Receptor Antagonist Picrotoxin Research Articles

Sleep-Enhancing Effect of Water Extract from Jujube (Zizyphus jujuba Mill.) Seeds Fermented by Lactobacillus brevis L32.

Although Ziziphus jujuba Mill (jujube) is used in folk medicine for hypnotic sedative, anxiolytic, and many other purposes, to date, only a few studies have revealed its sleep-promoting effects and related mechanisms. Currently, drugs used for the treatment of sleep disorders have various side effects, so it is essential to develop safe natural materials. Therefore, we evaluated the sleep-enhancing activity and mechanism of action of an aqueous extract of jujube seeds (ZW) fermented with Lactobacillus brevis L-32 in rodent models. The starch contained in ZW was removed by enzymatic degradation and fermented with L. brevis to obtain a fermented product (ZW-FM) with a high γ-aminobutyric acid (GABA) content. To evaluate the sleep-promoting effect of ZW-FM, pentobarbital-induced sleep tests were performed on ICR mice, and electroencephalography analysis was undertaken in Sprague Dawley rats. Additionally, the awakening relief effects of ZW-FM were confirmed in a caffeine-induced insomnia model. Finally, the mechanism of sleep enhancement by ZW-FM was analyzed using GABA receptor type A (GABAA) antagonists. The ZW-FM-treated groups (100 and 150 mg/kg) showed increased sleep time, especially the δ-wave time during non-rapid eye movement (NREM) sleep. In addition, the 150 mg/kg ZW-FM treatment group showed decreased sleep latency and increased sleep time in the insomnia model. In particular, NREM sleep time was increased and REM sleep time, which was increased by caffeine treatment, was decreased by ZW-FM treatment. ZW-FM-induced sleep increase was inhibited by the GABAA receptor antagonists picrotoxin, bicuculline, and flumazenil, confirming that the increase was the result of a GABAergic mechanism. These results strongly suggest that the increased GABA in water extract from jujube seeds fermented by L. brevis acts as a sleep-promoting compound and that the sleep-promoting activity is related to GABAA receptor binding.

Ethanol depresses neurons in the lateral parabrachial nucleus by potentiating pre- and postsynaptic GABAA receptors.

As a psychoactive substance, ethanol is widely used in people's life. However, the neuronal mechanisms underlying its sedative effect remain unclear. In this study, we investigated the effects of ethanol on the lateral parabrachial nucleus (LPB), which is a novel component related to sedation. Coronal brain slices (280 μm thick) containing the LPB were prepared from C57BL/6J mice. The spontaneous firing and membrane potential of LPB neurons, and GABAergic transmission onto these neurons were recorded using whole-cell patch-clamp recordings. Drugs were applied through superfusion. The LPB neurons exhibited a regular spontaneous discharge at a rate of 1.5-3 Hz without burst firing. Brief superfusion of ethanol (30, 60, and 120 mM) concentration-dependently and reversibly suppressed the spontaneous firing of the neurons in LPB. In addition, when synaptic transmission was blocked by tetrodotoxin (TTX) (1 μM), ethanol (120 mM) caused hyperpolarization of the membrane potential. Furthermore, superfusion of ethanol markedly increased the frequency and amplitude of spontaneous and miniature inhibitory postsynaptic currents, which were abolished in the presence of the GABAA receptor (GABAA-R) antagonist picrotoxin (100 μM). In addition, the inhibitory effect of ethanol on the firing rate of LPB neurons was completely abolished by picrotoxin. Ethanol inhibits the excitability of LPB neurons in mouse slices, possibly via potentiating GABAergic transmission onto the neurons at pre- and postsynaptic sites.

Aconitum Alkaloid Songorine Exerts Potent Gamma-Aminobutyric Acid-A Receptor Agonist Action In Vivo and Effectively Decreases Anxiety without Adverse Sedative or Psychomotor Effects in the Rat.

Songorine (SON) is a diterpenoid alkaloid from Aconitum plants. Preparations of Aconitum roots have been employed in traditional oriental herbal medicine, however, their mechanisms of action are still unclear. Since GABA-receptors are possible brain targets of SON, we investigated which subtypes of GABA-receptors contribute to the effects of SON, and how SON affects anxiety-like trait behavior and psychomotor cognitive performance of rats. First, we investigated the effects of microiontophoretically applied SON alone and combined with GABA-receptor agents picrotoxin and saclofen on neuronal firing activity in various brain areas. Next, putative anxiolytic effects of SON (1.0–3.0 mg/kg) were tested against the GABA-receptor positive allosteric modulator reference compound diazepam (1.0–5.0 mg/kg) in the elevated zero maze (EOM). Furthermore, basic cognitive effects were assessed in a rodent version of the psychomotor vigilance task (PVT). Local application of SON predominantly inhibited the firing activity of neurons. This inhibitory effect of SON was successfully blocked by GABA(A)-receptor antagonist picrotoxin but not by GABA(B)-receptor antagonist saclofen. Similar to GABA(A)-receptor positive allosteric modulator diazepam, SON increased the time spent by animals in the open quadrants of the EOM without any signs of adverse psychomotor and cognitive effects observed in the PVT. We showed that, under in vivo conditions, SON acts as a potent GABA(A)-receptor agonist and effectively decreases anxiety without observable side effects. The present findings facilitate the deeper understanding of the mechanism of action and the widespread pharmacological use of diterpene alkaloids in various CNS indications.

Detection of pentylenetetrazol-induced seizure activity in the 19-21 Hz beta range using a magnetic coil induction method.

A force transducer or automatic scoring system is not sufficient to detect small or fine seizure activity. To improve previous assessments of epileptic behavior, a novel coil method was developed to detect an early behavioral marker for epileptic seizures. The present study used the γ-aminobutyric acid (GABA) receptor antagonist pentylenetetrazol (PTZ) to induce seizure activity and epileptic behavior in mice. A coil method was used to detect motor seizures consisting of small amplitude 19-21 Hz muscle contractions. Seizure activity in the 19-21 Hz range detected by the coil method was positively correlated with generalized clonic seizures with a kangaroo posture after PTZ administration. GABA receptor agonist valproic acid and ethosuximide decreased PTZ-induced 19-21 Hz seizure activity. The pattern of the amplitude ratio (%) of 19-21 Hz seizure activity after administration of the GABAA/C receptor antagonist picrotoxin was similar to the group that was treated with PTZ but different from the group that was treated with the nonselective muscarinic receptor agonist pilocarpine. The coil method detected 19-21 Hz seizure activity after PTZ administration. However, the force transducer method did not detect 19-21 Hz seizure activity. The coil method was more sensitive than the force transducer method for detecting epileptic behaviors. The findings may indicate a novel behavioral marker that can be detected by the coil method to reveal epileptic seizures, thus improving our understanding of the brain mechanisms of action and specific brain waves that are associated with PTZ-induced 19-21 Hz seizure activity.

Exposure to GABAA Receptor Antagonist Picrotoxin in Pregnant Mice Causes Autism-Like Behaviors and Aberrant Gene Expression in Offspring.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that is characterized by impairments in social interaction and restricted/repetitive behaviors. The neurotransmitter γ-aminobutyric acid (GABA) through GABAA receptor signaling in the immature brain plays a key role in the development of neuronal circuits. Excitatory/inhibitory imbalance in the mature brain has been investigated as a pathophysiological mechanism of ASD. However, whether and how disturbances of GABA signaling in embryos that are caused by GABAA receptor inhibitors cause ASD-like pathophysiology are poorly understood. The present study examined whether exposure to the GABAA receptor antagonist picrotoxin causes ASD-like pathophysiology in offspring by conducting behavioral tests from the juvenile period to adulthood and performing gene expression analyses in mature mouse brains. Here, we found that male mice that were prenatally exposed to picrotoxin exhibited a reduction of active interaction time in the social interaction test in both adolescence and adulthood. The gene expression analyses showed that picrotoxin-exposed male mice exhibited a significant increase in the gene expression of odorant receptors. Weighted gene co-expression network analysis showed a strong correlation between social interaction and enrichment of the “odorant binding” pathway gene module. Our findings suggest that exposure to a GABAA receptor inhibitor during the embryonic period induces ASD-like behavior, and impairments in odorant function may contribute to social deficits in offspring.

GABAA Receptors in Astrocytes Are Targets for Commonly Used Intravenous and Inhalational General Anesthetic Drugs.

Background: Perioperative neurocognitive disorders (PNDs) occur commonly in older patients after anesthesia and surgery. Treating astrocytes with general anesthetic drugs stimulates the release of soluble factors that increase the cell-surface expression and function of GABAA receptors in neurons. Such crosstalk may contribute to PNDs; however, the receptor targets in astrocytes for anesthetic drugs have not been identified. GABAA receptors, which are the major targets of general anesthetic drugs in neurons, are also expressed in astrocytes, raising the possibility that these drugs act on GABAA receptors in astrocytes to trigger the release of soluble factors. To date, no study has directly examined the sensitivity of GABAA receptors in astrocytes to general anesthetic drugs that are frequently used in clinical practice. Thus, the goal of this study was to determine whether the function of GABAA receptors in astrocytes was modulated by the intravenous anesthetic etomidate and the inhaled anesthetic sevoflurane.Methods: Whole-cell voltage-clamp recordings were performed in astrocytes in the stratum radiatum of the CA1 region of hippocampal slices isolated from C57BL/6 male mice. Astrocytes were identified by their morphologic and electrophysiologic properties. Focal puff application of GABA (300 μM) was applied with a Picospritzer system to evoke GABA responses. Currents were studied before and during the application of the non-competitive GABAA receptor antagonist picrotoxin (0.5 mM), or etomidate (100 μM) or sevoflurane (532 μM).Results: GABA consistently evoked inward currents that were inhibited by picrotoxin. Etomidate increased the amplitude of the peak current by 35.0 ± 24.4% and prolonged the decay time by 27.2 ± 24.3% (n = 7, P < 0.05). Sevoflurane prolonged current decay by 28.3 ± 23.1% (n = 7, P < 0.05) but did not alter the peak amplitude. Etomidate and sevoflurane increased charge transfer (area) by 71.2 ± 45.9% and 51.8 ± 48.9% (n = 7, P < 0.05), respectively.Conclusion: The function of astrocytic GABAA receptors in the hippocampus was increased by etomidate and sevoflurane. Future studies will determine whether these general anesthetic drugs act on astrocytic GABAA receptors to stimulate the release of soluble factors that may contribute to PNDs.

Hippocampal Disinhibition Reduces Contextual and Elemental Fear Conditioning While Sparing the Acquisition of Latent Inhibition

Hippocampal neural disinhibition, i.e., reduced GABAergic inhibition, is a key feature of schizophrenia pathophysiology. The hippocampus is an important part of the neural circuitry that controls fear conditioning and can also modulate prefrontal and striatal mechanisms, including dopamine signaling, which play a role in salience modulation. Consequently, hippocampal neural disinhibition may contribute to impairments in fear conditioning and salience modulation reported in schizophrenia. Therefore, we examined the effect of ventral hippocampus (VH) disinhibition in male rats on fear conditioning and salience modulation, as reflected by latent inhibition (LI), in a conditioned emotional response (CER) procedure. A flashing light was used as the conditioned stimulus (CS), and conditioned suppression was used to index conditioned fear. In experiment 1, VH disinhibition via infusion of the GABA-A receptor antagonist picrotoxin before CS pre-exposure and conditioning markedly reduced fear conditioning to both the CS and context; LI was evident in saline-infused controls but could not be detected in picrotoxin-infused rats because of the low level of fear conditioning to the CS. In experiment 2, VH picrotoxin infusions only before CS pre-exposure did not affect the acquisition of fear conditioning or LI. Together, these findings indicate that VH neural disinhibition disrupts contextual and elemental fear conditioning, without affecting the acquisition of LI. The disruption of fear conditioning resembles aversive conditioning deficits reported in schizophrenia and may reflect a disruption of neural processing both within the hippocampus and in projection sites of the hippocampus.

A functional hiPSC-cortical neuron differentiation and maturation model and its application to neurological disorders.

SummaryThe maturation and functional characteristics of human induced pluripotent stem cell (hiPSC)-cortical neurons has not been fully documented. This study developed a phenotypic model of hiPSC-derived cortical neurons, characterized their maturation process, and investigated its application for disease modeling with the integration of multi-electrode array (MEA) technology. Immunocytochemistry analysis indicated early-stage neurons (day 21) were simultaneously positive for both excitatory (vesicular glutamate transporter 1 [VGlut1]) and inhibitory (GABA) markers, while late-stage cultures (day 40) expressed solely VGlut1, indicating a purely excitatory phenotype without containing glial cells. This maturation process was further validated utilizing patch clamp and MEA analysis. Particularly, induced long-term potentiation (LTP) successfully persisted for 1 h in day 40 cultures, but only achieved LTP in the presence of the GABAA receptor antagonist picrotoxin in day 21 cultures. This system was also applied to epilepsy modeling utilizing bicuculline and its correction utilizing the anti-epileptic drug valproic acid.

5-HT7 receptors enhance inhibitory synaptic input to principal neurons in the mouse basal amygdala

The basal amygdala (BA) has been implicated in encoding fear and its extinction. The level of serotonin (5-HT) in the BA increases due to arousal and stress related to aversive stimuli. The effects of 5-HT7 receptor (5-HT7R) activation and blockade on the activity of BA neurons have not yet been investigated. In the present study, a transgenic mouse line carrying green fluorescent protein (GFP) reporter gene was used to identify neurons that express the 5-HT7R. GFP immunoreactivity was present mainly in cells that also expressed GAD67 or parvalbumin (PV), the phenotypic markers for GABAergic interneurons. Most cells showing GFP fluorescence demonstrated firing patterns characteristic of BA inhibitory interneurons. Activation of 5-HT7Rs resulted in a depolarization and/or occurrence of spontaneous spiking activity of BA interneurons that was accompanied by an increase in the mean frequency and mean amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) recorded from BA principal neurons. These effects were blocked by a specific 5-HT7R antagonist, SB269970 and were absent in slices from 5-HT7R knockout mice. Activation of 5-HT7Rs also decreased the mean frequency of spontaneous excitatory postsynaptic currents (sEPSCs) recorded from BA principal neurons, which was blocked by the GABAA receptor antagonist picrotoxin. Neither inhibitory nor excitatory miniature postsynaptic currents (mIPSCs/mEPSCs) were affected by 5-HT7R activation. These results show that in the BA 5-HT7Rs stimulate an activity-dependent enhancement of inhibitory input from local interneurons to BA principal neurons and provide insights about the possible involvement of BA serotonergic receptors in neuronal mechanisms underlying fear memory.

The role of ligand-gated chloride channels in behavioural alterations at elevated CO2 in a cephalopod.

Projected future carbon dioxide (CO2) levels in the ocean can alter marine animal behaviours. Disrupted functioning of γ-aminobutyric acid type A (GABAA) receptors (ligand-gated chloride channels) is suggested to underlie CO2-induced behavioural changes in fish. However, the mechanisms underlying behavioural changes in marine invertebrates are poorly understood. We pharmacologically tested the role of GABA-, glutamate-, acetylcholine- and dopamine-gated chloride channels in CO2-induced behavioural changes in a cephalopod, the two-toned pygmy squid (Idiosepius pygmaeus). We exposed squid to ambient (∼450 µatm) or elevated (∼1000 µatm) CO2 for 7days. Squid were treated with sham, the GABAA receptor antagonist gabazine or the non-specific GABAA receptor antagonist picrotoxin, before measurement of conspecific-directed behaviours and activity levels upon mirror exposure. Elevated CO2 increased conspecific-directed attraction and aggression, as well as activity levels. For some CO2-affected behaviours, both gabazine and picrotoxin had a different effect at elevated compared with ambient CO2, providing robust support for the GABA hypothesis within cephalopods. In another behavioural trait, picrotoxin but not gabazine had a different effect in elevated compared with ambient CO2, providing the first pharmacological evidence, in fish and marine invertebrates, for altered functioning of ligand-gated chloride channels, other than the GABAAR, underlying CO2-induced behavioural changes. For some other behaviours, both gabazine and picrotoxin had a similar effect in elevated and ambient CO2, suggesting altered function of ligand-gated chloride channels was not responsible for these CO2-induced changes. Multiple mechanisms may be involved, which could explain the variability in the CO2 and drug treatment effects across behaviours.

A GABAB receptor antagonist rescues functional and structural impairments in the perirhinal cortex of a mouse model of CDKL5 deficiency disorder

CDKL5 (cyclin-dependent kinase-like 5) deficiency disorder (CDD) is a severe neurodevelopmental encephalopathy characterized by early-onset epilepsy and intellectual disability. Studies in mouse models have linked CDKL5 deficiency to defects in neuronal maturation and synaptic plasticity, and disruption of the excitatory/inhibitory balance. Interestingly, increased density of both GABAergic synaptic terminals and parvalbumin inhibitory interneurons was recently observed in the primary visual cortex of Cdkl5 knockout (KO) mice, suggesting that excessive GABAergic transmission might contribute to the visual deficits characteristic of CDD. However, the functional relevance of cortical GABAergic circuits abnormalities in these mutant mice has not been investigated so far. Here we examined GABAergic circuits in the perirhinal cortex (PRC) of Cdkl5 KO mice, where we previously observed impaired long-term potentiation (LTP) associated with deficits in novel object recognition (NOR) memory. We found a higher number of GABAergic (VGAT)-immunopositive terminals in the PRC of Cdkl5 KO compared to wild-type mice, suggesting that increased inhibitory transmission might contribute to LTP impairment. Interestingly, while exposure of PRC slices to the GABAA receptor antagonist picrotoxin had no positive effects on LTP in Cdkl5 KO mice, the selective GABAB receptor antagonist CGP55845 restored LTP magnitude, suggesting that exaggerated GABAB receptor-mediated inhibition contributes to LTP impairment in mutants. Moreover, acute in vivo treatment with CGP55845 increased the number of PSD95 positive puncta as well as density and maturation of dendritic spines in PRC, and restored NOR memory in Cdkl5 KO mice. The present data show the efficacy of limiting excessive GABAB receptor-mediated signaling in improving synaptic plasticity and cognition in CDD mice.

Increased Purkinje Cell Complex Spike and Deep Cerebellar Nucleus Synchrony as a Potential Basis for Syndromic Essential Tremor. A Review and Synthesis of the Literature.

We review advances in understanding Purkinje cell (PC) complex spike (CS) physiology that suggest increased CS synchrony underlies syndromic essential tremor (ET). We searched PubMed for papers describing factors that affect CS synchrony or cerebellar circuits potentially related to tremor. Inferior olivary (IO) neurons are electrically coupled, with the degree of coupling controlled by excitatory and GABAergic inputs. Clusters of coupled IO neurons synchronize CSs within parasagittal bands via climbing fibers (Cfs). When motor cortex is stimulated in rats at varying frequencies, whisker movement occurs at ~10Hz, correlated with synchronous CSs, indicating that the IO/CS oscillatory rhythm gates movement frequency. Intra-IO injection of the GABAA receptor antagonist picrotoxin increases CS synchrony, increases whisker movement amplitude, and induces tremor. Harmaline and 5-HT2a receptor activation also increase IO coupling and CS synchrony and induce tremor. The hotfoot17 mouse displays features found in ET brains, including cerebellar GluRδ2 deficiency and abnormal PC Cf innervation, with IO- and PC-dependent cerebellar oscillations and tremor likely due to enhanced CS synchrony. Heightened coupling within the IO oscillator leads, through its dynamic control of CS synchrony, to increased movement amplitude and, when sufficiently intense, action tremor. Increased CS synchrony secondary to aberrant Cf innervation of multiple PCs likely also underlies hotfoot17 tremor. Deep cerebellar nucleus (DCN) hypersynchrony may occur secondary to increased CS synchrony but might also occur from PC axonal terminal sprouting during partial PC loss. Through these combined mechanisms, increased CS/DCN synchrony may plausibly underlie syndromic ET.

Frequency- and state-dependent effects of hippocampal neural disinhibition on hippocampal local field potential oscillations in anesthetized rats.

Reduced inhibitory GABA function, so-called neural disinhibition, has been implicated in cognitive disorders, including schizophrenia and age-related cognitive decline. We previously showed in rats that hippocampal disinhibition by local microinfusion of the GABA-A receptor antagonist picrotoxin disrupted memory and attention and enhanced hippocampal multi-unit burst firing recorded around the infusion site under isoflurane anesthesia. Here, we analyzed the hippocampal local field potential (LFP) recorded alongside the multi-unit data. We predicted frequency-specific LFP changes, based on previous studies implicating GABA in hippocampal oscillations, with the weight of evidence suggesting that disinhibition would facilitate theta and disrupt gamma oscillations. Using a new semi-automated method based on the kurtosis of the LFP peak-amplitude distribution as well as on amplitude envelope thresholding, we separated three distinct hippocampal LFP states under isoflurane anesthesia: "burst" and "suppression" states-high-amplitude LFP spike bursts and the interspersed low-amplitudeperiods-and a medium-amplitude "continuous" state. The burst state showed greater overall power than suppression and continuous states and higher relative delta/theta power, but lower relative beta/gamma power. The burst state also showed reduced functional connectivity across the hippocampal recording area, especially around theta and beta frequencies. Overall neuronal firing was higher in the burst than the other two states, whereas the proportion of burst firing was higher in burst and continuous states than the suppression state. Disinhibition caused state- and frequency-dependent LFP changes, tending to increase power at lower frequencies (<20 Hz), but to decrease power and connectivity at higher frequencies (>20 Hz) in burst and suppression states. The disinhibition-induced enhancement of multi-unit bursting was also state-dependent, tending to be more pronounced in burst and suppression states than the continuous state. Overall, we characterized three distinct hippocampal LFP states in isoflurane-anesthetized rats. Disinhibition changed hippocampal LFP oscillations in a state- and frequency-dependent way. Moreover, the disinhibition-induced enhancement of multi-unit bursting was also LFP state-dependent.

The Role of Chloride in the Breathing‐related Response to Central Acidosis in Developing Birds

Rhythmic spontaneous neural activity (rSNA) is thought to contribute to the organization and development of motor circuits. In spinal motor networks, rSNA has a role in synapse refinement, axon trajectory, and the formation of neuromuscular junctions. In the zebra finch embryo, extracellular recordings of hindbrain breathing‐related motor activity also show that rSNA burst pattern, shape, and neurotransmitter signaling change in an age‐dependent manner. Further, the role of chloride‐mediated neurotransmission exhibits a switch in polarity such that GABAergic and glycinergic signaling transitions from excitatory to inhibitory over the course of 24–48 hours. Despite recognizing these ontogenetic changes in hindbrain rSNA, we are just beginning to explore whether the changing physiochemical environment within the eggshell shapes rhythmic motor activity. As a bird embryo grows within the egg, it experiences progressive alterations in respiratory gases (↓PO2, ↑PCO2), an emerging acidosis, as well as modifications to the anatomical structures that facilitate gas exchange. Data show that lowering and raising the pH of the superfusate bathing the isolated avian hindbrain influences breathing‐linked rSNA as a function of age. Embryos employing diffusive gas exchange within the egg show a decreased rate of rSNA, while freely breathing birds employing pulmonary gas exchange show an increased rate of cranial nerve motor output. Given these findings, our objective here was to test mechanisms contributing to this pH response. We speculate that chloride is an important player as a result of three observations: 1) chloride‐mediated neurotransmission shifts in polarity at nearly the same embryonic age as the switch in the rSNA response to low pH; 2) chloride transport is involved in pH homeostasis; 3) GABAA receptors conduct both chloride and hydrogen carbonate, which are tied to pH regulation. To explore the first point, we recorded rSNA and breathing‐related motor rhythms while lowering pH under chloride free conditions. Results show that the removal of chloride eliminates rSNA in embryonic days (E) 4–6 embryos. On E7‐10, chloride removal decreases the rate of motor output compared with low pH alone. Related to the second point, we treated the hindbrain with low pH and bumetanide, an antagonist for the transmembrane sodium‐potassium‐chloride cotransporter (NKCC1). Data show that, compared to controls, the instantaneous frequency increases in E7‐10 birds. To address the third point, we applied the GABAA receptor antagonist picrotoxin (PTX) during low pH treatments. Results show that PTX and low pH treatment decreases rSNA frequency to 49 ± 11% of control in &lt;E11 birds and increases frequency to 129 ± 20% of control at &gt;E11. Together, our results suggest that interfering with chloride gradients and blocking GABAA receptor activity during low pH treatment has consequences for rSNA that are similar to lowering pH alone. In contrast, the inhibition of NKCC1 with bumetanide seems to disrupt the rhythmic response to central acidosis in the zebra finch hindbrain, at least prior to the onset of breathing.Support or Funding InformationIdaho IDeA Network of Biomedical Research and Excellence Program Grant # P20 GM103408 and the National Institute of Neurological Disorders and Stroke via the AREA Program Grant # 1R15NS087521‐01A1.

Presynaptic glutamatergic transmission and feedback system of oxytocinergic neurons in the hypothalamus of a rat model of adjuvant arthritis.

The neurohypophysial hormone oxytocin (OXT) is synthesized in the hypothalamic paraventricular and supraoptic nuclei. Recently, some studies have considered OXT to be important in sensory modulation and that the OXT protein is upregulated by acute and chronic nociception. However, the mechanism by which OXT is upregulated in neurons is unknown. In this study, we examined the resting membrane potentials and excitatory postsynaptic currents in OXT-ergic neurons in the paraventricular nucleus in adjuvant arthritis rat model, a model of chronic inflammation, using whole-cell patch-clamping. Transgenic rats expressing OXT and monomeric red fluorescent protein 1 (mRFP1) fusion protein to visualize the OXT-ergic neurons were used, and the OXT-mRFP1 transgenic rat model of adjuvant arthritis was developed by injection of heat-killed Mycobacterium butyricum. Furthermore, the feedback system of synthesized OXT was also examined using the OXT receptor antagonist L-368,899. We found that the resting membrane potentials and frequency of miniature excitatory postsynaptic currents and spontaneous excitatory postsynaptic currents in OXT-monomeric red fluorescent protein 1 neurons in the paraventricular nucleus were significantly increased in adjuvant arthritis rats. Furthermore, L-368,899 dose-dependently increased the frequency of miniature excitatory postsynaptic currents and spontaneous excitatory postsynaptic currents in OXT-ergic neurons. Following bath application of the GABAA receptor antagonist picrotoxin and the cannabinoid receptor 1 antagonist AM 251, L-368,899 still increased the frequency of miniature excitatory postsynaptic currents. However, following bath application of the nitric oxide synthase inhibitor Nω-Nitro-L-arginine methyl ester hydrochloride, L-368,899 did not alter the miniature excitatory postsynaptic current frequency. Thus, it is suggested that OXT-ergic neuron activity is upregulated via an increase in glutamate release, and that the upregulated OXT neurons have a feedback system with released endogenous OXT. It is possible that nitric oxide, but not GABA, may contribute to the feedback system of OXT neurons in chronic inflammation.

GABA signaling affects motor function in the honey bee

GABA is the most common inhibitory neurotransmitter in both vertebrate and invertebrate nervous systems. In insects, inhibition plays important roles at the neuromuscular junction, in the regulation of central pattern generators, and in the modulation of information in higher brain processing centers. Additionally, increasing our understanding of the functions of GABA is important since GABAA receptors are the targets of several classes of pesticides. To investigate the role of GABA in motor function, honey bee foragers were injected with GABA or with agonists or antagonists specific for either GABAA or GABAB receptors. Compounds that activated either type of GABA receptor decreased activity levels. Bees injected with the GABAA receptor antagonist picrotoxin lost the ability to right themselves, whereas blockade of GABAB receptors led to increases in grooming. Injection with antagonists of either GABAA or GABAB receptors resulted in an increase in extended wing behavior, during which bees kept their wings out at right angles to their body rather than folded along their back. These data suggest that the GABA receptor types play distinct roles in behavior and that GABA may affect behavior at several different levels.

Mild chronic perturbation of inhibition severely alters hippocampal function

Pentameric GABAA receptors mediate a large share of CNS inhibition. The γ2 subunit is a typical constituent. At least 11 mutations in the γ2 subunit cause human epilepsies, making the role of γ2-containing receptors in brain function of keen basic and translational interest. How small changes to inhibition may cause brain abnormalities, including seizure disorders, is unclear. In mice, we perturbed fast inhibition with a point mutation T272Y (T6′Y in the second membrane-spanning domain) to the γ2 subunit. The mutation imparts resistance to the GABAA receptor antagonist picrotoxin, allowing verification of mutant subunit incorporation. We confirmed picrotoxin resistance and biophysical properties in recombinant receptors. T6′Y γ2-containing receptors also exhibited faster deactivation but unaltered steady-state properties. Adult T6′Y knockin mice exhibited myoclonic seizures and abnormal cortical EEG, including abnormal hippocampal-associated theta oscillations. In hippocampal slices, picrotoxin-insensitive inhibitory synaptic currents exhibited fast decay. Excitatory/inhibitory balance was elevated by an amount expected from the IPSC alteration. Partial pharmacological correction of γ2-mediated IPSCs with diazepam restored total EEG power toward baseline, but had little effect on the abnormal low-frequency peak in the EEG. The results suggest that at least part of the abnormality in brain function arises from the acute effects of truncated inhibition.

The influence of acizol into effects of picrotoxin, injected in rat’s neostriatum

The aim of the article. The article is devoted to investigation of of zinc donator acizol influence to rat’s behavior, broken by intrastriatal injection of GABA-A receptor antagonist picrotoxin.&#x0D; Materials and methods. Adult male Wistar rats with avoidance conditioning reflexes in “shuttle box” and free locomotor activity in “open field” were used. Daily microinjection of picrotoxin (2 mcg/1 mcl) bilateral into rostral neostriatum in term of 15 days were made. Zinc donator acizol was injected intraperitoneal (24 mg/kg).&#x0D; Results. Steady losses of avoidance conditioning and choreo-mioclonic hyperkinesis of limbs and body, similar with human Huntington’s chorea by picrotoxin were produced. Acizol is contribute to restore avoidance conditioning and to prevent the development of hyperkinesis or essentially extend latency and lover duration of it.&#x0D; Conclusion. With the early data obtained, there is reason to propose, that acizol, to increasing the zinc content in the body, especially in the brain, is recover damaged cognitive function and to prevent the picrotoxin-induced hyperkinesis. Acizol should be proposed as perspective drug in extrapyramidal hyperkinetic deviation treatment in human.

The Positive Allosteric Modulator of Alpha-2/3-Containing γ-Aminobutyric Acid Type A Receptors, KRM-II-81, Is Active in Pharmacoresistant Models of Epilepsy and in Human Epileptic Tissue

Abstract INTRODUCTION Epilepsy patients continue to suffer from the lack of efficacious medications. Recent attention has been directed toward the potential advantages of developing positive allosteric modulators of alpha-2/3-containing γ-aminobutyric acid type A (GABAA) receptors as antiepileptic drugs. A proof of principle has been reported with one such molecule in patients with photosensitive epilepsy. KRM-II-81 (5- (8-ethynyl-6- (pyridin-2-yl)-4H-benzo[f]imidazole[1,5-alpha][1,4]diazepin-3-yl)oxazole) is an orally-bioavailable compound recently designed for selectivity at alpha-2/3-containing GABAA receptors over the alpha-1-subtype involved in motor-impairing effects. KRM-II-81 has recently been reported to dampen seizure activity in rodents induced by acute and chronic seizure provocation. KRM-II-81 was often more efficacious than diazepam as an anticonvulsant while producing less motor impairment than diazepam. The reduced motor impact of KRM-II-81 is hypothesized to enable higher central target exposure and hence increased efficacy. METHODS The effects of KRM-II-81 were investigated in a mouse mesial temporal lobe model and a rat lamotrigine-resistant kindling model. We also explored the antiepileptic electrophysiological effects of KRM-II-81 in cortical slices from epileptic pediatric patients to help guide the development of novel compounds that might be valuable against antiepileptic drug-resistant epilepsies. RESULTS Mice with kainate-induced mesial temporal lobe seizures exhibited spontaneous recurrent hippocampal paroxysmal discharges (16.8 +/–2.5). KRM-II-81 significantly reduced the discharge frequency to 5.5 +/–1.4 after oral dosing at 15 mg/kg. KRM-II-81 also decreased convulsions in rats undergoing amygdala kindling in the presence of lamotrigine. In slices of epileptic cortex, KRM-II-81 produced a concentration-dependent dampening of network activity engendered by the GABAA receptor antagonist picrotoxin or the K+-channel modulator 4-aminopyridine. CONCLUSION This study provides increased levels of confidence regarding the unique anticonvulsant profile of KRM-II-81 and its potential as an improved antiepileptic drug. The data also help to solidify the veracity of alpha-2/3-containing GABAA receptors as a novel molecular target for antiepileptic drugs.

Propofol decreases the excitability of cholinergic neurons in mouse basal forebrain via GABAA receptors.

Propofol is an intravenous anesthetic that can active γ-aminobutyric acid A (GABAA) receptors and generate sedative-hypnotic effects. Propofol has been widely applied clinically to achieve sedation comparable to sleep in humans. The basal forebrain (BF) is a brain region that plays an important role in sleep-wake regulation. Previous studies suggest that propofol affects the sleep-wake circuit via the BF; however, the mechanism remains elusive. In the current study we investigated the effects of propofol on the inherent properties of cholinergic neurons and their ability to convert excitatory inputs into spikes in mouse BF slices using whole-cell patch clamp recordings. Bath application of propofol (10 μM) significantly elevated the threshold potentials (Vts), decreased the number of spikes in response to a depolarizing current injection, and augmented the inter-spike intervals (ISIs), energy barrier (Vts-Vrs), and absolute refractory periods (ARPs). These effects were eliminated by co-application of a GABAA receptor antagonist picrotoxin (50 μM). Altogether, our results reveal that propofol decreases the excitability of cholinergic neurons in mouse BF via GABAA receptors.