The Role And Mechanism Of Hyperpolarization-activated Cyclic Nucleotide-gated(HCN) Channels In Methamphetamine Addiction

In recent years, synthetic drugs, represented by methamphetamine, are gradually becoming the most popular drugs in China instead of classic drugs such as cocaine. Compared with classic drugs, synthetic drugs have higher potentials for abuse, and harm central nervous system more seriously. In addition, synthetic drugs addition results in serious problems of national security, social stability, population quality and public health. However, till now, the neurobiological mechanisms underlying synthetic drug addiction remain unclear, and neither effective target nor approved pharmacological therapeutic is available. Therefore, it is urgent to investigate the neurobiological mechanism and effective therapies for synthetic drugs addiction.The reward circuits are consisted of dopaminergic neurons projecting from the ventral tegmental area to the nucleus accumbens, the hippocampus, the prefrontal cortex and the amygdala, which are the most important neurobiological foundation of drug addiction. The dopaminergic projection from the ventral tegmental area to the nucleus accumbens is closely related to drugs’ positive reinforcing effects, which is key to initiate drug addiction. In addition, the dopaminergic neurons projecting from the ventral tegmental area to the hippocampus, the prefrontal cortex and the amygdala form complicated regulatory networks, regulating functions of glutamatergic neurons projecting to the ventral tegmental area and the nucleus accumbens, which are involved in drug-context associative learning. Addictive drugs could act on glutamatergic projections and increase addicts’ sensitivity to clues and context, which is the basis of compulsive drug use, and fundamental factor of relapse. The ventral tegmental area, the nucleus accumbens and the hippocampus play important roles in addiction.The strong rewarding effect of synthetic drug methamphetamine initiates behavioral alterations in methamphetamine addition. Methamphetamine could rapidly increase extracellular dopamine in the reward circuits, which is the material foundation of methamphetamine-induced reinforcing effects, learning of drug-context association, as well as the further addiction. It is the traditional view that methamphetamine mainly interacts with dopamine transporters and vesicular monoamine transporter-2 to inhibit dopamine uptake and facilitate dopamine reverse transport, thus increases extracellular dopamine. However, this hypothesis cannot explain all the experimental phenomena of methamphetamine addiction and neurotoxicity satisfactorily. Is there any other new mechanisms? Could methamphetamine affect neurons’ functions directly? This investigation might provide a new research direction and a new therapeutic strategy for methamphetamine addiction.Hyperpolarization-activated, cyclic nucleotide-gated(HCN) channels are ion channels that mediate the hyperpolarization-activated, non-selective cation current(Ih), and widely distributed in the central nervous system. The physiological functions of HCN channels include:(1) participating in the pacemaker activity of spontaneously activated neurons;(2) setting and controlling neuronal resting membrane potential;(3) regulation of membrane input resistance and the synaptic integration;(4) modulation of neurotransmission and synaptic plasticity. Accordingly, HCN channels are believed to be associated with important physiological functions such as emotion, cognition and neuroendocrine; and also involved in the pathophysiological processes of several central nervous system disorders including seizures, epilepsy, neuropathic pain, Parkinson disease and age-related working memory decline.Does methamphetamine act on HCN channels? Is it possible that methamphetamine acts on HCN channels to modulate neuronal excitability and synaptic transmission? In this paper, we performed the systematic research on the role of HCN channels in methamphetamine addiction and the possible neurobiological mechanisms for the first time. This study reveals a possible novel mechanism underlying methamphetamine-induced functional enhancement of the mesolimbic reward circuits, provides new clues to fully clarify the neurobiological mechanisms of methamphetamine addiction, and points out a novel direction for discovery of new targets, development of new drugs as well as prevention and treatment of methamphetamine addiction.Our findings are mainly as follows:1. The relationship between HCN channels and methamphetamine addiction a) The effect of HCN channels on methamphetamine-induced addictive behaviorUsing HCN channel blocker ZD7288, we determined the role of HCN channels in methamphetamine-induced addictive behavior on multiple animal models.i. Locomotor activity test Using locomotor activity test, the effect of intracerebroventricular(i.c.v.) injection of HCN channel blocker ZD7288 on methamphetamine’s psychostimulatory effects were determined in rats. 1) Acute i.c.v. injection of ZD7288(0.312, 0.625, 1.25, 2.5 μg) didn’t alter the basal locomotor activity in rats(P > 0.05, n = 6). 2) Acute i.c.v. injection of ZD7288(0.625, 1.25 μg) significantly reduced methamphetamine(1 mg/kg, i.p.)-induced hyperactivity in rats(P < 0.01, n = 8). These results showed that blockade of HCN channels reduced methamphetamine’s psychostimulatory effects(reinforcing effects) with no effects on basal locomotor activity, suggested that HCN channels are involved in methamphetamine’s psychostimulatory effects.ii. Self-administration experiments(1) Reinforcing effects Using Self-administration experiments, the effects of HCN channel blocker ZD7288 on methamphetamine’s reinforcing effects were determined in rats.(1) Under fixed ratio reinforcement, 1) Chronic i.c.v. injection of ZD7288(0.625 μg) significantly reduced the acquisition of methamphetamine(0.0125 mg/kg/infusion, i.v.)-induced self-administration(P < 0.01, n = 6-8); acute i.c.v. injection of ZD7288(0.3125, 0.625 μg) significantly reduced methamphetamine(0.0125 mg/kg/infusion, i.v.)-induced self-administration(P < 0.01, n = 8), but had no effects on methamphetamine(0.05 mg/kg/infusion, i.v.)-induced self-administration(P > 0.05, n = 4-6). 2) To determine loci of action in the rat brain, ZD7288 was microinjected into the nucleus accumbens for its vital role in reward system. Acute intra-accumbens microinjections of ZD7288(0.3125, 0.625 μg/side) significantly reduced methamphetamine(0.05 mg/kg/infusion, i.v.)-induced self-administration(P < 0.001, n = 10).(2) Under progressive ratio reinforcement, 1) Acute i.c.v. injection of ZD7288(0.3125, 0.625 μg/side) significantly reduced motivation for methamphetamine(0.05 mg/kg/infusion, i.v.)(P < 0.01, n = 11). 2) Acute intra-accumbens injection of ZD7288(0.3125, 0.625 μg/side) significantly reduced motivation for methamphetamine(0.05 mg/kg/infusion, i.v.)(P < 0.001, n = 9). These results suggested that during acquisition and maintenance, HCN channels are involved in reinforcing effects and motivation for methamphetamine, and that the nucleus accumbens might be the target region for HCN channels to affect the rewarding effects of methamphetamine.(2) Relapse 1) During extinction, chronic i.c.v. injection of ZD7288(0.625, 1.25 μg) didn’t alter methamphetamine(1 mg/kg, i.p.)-induced relapse(P > 0.05, n = 9-12). 2) Before priming, acute intra-accumbens microinjections of ZD7288(0.3125, 0.625 μg/side) had no effect on methamphetamine(1 mg/kg, i.p.)-induced relapse(P > 0.05, n = 8). 3) Before priming, acute intra-accumbens microinjections of ZD7288(0.3125, 0.625 μg/side) had no effect on cue-induced relapse(P > 0.05, n = 7-8). The i.c.v. injection of ZD7288 might be poorly specific to relapse circuits. HCN channels in the nucleus accumbens core may not be involved in the trigger of drug- or cue-induced relapse, but their roles in the adaptation deserved further research. In addition, different HCN channel subtypes may play different roles in relapse. Since ZD7288 is a non-selective blocker of HCN channels, its effects on methamphetamine-induced relapse seem little. Further studies are needed for different HCN channel subtypes in other brain regions, as well as for stress-induced relapse.iii. Conditioned place preference(CPP) experiments Chronic i.c.v. injection of ZD7288(0.625, 1.25 μg) significantly reduced the acquisition of methamphetamine(1 mg/kg, i.p.)-induced CPP, suggested that HCN channels are involved in methamphetamine-induced drug-context associative learning(P < 0.01, n = 10-13). b) The expression of HCN channels in the process of methamphetamine addiction We have found that HCN channels take part in modulating methamphetamine addition, then, how do HCN channels express during methamphetamine addiction? Using real-time RT-PCR, the expression of HCN channel subtypes was determinedduring maintenance, early withdrawal, late withdrawal and relapse of self-administration in m RNA level.Compared with the control rats without methamphetamine treatment, methamphetamine induced alterations of HCN1 and HCN2.1) Nucleus accumbens: HCN1 mRNA didn’t alter during maintenance, early withdrawal and late withdrawal(P > 0.05), but significantly increased in relapse(P < 0.05); HCN2 m RNA increased in maintenance(P < 0.01), returned to the normal level in early withdrawal(P > 0.05), increased again in late withdrawal(P < 0.05), and tended to rise in relapse(P > 0.05).(n = 5-8) 2) Hippocampus: HCN1 m RNA decreased in maintenance(P < 0.05), returned to the normal level in early withdrawal(P > 0.05), and tended to rise in late withdrawal(P > 0.05) and relapse(P > 0.05), but significantly raised in relapse compared to maintenance(P < 0.05); HCN2 m RNA tended to decrease in maintenance(P > 0.05) and early withdrawal(P > 0.05), tended to rise in late withdrawal(P > 0.05), and had no alteration in relapse(P > 0.05), but significantly raised in late withdrawal compared to maintenance(P < 0.05).(n = 4-7) 3) Prefrontal cortex: HCN1 m RNA significantly decreased in maintenance(P < 0.001), early withdrawal(P < 0.001), late withdrawal(P < 0.01) and relapse(P < 0.001); HCN2 m RNA tended to increase in maintenance(P > 0.05) and early withdrawal(P > 0.05), and continued to rise in late withdrawal(P < 0.05), but returned in relapse(P > 0.05).(n = 5-7) 4) Dorsal striatum: HCN1 m RNA significantly decreased in maintenance(P < 0.01), early withdrawal(P < 0.01), late withdrawal(P < 0.01) and relapse(P < 0.01); HCN2 m RNA tended to increase in maintenance(P > 0.05) but returned subsequently(P > 0.05).(n = 5-7)These results suggested that the expression of HCN1 and HCN2 m RNA altered during methamphetamine addiction, HCN channels may play roles in methamphetamine addiction. HCN2 in the nucleus accumbens raised in maintenance, which is consistent with the results in pharmacology of HCN channel blocker. Considering that HCN2 exciting neurons, HCN channels might modulate methamphetamine’s reinforcing effects through HCN2 in the nucleus accumbens. Certainly, further studies in the protein level are needed. In addition, our results showed that the same HCN channel subtype altered in the process of addiction, whichprovides new clues to clarify the whole mechanisms underlying methamphetamine addiction.2. The mechanisms underlying HCN channels modulating methamphetamine addiction We have found that HCN channels are involved in methamphetamine addiction. Then, how is the mechanism? This study tried to clarify the mechanism from neurobiochemistry and neuronal excitability. a) Neurobiochemistry Given that dopamine and glutamate increase in the nucleus accumbens is the material foundation of methamphetamine’s reinforcing effects and drug-context associative learning, is it possible that HCN channels modulate methamphetamine addiction through regulating neurotransmission in the nucleus accumbens? Using in vivo microdialysis, we determined the effect of blockade of HCN channels on neurotransmission in the nucleus accumbens. 1) Acute i.c.v. injection of ZD7288(0.625, 1.25 μg) significantly reduced methamphetamine(1 mg/kg, i.p.)-induced rapid elevation of extracellular dopamine in the nucleus accumbens(P < 0.01), but had no effects on the basal dopamine level in the nucleus accumbens(P > 0.05).(n = 5) 2) Acute i.c.v. injection of ZD7288(0.625, 1.25 μg) significantly reduced methamphetamine(1 mg/kg, i.p.)-induced chronic elevation of extracellular glutamate in the nucleus accumbens(P < 0.05), but had no effects on the basal glutamate level in the nucleus accumbens(P > 0.05).(n = 4-5) These results suggested that methamphetamine increased extracellular dopamine and glutamate in the nucleus accumbens through the activation of HCN channels, which is the material foundation of methamphetamine-induced addictive behavior. b) Neuronal excitability Then, how do HCN channels affect neurotransmission induced by methamphetamine? Using electrophysiological experiments, we investigated the mechanisms underlying HCN channels affecting methamphetamine addiction from neuronal excitability and synaptic plasticity. 1) In CA1 pyramidal neurons in hippocampal slices, methamphetamine(1 μM) induced a membrane depolarization accompanied by an increase in the firing rate(P < 0.05, n = 11). Methamphetamine-induced depolarization didn’t alter in the application of 1 μM TTX(P < 0.05, n = 11), but was abolished in the presence of ZD7288(10 μM) and TTX(1 μM)(P > 0.05, n = 11). These results suggested that methamphetamine enhanced neuron excitability through HCN channels. 2) In CA1 pyramidal neurons in hippocampal slices, the amplitude of HCN channel currents was enhanced by methamphetamine(0.1, 1 μM)(P < 0.001, n = 15), but reduced by methamphetamine(10 μM)(P < 0.001, n = 15). 3) In the presence of dopamine D1/D5 receptors antagonist SCH23390(1, 5, 10 μM), the enhancement of HCN channel currents induced by methamphetamine(1 μM) was partially reversed(P < 0.05, n = 7). 4) Similar to that in hippocampal CA1 pyramidal neurons, methamphetamine(0.1, 1 μM) enhanced the amplitude of HCN1 and HCN2 channel currents expressed in Xenopus oocytes(P < 0.05, n = 9-10), whereas methamphetamine at high concentration(10 μM) reduced the amplitude of HCN1 and HCN2 channel currents expressed in Xenopus oocytes(P < 0.001, n = 9-10). 5) Methamphetamine(1 mg/kg, i.p.) increased long-term potentiation(LTP) induction at Sc-CA1 excitatory synapses in vivo(P < 0.001, n = 12), acute microinjection of ZD7288(0.625 μg) into the hippocampal CA1 area prior to methamphetamine(1 mg/kg, i.p.) reduced LTP induction(P < 0.05, n = 12), suggested that methamphetamine-induced LTP enhancement depended on its facilitating HCN channels.These results suggested that methamphetamine enhanced neuron HCN channel currents, shifting the membrane potential in a more depolarized direction. We found for the first time that methamphetamine-induced enhancement of HCN channel currents was not totally in a dopamine-dependent manner; methamphetamine might also activate HCN channels directly. In addition, methamphetamine influenced hippocampal synaptic plasticity through activating HCN channels, thus induce drug-associated context learning.Taken together, we demonstrated for the first time that HCN channels were involved in methamphetamine addition. First, in multiple animal models of addiction, HCN channel blocker ZD7288 could reduce methamphetamine’s psychostimulatory, reinforcing effects, as well as drug-associated context learning. The nucleus accumbens might be a vital brain region for HCN channels’ involvement in psychostimulatory and reinforcing effects of methamphetamine addiction. Second, the expressions of HCN1 m RNA and HCN2 m RNA differ in distinct brain regions during methamphetamine addiction, while HCN2 m RNA in the nucleus accumbens expresses in accordance with the pharmacological effects of ZD7288 on addictive behavior. Last, in the research of neurobiological mechanisms, using in vivo microdialysis and electrophysiological experiments, we found for the first time that methamphetamine activated HCN channels in a dopamine-dependent manner and probably also in a direct fashion. On one hand, methamphetamine could act on dopamine transports to increase extracellular dopamine levels, thus activates dopamine D1/D5 receptors and elevates intracellular c AMP levels, results in c AMP-sensitive HCN channels. On the other hand, methamphetamine may also directly activate HCN channels. The activation of HCN channels enhances neuron excitability, leads to the enhancement of neurotransmission and LTP, affects neuronal synaptic plasticity, thus participates in methamphetamine’s psychostimulatory effects, reinforcing effects and drug-context associative learning, and finally results in methamphetamine addictive behavior. This study provides new clues for clarifying the neurobiological mechanisms underlying methamphetamine addiction, as well as novel theoretical foundation and experiment evidence for therapeutic strategies.