Figure 1.
Effects of VOCC toxins on oxytocin release from SONs in vitro.
Blocking N-type VOCC in SONs from adult (A) PDN-8 (B), thapsigargin- (TG)-treated adult rats (C) and TG-treated PDN-8 rats (D) shows a decrease in amplitude and duration of the potassium stimulated response. Stimulated release was enhanced after pretreatment with 0.2 µM TG as previously described (C D black lines) (Ludwig et al., 2002); this was prevented by blocking N-type Ca2+ channels (red lines). Effects of blocking different VOCC on the S2:S1 oxytocin response in SONs from adult and adult TG-treated rats (E) and PND-8 rats and TG-treated PDN-8 rats (F). Means±S.E.M., n = 6 per group, # P<0.05 vs first S1, * P<0.05 vs control, one-way ANOVA followed by Dunn's post-hoc test.
Figure 2.
Ca2+ channel distribution in tissue sections of SON.
(A) Immunostaining for Cav subunits (red staining) and oxytocin (A) or vasopressin (B) (green staining) in SON sections from PND-8, adult, and lactating rats. Average optical density for the subunits normalised by the optical density of (C) oxytocin and (D) vasopressin (n = 5). No labelling was detected when primary antibodies were omitted or incubated with a five-fold excess of control immunogen before being exposed to the tissues (not shown). Means±S.E.M. vs adult, # P<0.05 vs lactating rats, one-way ANOVA followed by Holm-Sidak post-hoc test. Scale bars, 20 µm.
Figure 3.
Ca2+ channel distribution in isolated magnocellular neurons.
Immunostaining for Cav α1 subunits (red) in acutely-dispersed oxytocin (A) and vasopressin neurons (B) (green). Immunostaining for all subunits was observed in the soma and dendrites, in both in the cytoplasm and at the plasma membrane. Immunostaining for oxytocin and Cav2.1 and 2.2 also showed strong perinuclear staining. The merged images show areas of coincidence in yellow (third column). Scatter plots (fourth column) show co-variance of subunit signal with the oxytocin signal for each voxel The residual map corresponds to weighted residuals from the line fit to the scatter plot, thus indicating fluorescent channel covariance (hue from –1 to 1, with cyan corresponding to zero residual). (C) Quantification of covariance data of oxytocin or vasopressin and one of the five α1-subunits using Pearson's coefficient values (n = 5 per group). Means±S.E.M. * P<0.05, one-way ANOVA followed by Holm-Sidak post-hoc test vs Cav2.2. Scale bars 10 µm.
Figure 4.
Ca2+ channel distribution in relation to the Golgi apparatus.
(A) Immunostaining for Cav α1 subunits (red) and the Golgi marker GM130 (green) in acutely-dispersed supraoptic neurons. There was strong co-localisation of Cav2.2 with GM130, a marker for the Golgi apparatus. No fluorescent labelling was detected when primary antibodies were omitted or incubated with a five-fold excess of control immunogen before being exposed to the cells (not shown). (C) Quantification of covariance data of GM130 and one of the five α1-subunits using Pearson's coefficient values (n = 5 per group) showed a significant difference between the co-localisation of GM130 and Cav2.2 versus the other α1-subunits. Means±S.E.M. * P<0.05, one-way ANOVA followed by Holm-Sidak post-hoc test vs Cav2.2. Scale bars 10 µm.
Figure 5.
Effects of channel toxins on Ca2+ currents in isolated SON neurons.
(A) Isolated magnocellular neurons were identified as either vasopressin or oxytocin by their presence or absence of the GFP signal. Scale bar = 10 µm. (B) The current-voltage relationships show no difference between whole-cell calcium currents (WCCC) of vasopressin and oxytocin neurons (n = 20 each). (C) Normalized conductance (G/Gmax, fitted using Boltzmann equation) showed no significant change with or without TG. (D) Examples of two neurons (both oxytocin neurons), one vehicle treated (•) and one 50 min after pre-treating with thapsigargin (○). The steady-state Ca2+ current is plotted against time following treatment with channel toxins (in order: 1 µM nicardipine; 200 nM ω-agatoxin IV; 500 nM ω-conotoxin GVIA). The current measured after each toxin was subtracted from that evoked before each treatment to determine the current carried by L-, P/Q- and N-type channels. The remaining current was attributed to R-type channels. (E) Examples of N-, L- and WCCC elicited by voltage steps in controls and TG treated cells. (F) The resulting current density for each current type normalised for the peak maximal current was averaged (n = 5) to give the average current density from neurons treated with vehicle or thapsigargin. (G) In a subsequent experiment L- or N-type channel toxins were administered, alternating the order (n = 5 for each condition). Mean±S.E.M are shown and compared by t-test. *P<0.05 vs control.