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Orexin-mediated restoration of hippocampal synaptic potentiation in mice with established cocaine-conditioned place preference

Lu GL, Lee MT, Chiou LC

Addict Biol, 2019 11;24(6):1153-1166.
PMID: 30276922 DOI: 10.1111/adb.12672

Orexins (also called hypocretins) are implicated in reward and addiction, but little is known about their role(s) in the association between hippocampal synaptic plasticity and drug preference. Previously, we found that exogenous orexin via OX1 and OX2 receptors can impair low frequency stimulation-induced depotentiation, i.e. restoring potentiation of excitatory synaptic transmission (re-potentiation) in mouse hippocampal slices. Here, we found this re-potentiation in hippocampal slices from mice that had acquired conditioned place preference (CPP) to cocaine. Both 10 and 20 mg/kg of cocaine induced similar magnitudes of CPP in mice and re-potentiation in their hippocampal slices, but differed in their susceptibility to TCS1102, a dual (OX1 and OX2 ) orexin receptor antagonist. TCS1102 significantly attenuated CPP and hippocampal re-potentiation induced by cocaine at 10 mg/kg but not at 20 mg/kg. Nonetheless, SCH23390, an antagonist of dopamine D1-like receptors (D1-likeRs), inhibited the effects induced by both doses of cocaine. SKF38393, a D1-likeR-selective agonist, also induced hippocampal re-potentiation in vitro. Interestingly, this effect was attenuated by TCS1102. Conversely, SCH23390 prevented orexin A-induced hippocampal re-potentiation. These results suggest that endogenous orexins are released in mice during cocaine-CPP acquisition, which sustains potentiated hippocampal transmission via OX1 /OX2 receptors and may contribute to the addiction memory of cocaine. This effect of endogenous orexins, however, may be substituted by dopamine that may dominate hippocampal re-potentiation and CPP via D1-likeRs when the reinforcing effect of cocaine is high.
Fulltext Alternative pain management via endocannabinoids in the time of the opioid epidemic: Peripheral neuromodulation and pharmacological interventions

Lee MT, Mackie K, Chiou LC

Br J Pharmacol, 2023 Apr;180(7):894-909.
PMID: 34877650 DOI: 10.1111/bph.15771

The use of opioids in pain management is hampered by the emergence of analgesic tolerance, which leads to increased dosing and side effects, both of which have contributed to the opioid epidemic. One promising potential approach to limit opioid analgesic tolerance is activating the endocannabinoid system in the CNS, via activation of CB1 receptors in the descending pain inhibitory pathway. In this review, we first discuss preclinical and clinical evidence revealing the potential of pharmacological activation of CB1 receptors in modulating opioid tolerance, including activation by phytocannabinoids, synthetic CB1 receptor agonists, endocannabinoid degradation enzyme inhibitors, and recently discovered positive allosteric modulators of CB1 receptors. On the other hand, as non-pharmacological pain relief is advocated by the US-NIH to combat the opioid epidemic, we also discuss contributions of peripheral neuromodulation, involving the electrostimulation of peripheral nerves, in addressing chronic pain and opioid tolerance. The involvement of supraspinal endocannabinoid systems in peripheral neuromodulation-induced analgesia is also discussed. LINKED ARTICLES: This article is part of a themed issue on Advances in Opioid Pharmacology at the Time of the Opioid Epidemic. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v180.7/issuetoc.
Median Nerve Stimulation as a Nonpharmacological Approach to Bypass Analgesic Tolerance to Morphine: A Proof-of-Concept Study in Mice

Lee MT, Chen YH, Mackie K, Chiou LC

J Pain, 2021 03;22(3):300-312.
PMID: 33069869 DOI: 10.1016/j.jpain.2020.09.003

Analgesic tolerance to opioids contributes to the opioid crisis by increasing the quantity of opioids prescribed and consumed. Thus, there is a need to develop non-opioid-based pain-relieving regimens as well as strategies to circumvent opioid tolerance. Previously, we revealed a non-opioid analgesic mechanism induced by median nerve electrostimulation at the overlaying PC6 (Neiguan) acupoint (MNS-PC6). Here, we further examined the efficacy of MNS-PC6 in morphine-tolerant mice with neuropathic pain induced by chronic constriction injury (CCI) of the sciatic nerve. Daily treatments of MNS-PC6 (2 Hz, 2 mA), but not electrostimulation at a nonmedian nerve-innervated location, for a week post-CCI induction significantly suppressed established mechanical allodynia in CCI-mice in an orexin-1 (OX1) and cannabinoid-1 (CB1) receptor-dependent fashion. This antiallodynic effect induced by repeated MNS-PC6 was comparable to that induced by repeated gabapentin (50 mg/kg, i.p.) or single morphine (10 mg/kg, i.p.) treatments, but without tolerance, unlike repeated morphine-induced analgesia. Furthermore, single and repeated MNS-PC6 treatments remained fully effective in morphine-tolerant CCI-mice, also in an OX1 and CB1 receptor-dependent fashion. In CCI-mice receiving escalating doses of morphine for 21 days (10, 20 and 50 mg/kg), single and repeated MNS-PC6 treatments remained fully effective. Therefore, repeated MNS-PC6 treatments induce analgesia without tolerance, and retain efficacy in opioid-tolerant mice via a mechanism that involves OX1 and CB1 receptors. This study suggests that MNS-PC6 is an alternative pain management strategy that maybe useful for combatting the opioid epidemic, and opioid-tolerant patients receiving palliative care. PERSPECTIVE: Median nerve stimulation relieves neuropathic pain in mice without tolerance and retains efficacy even in mice with analgesic tolerance to escalating doses of morphine, via an opioid-independent, orexin-endocannabinoid-mediated mechanism. This study provides a proof of concept for utilizing peripheral nerve stimulating devices for pain management in opioid-tolerant patients.
Stress induces reinstatement of extinguished cocaine conditioned place preference by a sequential signaling via neuropeptide S, orexin, and endocannabinoid

Chou YH, Hor CC, Lee MT, Lee HJ, Guerrini R, Calo G, et al.

Addict Biol, 2020 Oct 19.
PMID: 33078457 DOI: 10.1111/adb.12971

Neurons containing neuropeptide S (NPS) and orexins are activated during stress. Previously, we reported that orexins released during stress, via orexin OX1 receptors (OX1 Rs), contribute to the reinstatement of cocaine seeking through endocannabinoid/CB1 receptor (CB1 R)-mediated dopaminergic disinhibition in the ventral tegmental area (VTA). Here, we further demonstrated that NPS released during stress is an up-stream activator of this orexin-endocannabinoid cascade in the VTA, leading to the reinstatement of cocaine seeking. Mice were trained to acquire cocaine conditioned place preference (CPP) by context-pairing cocaine injections followed by the extinction training with context-pairing saline injections. Interestingly, the extinguished cocaine CPP in mice was significantly reinstated by intracerebroventricular injection (i.c.v.) of NPS (1 nmol) in a manner prevented by intraperitoneal injection (i.p.) of SHA68 (50 mg/kg), an NPS receptor antagonist. This NPS-induced cocaine reinstatement was prevented by either i.p. or intra-VTA microinjection (i.vta.) of SB-334867 (15 mg/kg, i.p. or 15 nmol, i.vta.) and AM 251 (1.1 mg/kg, i.p. or 30 nmol, i.vta.), antagonists of OX1 Rs and CB1 Rs, respectively. Besides, NPS (1 nmol, i.c.v.) increased the number of c-Fos-containing orexin neurons in the lateral hypothalamus (LH) and increased orexin-A level in the VTA. The latter effect was blocked by SHA68. Furthermore, a 30-min restraint stress in mice reinstated extinguished cocaine CPP and was prevented by SHA68. These results suggest that NPS is released upon stress and subsequently activates LH orexin neurons to release orexins in the VTA. The released orexins then reinstate extinguished cocaine CPP via an OX1 R- and endocannabinoid-CB1 R-mediated signaling in the VTA.
Fulltext Age-Dependent Anti-migraine Effects of Valproic Acid and Topiramate in Rats

Huang P, Kuo PH, Lee MT, Chiou LC, Fan PC

Front Pharmacol, 2018;9:1095.
PMID: 30319425 DOI: 10.3389/fphar.2018.01095

Background: Valproic acid (VPA) and topiramate (TPM), initially developed as antiepileptics, are approved for migraine prophylaxis in adults but not children. The differences in their antimigraine mechanism(s) by age remain unclear. Methods: A migraine model induced by intra-cisternal (i.c.) capsaicin instillation in pediatric (4-5 weeks) and adult (8-9 weeks) rats was pretreated with VPA (30, 100 mg/kg) or TPM (10, 30, 100 mg/kg). Noxious meningeal stimulation by the irritant capsaicin triggered trigeminovascular system (TGVS) activation mimicking migraine condition, which were assessed peripherally by the depletion of calcitonin gene-related peptide (CGRP) in sensory nerve fibers of the dura mater, the increased CGRP immunoreactivity at trigeminal ganglia (TG) and centrally by the number of c-Fos-immunoreactive (c-Fos-ir) neurons in the trigeminocervical complex (TCC). Peripherally, CGRP released from dural sensory nerve terminals of TG triggered pain signal transmission in the primary afferent of trigeminal nerve, which in turn caused central sensitization of the TGVS due to TCC activation and hence contributed to migraine. Results: In the VPA-treated group, the central responsiveness expressed by reducing the number of c-Fos-ir neurons, which had been increased by i.c. capsaicin, was significant in pediatric, but not adult, rats. Inversely, VPA was effective in peripheral inhibition of elevated CGRP immunoreactivity in the TG and CGRP depletion in the dura mater of adult, but not pediatric, rats. In TPM group, the central responsiveness was significant in both adult and pediatric groups. Peripherally, TPM significantly inhibited capsaicin-induced CGRP expression of TG in adult, but not pediatric, rats. Interestingly, the capsaicin-induced depletion of CGRP in dura was significantly rescued by TPM at high doses in adults, but at low dose in pediatric group. Conclusion: These results suggest VPA exerted peripheral inhibition in adult, but central suppression in pediatric migraine-rats. In contrast, TPM involves both central and peripheral inhibition of migraine with an optimal therapeutic window in both ages. These findings may clarify the age-dependent anti-migraine mechanism of VPA and TPM, which may guide the development of new pediatric anti-migraine drugs in the future.
Fulltext Plasma calcitonin gene-related peptide: A potential biomarker for diagnosis and therapeutic responses in pediatric migraine

Fan PC, Kuo PH, Lee MT, Chang SH, Chiou LC

Front Neurol, 2019;10:10.
PMID: 30733702 DOI: 10.3389/fneur.2019.00010

Background: Plasma calcitonin gene-related peptide (CGRP) plays a key role in the migraine pathophysiology. This study aimed to investigate its role in predicting diagnosis and outcome of pharmacotherapy in pediatric migraine. Methods: We prospectively recruited 120 subjects, who never took migraine-preventive agents in a pediatric clinic, including 68 patients with migraine, 30 with non-migraine headache (NM), and 22 non-headache (NH) age-matched controls. Short-term therapeutic response was measured for at least 2 weeks after the start of therapy. Responders were defined with >50% headache reduction. Plasma CGRP concentrations were measured by ELISA. Results: In the migraine group, more patients required acute therapy, as compared to the NM group (62/68, 91% vs. 5/30, 15%, p = 0.001). The mean plasma CGRP level in migraineurs either during (291 ± 60 pg/ml) or between (240 ± 48) attacks was higher than in NM patients (51 ± 5 pg/ml, p = 0.006 and 0.018, respectively) and NH controls (53 ± 6 pg/ml, p = 0.016 and 0.045, respectively). Forty-seven patients (69%) needed preventive treatments and had higher plasma CGRP levels (364 ± 62 pg/ml, n = 47) than those not (183 ± 54 pg/ml, n = 21) (p = 0.031). Topiramate responders had higher plasma CGRP levels than non-responders (437 ± 131 pg/ml, n = 14 vs. 67 ± 19 pg/ml, n = 6, p = 0.021). Survival curves of plasma CGRP levels also showed those with higher CGRP levels responded better to topiramate. Differences were not found in the other preventives. Conclusion: The plasma CGRP level can differentiate migraine from non-migraine headache. It may also serve as a reference for the therapeutic strategy since it is higher in patients requiring migraine prevention and responsive to short-term topiramate treatment. These results are clinically significant, especially for the young children who cannot clearly describe their headache symptoms and may provide new insights into the clinical practice for the diagnosis and treatment of pediatric migraine.
Study site: Paediatric outpatient clinic,National Taiwan University Hospital (NTUH), Taiwan
Pivotal Role of Slitrk1 in Adult Striatal Cholinergic Neurons in Mice: Implication in Tourette Syndrome

Du JC, Chang MH, Yeh CJ, Lee MT, Lee HJ, Chuang SH, et al.

Ann Neurol, 2023 Sep 30.
PMID: 37776102 DOI: 10.1002/ana.26805

OBJECTIVE: The SLIT and NTRK-like 1 (SLITRK1) gene mutation and striatal cholinergic interneurons (ChIs) loss are associated with Tourette syndrome (TS). ChIs comprise only 1 to 2% of striatal neurons but project widely throughout the stratum to impact various striatal neurotransmission, including TS-related dopaminergic transmission. Here, we link striatal Slitrk1, ChI function, and dopaminergic transmission and their associations with TS-like tic behaviors.
METHODS: Slitrk1-KD mice were induced by bilaterally injecting Slitrk1 siRNA into their dorsal striatum. Control mice received scrambled siRNA injection. Their TS-like tic behaviors, prepulse inhibition, sensory-motor function and dopamine-related behaviors were compared. We also compared dopamine and ACh levels in microdialysates, Slitrk protein and dopamine transporter levels, and numbers of Slitrk-positive ChIs and activated ChIs in the striatum between two mouse groups, and electrophysiological properties between Slitrk-positive and Slitrk-negative striatal ChIs.
RESULTS: Slitrk1-KD mice exhibit TS-like haloperidol-sensitive stereotypic tic behaviors, impaired prepulse inhibition, and delayed sensorimotor response compared with the control group. These TS-like characteristics correlate with lower striatal Slitrk1 protein levels, fewer Slitrk1-containing ChIs, and fewer activated ChIs in Slitrk1-KD mice. Based on their electrophysiological properties, Slitrk1-negative ChIs are less excitable than Slitrk1-positive ChIs. Slitrk1-KD mice have lower evoked acetylcholine and dopamine levels, higher tonic dopamine levels, and downregulated dopamine transporters in the striatum, increased apomorphine-induced climbing behaviors, and impaired methamphetamine-induced hyperlocomotion compared with controls.
INTERPRETATION: Slitrk1 is pivotal in maintaining striatal ChIs activity and subsequent dopaminergic transmission for normal motor functioning. Furthermore, conditional striatal Slitrk1-KD mice may serve as a translational modality with aspects of TS phenomenology. ANN NEUROL 2023.
α6-Containing GABAA Receptors: Functional Roles and Therapeutic Potentials

Sieghart W, Chiou LC, Ernst M, Fabjan J, M Savić M, Lee MT

Pharmacol Rev, 2022 Jan;74(1):238-270.
PMID: 35017178 DOI: 10.1124/pharmrev.121.000293

GABAA receptors containing the α6 subunit are highly expressed in cerebellar granule cells and less abundantly in many other neuronal and peripheral tissues. Here, we for the first time summarize their importance for the functions of the cerebellum and the nervous system. The cerebellum is not only involved in motor control but also in cognitive, emotional, and social behaviors. α6βγ2 GABAA receptors located at cerebellar Golgi cell/granule cell synapses enhance the precision of inputs required for cerebellar timing of motor activity and are thus involved in cognitive processing and adequate responses to our environment. Extrasynaptic α6βδ GABAA receptors regulate the amount of information entering the cerebellum by their tonic inhibition of granule cells, and their optimal functioning enhances input filtering or contrast. The complex roles of the cerebellum in multiple brain functions can be compromised by genetic or neurodevelopmental causes that lead to a hypofunction of cerebellar α6-containing GABAA receptors. Animal models mimicking neuropsychiatric phenotypes suggest that compounds selectively activating or positively modulating cerebellar α6-containing GABAA receptors can alleviate essential tremor and motor disturbances in Angelman and Down syndrome as well as impaired prepulse inhibition in neuropsychiatric disorders and reduce migraine and trigeminal-related pain via α6-containing GABAA receptors in trigeminal ganglia. Genetic studies in humans suggest an association of the human GABAA receptor α6 subunit gene with stress-associated disorders. Animal studies support this conclusion. Neuroimaging and post-mortem studies in humans further support an involvement of α6-containing GABAA receptors in various neuropsychiatric disorders, pointing to a broad therapeutic potential of drugs modulating α6-containing GABAA receptors. SIGNIFICANCE STATEMENT: α6-Containing GABAA receptors are abundantly expressed in cerebellar granule cells, but their pathophysiological roles are widely unknown, and they are thus out of the mainstream of GABAA receptor research. Anatomical and electrophysiological evidence indicates that these receptors have a crucial function in neuronal circuits of the cerebellum and the nervous system, and experimental, genetic, post-mortem, and pharmacological studies indicate that selective modulation of these receptors offers therapeutic prospects for a variety of neuropsychiatric disorders and for stress and its consequences.
The deuterated pyrazoloquinolinone targeting α6 subunit-containing GABAA receptor as novel candidate for inhibition of trigeminovascular system activation: implication for migraine therapy

Fan PC, Chiou LC, Lai TH, Sharmin D, Cook J, Lee MT

Front Pharmacol, 2024;15:1451634.
PMID: 39253381 DOI: 10.3389/fphar.2024.1451634

INTRODUCTION: The α6 subunit-containing GABAA receptors (α6GABAARs) are highly expressed in the trigeminal ganglia (TG), the sensory hub of the trigeminovascular system (TGVS). Hypo-GABAergic transmission in the TG was reported to contribute to migraine-related behavioral and histopathological phenotypes. Previously, we found that Compound 6, an α6GABAAR-selective positive allosteric modulator (PAM), significantly alleviated TGVS activation-induced peripheral and central sensitization in a capsaicin-induced migraine-mimicking model.
METHODS: Here, we tested whether the deuterated analogues of Compound 6, namely DK-1-56-1 and RV-I-29, known to have longer half-lives than the parent compound, can exert a similar therapeutic effect in the same model. The activation of TGVS was triggered by intra-cisternal (i.c.) instillation of capsaicin in male Wistar rats. Centrally, i.c. capsaicin increased the quantity of c-Fos-immunoreactive (c-Fos-ir) neurons in the trigeminal cervical complex (TCC). Peripherally, it increased the calcitonin gene-related peptide immunoreactivity (CGRP-ir) in TG, and caused CGRP release, leading to CGRP depletion in the dura mater.
RESULTS: DK-I-56-1 and RV-I-29, administered intraperitoneally (i.p.), significantly ameliorated the TCC neuronal activation, TG CGRP-ir elevation, and dural CGRP depletion induced by capsaicin, with DK-I-56-1 demonstrating better efficacy. The therapeutic effects of 3 mg/kg DK-I-56-1 are comparable to that of 30 mg/kg topiramate. Notably, i.p. administered furosemide, a blood-brain-barrier impermeable α6GABAAR-selective antagonist, prevented the effects of DK-I-56-1 and RV-I-29. Lastly, orally administered DK-I-56-1 has a similar pharmacological effect.
DISCUSSION: These results suggest that DK-I-56-1 is a promising candidate for novel migraine pharmacotherapy, through positively modulating TG α6GABAARs to inhibit TGVS activation, with relatively favourable pharmacokinetic properties.
Fulltext The α6 subunit-containing GABAA receptor: A novel drug target for inhibition of trigeminal activation

Fan PC, Lai TH, Hor CC, Lee MT, Huang P, Sieghart W, et al.

Neuropharmacology, 2018 09 15;140:1-13.
PMID: 30016665 DOI: 10.1016/j.neuropharm.2018.07.017

Novel treatments against migraine are an urgent medical requirement. The α6 subunit-containing GABAA receptors (α6GABAARs) are expressed in trigeminal ganglia (TG), the hub of the trigeminal vascular system (TGVS) that is involved in the pathogenesis of migraine. Here we reveal an unprecedented role of α6GABAARs in ameliorating TGVS activation using several pharmacological approaches in an animal model mimicking pathological changes in migraine. TGVS activation was induced by intra-cisternal (i.c.) instillation of capsaicin in Wistar rats. Centrally, i.c. capsaicin activated the trigeminal cervical complex (TCC) measured by the increased number of c-Fos-immunoreactive (c-Fos-ir) TCC neurons. Peripherally, it elevated calcitonin gene-related peptide immunoreactivity (CGRP-ir) in TG and depleted CGRP-ir in the dura mater. Pharmacological approaches included a recently identified α6GABAAR-selective positive allosteric modulator (PAM), the pyrazoloquinolinone Compound 6, two α6GABAAR-active PAMs (Ro15-4513 and loreclezole), an α6GABAAR-inactive benzodiazepine (diazepam), an α6GABAAR-selective antagonist (furosemide), and a clinically effective antimigraine agent (topiramate). We examined effects of these compounds on both central and peripheral TGVS responses induced by i.c. capsaicin. Compound 6 (3-10 mg/kg, i.p.) significantly attenuated the TCC neuronal activation and TG CGRP-ir elevation, and dural CGRP depletion induced by capsaicin. All these effects of Compound 6 were mimicked by topiramate, Ro15-4513 and loreclezole, but not by diazepam. The brain-impermeable furosemide antagonized the peripheral, but not central, effects of Compound 6. These results suggest that the α6GABAAR in TG is a novel drug target for TGVS activation and that α6GABAAR-selective PAMs have the potential to be developed as a novel pharmacotherapy for migraine.
Fulltext Neuropeptide S-initiated sequential cascade mediated by OX1, NK1, mGlu5 and CB1 receptors: a pivotal role in stress-induced analgesia

Lee MT, Chiu YT, Chiu YC, Hor CC, Lee HJ, Guerrini R, et al.

J Biomed Sci, 2020 Jan 09;27(1):7.
PMID: 31915019 DOI: 10.1186/s12929-019-0590-1

BACKGROUND: Stress-induced analgesia (SIA) is an evolutionarily conserved phenomenon during stress. Neuropeptide S (NPS), orexins, substance P, glutamate and endocannabinoids are known to be involved in stress and/or SIA, however their causal links remain unclear. Here, we reveal an unprecedented sequential cascade involving these mediators in the lateral hypothalamus (LH) and ventrolateral periaqueductal gray (vlPAG) using a restraint stress-induced SIA model.
METHODS: Male C57BL/6 mice of 8-12 week-old were subjected to intra-cerebroventricular (i.c.v.) and/or intra-vlPAG (i.pag.) microinjection of NPS, orexin-A or substance P alone or in combination with selective antagonists of NPS receptors (NPSRs), OX1 receptors (OX1Rs), NK1 receptors (NK1Rs), mGlu5 receptors (mGlu5Rs) and CB1 receptors (CB1Rs), respectively. Antinociceptive effects of these mediators were evaluated via the hot-plate test. SIA in mice was induced by a 30-min restraint stress. NPS levels in the LH and substance P levels in vlPAG homogenates were compared in restrained and unrestrained mice.
RESULTS: NPS (i.c.v., but not i.pag.) induced antinociception. This effect was prevented by i.c.v. blockade of NPSRs. Substance P (i.pag.) and orexin-A (i.pag.) also induced antinociception. Substance P (i.pag.)-induced antinociception was prevented by i.pag. Blockade of NK1Rs, mGlu5Rs or CB1Rs. Orexin-A (i.pag.)-induced antinociception has been shown previously to be prevented by i.pag. blockade of OX1Rs or CB1Rs, and here was prevented by NK1R or mGlu5R antagonist (i.pag.). NPS (i.c.v.)-induced antinociception was prevented by i.pag. blockade of OX1Rs, NK1Rs, mGlu5Rs or CB1Rs. SIA has been previously shown to be prevented by i.pag. blockade of OX1Rs or CB1Rs. Here, we found that SIA was also prevented by i.c.v. blockade of NPSRs or i.pag. blockade of NK1Rs or mGlu5Rs. Restrained mice had higher levels of NPS in the LH and substance P in the vlPAG than unrestrained mice.
CONCLUSIONS: These results suggest that, during stress, NPS is released and activates LH orexin neurons via NPSRs, releasing orexins in the vlPAG. Orexins then activate OX1Rs on substance P-containing neurons in the vlPAG to release substance P that subsequently. Activates NK1Rs on glutamatergic neurons to release glutamate. Glutamate then activates perisynaptic mGlu5Rs to initiate the endocannabinoid retrograde inhibition of GABAergic transmission in the vlPAG, leading to analgesia.
Fulltext Cerebellar α6GABAA Receptors as a Therapeutic Target for Essential Tremor: Proof-of-Concept Study with Ethanol and Pyrazoloquinolinones

Huang YH, Lee MT, Hsueh HY, Knutson DE, Cook J, Mihovilovic MD, et al.

Neurotherapeutics, 2023 Mar;20(2):399-418.
PMID: 36696034 DOI: 10.1007/s13311-023-01342-y

Ethanol has been shown to suppress essential tremor (ET) in patients at low-to-moderate doses, but its mechanism(s) of action remain unknown. One of the ET hypotheses attributes the ET tremorgenesis to the over-activated firing of inferior olivary neurons, causing synchronic rhythmic firings of cerebellar Purkinje cells. Purkinje cells, however, also receive excitatory inputs from granule cells where the α6 subunit-containing GABAA receptors (α6GABAARs) are abundantly expressed. Since ethanol is a positive allosteric modulator (PAM) of α6GABAARs, such action may mediate its anti-tremor effect. Employing the harmaline-induced ET model in male ICR mice, we evaluated the possible anti-tremor effects of ethanol and α6GABAAR-selective pyrazoloquinolinone PAMs. The burrowing activity, an indicator of well-being in rodents, was measured concurrently. Ethanol significantly and dose-dependently attenuated action tremor at non-sedative doses (0.4-2.4 g/kg, i.p.). Propranolol and α6GABAAR-selective pyrazoloquinolinones also significantly suppressed tremor activity. Neither ethanol nor propranolol, but only pyrazoloquinolinones, restored burrowing activity in harmaline-treated mice. Importantly, intra-cerebellar micro-injection of furosemide (an α6GABAAR antagonist) had a trend of blocking the effect of pyrazoloquinolinone Compound 6 or ethanol on harmaline-induced tremor. In addition, the anti-tremor effects of Compound 6 and ethanol were synergistic. These results suggest that low doses of ethanol and α6GABAAR-selective PAMs can attenuate action tremor, at least partially by modulating cerebellar α6GABAARs. Thus, α6GABAARs are potential therapeutic targets for ET, and α6GABAAR-selective PAMs may be a potential mono- or add-on therapy.
Fulltext α6GABAA Receptor Positive Modulators Alleviate Migraine-like Grimaces in Mice via Compensating GABAergic Deficits in Trigeminal Ganglia

Tzeng HR, Lee MT, Fan PC, Knutson DE, Lai TH, Sieghart W, et al.

Neurotherapeutics, 2021 Jan;18(1):569-585.
PMID: 33111258 DOI: 10.1007/s13311-020-00951-1

Migraine is caused by hyperactivity of the trigeminovascular system, where trigeminal ganglia (TG) play an important role. This hyperactivity might originate from an underfunctional GABAergic system in TG. To investigate this possibility, we adapted a mouse model of migraine by inducing migraine-like grimaces in male mice via repeated injections of nitroglycerin (NTG, 10 mg/kg, i.p.), once every 2 days, for up to 5 sessions. Migraine-like facial pain scores were measured using the mouse grimace scale. Repeated NTG treatments in mice caused significant increases in migraine-like grimaces that were aborted and prevented by two anti-migraine agents sumatriptan and topiramate, respectively. After 5 sessions of NTG injections, the GABA-synthesizing enzyme, 65-kDa glutamate decarboxylase (GAD65), but not the GABA transporter 1 (GAT1) or the α6 subunit-containing GABAA receptors (α6GABAARs), was downregulated in mouse TG tissues. Taking advantage of the unaffected TG α6GABAAR expression in NTG-treated mice, we demonstrated that an α6GABAAR-selective positive allosteric modulator (PAM), DK-I-56-1, exhibited both abortive and prophylactic effects, comparable to those of sumatriptan and topiramate, respectively, in this migraine-mimicking mouse model. The brain-impermeable furosemide significantly prevented the effects of DK-I-56-1, suggesting its peripheral site of action, likely via preventing α6GABAAR modulation in TG. Results suggest that a decreased GABA synthesis caused by the reduced GAD65 expression in TG contributes to the trigeminovascular activation in this repeated NTG-induced migraine-mimicking model and that the unaltered α6GABAARs in TG are potential targets for migraine treatment. Thus, α6GABAAR-selective PAMs are potential anti-migraine agents for both abortive and preventive therapies.
Fulltext Median nerve stimulation induces analgesia via orexin-initiated endocannabinoid disinhibition in the periaqueductal gray

Chen YH, Lee HJ, Lee MT, Wu YT, Lee YH, Hwang LL, et al.

Proc Natl Acad Sci U S A, 2018 11 06;115(45):E10720-E10729.
PMID: 30348772 DOI: 10.1073/pnas.1807991115

Adequate pain management remains an unmet medical need. We previously revealed an opioid-independent analgesic mechanism mediated by orexin 1 receptor (OX1R)-initiated 2-arachidonoylglycerol (2-AG) signaling in the ventrolateral periaqueductal gray (vlPAG). Here, we found that low-frequency median nerve stimulation (MNS) through acupuncture needles at the PC6 (Neiguan) acupoint (MNS-PC6) induced an antinociceptive effect that engaged this mechanism. In mice, MNS-PC6 reduced acute thermal nociceptive responses and neuropathy-induced mechanical allodynia, increased the number of c-Fos-immunoreactive hypothalamic orexin neurons, and led to higher orexin A and lower GABA levels in the vlPAG. Such responses were not seen in mice with PC6 needle insertion only or electrical stimulation of the lateral deltoid, a nonmedian nerve-innervated location. Directly stimulating the surgically exposed median nerve also increased vlPAG orexin A levels. MNS-PC6-induced antinociception (MNS-PC6-IA) was prevented by proximal block of the median nerve with lidocaine as well as by systemic or intravlPAG injection of an antagonist of OX1Rs or cannabinoid 1 receptors (CB1Rs) but not by opioid receptor antagonists. Systemic blockade of OX1Rs or CB1Rs also restored vlPAG GABA levels after MNS-PC6. A cannabinoid (2-AG)-dependent mechanism was also implicated by the observations that MNS-PC6-IA was prevented by intravlPAG inhibition of 2-AG synthesis and was attenuated in Cnr1-/- mice. These findings suggest that PC6-targeting low-frequency MNS activates hypothalamic orexin neurons, releasing orexins to induce analgesia through a CB1R-dependent cascade mediated by OX1R-initiated 2-AG retrograde disinhibition in the vlPAG. The opioid-independent characteristic of MNS-PC6-induced analgesia may provide a strategy for pain management in opioid-tolerant patients.

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