Figure 4. Astrocyte Dependence of the Norepinephrine Regulation of CRH Neurons.

(A) The norepinephrine-induced increase in sEPSC frequency is inhibited by preincubation of slices in the gliotoxin fluorocitric acid (FCA) (n = 13 cells).

(B) Mean sEPSC frequency changes in norepinephrine relative to baseline in control slices and slices incubated in FCA (n = 17 and 13 cells).

(C) The norepinephrine-induced increase in sEPSC frequency was blocked by the purinergic receptor antagonist PPADS (n = 7 cells) and by the P2x-selective antagonist TNP-ATP (n = 5 cells).

(D) The norepinephrine-induced facilitation of sIPSC frequency was blocked by preincubation of slices in the gliotoxin FCA, but the norepinephrine-induced suppression of sIPSCs was retained following FCA preincubation (n = 10 cells).

(E) Mean norepinephrine-induced increase (NE(+)) and decrease (NE(−)) in sIPSC frequency relative to baseline following FCA treatment. The norepinephrine-induced increase in sIPSC frequency (NE(+)), but not the decrease in sIPSC frequency (NE(−)), was blocked by FCA preincubation (n = 9–11 cells/group).

(F) The norepinephrine-induced increase in sIPSC frequency was blocked by the P2X-selective purinergic receptor antagonist TNP-ATP but the norepinephrine-induced decrease in sIPSC frequency was not (n = 6 cells).

(G) Fluorescence image showing the glial Ca²⁺ response to norepinephrine in the PVN with the glia-specific Rhod-2/AM Ca²⁺ indicator. The changes in Ca²⁺ signal in 4 of the cells in the slice (arrows) are shown in the image and in the fluorescence recordings (ΔF/F₀) on the right.

(H) The glial Ca²⁺ response to VP imaged with the glia-specific Rhod-2/AM Ca²⁺ indicator.

(I) Quantification of the glial Ca²⁺ responses to norepinephrine (NE) (n = 30 cells), which was blocked by the VP receptor blocker Manning compound (NE/MC) (n = 13 cells), and to VP (n = 9 cells).

(J) The norepinephrine-induced increase in sEPSC frequency (control/NE) was retained in CRH neurons from dnSNARE mice maintained on the dox diet (on-dox/NE) (n = 6 cells) but was lost in CRH neurons of mice taken off the dox diet (off-dox/NE) (n = 7 cells).

(K) The norepinephrine-induced increase in sIPSC frequency was blocked, but the sIPSC suppression was not affected, in CRH neurons from mice taken off the dox diet (off-dox/NE) (n = 10 cells). *p < 0.05; **p < 0.01.