Forebrain HCN1 channels contribute to hypnotic actions of ketamine

Abstract

Background: Ketamine is a commonly used anesthetic, but the mechanistic basis for its clinically relevant actions remains to be determined. The authors previously showed that HCN1 channels are inhibited by ketamine and demonstrated that global HCN1 knockout mice are twofold less sensitive to hypnotic actions of ketamine. Although that work identified HCN1 channels as a viable molecular target for ketamine, it did not determine the relevant neural substrate.

Methods: To localize the brain region responsible for HCN1-mediated hypnotic actions of ketamine, the authors used a conditional knockout strategy to delete HCN1 channels selectively in excitatory cells of the mouse forebrain. A combination of molecular, immunohistochemical, and cellular electrophysiologic approaches was used to verify conditional HCN1 deletion; a loss-of-righting reflex assay served to ascertain effects of forebrain HCN1 channel ablation on hypnotic actions of ketamine.

Results: In conditional knockout mice, HCN1 channels were selectively deleted in cortex and hippocampus, with expression retained in cerebellum. In cortical pyramidal neurons from forebrain-selective HCN1 knockout mice, effects of ketamine on HCN1-dependent membrane properties were absent; notably, ketamine was unable to evoke membrane hyperpolarization or enhance synaptic inputs. Finally, the EC50 for ketamine-induced loss-of-righting reflex was shifted to significantly higher concentrations (by approximately 31%).

Conclusions: These data indicate that forebrain principal cells represent a relevant neural substrate for HCN1-mediated hypnotic actions of ketamine. The authors suggest that ketamine inhibition of HCN1 shifts cortical neuron electroresponsive properties to contribute to ketamine-induced hypnosis.

Figure 1. HCN1 is selectively deleted from forebrain neurons in CaMKCre:HCN1^f/f mice

A. HCN1 channel expression was assessed by immunohistochemistry in the cortex, hippocampus and cerebellum from wild type (HCN1^+/+) or floxed (HCN1^f/f) mice, from global HCN1 knockout mice (HCN1^−/−) and from floxed mice expressing Cre recombinase under the CaMKIIα promoter (CaMKCre:HCN1^f/f). The characteristic HCN1 expression in apical dendrites of the cortex and in stratum lacunosum-moleculare of CA1 in the hippocampus (see arrows) that is typical of wild type mice was also present in HCN1^f/f mice, but was absent in both HCN1^−/− and CaMKCre:HCN1^f/f mice. The insets highlight dendritic staining, at 4x magnification). Note that HCN1 expression was preserved in the cerebellum of all mice except the global knockout. B. qRT-PCR shows that HCN1 transcript levels were reduced by over 90% in both the cortex and hippocampus from CaMKCre:HCN1^f/f mice by comparison to HCN1^f/f mice, but HCN1 expression in the cerebellum was not different. n=5, P<0.05 by two-way ANOVA, * HCN1^f/f:cre vs. HCN1^f/f. qRT-PCR: quantitative real-time polymerase chain reaction.

Figure 2. I_h is diminished and effects of ketamine on I_h are reduced in cortical pyramidal neurons from CaMKCre:HCN1^f/f mice

A. Sample voltage clamp recordings of I_h in cortical pyramidal neurons from HCN1^f/f and CaMKCre:HCN1^f/f mice under control conditions (upper), during exposure to ketamine (20 μM, middle) and following treatment with the I_h blocker, ZD-7288 (50 μM, lower). B. Averaged data (± SE) depicting I_h amplitude (at −118 mV) and V of activation of I_h in cortical pyramidal neurons from HCN1^f/f mice and CaMKCre:HCN1^f/f mice. These data show that I_h was smaller with a more hyperpolarized V in cortical pyramidal neurons from CaMKCre:HCN1^f/f mice; they also reveal that the ketamine-induced decrease in current amplitude and hyperpolarizing shift in V observed in cells from HCN1^f/f mice were absent in CaMKCre:HCN1^f/f mice. Note that ZD7288 completely blocked I_h in cells from both genotypes, so amplitude and V data after ZD7288 is not presented in B. n=5 & 7, P<0.05, * ketamine vs. control; ‡, HCN1^f/f:cre vs. HCN1^f/f.

Figure 3. HCN1 deletion from cortical pyramidal neurons of CaMKCre:HCN1 ^f/f mice alters basal membrane properties and occludes ketamine modulation

A. Sample current clamp recordings from cortical pyramidal neurons from HCN1^f/f and CaMKCre:HCN1^f/f mice under control conditions (upper) and during exposure to ketamine (20 μM, middle) and ZD-7288 (50 μM, lower). The depolarizing membrane sag observed during membrane hyperpolarization, a phenomenon attributed to I_h, is indicated by the arrow. Tetrodotoxin was not applied in these experiments. B. Averaged data (± SE) show that membrane potential was more hyperpolarized, R_N was higher and sag was reduced in cortical pyramidal neurons from CaMKCre:HCN1^f/f mice. Ketamine hyperpolarized membrane potential, increased R_N and decreased sag only in cells from HCN1^f/f mice. n=5 & 5, P<0.05, * ketamine vs. control; ‡, HCN1^f/f:cre vs. HCN1^f/f.

Figure 4. Ketamine fails to enhance EPSP temporal summation in cortical pyramidal neurons from CaMKCre:HCN1 ^f/f mice

A. Sample voltage traces show EPSP recordings in cortical pyramidal neuron from HCN1^f/f (left) and CaMKCre:HCN1^f/f mice (right) in response to 40 Hz stimulation under control conditions, and during exposure to ketamine (20 μM). Lower panels: EPSPs were aligned to initial membrane potential and normalized to the amplitude of the first EPSP in the train in order to highlight ketamine effects on temporal summation. B. Averaged EPSP summation ratio (EPSP5/EPSP1) for HCN1^f/f and CaMKCre:HCN1^f/f mice under the indicated conditions. Ketamine enhanced EPSP summation in cortical neurons from HCN1^f/f animals, but not from CaMKCre:HCN1^f/f mice. *, n=5 & 5, P<0.05 by two-way RM-ANOVA. Tetrodotoxin was not applied in these experiments. EPSP: excitatory postsynaptic potential.

Figure 5. Deletion of HCN1 channels from forebrain reduced sensitivity of mice to hypnotic actions of ketamine

A & B. Mice were injected with incrementing concentrations of ketamine (5–20 mg/kg, i.v.) and the fraction of HCN1^f/f and CaMKCre:HCN1^f/f mice that failed to right themselves (loss-of-righting reflex; LORR) was determined as a measure of hypnosis. CaMKCre:HCN1^f/f mice were less sensitive to hypnotic effects of ketamine, as indicated by increased EC₅₀ for ketamine-induced loss-of-righting reflex (A; F_1,328=33.2, P<0.0001 by ANOVA) and reduced duration of the loss-of-righting reflex (B; genotype main effect, F_1,211=10.4, P<0.002 by two-way ANOVA). C. The latency for mice to remove tail from a radiant heat source was essentially identical in HCN1^f/f and CaMKCre:HCN1^f/f mice under control conditions, and ketamine (20 mg/kg, i.v.) evoked a similar transient increase in latency in both sets of mice. D. The duration that HCN1^f/f and CaMKCre:HCN1^f/f mice were able to stay on an accelerating rotarod was not different before or immediately after treatment with a sub-anesthetic dose of ketamine (2.5 mg/kg, i.v.). n=31 &52, P<0.05, * HCN1^f/f:cre vs. HCN1^f/f.