Activation of CRH receptor type 1 expressed on glutamatergic neurons increases excitability of CA1 pyramidal neurons by the modulation of voltage-gated ion channels

Abstract

Corticotropin-releasing hormone (CRH) plays an important role in a substantial number of patients with stress-related mental disorders, such as anxiety disorders and depression. CRH has been shown to increase neuronal excitability in the hippocampus, but the underlying mechanisms are poorly understood. The effects of CRH on neuronal excitability were investigated in acute hippocampal brain slices. Population spikes (PS) and field excitatory postsynaptic potentials (fEPSP) were evoked by stimulating Schaffer-collaterals and recorded simultaneously from the somatic and dendritic region of CA1 pyramidal neurons. CRH was found to increase PS amplitudes (mean ± Standard error of the mean; 231.8 ± 31.2% of control; n = 10) while neither affecting fEPSPs (104.3 ± 4.2%; n = 10) nor long-term potentiation (LTP). However, when Schaffer-collaterals were excited via action potentials (APs) generated by stimulation of CA3 pyramidal neurons, CRH increased fEPSP amplitudes (119.8 ± 3.6%; n = 8) and the magnitude of LTP in the CA1 region. Experiments in slices from transgenic mice revealed that the effect on PS amplitude is mediated exclusively by CRH receptor 1 (CRHR1) expressed on glutamatergic neurons. The effects of CRH on PS were dependent on phosphatase-2B, L- and T-type calcium channels and voltage-gated potassium channels but independent on intracellular Ca(2+)-elevation. In patch-clamp experiments, CRH increased the frequency and decay times of APs and decreased currents through A-type and delayed-rectifier potassium channels. These results suggest that CRH does not affect synaptic transmission per se, but modulates voltage-gated ion currents important for the generation of APs and hence elevates by this route overall neuronal activity.

Keywords: CRH; CRH receptor; neuronal excitability; potassium channels; protein kinases.

Figure 1

When Schaffer-collaterals are electrically stimulated CRH increases the population spike (PS) amplitude without affecting field excitatory postsynaptic potentials (fEPSPs). Acute sagittal hippocampal brain slices were obtained from male CD-1 mice (P30–P50). Dual recording was performed by positioning one electrode in the stratum radiatum of the CA1 region for monitoring fEPSPs (SC-stim-fEPSPs; white symbols) and the second electrode was positioned in the pyramidal layer for monitoring PS (black symbols). After 10 min of reaching a stable baseline, CRH (125 nM) was applied to the perfusion medium. CRH increases PS amplitudes to 231.8 ± 31.2% of baseline level (n = 10; p < 0.05) without affecting fEPSP slope (104.3 ± 4.2% of control; n = 10; p > 0.05). In a subset of experiments the stimulation electrode was positioned in the CA3 pyramidal layer and fEPSPs were recorded from the CA1 stratum radiatum (CA3-stim-fEPSPs; gray symbols). This stimulation technique allows the inclusion of an action potential generation site and, consequently, transmitter release at CA3-CA1 synapses is dependent on somatic action potential initiation and axonal signal propagation. Under these conditions, CRH (125 nM) significantly increased CA3-stim-fEPSP amplitudes to 119.8 ± 3.6% of control (n = 8; p < 0.05). Each data point represents the mean ± SEM of 4 consecutive fEPSP/PS amplitude measurements normalized to the last 5 min before CRH application. Insets show representative recording traces; § stimulation artifact. Black bar indicates presence of CRH.

Figure 2

The CRH-mediated effect depends on CRH receptor type 1 (CRHR1) expression on glutamatergic neurons. Slices were prepared from conditional knock-out mice lacking CRHR1 either on glutamatergic neurons (CRHR1^Glu-CKO) or on GABAergic neurons (CRHR1^GABA-CKO). CRHR1^flox/flox mice littermates served as control.CRH increases PS amplitude in slices from CRHR1^ctrl to 154.7 ± 10.2% (n = 7; p < 0.05; black dots) and CRHR1^GABA-CKO (144.5 ± 9.5%; n = 7; p < 0.05; gray dots), but showed no effect in CA1 hippocampal neurons of CRHR1^Glu-CKO (98.2 ± 2.0%; n = 7; p < 0.05; white dots). Insets show representative recording traces. § stimulation artifact. Black bar represents presence of CRH.

Figure 3

(A) The effect of CRH on CA1-LTP depends on the site of electrical stimulation. When SC-stim-fEPSPs were recorded, CRH did not change LTP induced by HFS (control: 173.3 ± 19.8 %; n = 5, in the presence of CRH: 171.3 ± 8.2%; n = 5, p > 0.05). However, when HFS was delivered to the CA3 stratum pyramidale, (B) CRH increased the potentiation of CA3-stim-fEPSPs from 154.8 ± 5.0% (n = 6) under control conditions to 180.6 ± 8.1% (n = 6; p < 0.05 for control vs. CRH). (C) The effect of CRH on the population spike amplitude is dependent on phosphatase 2B (PP-2B), voltage-gated potassium channels (K_V) and T- and L-type calcium channels. PS were recorded from the CA1 pyramidal layer upon electrical stimulation of the Schaffer-collaterals. The inhibition of PKA, PKC, and CaMKII with Kn252a (500 nM), H7 (100 μM), and H89 (10 μM), respectively did not affect the CRH-mediated effect on PS amplitude. In the presence of the T- and L-type calcium channel antagonist nifedipine (nif) and Ni²⁺, respectively, CRH failed to enhance PS amplitudes (105.9 ± 7.6%; n = 6; p > 0.05). CRH did neither increase PS amplitudes when PP-2B was inhibited with cycA (113.1 ± 7.3%; n = 6; p > 0.05) nor when voltage-gated potassium channels were antagonized with 4-aminopyridine (4-AP; 113.5 ± 6.2%; n = 6; p > 0.05). ^**p < 0.01; ^***p < 0.001.

Figure 4

CRH increases intracellular calcium influx in CA1 pyramidal neurons upon electrical stimulation of the Schaffer-collaterals. (A) Cells were loaded with the calcium sensitive dye oregon green-AM. After 1 h of incubation, the SC were electrically stimulated every 15 s and calcium influx was recorded simultaneously (sampling rate 20 Hz; duration 1.5 s). In the presence of CRH the intracellular Ca²⁺⁻influx during synaptic activity increased to 231.7 ± 13.8% of control (n = 4; p < 0.05). Right insets: Videomicroscopic photograph of the CA1 region of the hippocampus and fluorescence recorded under control conditions (middle) and in the presence of 125 nM CRH (lower inset). (B) Barium currents through T- and L-type calcium channels were recorded in single-cell voltage-clamp recordings. CRH did neither increase L-currents (96.8 ± 6.4% of control; n = 6; p > 0.05) nor T-currents (103.8 ± 16.4% of control; n = 7; p > 0.05). (C) PS amplitudes recorded from Ca_V1.2^CNS-CKO animals were increased to 197.3 ± 16.0% of control (n = 3; p < 0.05). The effect of CRH on PS amplitude in CNS-CKO animals was diminished in the presence of the calcium channel antagonists nifedipine (132.3 ± 10.0% of control; n = 4; p < 0.05) or Ni²⁺ (119.6 ± 5.9% of control; n = 5; p < 0.05). ^*p < 0.05; ^**p < 0.01.

Figure 5

CRH does not modulate Na_V channels but increases spiking frequency and prolongs action potentials. Sodium currents were elicited by depolarizing the membrane from −70 to −20 mV (ΔU = 2 mV). (A) The normalized amplitude of Na⁺-channel current amplitudes was plotted against the membrane potential. Na_V channels showed half-maximum activation (V_1/2) of 57.4 ± 0.2 mV (n = 7) under control conditions. In the presence of CRH (125 nM) V_1/2 was not significantly different (52.8 ± 0.3; n = 7; p > 0.05). (B) Action potentials were elicited by inducing a depolarizing current of +130 pA and had a mean frequency of 20.9 ± 1.0 Hz. Application of CRH resulted in a significant increase in the frequency (23.2 ± 0.6 Hz; n = 8; p < 0.05). (C) Compared to control, CRH prolonged the half-width of APs from 3.0 ± 0.0 to 3.2 ± 0.0 ms (n = 8; p < 0.05). (D) Current-voltage relationships revealed that CRH does not change membrane resistance in CA1 pyramidal neurons. ^* p < 0.05.

Figure 6

CRH reduces currents through A-type (I_A) and delayed rectifier (I_K) potassium channels. In the voltage clamp mode, currents through voltage gated potassium channels were recorded upon stepwise depolarization of CA1 pyramidal neurons from −50 to +40 mV (ΔU = 10 mV). (A) Representative recording traces under control conditions (black traces) and in the presence of CRH 125 nM (gray traces). (B) A preceding hyperpolarizing step (100 ms) de-inactivates fast I_A and allows the graphical subtraction of currents with and without the preceding hyperpolarizing step (C), I_A currents can be analyzed. (C) Representative recording traces of I_A currents under control conditions (black) and in the presence of CRH 125 nM (gray). (D) After application of CRH, currents through delayed rectifier potassium channels (I_K) were reduced by 14.2 ± 5.7% to 30.4 ± 6.6% dependent on the voltage step. (E) CRH significantly reduced I_A currents ranging from 30.1 ± 6.0% to 83.2 ± 26.8% depending on the voltage step (n = 5; p < 0.05). ^*p < 0.05; ^**p < 0.01. Insets show representative recording traces; leak subtraction was performed with the P/4 subtraction protocol.