Muscimol Research Articles

Acute physical exercise improves recognition memory via locus coeruleus activation but not via ventral tegmental area activation

Both animals and humans have been studied to explore the impact of acute physical exercise (PE) on memory. In rats, a single session of PE enhances the persistence of novel object recognition (NOR) memory, which depends on dopamine and noradrenaline activity in the hippocampus. However, limited research has examined the involvement of other brain regions in this phenomenon. In this study, we investigated the role of the ventral tegmental area (VTA) and locus coeruleus (LC) in modulating the persistence of NOR memory induced by acute PE. After NOR training, some animals underwent a 30 min treadmill PE session, followed by infusion of either vehicle (VEH) or muscimol (MUS) in either the VTA or LC. Other animals did not undergo PE and only received VEH, MUS, or NMDA within the same time window. We evaluated memory recall 1, 7, and 14 days later. Acute PE promoted memory persistence for up to 14 days afterward, similar to NMDA glutamatergic stimulation of the VTA or LC. Moreover, only the LC region was required for the memory improvement induced by acute PE since blocking this region with MUS impaired NOR encoding. Our findings suggest that acute PE can improve learning within a closed time window, and this effect depends on LC, but not VTA, activity.

Analysis of the Ibotenic Acid, Muscimol, and Ergosterol Content of an Amanita Muscaria Hydroalcoholic Extract with an Evaluation of Its Cytotoxic Effect against a Panel of Lung Cell Lines In Vitro.

The fungus Amanita muscaria is universally recognizable for its iconic appearance; it is also widely regarded as poisonous, inedible, and even deadly. In spite of that, there have been documented cases of use of A. muscaria-containing preparations against various diseases, including cancer, to no apparent ill effect. The search for compounds that can be used to treat cancer among various plants and fungi has been intensifying in recent years. In light of this, we describe an HPLC HILIC analytical method for the evaluation of the content of the anticancer compound ergosterol (ERG) and the neuroactive alkaloids ibotenic acid (IBO) and muscimol (MUS) that contribute significantly to the unpleasant physiological syndrome associated with A. muscaria consumption. A 'homemade' A. muscaria tincture made using 80-proof rye vodka as the solvent, an A. muscaria extract made with a standardized water-ethanol solution as the solvent, and fractions obtained from the second extract via liquid-liquid extraction with nonpolar solvents were analyzed. The study also presents the results of capillary zone electrophoresis with contactless conductivity detection and UHPLC-MS/MS analyses of the IBO and MUS content of the two native A. muscaria extracts and an evaluation of the standardized extract's cytotoxic effect against a small panel of lung cell cultures in vitro. Our results show that the standardized extract has a significant cytotoxic effect and does not contain the compounds of interest in any significant quantity.

Determination of ibotenic acid and muscimol in species of the genus Amanita section Amanita from China

The genus Amanita sect. Amanita harbors approximately 150 species in the world, and 27 species have been recognized in China. Some of the species in China have continuously caused poisoning. The responsible toxins should be ibotenic acid (IBO) and muscimol (MUS). However, species of the section Amanita containing IBO and MUS and their systematic positions are unclear. In this study, the contents of IBO and MUS in 84 samples of 24 species in section Amanita were detected using UPLC‒MS/MS, and the distribution of toxin-containing species in the molecular phylogeny was analyzed by the combined (ITS, nrLSU, RPB2, TUB2 and TEF1-α) dataset using maximum likelihood (ML) analysis and Bayesian inference (BI). Our results indicated that 10 of the 24 species contained IBO and MUS ranging from 0.6125 to 32.0932 and 0.0056–5.8685 g/kg dry weight, respectively. Among these 10 species, the toxins of eight species, including Amanita altipes, A. concentrica, A. flavopantherina, A. griseopantherina, A. pseudopantherina, A. rubrovolvata, A. subglobosa and A. sychnopyramis, were detected for the first time. In addition, the IBO and MUS contents of A. subglobosa in different growth stages showed that both toxins decreased in the mature stage. The phylogenetic analysis showed that all species of sect. Amanita from China were divided into 5 groups. And IBO- and MUS-containing species were gathered in clades Ⅰ and Ⅳ, but not all of the species in the two clades contain the toxins. No presence of IBO and MUS in the species of clades Ⅱ, Ⅲ and Ⅴ were confirmed.

Comparative study between radiofrequency-induced and muscimol-induced inhibition of cultured networks of cortical neuron.

Previous studies have shown that spontaneously active cultured networks of cortical neuron grown planar microelectrode arrays are sensitive to radiofrequency (RF) fields and exhibit an inhibitory response more pronounced as the exposure time and power increase. To better understand the mechanism behind the observed effects, we aimed at identifying similarities and differences between the inhibitory effect of RF fields (continuous wave, 1800 MHz) to the γ-aminobutyric acid type A (GABAA) receptor agonist muscimol (MU). Inhibition of the network bursting activity in response to RF exposure became apparent at an SAR level of 28.6 W/kg and co-occurred with an elevation of the culture medium temperature of ~1°C. Exposure to RF fields preferentially inhibits bursting over spiking activity and exerts fewer constraints on neural network bursting synchrony, differentiating it from a pharmacological inhibition with MU. Network rebound excitation, a phenomenon relying on the intrinsic properties of cortical neurons, was observed following the removal of tonic hyperpolarization after washout of MU but not in response to cessation of RF exposure. This implies that hyperpolarization is not the main driving force mediating the inhibitory effects of RF fields. At the level of single neurons, network inhibition induced by MU and RF fields occurred with reduced action potential (AP) half-width. As changes in AP waveform strongly influence efficacy of synaptic transmission, the narrowing effect on AP seen under RF exposure might contribute to reducing network bursting activity. By pointing only to a partial overlap between the inhibitory hallmarks of these two forms of inhibition, our data suggest that the inhibitory mechanisms of the action of RF fields differ from the ones mediated by the activation of GABAA receptors.

Nucleus reuniens inactivation does not impair consolidation or reconsolidation of fear extinction.

Recent data reveal that the thalamic nucleus reuniens (RE) has a critical role in the extinction of conditioned fear. Muscimol (MUS) infusions into the RE impair within-session extinction of conditioned freezing and result in poor long-term extinction memories in rats. Although this suggests that RE inactivation impairs extinction learning, it is also possible that it is involved in the consolidation of extinction memories. To examine this possibility, we examined the effects of RE inactivation on the consolidation and reconsolidation of fear extinction in male and female rats. Twenty-four hours after auditory fear conditioning, rats underwent an extinction procedure (45 CS-alone trials) in a novel context and were infused with saline (SAL) or MUS within minutes of the final extinction trial. Twenty-four hours later, conditioned freezing to the extinguished CS was assessed in the extinction context. Postextinction inactivation of the RE did not affect extinction retrieval. In a second experiment, rats underwent extinction training and, 24 h later, were presented with a single CS to reactivate the extinction memory; rats were infused with SAL or MUS immediately after the reactivation session. Pharmacological inactivation of the RE did not affect conditioned freezing measured in a drug-free retrieval test the following day. Importantly, we found in a subsequent test that MUS infusions immediately before retrieval testing increased conditioned freezing and impaired extinction retrieval, as we have previously reported. These results indicate that although RE inactivation impairs the expression of extinction, it does not impair either the consolidation or reconsolidation of extinction memories. We conclude that the RE may have a critical role in suppressing context-inappropriate fear memories in the extinction context.

Inhibition of the ventral midline thalamus does not alter encoding, short-term holding or retrieval of spatial information in rats performing a water-escape working memory task

Working memory (WM) is a function operating in three successive phases: encoding (sample trial), holding (delay), and retrieval (test trial) of information. Studies point to a possible implication of the thalamic reuniens nucleus (Re) in spatial WM (SWM). In which of the aforementioned 3 phases the Re has a function is largely unknown. Recently, in a delayed SWM water-escape task, we found that performance during the retrieval trial correlated positively with c-Fos expression in the Re nucleus, suggesting participation in retrieval. Here, we used the same task and muscimol (MUSC) inhibition or DREADD(hM4Di)-mediated inhibition of the Re during information encoding, right thereafter (thereby affecting the holding phase), or during the retrieval trial. A 6-hour delay separated encoding from retrieval. Concerning SWM, MUSC in the Re nucleus did not alter performance, be it during or after encoding, or during evaluation. CNO administered before encoding in DREADD-expressing rats was also ineffective, although CNO-induced inhibition disrupted set shifting performance, as found previously (Quet et al., Brain Struct Function 225, 2020), thereby validating DREADD efficiency. These findings are the first that do not support an implication of the Re nucleus in SWM. As most previous studies used T-maze alternation tasks, which carry high proactive interference risks, an important question to resolve now is whether the Re nucleus is required in (T-maze alternation) tasks using very short information-holding delays (seconds to minutes), and less so in other short-term spatial memory tasks with longer information holding intervals (hours) and therefore reduced interference risks.

GABAergic neurons in the nucleus accumbens regulate hedonic food intake via orexin-A expression in the lateral hypothalamus.

Objective(s):To investigate the regulatory effects of the nucleus accumbens (NAcSh)-lateral hypothalamus (LHA) GABAergic neural pathway on palatable food (PF) intake via orexin-A expression in diet-induced obesity (DIO) rats. Materials and Methods:NAcSh-LHA GABAergic pathways were observed by fluorogold retrograde tracing combined with fluorescence immunohistochemistry, and the regulatory effects of this neural pathway on PF intake were detected after 1) microinjection of GABA-A receptor agonist muscimol (MUS) or antagonist bicuculine (BIC) into LHA, 2) electrical stimulation NAcSh, and 3) blocking the orexin-A receptor by icv SB334867. Results:Compared with rats on a normal diet (ND), NAcSh-LHA GABAergic neurons in the DIO rats were significantly decreased, and orexin-A expression in LHA significantly increased (P<0.05). Microinjection of MUS into LHA significantly decreased the PF intake in both ND and DIO rats (P<0.05), and BIC could markedly increase the PF intake in the ND rats (P<0.05), but not the DIO rats (P>0.05). After NAcSh electrical stimulation or SB334867 ICV injection, the PF intake was significantly decreased in the DIO rats (P<0.05), and there was no significant difference after preadministration of BIC into LHA (P>0.05). Conclusion:This GABAergic pathway could regulate the expression of orexin-A in LHA and PF intake. Orexin-A neurons in LHA of DIO rats might be less sensitive to GABAergic signals and may consequently lead to more hedonic food intake.

Determination of ibotenic acid and muscimol in plasma by liquid chromatography-triple quadrupole mass spectrometry with bimolecular dansylation

Ibotenic acid (IBA) is an amino acid and muscimol (MUS) is the decarboxyl derivative of IBA. They are mushroom neurotoxins with high polarity and low molecular weight. Only one transition (159–>113 for IBA and 115->98 for MUS) can be found when directly measured by high performance liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS). Therefore, the identification and quantification of trace amount of the toxins in biomaterial are difficult. A highly sensitive and accurate analytical method for IBA and MUS in plasma was developed by LC-MS/MS with the application of bimolecular dansylation and internal standard calibration. Acetonitrile was used for protein precipitation and for toxin extraction from plasma. The toxins and internal standards (L-tyrosine-13C9,15N for IBA and tyramine-d4 for MUS) were derivatized with dansyl chloride (DNSCl). The reaction conditions of the bimolecular dansylation were optimized and the fragmentation pathways of the derivatives in MS/MS were studied. Method validation was carried out according to the Bioanalytical Method Validation Guidance for Industry (FDA, USA, 2018). The limits of detection for IBA and MUS in plasma were 0.3 ng mL−1 and 0.1 ng mL−1, respectively. The linear ranges in plasma were 1–500 ng mL−1 and 1–200 ng mL−1 with the correlation coefficients of 0.998 and 0.999 for IBA and MUS, respectively. The recoveries at three spiked levels were 90.7–111.4% with relative standard deviations (RSDs) of 6.4–10.3% for IBA and the results were 85.1–94.2% with RSDs of 5.0–8.9% for MUS. The toxin levels in patients’ plasma samples under different poisoning degree were presented.

Direct Analysis of Psilocin and Muscimol in Urine Samples Using Single Drop Microextraction Technique In-Line with Capillary Electrophoresis.

The fully automated system of single drop microextraction coupled with capillary electrophoresis (SDME-CE) was developed for in-line preconcentration and determination of muscimol (MUS) and psilocin (PSC) from urine samples. Those two analytes are characteristic active metabolites of Amanita and Psilocybe mushrooms, evoking visual and auditory hallucinations. Study analytes were selectively extracted from the donor phase (urine samples, pH 4) into the organic phase (a drop of octanol layer), and re-extracted to the acidic acceptor (background electrolyte, BGE), consisting of 25 mM phosphate buffer (pH 3). The optimized conditions for the extraction procedure of a 200 µL urine sample allowed us to obtain more than a 170-fold enrichment effect. The calibration curves were linear in the range of 0.05–50 mg L−1, with the correlation coefficients from 0.9911 to 0.9992. The limit of detections was determined by spiking blank urine samples with appropriate standards, i.e., 0.004 mg L−1 for PSC and 0.016 mg L−1 for MUS, respectively. The limits of quantification varied from 0.014 mg L−1 for PSC and 0.045 mg L−1 for MUS. The developed method practically eliminated the sample clean-up step, which was limited only to simple dilution (1:1, v/v) and pH adjustment.

GABAA Receptor β2E155 Residue Located at the Agonist-Binding Site Is Involved in the Receptor Gating.

GABAA receptors (GABAARs) play a crucial role in mediating inhibition in the adult brain. In spite of progress in describing (mainly) the static structures of this receptor, the molecular mechanisms underlying its activation remain unclear. It is known that in the α1β2γ2L receptors, the mutation of the β2E155 residue, at the orthosteric binding site, strongly impairs the receptor activation, but the molecular and kinetic mechanisms of this effect remain elusive. Herein, we investigated the impact of the β2E155C mutation on binding and gating of the α1β2γ2L receptor. To this end, we combined the macroscopic and single-channel analysis, the use of different agonists [GABA and muscimol (MSC)] and flurazepam (FLU) as a modulator. As expected, the β2E155C mutation caused a vast right shift of the dose–response (for GABA and MSC) and, additionally, dramatic changes in the time course of current responses, indicative of alterations in gating. Mutated receptors showed reduced maximum open probability and enhanced receptor spontaneous activity. Model simulations for macroscopic currents revealed that the primary effect of the mutation was the downregulation of the preactivation (flipping) rate. Experiments with MSC and FLU further confirmed a reduction in the preactivation rate. Our single-channel analysis revealed the mutation impact mainly on the second component in the shut times distributions. Based on model simulations, this finding further confirms that this mutation affects mostly the preactivation transition, supporting thus the macroscopic data. Altogether, we provide new evidence that the β2E155 residue is involved in both binding and gating (primarily preactivation).

Three-dimensional (3D) brain microphysiological system for organophosphates and neurochemical agent toxicity screening.

We investigated a potential use of a 3D tetraculture brain microphysiological system (BMPS) for neurotoxic chemical agent screening. This platform consists of neuronal tissue with extracellular matrix (ECM)-embedded neuroblastoma cells, microglia, and astrocytes, and vascular tissue with dynamic flow and membrane-free culture of the endothelial layer. We tested the broader applicability of this model, focusing on organophosphates (OPs) Malathion (MT), Parathion (PT), and Chlorpyrifos (CPF), and chemicals that interact with GABA and/or opioid receptor systems, including Muscimol (MUS), Dextromethorphan (DXM), and Ethanol (EtOH). We validated the BMPS platform by measuring the neurotoxic effects on barrier integrity, acetylcholinesterase (AChE) inhibition, viability, and residual OP concentration. The results show that OPs penetrated the model blood brain barrier (BBB) and inhibited AChE activity. DXM, MUS, and EtOH also penetrated the BBB and induced moderate toxicity. The results correlate well with available in vivo data. In addition, simulation results from an in silico physiologically-based pharmacokinetic/pharmacodynamic (PBPK/PD) model that we generated show good agreement with in vivo and in vitro data. In conclusion, this paper demonstrates the potential utility of a membrane-free tetraculture BMPS that can recapitulate brain complexity as a cost-effective alternative to animal models.

GABAergic Control of Nigrostriatal and Mesolimbic Dopamine in the Rat Brain.

Purpose: The present study assessed the effects of the GABAA receptor (R) agonist muscimol (MUS), and the GABAAR antagonist bicuculline (BIC) on neocortical and subcortical radioligand binding to dopamine D2/3Rs in relation to motor and exploratory behaviors in the rat.Methods: D2/3R binding was measured with small animal SPECT in baseline and after challenge with either 1 mg/kg MUS or 1 mg/kg BIC, using [123I]IBZM as radioligand. Motor/exploratory behaviors were assessed for 30 min in an open field prior to radioligand administration. Anatomical information was gained with a dedicated small animal MRI tomograph. Based on the Paxinos rat brain atlas, regions of interest were defined on SPECT-MRI overlays. Estimations of the binding potentials in baseline and after challenges were obtained by computing ratios of the specifically bound compartments to the cerebellar reference region.Results: After MUS, D2/3R binding was significantly reduced in caudateputamen, nucleus accumbens, thalamus, substania nigra/ventral tegmental area, and posterior hippocampus relative to baseline (0.005 ≤ p ≤ 0.012). In all these areas, except for the thalamus, D2/3R binding was negatively correlated with grooming in the first half and positively correlated with various motor/exploratory behaviors in the second half of the testing session. After BIC, D2/3R binding was significantly elevated in caudateputamen (p = 0.022) and thalamus (p = 0.047) relative to baseline. D2/3R binding in caudateputamen and thalamus was correlated negatively with sitting duration and sitting frequency and positively with motor/exploratory behaviors in the first half of the testing time.Conclusions: Findings indicate direct GABAergic control over nigrostriatal and mesolimbic dopamine levels in relation to behavioral action. This may be of relevance for neuropsychiatric conditions such as anxiety disorder and schizophrenia, which are characterized by both dopaminergic and GABAergic dysfunction.

Salicylate-Induced Suppression of Electrically Driven Activity in Brain Slices from the Auditory Cortex of Aging Mice.

The prevalence of tinnitus is known to increase with age. The age-dependent mechanisms of tinnitus may have important implications for the development of new therapeutic treatments. High doses of salicylate can be used experimentally to induce transient tinnitus and hearing loss. Although accumulating evidence indicates that salicylate induces tinnitus by directly targeting neurons in the peripheral and central auditory systems, the precise effect of salicylate on neural networks in the auditory cortex (AC) is unknown. Here, we examined salicylate-induced changes in stimulus-driven laminar responses of AC slices with salicylate superfusion in young and aged senescence-accelerated-prone (SAMP) and -resistant (SAMR) mice. Of the two strains, SAMP1 is known to be a more suitable model of presbycusis. We recorded stimulus-driven laminar local field potential (LFP) responses at multi sites in AC slice preparations. We found that for all AC slices in the two strains, salicylate always reduced stimulus-driven LFP responses in all layers. However, for the amplitudes of the LFP responses, the two senescence-accelerated mice (SAM) strains showed different laminar properties between the pre- and post-salicylate conditions, reflecting strain-related differences in local circuits. As for the relationships between auditory brainstem response (ABR) thresholds and the LFP amplitude ratios in the pre- vs. post-salicylate condition, we found negative correlations in layers 2/3 and 4 for both older strains, and in layer 5 (L5) in older SAMR1. In contrast, the GABAergic agonist muscimol (MSC) led to positive correlations between ABR thresholds and LFP amplitude ratios in the pre- vs. post-MSC condition in younger SAM mice from both strains. Further, in younger mice, salicylate decreased the firing rate in AC L4 pyramidal neurons. Thus, salicylate can directly reduce neural excitability of L4 pyramidal neurons and thereby influence AC neural circuit activity. That we observed age-dependent effects of salicylate and varied GABAergic sensitivity in the AC among mouse strains with hearing loss implies that potential therapeutic mechanisms for tinnitus may operate differently in young vs. aged subjects. Therefore, scientists developing new therapeutic modalities for tinnitus treatment should consider using both aged and young animals.

Evidence for Tonic Control by the GABAA Receptor of Extracellular D-Serine Concentrations in the Medial Prefrontal Cortex of Rodents.

Endogenous D-serine is a putative dominant co-agonist for the N-methyl-D-aspartate glutamate receptor (NMDAR) in the mammalian forebrain. Although the NMDAR regulates the higher order brain functions by interacting with various neurotransmitter systems, the possible interactions between D-serine and an extra-glutamatergic system largely remain elusive. For the first time, we show in the rat and mouse using an in vivo microdialysis technique that the extracellular D-serine concentrations are under tonic increasing control by a major inhibitory transmitter, GABA, via the GABAA (GABAAR) in the medial prefrontal cortex (mPFC). Thus, an intra-mPFC infusion of a selective GABAAR antagonist, bicuculline (BIC), caused a concentration-dependent and reversible decrease in the extracellular levels of D-serine in the rat mPFC without affecting those of another intrinsic NMDAR coagonist, glycine and an NMDAR agonist, L-glutamate. The decreasing effects of BIC were eliminated by co-infusion of a selective GABAA agonist, muscimol (MUS) and were mimicked by a GABAA antagonist, gabazine (GBZ). In contrast, selective blockade of the GABAB or homomeric ρGABAA (formerly GABAC) receptor by saclofen or (1,2,5,6-tetrahydropyridin-4-yl)-methylphosphinic acid (TPMPA), respectively, failed to downregulate the prefrontal extracellular D-serine levels. Moreover, the local BIC application attenuated the ability of NMDA given to the mPFC to increase the cortical extracellular concentrations of taurine, indicating the hypofunction of the NMDAR. Finally, in the mouse mPFC, the reduction of the extracellular D-serine levels by a local injection of BIC into the prefrontal portion was replicated, and was precluded by inhibition of the neuronal or glial activity by co-local injection with tetrodotoxin (TTX) or fluorocitrate (Fluo), respectively. These findings suggest that the GABAAR-mediated regulation of the D-serine signaling may exert fine-tuning of the NMDAR function and require both neuronal and glial activities in the mammalian mPFC.

Role of spinal GABAA receptors in preoperative anxiety-induced postoperative hyperalgesia in rats

Objective To investigate the role of spinal γ-aminobutyric acid A(GABAA)receptors in preoperative anxiety-induced postoperative hyperalgesia in rats. Methods Forty-two pathogen-free healthy male Sprague-Dawley rats, aged 6-8 weeks, weighing 200-250 g, were randomly divided into 5 groups using a random number table: control group(group C, n=10), operation group(group O, n=10), preoperative anxiety group(group PA, n=10), normal saline group(group NS, n=6)and muscimol group(group M, n=6). The rats were exposed to single prolonged stress to establish the model of preoperative anxiety.The rats were only anaesthetized with sevoflurane in group C. The rats were anesthetized with sevoflurane and underwent an incision operation on the right hindlimb in group O. In group PA, the rats underwent an incision operation on the right hindlimb after establishment of preoperative anxiety model.In group NS, normal saline 10 μl was injected intrathecally at 30 min before establishment of the model, at 30 min before operation, and on postoperative days 1, 2 and 3.In group M, 0.01 μg/μl GABAA receptor agonist muscimol 10 μl was injected intrathecally at the time points mentioned above.The mechanical paw withdrawal threshold(MWT)was measured at 24 h before establishment of the model(baseline)and on postoperative days 1, 2, 3, 5 and 7.In C, O and PA groups, the lumbar segments(L3-5)of the spinal cord were removed after measurement of the pain threshold on postoperative days 1 and 7 for determination of GABAA receptor expression by Western blot. Results Compared with group C and the baseline values, the MWT was significantly decreased on postoperative days 1-3, and the expression of GABAA receptors was significantly down-regulated on postoperative day 1 in group O(P<0.05). Compared with group O, the MWT was significantly decreased on postoperative days 1-7, and the expression of GABAA receptors was significantly down-regulated on postoperative days 1 and 7 in group PA(P<0.05). Compared with group PA, the MWT was significantly increased on postoperative days 1-7 in group M(P<0.05). Conclusion The mechanism underlying preoperative anxiety-induced postoperative hyperalgesia is probably related to inhibition of spinal GABAA receptor function in rats. Key words: Anxiety; Hyperalgesia; Receptors, GABA-A; Spinal cord

The lateral parabrachial nucleus is actively involved in the acquisition of fear memory in mice.

BackgroundPavlovian fear conditioning is a form of learning accomplished by associating a conditioned stimulus (CS) and an unconditioned stimulus (US). While CS–US associations are generally thought to occur in the amygdala, the pathway mediating US signal processing has only been partially identified. The external part of the pontine lateral parabrachial nucleus (elPB) is well situated for providing US nociceptive information to the central amygdala (CeA), which was recently revealed to play a primary role in fear acquisition. Therefore, we manipulated the elPB activity to examine its role in the regulation of fear learning.ResultsFirst, we transiently inactivate the elPB during the acquisition of fear memory. Mice received bilateral elPB injections of the GABAA agonist muscimol (MUS) or phosphate-buffered saline (drug control), with bilateral misplacement of MUS defined as a placement control group. After the injection, mice were conditioned with a pure tone and foot-shock. On a memory retrieval test on day 2, the freezing ratio was significantly lower in the MUS group compared with that in the drug control or placement control groups. A second retrieval test using a pip tone on day 4 following de novo training on day 3, resulted in significant freezing with no group differences, indicating integrity of fear learning and a temporary limited effect of MUS. Next, we examined whether selectively activating the elPB-CeC pathway is sufficient to induce fear learning when paired with CS. Mice with channelrhodopsin2 (ChR2) expressed in the elPB received a pure tone (CS) in association with optical stimulation in the CeA (CS-LED paired group). On the retrieval test, CS-LED paired mice exhibited significantly higher freezing ratios evoked by CS presentation compared with both control mice receiving optical stimulation immediately after being placed in the shock chamber and exposed to the CS much later (immediate shock group) and those expressing only GFP (GFP control group). These results suggest that selective stimulation of the elPB-CeC pathway substitutes for the US to induce fear learning.ConclusionsThe elPB activity is necessary and sufficient to trigger fear learning, likely as a part of the pathway transmitting aversive signals to the CeA.

Muscimol infusions to infralimbic cortex impair extinction but not acquisition of a trace eyeblink conditioned response in rabbits.

This study assessed the effects of reversible lesions with microinfusions of the GABAA agonist muscimol (MUSC) to infralimbic cortex (IL; Brodmann’s area 25) of the medial prefrontal cortex (mPFC) on trace eyeblink conditioning and extinction in rabbits (Oryctolagus cuniculus). Four groups were tested: rabbits receiving MUSC infusions 5 min before acquisition and extinction sessions (MUSC/MUSC group), rabbits receiving vehicle (VEH) 5 min before acquisition and extinction sessions (VEH/VEH group), rabbits receiving MUSC before acquisition and VEH before extinction sessions (MUSC/VEH group), and rabbits receiving VEH before acquisition and MUSC before extinction sessions (VEH/MUSC). Results revealed that MUSC infusions to IL had no effects on acquisition but retarded extinction when injected before either acquisition or extinction sessions. These findings are among the first to demonstrate that MUSC infusions to the IL disrupt extinction of an EB CR in rabbits, and are in agreement with data from rodent studies noting the critical role of IL for the extinction of aversive fear-conditioned responses.

Basolateral amygdala activity is required for enhancement of memory consolidation produced by histone deacetylase inhibition in the hippocampus

Histone acetylation, a type of chromatin modification that allows increased gene transcription and can be pharmacologically promoted by histone deacetylase (HDAC) inhibitors (HDACis), has been consistently associated with promoting memory formation in the hippocampus. The basolateral nucleus of the amygdala (BLA) is a brain area crucially involved in enabling hormones and drugs to influence memory formation. Here, we show that BLA activity is required for memory enhancement by intrahippocampal administration of an HDACi. Two different HDACis, sodium butyrate (NaB) and trichostatin A (TSA), differentially enhanced the retention of memory for inhibitory avoidance (IA) when administered to the dorsal hippocampus after training. TSA showed a biphasic pattern of response during consolidation, in which infusions given immediately or 3h after training produced memory enhancement, whereas no effect was observed when it was infused 1.5 or 6h posttraining. Muscimol (MUS)-induced unilateral functional inactivation of the BLA prevented the enhancement of memory retention produced by posttraining infusion of TSA into the ipsilateral hippocampus. TSA did not affect IA extinction or reconsolidation. These results indicate that HDACis can increase IA memory retention when given into the hippocampus, and, most importantly, BLA activity is necessary for enabling HDACi-induced influences on memory formation.

The basolateral amygdala determines the effects of fear memory on sleep in an animal model of PTSD

Fear conditioning [inescapable shock training (ST)] and fearful context re-exposure (CR) alone can produce significant fear indicated by increased freezing and reductions in subsequent rapid eye movement (REM) sleep. Damage to or inactivation of the basolateral nucleus of the amygdala (BLA) prior to or after ST or prior to CR generally has been found to attenuate freezing in the shock training context. However, no one has examined the impact of BLA inactivation on fear-induced changes in sleep. Here, we used the GABAA agonist, muscimol (MUS), to inactivate BLA prior to ST, the period when fear is learned, and assessed sleep after ST and sleep and freezing after two CR sessions. Wistar rats (n = 14) were implanted with electrodes for recording sleep and with cannulae aimed bilaterally into BLA. After recovery, the animals were habituated to the injection procedure (handling) over 2 consecutive days and baseline sleep following handling was recorded. On experimental day 1, the rats were injected (0.5 μl) into BLA with either MUS (1.0 μM; n = 7) or vehicle (distilled water, n = 7) 30 min prior to ST (20 footshocks, 0.8 mA, 0.5-s duration, 60-s interstimulus interval). On experimental days 7 and 21, the animals experienced CR (CR1 and CR2, respectively) alone. Electroencephalogram and electromyogram were recorded for 8 h on each day, and the recording was scored for non-rapid eye movement sleep, REM sleep, and wakefulness. Freezing was examined during CR1 and CR2. MUS microinjections into BLA prior to ST blocked the post-training reduction in REM sleep seen in vehicle-treated rats. Furthermore, in MUS-treated rats, REM sleep after CR1 and CR2 was at baseline levels and freezing was significantly attenuated. Thus, BLA inactivation prior to ST blocks the effects of footshock stress on sleep and reduces fear memory, as indicated by the lack of freezing and changes in sleep after CR. These data indicate that BLA is an important regulator of stress-induced alterations in sleep and an important site for forming fear memories that can alter sleep.

Burst and principal components analyses of MEA data for 16 chemicals describe at least three effects classes

Microelectrode arrays (MEAs) can be used to detect drug and chemical induced changes in neuronal network function and have been used for neurotoxicity screening. As a proof-of-concept, the current study assessed the utility of analytical “fingerprinting” using principal components analysis (PCA) and chemical class prediction using support vector machines (SVMs) to classify chemical effects based on MEA data from 16 chemicals. Spontaneous firing rate in primary cortical cultures was increased by bicuculline (BIC), lindane (LND), RDX and picrotoxin (PTX); not changed by nicotine (NIC), acetaminophen (ACE), and glyphosate (GLY); and decreased by muscimol (MUS), verapamil (VER), fipronil (FIP), fluoxetine (FLU), chlorpyrifos oxon (CPO), domoic acid (DA), deltamethrin (DELT) and dimethyl phthalate (DMP). PCA was performed on mean firing rate, bursting parameters and synchrony data for concentrations above each chemical's EC50 for mean firing rate. The first three principal components accounted for 67.5, 19.7, and 6.9% of the data variability and were used to identify separation between chemical classes visually through spatial proximity. In the PCA, there was clear separation of GABAA antagonists BIC, LND, and RDX from other chemicals. For the SVM prediction model, the experiments were classified into the three chemical classes of increasing, decreasing or no change in activity with a mean accuracy of 83.8% under a radial kernel with 10-fold cross-validation. The separation of different chemical classes through PCA and high prediction accuracy in SVM of a small dataset indicates that MEA data may be useful for separating chemicals into effects classes using these or other related approaches.