Corticotropin-releasing factor increases mouse ventral tegmental area dopamine neuron firing through a protein kinase C-dependent enhancement of Ih

Abstract

Stress induces the release of the peptide corticotropin-releasing factor (CRF) into the ventral tegmental area (VTA), and also increases dopamine levels in brain regions receiving dense VTA input. Therefore, stress may activate the mesolimbic dopamine system in part through the actions of CRF in the VTA. Here, we explored the mechanism by which CRF affects VTA dopamine neuron firing. Using patch-clamp recordings from brain slices we first determined that the presence of I(h) is an excellent predictor of dopamine content in mice. We next showed that CRF dose-dependently increased VTA dopamine neuron firing, which was prevented by antagonism of the CRF receptor-1 (CRF-R1), and was mimicked by CRF-R1 agonists. Inhibition of the phospholipase C (PLC)-protein kinase C (PKC) signalling pathway, but not the cAMP-protein kinase A (PKA) signalling pathway, prevented the increase in dopamine neuron firing by CRF. Furthermore, the effect of CRF on VTA dopamine neurons was not attenuated by blockade of I(A), I(K(Ca)) or I(Kir), but was completely eliminated by inhibition of I(h). Although cAMP-dependent modulation of I(h) through changes in the voltage dependence of activation is well established, we surprisingly found that CRF, through a PKC-dependent mechanism, enhanced I(h) independent of changes in the voltage dependence of activation. Thus, our results demonstrated that CRF acted on the CRF-R1 to stimulate the PLC-PKC signalling pathway, which in turn enhanced I(h) to increase VTA dopamine neuron firing. These findings provide a cellular mechanism of the interaction between CRF and dopamine, which can be involved in promoting the avoidance of threatening stimuli, the pursuit of appetitive behaviours, as well as various psychiatric conditions.

Figure 1

CRF increased the firing rate of VTA dopamine neurons in the mouse The presence of I_h predicted dopamine content in mouse VTA neurons. A1, example neuron where a 250 ms hyperpolarizing voltage step from −60 mV to −120 mV elicited a slowly developing inward current. The magnitude of I_h was calculated by subtracting the instantaneous current (IS) from the steady-state current (SS) achieved during the voltage step. Scale bar vertical is 500 pA and horizontal is 100 ms. A2, 53/54 recorded neurons (red) with I_h co-localized with tyrosine hydroxylase immunohistochemical staining (green). A3, 1/54 recorded neurons with I_h did not co-localize with tyrosine hydroxylase. B, example neuron showing enhancement of VTA dopamine neuron firing by 10 min application of 1 μm CRF. Inset scale bar vertical is 20 mV and horizontal is 2.5 s. C, average effect of 1 μm CRF application (10 min) on firing rate of VTA dopamine neurons (n = 14). D, significant maximal increases in firing rate on dopamine neurons were observed with 1 μm CRF (n = 14) and 500 nm CRF (n = 6), but not with 100 nm CRF (n = 5). **P < 0.01, ***P < 0.001.

Figure 2

CRF increased the firing of VTA dopamine neurons through the CRF-R1 A, the CRF-R1 agonist, oCRF (1 μm), increased the firing rate of VTA dopamine neurons (n = 7), while B, the CRF-R2 agonist, urocortin II (UCN II, 1 μm) did not (n = 7). CRF receptor antagonists were applied for 5 min prior to and during CRF exposure. C, the non-specific CRF receptor antagonist (d-Phe-CRF, 1 μm, black circles, n = 7) and the CRF-R1 antagonist (CP-154,526, 3 μm, red squares, n = 7), but not the CRF-R2 antagonist (AS-30, 250 nm, blue triangles, n = 7) prevented the increase in firing by CRF. D, summary of the effects of various CRF receptor agonists and antagonists on maximum changes in firing. ***P < 0.001 from baseline firing. ##P < 0.01, #P < 0.05, respectively, reduced from CRF alone. E, CRF increased the firing of VTA dopamine neurons in CRF-R1^+/+ mice (black circles, n = 5) and in CRF-R1^+/− mice (red squares, n = 6), though to a lesser degree than in CRF-R1^+/+ mice, and did not affect firing in CRF-R1^−/– mice (blue triangles, n = 4. F, CRF enhanced the firing of VTA dopamine neurons to similar levels in CRF-R2^+/+ (black circles, n = 7), CRF-R2^+/− (red squares, n = 10) animals and CRF-R2^−/– mice (blue triangles, n = 5).

Figure 3

PLC and PKC are required for CRF to increase the firing of VTA dopamine neurons Inhibitors to intracellular signalling pathways were included in the internal recording solution. A, 100 μm Rp-cAMPs (red squares, n = 8) or 20 μm PKI (blue triangles, n = 7), did not prevent the effect of CRF on the firing of VTA dopamine neurons. B, U-73122 (1 μm, red squares, n = 7) and BIS (1 μm, blue triangles, n = 8), both blocked the increase in VTA dopamine neuron firing by CRF. C, summary of the effects of various intracellular signalling pathway inhibitors on the maximal change in firing by CRF. ***P < 0.001 relative to CRF alone. **P < 0.01 relative to CRF alone.

Figure 4

I_h is required for CRF to increase VTA dopamine neuron firing A, overlay of action potentials from an example VTA dopamine neuron during baseline (black) and 30 μm ZD-7288 application (red) that highlights the increase of the AHP after I_h inhibition. Scale bar vertical is 30 mV and horizontal is 25 ms. Example (B) and summary of 5 neurons (C) demonstrating that I_h inhibition with continuous application of 1 μm ZD-7288 prevented the increase in firing rate by CRF in VTA dopamine neurons. D, pharmacological blockade of I_K(Ca), I_Kir or slow I_A did not prevent the CRF-induced increase in firing. In contrast, inhibition of I_h prevented the increase in dopamine neuron firing by CRF. ***P < 0.001 relative to peak increase in firing by CRF alone.

Figure 5

CRF enhanced I_h without affecting the voltage dependence of activation for I_h in VTA dopamine neurons A, hyperpolarizing voltage steps (500 ms) from a holding potential of −60 mV to −80, −100 and −120 mV activated the slowly developing I_h (black), which was increased by CRF (red). Presented are raw traces (Aa), subtraction of traces (Ab), and the time course of this reversible effect for the step to −120 mV (Ac). Scale bar horizontal is 100 ms and vertical is 400 pA (Aa) or 100 pA (Ab). B, summary of the CRF enhancement of I_h at all voltage steps tested (n = 7). C and D, CRF did not alter the voltage dependence of activation for I_h. C, example neuron demonstrating that CRF did not alter the tail current elicited by the offset of hyperpolarizing voltage steps (1 s) to −60 mV from −130, −90, −80 and −60 mV. Scale bar horizontal is 250 ms and vertical is 200 pA. D, summary of the effect of CRF on I_h tail currents in 8 neurons.

Figure 6

CRF enhanced I_h through a PKC-dependent mechanism in VTA dopamine neurons A, example neuron demonstrating that 1 μm BIS in the internal recording solution prevented an increase in I_h by CRF. Shown are hyperpolarizing voltage steps (500 ms) to −80, −100 and −120 mV from a holding potential of −60 mV (Aa) and the subtraction of traces between treatments for each voltage step (Ab). Under these conditions, CRF did not affect the voltage dependence of activation for I_h (refer to Supplemental Fig. 4A and B). Scale bar horizontal is 100 ms and vertical is 400 pA (Aa) or 100 pA (Ab). B, BIS reduced the maximal effect of CRF on I_h for the voltage step measured at −120 mV (n = 7). **P < 0.01 relative to control internal. C, example neuron demonstrating that PDBU enhanced I_h. Shown are hyperpolarizing voltage steps (500 ms) to −80, −100 and −120 mV from a holding potential of −60 mV (Ca) and the subtraction of traces between treatments for each voltage step (Cb). Scale bar horizontal is 100 ms and vertical is 400 pA (Ca) or 100 pA (Cb). D, PDBU enhanced I_h at a range of voltages tested (n = 8). E and F, example neuron (E) and summary of 8 neurons (F) showing that 500 nm PDBU did not change the tail current elicited by the offset of hyperpolarizing voltage steps (1 s) from −130, −90, −80 and −60 mV to the holding potential of −60 mV. Scale bar horizontal is 250 ms and vertical is 200 pA.

Figure 7

Inhibition of cAMP-dependent processes shifts the voltage dependence of activation for I_h, but does not prevent CRF from enhancing I_h in VTA dopamine neurons A, examples of tail currents elicited by the offset of hyperpolarizing voltage steps (1 s) from −130, −90, −80 or −60 mV to the holding potential of −60 mV with Rp-cAMPs in the internal solution and before (baseline) or after addition of CRF. Scale bar horizontal is 250 ms and vertical is 200 pA. B, Rp-cAMPs did induce a significant hyperpolarizing shift in the baseline V_1/2 relative to recordings with the control recording. However, identical to control conditions, CRF did not further alter the voltage dependence of activation of I_h during recordings with the Rp-cAMPs internal solution. C and D, in addition, Rp-cAMPs in the internal recording solution did not prevent the CRF-mediated enhancement of I_h. C, example neuron demonstrating that Rp-cAMPs did not prevent an increase in I_h by CRF. Shown are hyperpolarizing voltage steps (500 ms) to −80, −100 and −120 mV from a holding potential of −60 mV (Ca) and the subtraction of traces between treatments for each voltage step (Cb). Scale bar horizontal is 100 ms and vertical is 400 pA (Ca) or 100 pA (Cb). D, Rp-cAMPs did not alter the maximal effect of CRF on I_h, measured at the voltage step to −120 mV relative to recordings with the control internal.