Ze-Jun Wang

Cannabinoid Receptor-Mediated Regulation of Neuronal Activity and Signaling in Glomeruli of the Main Olfactory Bulb

Cannabinoid receptors (CB1Rs) are present in glomeruli of the main olfactory bulb. The functions of CB1Rs and their endogenous activators, endocannabinoids, for glomerular signaling are unknown. Glomeruli contain at least three types of neurons: periglomerular (PG), external tufted (ET), and short-axon (SA) cells. PG cells form inhibitory GABAergic dendrodendritic synapses with ET cells. ET cells form excitatory glutamatergic dendrodendritic synapses with PG and SA cells. In mouse brain slices, we used whole-cell patch-clamp recordings to study the role of CB1Rs in regulating PG and ET cells. Cannabinoids displayed strong, direct inhibitory effects on PG cells and weak effects on ET cells. Single pulses or a train of pulses of depolarizing current injected into an ET cell evoked suppression of IPSCs. This suggests retrograde endocannabinoid signaling, namely, depolarization-induced suppression of inhibition (DSI) in ET cells. Our results support the hypothesis that burst firing of ET cells triggers the release of endocannabinoids which in turn directly inhibit PG cells and reduce GABA release from PG cells. This, in turn, can result in a transient reduction of PG cell inhibitory input to ET cells.

Ginseng derivative ocotillol enhances neuronal activity through increased glutamate release: a possible mechanism underlying increased spontaneous locomotor activity of mice

Ginsenosides are the main active ingredients in ginseng and have recently been reported to have beneficial effects on the CNS. Ocotillol is a derivate of pseudoginsenoside-F11, which is an ocotillol-type ginsenoside found in American ginseng. We examined the effects of ocotillol (a) on neuronal activity of projection neurons, mitral cells (MC), in a mouse olfactory bulb brain slice preparation using whole-cell patch-clamp recording, and (b) on animal behavior by measuring locomotor activity of mice in vivo. Ocotillol displayed an excitatory effect on spontaneous action potential firing and depolarized the membrane potential of MCs. The effect was concentration-dependent, with an EC(50) of 4 μM. In the presence of blockers of ionotropic glutamatergic and GABAergic synaptic transmission (6-cyano-7-nitroquinoxaline-2,3-dione [CNQX], 10 μM; D-AP5, 50 μM; gabazine, 5 μM), the excitatory effect of ocotillol on firing was abolished. Further experiments showed that the ocotillol-induced neuronal excitation persisted in the presence of GABA(A) receptor antagonist gabazine but was eliminated by applying AMPA/kainate receptor antagonist CNQX and N-methyl-d-aspartate (NMDA) receptor antagonist D-AP5, suggesting that ionotropic glutamate transmission was involved in mediating the effects of ocotillol. Bath application of ocotillol evoked an inward current as well as an increased frequency of spontaneous glutamatergic excitatory postsynaptic currents (EPSCs). Both the inward current and sEPSCs could be blocked by ionotropic glutamate receptor antagonists CNQX and D-AP5. These results indicate that the excitatory action of ocotillol on MCs was mediated by enhanced glutamate release. Behavioral experiments demonstrated that ocotillol increased locomotor activities of mice. Our results suggest that ocotillol-evoked neuronal excitability was mediated by increased release of glutamate, which may be responsible for the increased spontaneous locomotor activities in vivo.

Inhibition of Nav1.7 channels by methyl eugenol as a mechanism underlying its antinociceptive and anesthetic actions.

Aim:Methyl eugenol is a major active component extracted from the Chinese herb Asari Radix et Rhizoma, which has been used to treat toothache and other pain. Previous in vivo studies have shown that methyl eugenol has anesthetic and antinociceptive effects. The aim of this study was to determine the possible mechanism underlying its effect on nervous system disorders.Methods:The direct interaction of methyl eugenol with Na+ channels was explored and characterized using electrophysiological recordings from Nav1.7-transfected CHO cells.Results:In whole-cell patch clamp mode, methyl eugenol tonically inhibited peripheral nerve Nav1.7 currents in a concentration- and voltage-dependent manner, with an IC50 of 295 μmol/L at a −100 mV holding potential. Functionally, methyl eugenol preferentially bound to Nav1.7 channels in the inactivated and/or open state, with weaker binding to channels in the resting state. Thus, in the presence of methyl eugenol, Nav1.7 channels exhibited reduced availability for activation in a steady-state inactivation protocol, strong use-dependent inhibition, enhanced binding kinetics, and slow recovery from inactivation compared to untreated channels. An estimation of the affinity of methyl eugenol for the resting and inactivated states of the channel also demonstrated that methyl eugenol preferentially binds to inactivated channels, with a 6.4 times greater affinity compared to channels in the resting state. The failure of inactivated channels to completely recover to control levels at higher concentrations of methyl eugenol implies that the drug may drive more drug-bound, fast-inactivated channels into drug-bound, slow-inactivated channels.Conclusion:Methyl eugenol is a potential candidate as an effective local anesthetic and analgesic. The antinociceptive and anesthetic effects of methyl eugenol result from the inhibitory action of methyl eugenol on peripheral Na+ channels.

A Substituted Anilino Enaminone Acts as a Novel Positive Allosteric Modulator of GABA A Receptors in the Mouse Brain

A small library of anilino enaminones was analyzed for potential anticonvulsant agents. We examined the effects of three anilino enaminones on neuronal activity of output neurons, mitral cells (MC), in an olfactory bulb brain slice preparation using whole-cell patch-clamp recording. These compounds are known to be effective in attenuating pentylenetetrazol-induced convulsions. Among the three compounds tested, 5-methyl-3-(4-trifluoromethoxy-phenylamino)-cyclohex-2-enone (KRS-5Me-4-OCF3) showed potent inhibition of MC activity with an EC50 of 24.5 μM. It hyperpolarized the membrane potential of MCs accompanied by suppression of spontaneous firing. Neither ionotropic glutamate receptor blockers nor a GABAB receptor blocker prevented the KRS-5Me-4-OCF3-evoked inhibitory effects. In the presence of GABAA receptor antagonists, KRS-5Me-4-OCF3 completely failed to evoke inhibition of MC spiking activity, suggesting that KRS-5Me-4-OCF3-induced inhibition may be mediated by direct action on GABAA receptors or indirect action through the elevation of tissue GABA levels. Neither vigabatrin (a selective GABA-T inhibitor) nor 1,2,5,6-tetrahydro-1-[2-[[(diphenylmethylene)amino]oxy]ethyl]-3-pyridinecarboxylic acid hydrochloride (NNC-711) (a selective inhibitor of GABA uptake by GABA transporter 1) eliminated the effect of KRS-5ME-4-OCF3 on neuronal excitability, indicating that the inhibitory effect of the enaminone resulted from direct activation of GABAA receptors. The concentration-response curves for GABA are left-shifted by KRS-5Me-4-OCF3, demonstrating that KRS-5Me-4-OCF3 enhanced GABA affinity and acted as a positive allosteric modulator of GABAA receptors. The effect of KRS-5Me-4-OCF3 was blocked by applying a benzodiazepine site antagonist, suggesting that KRS-5Me-4-OCF3 binds at the classic benzodiazepine site to exert its pharmacological action. The results suggest clinical use of enaminones as anticonvulsants in seizures and as a potential anxiolytic in mental disorders.

Identification of both GABAA receptors and voltage-activated Na(+) channels as molecular targets of anticonvulsant α-asarone.

Alpha (α)-asarone, a major effective component isolated from the Chinese medicinal herb Acorus tatarinowii, is clinically used as medication for treating epilepsy, cough, bronchitis, and asthma. In the present study, we demonstrated that α-asarone targets central nervous system GABAA receptor as well as voltage-gated Na+ channels. Using whole-cell patch-clamp recording, α-asarone inhibited spontaneous firing of output neurons, mitral cells (MCs), in mouse olfactory bulb brain slice preparation and hyperpolarized the membrane potential of MCs. The inhibitory effect of α-asarone persisted in the presence of ionotropic glutamate receptor blockers but was eliminated after adding a GABAA receptor blocker, suggesting that GABAA receptors mediated the inhibition of MCs by α-asarone. This hypothesis was supported by the finding that α-asarone evoked an outward current, but did not influence inhibitory postsynaptic currents (IPSCs). In addition to inhibiting spontaneous firing, α-asarone also inhibited the Nav1.2 channel, a dominant rat brain Na+ channel subtype. The effects of α-asarone on a defined Nav1.2 were characterized using transfected cells that stably expressed the Nav1.2 channel isoform. α-Asarone displayed strong tonic inhibition of Nav1.2 currents in a concentration- and membrane potential-dependent fashion. α-Asarone reduced channel availability in steady-state inactivation protocols by enhancing or stabilizing Na+ channel inactivation. Both Na+ channel blockade and activation of GABAA receptors provide a possible mechanism for the known anti-epileptic effects of α-asarone. It also suggests that α-asarone could benefit patients with cough possibly through inhibiting a Na+ channel subtype to inhibit peripheral and/or central sensitization of cough reflexes.

Resibufogenin and Cinobufagin Activate Central Neurons through an Ouabain-Like Action

Cinobufagin and resibufogenin are two major effective bufadienolides of Chan su (toad venom), which is a Chinese medicine obtained from the skin venom gland of toads and is used as a cardiotonic and central nervous system (CNS) respiratory agent, an analgesic and anesthetic, and as a remedy for ulcers. Many clinical cases showed that Chan su has severe side-effects on the CNS, causing shortness of breath, breathlessness, seizure, coma and cardiac arrhythmia. We used whole-cell recordings from brain slices to determine the effects of bufadienolides on excitability of a principal neuron in main olfactory bulb (MOB), mitral cells (MCs), and the cellular mechanism underlying the excitation. At higher concentrations, cinobufagin and resibufogenin induced irreversible over-excitation of MCs indicating a toxic effect. At lower concentrations, they concentration-dependently increased spontaneous firing rate, depolarized the membrane potential of MCs, and elicited inward currents. The excitatory effects were due to a direct action on MCs rather than an indirect phasic action. Bufadienolides and ouabain had similar effects on firing of MCs which suggested that bufadienolides activated neuron through a ouabain-like effect, most likely by inhibiting Na+/K+-ATPase. The direct action of bufadienolide on brain Na+ channels was tested by recordings from stably Nav1.2-transfected cells. Bufadienolides failed to make significant changes of the main properties of Nav1.2 channels in current amplitude, current-voltage (I-V) relationships, activation and inactivation. Our results suggest that inhibition of Na+/K+-ATPase may be involved in both the pharmacological and toxic effects of bufadienolide-evoked CNS excitation.

Cellular Mechanisms of Action of Drug Abuse on Olfactory Neurons.

Cannabinoids (Δ9-tetrahydrocannabinol) are the active ingredient of marijuana (cannabis) which is the most commonly abused illicit drug in the USA. In addition to being known and used as recreational drugs, cannabinoids are produced endogenously by neurons in the brain (endocannabinoids) and serve as important signaling molecules in the nervous system and the rest of the body. Cannabinoids have been implicated in bodily processes both in health and disease. Recent pharmacological and physiological experiments have described novel aspects of classic brain signaling mechanisms or revealed unknown mechanisms of cellular communication involving the endocannabinoid system. While several forms of signaling have been described for endocannabinoids, the most distinguishing feature of endocannabinoids is their ability to act as retrograde messengers in neural circuits. Neurons in the main olfactory bulb express high levels of cannabinoid receptors. Here, we describe the cellular mechanisms and function of this novel brain signaling system in regulating neural activity at synapses in olfactory circuits. Results from basic research have the potential to provide the groundwork for translating the neurobiology of drug abuse to the realm of the pharmacotherapeutic treatment of addiction, specifically marijuana substance use disorder.

Allosteric Modulation of GABAA Receptors by an Anilino Enaminone in an Olfactory Center of the Mouse Brain.

In an ongoing effort to identify novel drugs that can be used as neurotherapeutic compounds, we have focused on anilino enaminones as potential anticonvulsant agents. Enaminones are organic compounds containing a conjugated system of an amine, an alkene and a ketone. Here, we review the effects of a small library of anilino enaminones on neuronal activity. Our experimental approach employs an olfactory bulb brain slice preparation using whole-cell patch-clamp recording from mitral cells in the main olfactory bulb. The main olfactory bulb is a key integrative center in the olfactory pathway. Mitral cells are the principal output neurons of the main olfactory bulb, receiving olfactory receptor neuron input at their dendrites within glomeruli, and projecting glutamatergic axons through the lateral olfactory tract to the olfactory cortex. The compounds tested are known to be effective in attenuating pentylenetetrazol (PTZ) induced convulsions in rodent models. One compound in particular, KRS-5Me-4-OCF3, evokes potent inhibition of mitral cell activity. Experiments aimed at understanding the cellular mechanism underlying the inhibitory effect revealed that KRS-5Me-4-OCF3 shifts the concentration-response curve for GABA to the left. KRS-5Me-4-OCF3 enhances GABA affinity and acts as a positive allosteric modulator of GABAA receptors. Application of a benzodiazepine site antagonist blocks the effect of KRS-5Me-4-OCF3 indicating that KRS-5Me-4-OCF3 binds at the classical benzodiazepine site to exert its pharmacological action. This anilino enaminone KRS-5Me-4-OCF3 emerges as a candidate for clinical use as an anticonvulsant agent in the battle against epileptic seizures.

Essential Oils and Their Constituents Targeting the GABAergic System and Sodium Channels as Treatment of Neurological Diseases

Essential oils and the constituents in them exhibit different pharmacological activities, such as antinociceptive, anxiolytic-like, and anticonvulsant effects. They are widely applied as a complementary therapy for people with anxiety, insomnia, convulsion, pain, and cognitive deficit symptoms through inhalation, oral administration, and aromatherapy. Recent studies show that essential oils are emerging as a promising source for modulation of the GABAergic system and sodium ion channels. This review summarizes the recent findings regarding the pharmacological properties of essential oils and compounds from the oils and the mechanisms underlying their effects. Specifically, the review focuses on the essential oils and their constituents targeting the GABAergic system and sodium channels, and their antinociceptive, anxiolytic, and anticonvulsant properties. Some constituents target transient receptor potential (TRP) channels to exert analgesic effects. Some components could interact with multiple therapeutic target proteins, for example, inhibit the function of sodium channels and, at the same time, activate GABAA receptors. The review concentrates on perspective compounds that could be better candidates for new drug development in the control of pain and anxiety syndromes.

6 Hz Active Anticonvulsant Fluorinated N-Benzamide Enaminones and Their Inhibitory Neuronal Activity

A small library of novel fluorinated N-benzamide enaminones were synthesized and evaluated in a battery of acute preclinical seizure models. Three compounds (GSA 62, TTA 35, and WWB 67) were found to have good anticonvulsant activity in the 6-Hz ‘psychomotor’ 44-mA rodent model. The focus of this study was to elucidate the active analogs’ mode of action on seizure-related molecular targets. Electrophysiology studies were employed to evaluate the compounds’ ability to inhibit neuronal activity in central olfactory neurons, mitral cells, and sensory-like ND7/23 cells, which express an assortment of voltage and ligand-gated ion channels. We did not find any significant effects of the three compounds on action potential generation in mitral cells. The treatment of ND7/23 cells with 50 µM of GSA 62, TTA 35, and WWB 67 generated a significant reduction in the amplitude of whole-cell sodium currents. Similar treatment of ND7/23 cells with these compounds had no effect on T-type calcium currents, indicating that fluorinated N-benzamide enaminone analogs may have a selective effect on voltage-gated sodium channels, but not calcium channels.