−58.0 ± 5.2 mV) and in the presence of somatostatin (−67.4 ± 6.5 mV). Somatostatin caused statistically significant hyperpolarization (t₇ = 3.935, p = 0.0056, two-sided paired t-test, n = 8) in the dynorphin cells. C. Subthreshold currents measured in response to a voltage step protocol (−90 to −50 mV, 5 mV increments, 500 ms duration) while the cell was held at −60 mV under control conditions and in the presence of somatostatin. A reduction in the input resistance and an outward shift of the measured current during somatostatin application are apparent in this example trace. D. A plot of the values for each cell (circles, n = 8) before and during somatostatin application shows that all of the neurons have their input resistance reduced during somatostatin application. The mean values (gray bar ± SD) are shown for control (858.7 ± 380.7 MΩ) and somatostatin-treated conditions (516.9 ± 288.7 MΩ). A two-sided paired t-test indicates that this effect is significant (t₇ = 6.243, p = 0.00043). E. The current-voltage (I-V) relationship was plotted using the voltage step protocol (as in C) in control conditions and in the presence of somatostatin. The I-V relationship in control conditions was then subtracted from that in the presence of somatostatin, which demonstrates the I-V relationship for the current responsible for the somatostatin-induced hyperpolarization. This current appears to reverse at −80.6 mV, which suggests that activation of somatostatin receptors was coupled to a downstream effect of opening G-protein coupled inwardly rectifying potassium (GIRK) channels. This intracellular coupling mechanism was previously suggested for another population of sst_2a-expressing inhibitory interneurons in the mouse superficial dorsal horn³. Data were obtained from 8 cells and are shown as mean ± standard deviation. F. Patterns of action potential firing in control conditions (top) and during somatostatin application (bottom). Action potentials were evoked by injecting square current pulses (1 s). Of the 8 cells tested, 5 initially exhibited a tonic firing pattern while 3 cells showed an initial bursting pattern. However, after somatostatin application, only 1 cell continued to show tonic firing and the remaining 7 cells exhibited an initial bursting pattern. Note that the hyperpolarizing effect of somatostatin was counteracted by adding bias currents to the recorded cell to restore the membrane potential to around −60 mV. This conversion in firing patterns after somatostatin application indicates that not only the resting membrane potential, but also the ability of the cell to generate action potentials, was inhibited by somatostatin. G-I. Confocal optical section through the cell body of one of the recorded neurons, showing expression of GFP (green) and avidin-rhodamine (magenta), which was used to detect Neurobiotin. The recorded cell is GFP⁺. J. The morphology of the cell seen in a projection of 55 optical sections at 1 μm z-separation. Scale bars I = 20 μm, J = 50 μm. 3.Iwagaki, N., Garzillo, F., Polgar, E., Riddell, J.S. & Todd, A.J. Neurochemical characterisation of lamina II inhibitory interneurons that express GFP in the PrP-GFP mouse. Mol Pain 9, 56 (2013).