Figure 7. In vitro LMWH, but not HMWH, increases DRG neuron excitability. A, Control group. B, LMWH group. C, HMWH group. Left columns: Images of neurons, obtained by transmitted light (differential interference contrast, left images) or fluorescence microscopy (right images, in which the darker regions correspond to more intense IB4 binding). A, B, IB4+ neurons stained with IB4 conjugated with AlexaFluor-488 dye. C, The lack of staining determines the IB4− neuron. Arrows indicate the neurons from which the electrophysiological recordings of APs (middle and right columns) were obtained. A–C, Middle columns: The two sets of traces represent AP generation before (left) and after (right) intervention. Black traces represent AP generation in response to rheobase current injection. Gray traces represent the responses to stimulation below rheobase (no AP generation). The magnitude of current pulses is indicated above the inset boxes (gray). Right column: Traces represent the AHP development and recovery after AP induced by 1 ms current pulse before (black traces) and after (gray traces) intervention. Gray boxes represent stimulation profile in current-clamp mode. D, Pooled relative changes of AP parameters after application of perfusion solution alone (white bars), LMWH (black bars), or HMWH (gray bars). Rheobase (left) was significantly decreased in the LMWH group compared with the control and HMWH groups (F(2,28) = 11, p = 0.0003, one-way ANOVA, followed by Bonferroni post hoc test; t(22) = 3.5, **p < 0.01, when LMWH and control groups are compared, t(20) = 4.2, ###p < 0.001, when LMWH and HMWH groups are compared). In addition, there were no significant differences from baseline in the control and HMWH groups (control group: t(8) = 1.3, p = 0.2; HMWH group: t(6) = 0.0, p = 1.0, compared with 0 by one-sample Student's t test). This suggests that LMWH increases neuronal excitability by reducing rheobase, whereas HMWH does not alter rheobase. Rheobase baseline was 650 ± 130 pA (n = 32). AP threshold potential (middle) was not significantly different between control, LMWH, and HMWH groups, as well as from baseline in each group (F(2,26) = 1.5, p = 0.25, one-way ANOVA; t(6) = 1.2, p = 0.3, for the control group; t(14) = 0.2, p = 0.8, for the LMWH group; t(6) = 1.2, p = 0.3, for the HMWH group, compared with 0 by one-sample Student's t test), suggesting that neither LMWH nor HMWH alters AP threshold potential significantly, and the reduction of rheobase is not due to a shift of AP threshold potential. AP threshold potential baseline was −32 ± 3 mV (n = 29). Of note, as this value is negative, relative decrease makes it “more positive” (i.e., increases threshold, implying less excitability), and vice versa. Therefore, the vertical axis is intentionally inverted to avoid confusion. Although not significant, there is a tendency to a positive shift of AP threshold potential in HMWH group. Peak AHP (right) was significantly increased in the LMWH group, compared with the control and HMWH groups (F(2,16) = 5.0, p = 0.02, one-way ANOVA, followed by Dunnett's post hoc test: q = 2.5, *p < 0.05, when LMWH and control groups are compared; q = 2.8, #p < 0.05, when LMWH and HMWH groups are compared). In addition, there were no significant differences from baseline in the control and HMWH groups (control group: t(3) = 0.06, p = 0.96; HMWH group: t(7) = 0.4, p = 0.7, compared with 0 by one-sample Student's t test). This indicates that LMWH, but not HMWH, influences the repolarization phase of the AP, which can impact the generation of following APs. AHP baseline was 5.4 ± 0.5 mV (n = 19). In all experiments, readings were obtained 10 min after the application of perfusion solution alone (A, control; D, white bars), LMWH (B, and black bars in D) or HMWH (C, and gray bars in D) had started, when a stable effect was achieved. To avoid effects of washout, the concentration of LMWH and HMWH (0.2 mg/ml) was kept constant during recording. Of note, both IB4+ and IB4− neurons were evaluated. There was no significant difference between them in the effects of LMWH or HMWH on either parameter (data not shown). D, Left, Control group, n = 9 neurons; LMWH group, n = 15 neurons; HMWH group, n = 7 neurons. Middle, Control group, n = 7 neurons; LMWH group, n = 15 neurons; HMWH group, n = 7 neurons. Right, Control group, n = 4 neurons; LMWH group, n = 7 neurons; HMWH group, n = 8 neurons.