The sleep-modulating peptide cortistatin augments the h-current in hippocampal neurons

Abstract

Cortistatin (CST) is a sleep-modulating peptide found exclusively in the brain. Although CST is closely related to somatostatin (SST) and binds to SST receptors, CST has effects on sleep and neuronal activity in cortex and hippocampus that differ from SST. To uncover the cellular mechanisms affected by CST, we studied the electrophysiological postsynaptic effects of CST and assessed its interaction with SST on hippocampal CA1 pyramidal neurons. CST altered intrinsic membrane properties and occluded SST effects, indicating that both peptides similarly augment the sustained K+ M- and leak-currents (IM and IK(L)). In the presence of SST, however, CST elicited an additional inwardly rectifying component in the hyperpolarized range. This effect was unaffected by barium, used to block K+ currents, but was completely prevented by the selective h-current (Ih) blocker ZD7288. CST, but not SST, selectively increased Ih in a concentration-dependent manner by augmenting its maximum conductance. CST did not shift the Ih activation curve, and the peptide effect was unaffected by a membrane-permeable analog of cAMP. We conclude that CST and SST similarly increase K+ conductances in hippocampal neurons, most likely by activating SST receptors. However, CST additionally augments Ih, a voltage-dependent current that plays a key role in the modulation of synaptic integration and regulates oscillatory activity. Our results indicate that CST targets a specific conductance unaffected by SST to modulate cellular mechanisms implicated in sleep regulation.

Figure 1.

Cortistatin postsynaptically inhibits CA1 pyramidal neurons. A, Continuous current recording of a neuron held at -65 mV, near its RMP of -69 mV. Superfusion of 1 μm CST (bar, adjusted to effect onset) elicited a slowly developing outward current associated with an increased input conductance. During washout, current values returned to control level (dashed line). Downward deflections are currents elicited by 10 mV hyperpolarizing voltage steps. B, Selected current traces of a representative neuron held at -60 mV and subjected to three different voltage steps sequentially applied and superimposed at each condition (voltage protocol on the right). CST (1 μm) elicited an outward steady-state current at depolarized potentials and an inward current at hyperpolarized potentials. Dotted lines indicate control condition levels; RMP was -68 mV. C, Average of the net steady-state currents (subtracted from control) from 15 neurons. The CST effect showed outward rectification at potentials positive to -70 mV and inward rectification at potentials negative to -80 mV. The reversal potential of -86 mV suggested that CST did not solely affect K⁺ conductances.

Figure 2.

Cortistatin elicits an additional effect at hyperpolarized potentials. A, Net steady-state currents obtained from eight neurons exposed to 1 μm CST followed by 1 μm SST in the continued presence of CST. SST did not elicit an additional effect after the CST response was established. B, The reverse sequence of application (SST followed by CST) revealed that CST elicited an additional effect at potentials negative to -50 mV (n = 7). This inward component was voltage dependent and increased with hyperpolarization.

Figure 3.

Cortistatin-specific current and conductance. A, CST-specific component isolated by subtracting the currents obtained in SST plus CST from those obtained in SST alone (as in Fig. 2 B; n = 7). CST elicited an inwardly rectifying current that activated at -54 mV. Curve was obtained by polynomial fit. B, The CST-induced conductance increase, ΔG_CST, was calculated as I_CST/(V - V_rev), where I_CST is the CST-induced current, and V - V_rev is the driving force (V indicates command potential, and V_rev indicates reversal potential). The ΔG_CST appeared to reach a maximum at -140 mV and yielded a half-maximum activation potential of -94 mV (dashed line).

Figure 4.

Cortistatin increases I_h. A, Neuron stepped from -58 to -98 mV. SST elicited an outward current at -58 mV (dotted line is control level) but had no effect at -98 mV (dashed line), the reversal potential for K⁺. The addition of CST had no effect at -58 mV (threshold of I_h activation) but induced an inward current concomitant with an I_h augmentation at -98 mV. The bottom inset shows the I_h relaxations (identified with letters) magnified and superimposed. RMP was -69 mV. B, Plot average of the I_h relaxation amplitude (n = 6; curve was obtained by polynomial fit). SST alone did not affect I_h, but CST applied in the presence of SST increased I_h at all potentials. C, Plot average of the I_M relaxation amplitude (n = 6; polynomial fit). SST augmented I_M, and addition of CST did not further alter I_M. D, Concentration-response curve of the I_h relaxation augmentation by CST. The threshold response was below 0.1 μm, and the maximal effect was obtained with 1 μm to augment I_h by 26%. The apparent EC₅₀ was 0.30 ± 0.05 μm (dashed line). Curve was obtained by sigmoidal (logistic) fit; number of cells at each concentration are in parentheses.

Figure 5.

Characterization of the cortistatin effect in barium. A, Neuron was held at -61 mV and subjected to three different voltage steps (protocol at right). Application of 1 mm Ba²⁺ blocked K⁺ currents and decreased input conductance. Addition of 1 μm CST elicited an inward steady-state current at hyperpolarized potentials (dashed line is pre-peptide level) but had no effect at depolarized potentials. RMP was -70 mV before Ba²⁺. B, Net steady-state currents obtained from seven neurons exposed to CST in presence of Ba²⁺. CST elicited a voltage-dependent inwardly rectifying current that activated at -52 mV. C, Averaged I_h amplitudes in 10 cells exposed to CST in the presence of Ba²⁺. The peptide increased I_h to 135% of control.

Figure 6.

The I_h blocker ZD7288 prevents the CST effect. A, Neuron was bathed in 1 mm Ba²⁺, held at -64 mV, and subjected to three different voltage steps (protocol at right). ZD7288 (100 μm) decreased the input conductance and concomitantly blocked I_h. Further addition of CST had no effect. The bottom inset shows I_h relaxations magnified and superimposed. RMP was -71 mV before Ba²⁺. B, Averaged I_h amplitude (n = 4; 1 mm Ba²⁺). ZD7288 obliterated I_h, and subsequent addition of CST had no effect. C, Net steady-state currents obtained from two neuronal samples exposed to CST in the absence or presence of ZD7288 (n = 7 and 4, respectively; 1 mm Ba²⁺). With functional h-channels, CST elicited a large voltage-dependent effect. With I_h blocked by ZD7288, CST had a small nonspecific effect.

Figure 7.

Cortistatin does not alter I_h activation curve. A, A two-step protocol was used to study I_h activation. The prepulse (first step) was incremented by -11 mV from a holding potential of -44 mV. From each prepulse potential, I_h was fully activated with a second step to -143 mV. Current traces were leak corrected, 1 mm Ba²⁺ was used throughout, and RMP was -70 mV before Ba²⁺. B, Magnification of the current traces obtained at -143 mV. CST elicited an inward steady-state current associated with I_h augmentation from all prepulse potentials. C, Averaged activation curves constructed from I_h amplitudes obtained at full activation potential (second step) relative to the prepulse potential (n = 5). The graph represents the difference to the maximal current amplitude after normalization (1 - I_h/I_h max, at each condition). Data were fitted using a Boltzmann function. Cortistatin induced a minimal shift of +1.5 mV in the activation curve.

Figure 8.

CST augments I_h independently of cAMP. A, Left, Current recordings from a neuron held at -45 mV and sequentially stepped to -91 mV (I_h mid-activation) and -144 mV (full activation). Application of 1 mm 8Br-cAMP increased I_h by 13% at -91 mV but decreased it by 9% at -144 mV, suggesting a shift of the activation curve. Additional application of CST in the presence of 8Br-cAMP increased I_h at both potentials (128 and 133% of 8Br-cAMP values). Right, The same neuron was single stepped from -45 to -144 mV to fully activate I_h at once. 8Br-cAMP had little effect on I_h (104% of control), whereas subsequent addition of CST increased I_h to 127% of 8Br-cAMP values. Numbers refer to drug condition; current traces were leak corrected, 1 mm Ba²⁺ was used throughout, and RMP was -68 mV before Ba²⁺. B, Averaged activation curves obtained from six neurons. 8Br-cAMP shifted the activation curve by +5 mV, and subsequent addition of CST did not further affect the curve.