We took interest in the regulation of TREK1 by Gi-coupled GPCRs, since several transmitter-gated versions of these are found in the hippocampus (Padgett and Slesinger, 2010). Postsynaptically,

hippocampal GABAB receptors can inhibit calcium channels (Mintz and Bean, 1993), but they are primarily known to enhance the potassium channels that underlie the slow inhibitory postsynaptic potential (IPSP). The slow IPSP is known to involve G protein-coupled inwardly rectifying potassium (Kir3) channels (Lüscher et al., 1997). Baclofen is generally used to study the GABAB response (Dutar and Nicoll, 1988). Using baclofen, Koyrakh and colleagues showed evidences for an additional unidentified GABAB channel target (Koyrakh et al., 2005). Our PCS approach enables us to identify this channel as TREK1. As GSK1349572 in vitro with Kir3 channels, TREK1 is also postsynaptic (Sandoz et al., 2008), where it is complexed with the postsynaptic machinery via interaction with AKAP150 (Sandoz et al., 2006). This is the second case

where a 2P potassium channel has been implicated in GABAergic signaling, since TREK2 appears to mediate a different and much slower IPSP in entorhinal cortex (Deng et al., 2009). These findings suggest that 2P potassium channels may have a broad role in synaptic signaling in the brain. It breaks with the traditional learn more notions that Kir3 channels are the sole targets of postsynaptic GABAB receptors and that 2P-potassium channels serve simply as leak channels in the hippocampus. Our PCS approach offers an affordable and powerful strategy for identifying the molecular basis of unknown ionic currents and for obtaining a pharmacological foothold in multisubunit signaling proteins. Cysteine mutations were introduced into mTREK1 cDNA in the pIRES2EGFP

expression vector using the QuickChange mutagenesis kit (Agilent). The PCR protocol used was 1 cycle (95°, 30 s), 16 cycles (95°, 30 s; 55°, 1 min; 68°, 12 min). TREK1-PCS has been made by PCR and introduced in pIRES2EGFP expression vector. HEK293 Cells were transiently cotransfected using Lipofectamine 2000 (Invitrogen) with TREK1 mutants or TREK1-PCS. For Histidine ammonia-lyase coexpression, TREK1 or TREK1-PCS are cotransfected with a ratio of 1:3 to 1:5 with 1.6 μg of DNA total per 18-mm-diameter coverslip. Hippocampal neurons were transfected using the calcium phosphate method. Each 12 mm coverslip received 1.1 μg of TREK1-PCS DNA and 0.2 μg of Tomato DNA. HEK293 cells were maintained in DMEM with 5% FBS on poly-L-lysine-coated glass coverslips. Dissociated hippocampal neurons were obtained from postnatal rats (P0-1) and plated at 75,000 cells/coverslip on poly-L-lysine-coated glass coverslips (12 mM). Neurons were maintained in media containing MEM supplemented with 5% fetal bovine serum, B27 (Invitrogen), and GlutaMAX (Invitrogen). HEK293 cell electrophysiology was performed 24–72 hr after transfection solution containing (in mM): 145 mM NaCl, 4 mM KCl, 1 mM MgCl2, 2 mM CaCl2, and 10 mM HEPES.

The dose of euthanasia solution was based on the body weight determined on the day of euthanasia. Dogs were fasted 12 h immediately prior to euthanasia in order to decrease the amount of material in the intestinal tract. The abdominal cavity was opened along the ventral midline and double ligatures were placed at the cardia of

the stomach and at the distal rectum prior to removal of the gastrointestinal tract from the abdominal cavity. The stomach was opened along the greater curvature and the contents collected in a suitable container. The mucosa was thoroughly scrubbed, rinsed with water and the washings combined with the gastric contents. The mucosa was inspected selleck products and any attached parasites were removed and preserved in 10% formalin or other suitable preservative. In a similar manner, the small and large intestines were opened along their entire length and the contents and mucosa were collected, thoroughly rinsed with water and the scrapings/washings from the mucosa were combined. The mucosa was inspected and any attached

parasites were removed and preserved in 10% formalin. The gastric and intestinal contents DZNeP and rinsing fluids were washed over appropriately sized sieve(s) and any material that retained in the sieve(s) was preserved in 10% formalin. Worm recovery and identification were performed by appropriately trained and experienced individuals. Prior to examining the preserved worms and washings, formalin was removed by sedimentation and/or washing. Recovered worms were examined

microscopically, identified to genus Pramipexole and species based on characteristics of the buccal plates. The number of adult A. braziliense recovered from each dog was recorded along with the number of other adult worms recovered from each dog. Twelve dogs were randomized to each group to increase the probability of having at least six dogs adequately infected in the placebo control group indicating an adequate test. The individual dog was the experimental unit. Efficacy was determined on the basis of the percent reduction in adult worm counts in the treated groups compared to the placebo control group. Arithmetic and geometric means of all A. braziliense counts were calculated. Percent effectiveness was calculated using the following formula: Percent Effectiveness=100*cc−ctccwhere cc = geometric mean number of adult A. braziliense in the control group and ct = geometric mean number of adult A. braziliense in the treatment group. There were separate calculations for each treatment group. Since the calculation of the geometric mean involved taking the logarithm of the A. braziliense count of each animal, a “1” was added to the A. braziliense count for every animal in every group since there were A. braziliense counts equal to zero. This constant (1) was subtracted from the resultant calculated geometric mean prior to calculating percent effectiveness. Percent effectiveness based on ≥90% reduction in A.

Human genetic studies provide only limited support KU-55933 research buy for a link between angiogenic factors and AD so far (Ruiz de Almodovar et al., 2009). In ALS, VEGF

gene haplotypes that lower VEGF levels are associated with an increased risk in genetically homogeneous populations, while a meta-analysis of over 7,000 individuals documented an increased risk of “at-risk” VEGF gene variations in males (Lambrechts et al., 2009). VEGF levels in the cerebrospinal fluid of ALS patients are decreased, which could relate to impaired VEGF mRNA translation due to mutant SOD1. ALS can also result from mutations in angiogenin, another putative angiogenic factor (Li and Hu, 2010). In the healthy adult, cerebral vessels are quiescent and constitute a guardian for the CNS microenvironment. However, abnormal molecular regulation of vessel quiescence can lead to abnormal vessel growth, all or not accompanied with leakiness. In many cases, these lesions occur sporadically and their underlying molecular basis remains elusive. We will therefore highlight two prototypic monogenic hereditary cerebrovascular diseases characterized by vascular malformations for which novel molecular insight have been obtained, but Table 1 lists a more complete overview

of the known monogenic cerebrovascular anomalies. Human hereditary teleangiectasia (HHT), also known as Rendu-Osler-Weber disease, is an autosomal-dominant inherited vascular dysplasia causing arteriovenous malformations (AVMs) and teleangiectasias in the brain and other organs (Shovlin, 2010). Typical for AVMs are the presence of arteriovenous shunts without intervening capillary bed, and the presence of dilated LY294002 in vitro tortuous veins that, despite perfusion at arterial pressure, fail to become “arterialized” but maintain walls with venous molecular signature and appearance. Like CCMs (see below), they can cause neurological symptoms of varying severity and expressivity including headache,

focal neurological deficits, seizures, and hemorrhagic stroke. Autosomal dominant mutations have been identified in three genes—i.e., ENG encoding endoglin, ACVRL1 encoding isometheptene activin receptor-like kinase 1 (ALK1), and SMAD4 encoding SMAD4, all functioning in TGFβ signaling ( Pardali et al., 2010). The TGFβ pathway controls vessel wall stability and balances the angiogenic response during vascular remodeling. How haploinsufficient TGFβ signaling gives rise to vascular malformations is incompletely understood, though reduced mural cell coverage together with increased EC growth may cause vessel dilatation without accompanying maturation, resulting in deregulated vessel remodeling and formation of fast-flow arteriovenous shunts (Shovlin, 2010). A second hit, i.e., injury, induction of vessel growth, inflammation or hemodynamic overload, or other stimuli, is likely required to initiate focally a deregulated angiogenic response leading to AVM formation.

Calcium transients were calculated as ΔG/R = (G(t) – G0)/R (Yasuda et al., 2004), where G is the green fluorescent

signal of Oregon Green BAPTA-2 (G0 = baseline signal) and R is the red fluorescent signal of Alexa click here 633. CF stimulation (2 pulses; 50 ms interval) evoked complex spikes (Figures 8B and 8C) which were associated with widespread calcium transients that could be recorded throughout large parts of the dendritic tree (Figure 8D). To trigger excitability changes, we applied the local 50 Hz PF tetanization (weak protocol) as used in the triple-patch recordings. A first region of interest (ROI) for calcium measurements was chosen within a distance of ≤ 10 μm from the stimulus electrode. This ROI-1 represents the conditioned site. Additional ROIs were selected at greater distances, http://www.selleckchem.com/products/kpt-330.html with values determined relative to the center of ROI-1 (measured along the axis of the connecting dendritic branch). As shown in Figures 8E and 8F, local 50 Hz PF tetanization caused a pronounced calcium

transient in ROI-1, but not at two ROIs that were located at distances of 29.8 and 50.2 μm, respectively, from ROI-1 (Figure 8A). Following tetanization, CF-evoked calcium transients recorded at ROI-1 were enhanced, but calcium signals monitored at ROIs 2 and 3 were not (Figure 8D). On average, PF tetanization resulted in an increase in the peak amplitude and the area under the curve of calcium transients recorded at ROI-1 (peak: 130.5% ± 9.0%; p = 0.010; area: 165.7% ± 13.1%; p = 0.001; Terminal deoxynucleotidyl transferase n = 9; t = 10–15 min; Figures 8G–8I), but not at ROIs that were 30–60 μm away from ROI-1 (peak: 90.7% ± 5.8%; p = 0.020; area: 100.6% ± 8.1%; p = 0.925; n = 9; Figures 8G–8I). At

intermediate distances (10–30 μm), peak calcium transients were not significantly affected, while the area under the curve was increased (peak: 110.9% ± 11.0%; p = 0.366; area: 137.3% ± 13.8%; p = 0.049; n = 9; Figure 8I). Thus, consistent with the triple-patch recordings, the imaging data show that dendritic plasticity may be restricted to the activated areas of the dendritic tree. We have shown that synaptic or nonsynaptic stimulation protocols trigger plasticity of IE in the dendrites of cerebellar Purkinje cells. This amplification of dendritic signaling reflects downregulation of SK2 channel activity and can occur in a compartment-specific manner. Importantly, depolarizing current injections, nonsynaptic stimulations, enhance the amplitude of passively propagated Na+ spikes, a nonsynaptic response. This demonstrates that the underlying mechanism is an alteration of intrinsic Purkinje cell properties. The amplification of dendritic CF responses is likely to affect Purkinje cell output. CF signaling elicits widespread dendritic calcium transients, which, in PF-contacted spines, reach supralinear levels when PF and CF synapses are coactivated (Wang et al., 2000).

, 2013). In the case of BDNF, it is interesting to note that postsynaptic release of BDNF promotes the formation of perisomatic PV+ synapses in the cortex (Hong et al., 2008, Huang et al., 1999, Jiao et al., 2011 and Kohara et al., 2007). We therefore propose that BDNF signaling in the Selleck EPZ 6438 BA supports fear

extinction by increasing the number of perisomatic PV+ synapses around BA fear neurons, which is predicted to increase perisomatic inhibition (Gittis et al., 2011 and Kohara et al., 2007). A better understanding of the molecular mechanisms used by BDNF to increase PV+ perisomatic synapse numbers could lead to new therapeutic targets for the treatment of fear disorders. Though BDNF acts on many types of synapses, both inhibitory Vemurafenib and excitatory, it seems to use different signaling pathways within each type of synapse (Gottmann et al., 2009 and Matsumoto et al., 2006). It is therefore feasible that targets will be identified that specifically modulate the effect of BDNF on perisomatic inhibitory synapses. A potential role for inhibitory synapse plasticity in shaping patterns

of neural circuit activation has recently become more appreciated (Kullmann et al., 2012). Inhibitory interneurons can be highly interconnected, resulting in synchronized firing (Bartos et al., 2007), Galactosylceramidase and are in many brain regions outnumbered by excitatory neurons, with a single interneuron contacting as many as a 1,000 excitatory neurons (Miles et al., 1996). These traits make inhibitory interneurons seem ill-suited to exert finely targeted effects on individual excitatory neurons. The discovery of various forms of inhibitory synapse plasticity has made clear how inhibitory interneurons can specifically modulate the activation of individual target neurons (Kullmann et al., 2012). Perisomatic inhibitory synapses are especially well-positioned to enable this “personalized inhibition” by using their ability to suppress action potentials in the target neuron (Miles et al., 1996),

thereby functioning as a brake that keeps the excitatory “gas pedal” in check. If perisomatic synapses indeed participate in the fine-tuned sculpting of patterns of neural circuit activation, then they should be subjected to forms of target-specific plasticity so that two excitatory neurons receiving perisomatic synapses from the same cluster of interneurons can be differentially inhibited. Recently, target-specific properties have been reported for perisomatic PV+ synapses in the striatum (Gittis et al., 2011) and for perisomatic CCK+ synapses in the entorhinal cortex (Varga et al., 2010). Our study adds to the understanding of perisomatic synapse dynamics in three ways.

Moreover, the application of genome-wide tools to this problem will enable examination of DNA methylation patterns HSP mutation in a much wider pool of genes, which is currently lacking. Finally, chromatin immunoprecipitation (ChIP) procedures also allow detection

of methyl-DNA binding proteins and specific histone modifications at the level of these and other specific gene loci. Additionally, new molecular biological methods have emerged for identifying changes in DNA methylation at the single-cell and single-allele level. Bisulfite sequencing, considered the “gold standard” for assaying DNA methylation, provides single-nucleotide information about a cytosine’s methylation state. Global analysis of all DNA from a given brain region cannot distinguish between DNA methylation changes in different cell types Selleckchem BIBF-1120 (e.g., neurons versus glial cells, glutamatergic versus GABAergic cells, etc.), which is a current limitation. However, bacterial

subcloning of single pieces of DNA, which originate from single alleles within a single cell, allows isolation of DNA from single CNS cells. Thus, direct bisulfite sequencing combined with DNA subcloning enables quantitative interrogation of single-allele changes in methylation, at the single nucleotide level, in single cells from brain tissue ( Miller et al., 2010). Such an approach may be especially powerful for interrogating the sparsely encoded, environmentally induced neuronal changes that occur during learning and memory. Overall,

these recent and emerging techniques pave the way for substantive experimental interrogation of experience-driven epigenetic changes, potentially aiding in the identification of an epigenetic code, that underlie memory formation. The ultimate challenge for future studies will be to determine in a comprehensive fashion how DNA methylation and chromatin remodeling at the single-cell level is regulated and translated into changes in neural circuit function and behavior in the context of learning and memory. The MAPK cascade was first established as the prototypic regulator of cell division and differentiation in nonneuronal cells (Bading and Greenberg, 1991, English and Sweatt, 1996, Fiore et al., 1993 and Murphy et al., 1994). Lepirudin The prominent expression and activation of MAPKs in the mature nervous system, particularly in the hippocampus, prompted researchers to question the role of the MAPK cascade in terminally differentiated, nondividing neurons in the brain (Bading and Greenberg, 1991 and English and Sweatt, 1996). It was speculated that the cascade might have been co-opted in the mature nervous system to subserve synaptic plasticity and memory formation, thereby proposing a mechanism of molecular homology between cellular development and learning and memory (Atkins et al.

, 2006; Fernandez-Alfonso

and Ryan, 2008; Fredj and Burrone, 2009; Li et al., 2005). The use of a ratiometric indicator enabled us to perform baseline measurements, tests of bafilomycin action, release measurements, and indicator calibration sequentially on different sets of boutons (Figure 5A). To ensure that bafilomycin had diffused into the tissue and taken effect, we repeatedly tested reacidifiaction on a set of “sentinel” boutons that were not used for pool quantification (Figure 5B). After successful block of reacidification, saturating stimulation (200 + 1,200 APs) ensured that all release-competent vesicles along the axon had been released at least once, resulting in an increased G/R fluorescence ratio (the “recycling ratio”). this website NH4Cl was applied at the end of the experiment to obtain the “calibration ratio” (Gmax/R). To our surprise, chemical

alkalization GPCR Compound Library did not further increase the average G/R ratio in mature SC boutons, indicating that electrical stimulation had triggered complete turnover of essentially all vesicles ( Figure 5C). To validate our calibration approach, we employed an independent alkalization strategy using the protonophores nigericin (10 μM) and monensin (40 μM) in an external solution mimicking intracellular ion concentration and synaptic cleft pH ( Fernandez-Alfonso and Ryan, 2008). Recycling pool sizes obtained in these experiments were not different from NH4Cl calibration experiments (p = 0.84, data not shown). We therefore conclude that, within the limits of our technique, the recycling pool encompasses essentially all vesicles at mature SC boutons (104% ± 9%, n = 8 cells; Figure 5F). In a third set of experiments, we performed all steps of the alkaline trapping experiment on the same set of boutons. This strategy, which is standard for FGD2 dissociated culture, is not optimal for slice culture because reliable measurements could only be obtained from a small number of closely spaced SC boutons (4–10 versus 13–50 boutons/cell). Again, the relative size

of the recycling pool was close to maximal (89% ± 5%, n = 3 cells, p = 0.36) ( Figure S4). In immature hippocampal slice cultures (DIV 5–7), we found a significantly smaller recycling pool (65% ± 11%, p = 0.018) ( Figures 5D and 5F), indicating that the elimination of the resting pool is a developmental phenomenon. Synapses between dissociated hippocampal neurons had an even smaller recycling pool (45% ± 4%, p = 0.0009, Figures 5E and 5F) and recycling pool sizes of individual boutons were more variable (CV: 0.54 ± 0.04 versus 0.35 ± 0.07, p = 0.046). Differences in vesicle partitioning also explain why we found a size-dependence of the RF at SC synapses ( Figure 3) but not at boutons in dissociated culture (see above). Here, any clear dependency between total vesicle number and RF is likely obscured by the large and highly variable resting pool size ( Branco et al.

Unpaired t tests were used to compare the significance between the latencies in different genotypes. Locomotor activity was measured by scoring beam breaks in activity chambers (San Diego Instruments). Prior to open-field tests, animals were handled for 2 consecutive days. Standard rat cages were used as the novel open field for the mice tested. Locomotor activities were recorded LDN193189 for 1 hr and scored for both 5 min and 1 hr. Unpaired t tests were used to compare the significance in fine movements, ambulatory movements, and rearing between the different

genotypes. Mice were placed on a food-deprivation schedule to reduce their weight to 80%–85% of their baseline weight. They were fed for 2 hr with mouse chow in their home cages each day after training. Water was available at all times in the home cages. Training and testing took place in eight Med Associates operant chambers (21.6 cm length × 17.8 cm width × 12.7 cm height) housed in boxes with sound-attenuating walls. Each chamber was equipped with a food magazine, two retractable levers, one on each side of the magazine, and a 3 W, 24V house light mounted on the same wall, but above the food magazine. Bio-Serv 20 mg pellets from a dispenser into the magazine were used as reward. The software Med-PC-IV from Med Associates was used for equipment control and behavior recording. At the beginning of

each session, the house light was turned on and the lever inserted. At the end of Regorafenib each session, the light was turned off and the lever retracted. Mice were trained in an initial lever-press training consisting of 4 consecutive days of CRF, during which the mice received a pellet for each lever press. A session would end after 60 min

or after the mouse had collected 30 rewards, whichever came first. After CRF, mice were trained with RI schedules to generate habitual lever pressing (Dickinson et al., 1983). The training started with 2 days on RI 30 s, with a 0.1 probability of reward availability every 3 s contingent on lever press, and followed by 6 days on the 60 s interval schedule, PERK inhibitor with a 0.1 probability of reward availability every 6 s contingent on lever pressing. Repeated-measures ANOVA was used to compare lever press between the different genotypes. A specific satiety procedure was used for outcome devaluation. Mice were given unlimited access within a fixed duration to either the mouse chow to which they had been exposed in their home cages (nondevalued condition/control), or the purified pellets they normally earned during lever-press sessions (devalued condition). The mouse chow served as a control for overall level of satiety. This procedure controls the overall level of satiety and motivational state, while altering the current value of a specific reward. Immediately after 1 hr of unlimited exposure to the pellets or chow, the mice were subjected to a 5 min long probe test.

05%) or sulfarhodamine (0.01%) included in the recording electrode. To record tonic GABAergic currents, we voltage clamped cells at 0 mV in the presence of 5 μM GABA. The resting potential was determined in the current-clamp GW786034 nmr mode with zero holding current. Input resistance was also measured in current clamp from voltage changes due to current injections. To record transient GABA-evoked currents in dendrites and axon terminals of rod DBCs, we voltage clamped cells to the reversal potential for cations (0 mV), and currents

were evoked by 30 ms GABA puffs (100–300 μM) onto dendrites or their axon terminals. Immunostaining was performed essentially as described (Herrmann et al., 2010). To analyze light-dependent Chk inhibitor GABA immunostaining in retinal neurons, we exposed dark-adapted mice for 5 min to background illumination of varying intensities. Mice were sacrificed, and retinas were fixed and stained with a mixture of anti-GABA and anti-calbindin antibodies for 3 days. Following incubation with secondary antibodies for visualization of GABA and calbindin immunostaining in different color channels, flat-mounted retinas were analyzed by confocal microscopy. To quantify the light-dependent dynamics of GABA staining in horizontal cells,

we first selected a single optical section representing the outer plexiform layer and displaying the most intense calbindin immunostaining. We next measured the intensity of GABA immunostaining colocalizing with calbindin staining in the same section. We thank M.E. Burns for critically reading an earlier version of the manuscript. This work was supported by the NIH grants EY10336 (V.Y.A.),

MH073853 (M.C.), EY06671 (L.J.F.), EY014701 (M.A.M.), EY5722 (to Duke University), and RPB (M.A.M.). “
“Rodents move their large whiskers, also called facial vibrissae, through space to locate and identify objects (Carvell and Simons, 1990, Hutson and Masterton, 1986, Knutsen et al., 2006, Krupa et al., 2001 and O’Connor et al., 2010a). Conversely, whisker movements are guided by sensory feedback (Mitchinson et al., 2007 and Nguyen and Kleinfeld, 2005). These interactions between sensory and motor systems are crucial for haptic perception (Diamond et al., 2008, Gibson, 1962 and Wolpert et al., 1995). Sensorimotor integration in Endonuclease whisker-based somatosensation is mediated by brain structures that form a series of nested loops, at the levels of the brainstem, thalamus, and cerebral cortex (Diamond et al., 2008 and Kleinfeld et al., 1999). Little is known about the cellular architecture of these different loops. A prominent loop occurs at the level of the cerebral cortex (Aronoff et al., 2010, Chakrabarti and Alloway, 2006, Donoghue and Parham, 1983, Ferezou et al., 2007, Hoffer et al., 2003, Izraeli and Porter, 1995, Miyashita et al., 1994, Porter and White, 1983, Veinante and Deschênes, 2003, Vogt and Pandya, 1978, Welker et al., 1988 and White and DeAmicis, 1977).

Taken together, these data indicate that while the iPN contribution to the lateral horn IA response was abolished as a

result of mACT transection, there was an additional, highly significant gain of IA response in the vlpr neurons after mACT transection. This suggests that the vlpr response to IA stimulation is normally inhibited by iPN projections through the mACT. To test whether GABA release PD0325901 manufacturer mediates the observed inhibitory signals from the mACT onto the vlpr lateral horn neurons, we perturbed GABA synthesis from iPNs by introducing UAS-Gad1-RNAi in conjunction with UAS-Dicer2 into our imaging flies (Mz699-GAL4, UAS-GCaMP3) to knock down glutamic acid decarboxylase 1 (Gad1), the critical enzyme responsible for GABA biosynthesis ( Küppers et al., 2003). Immunostaining revealed no detectable GABA in 49 out of 51 Mz699+ neurons under the experimental condition ( Figure 3B; compared to control in Figure 3A). Although the Gad1 RNAi transgene was also expressed in Mz699+ vlpr

neurons, these neurons should be unaffected since they were not GABAergic ( Figure 1G). Control flies (no UAS-Gad1-RNAi) exhibited general elevation and a spatial pattern change of IA response in the lateral horn after mACT Crenolanib mouse transection ( Figure 3C2) compared with before ( Figure 3C1), as we have described ( Figure 2). However, Gad1 knockdown in iPNs resulted in a robust lateral horn IA response in intact flies, with a spatial pattern that resembled IA response after mACT transection ( Figure 3D1). Specifically, in intact Gad1 knockdown flies, IA robustly activated the ventral lateral horn near the vlpr dendrite entry site ( Figure 3D1, white arrow), a region that normally exhibited robust IA response only after transection in control flies.

mACT transection no longer resulted in significant spatial pattern changes, as shown by the representative images ( Figures 3D2 and 3D3) and by a higher correlation coefficient of spatial patterns before and after mACT transection compared with controls ( Figure 3E). Using ROIs defined by after-transection patterns to isolate vlpr responses, we found a statistically significant interaction between the fly genotype and mACT transection. Separate statistical tests on the ablation effect showed no statistically significant change the in Gad1 knockdown flies before and after mACT transection, in contrast to the increase of IA response in control animals after mACT transection ( Figure 3F). Together, these experiments indicate that GABAergic inhibition from the mACT is largely responsible for the suppression of IA responses of vlpr neurons under physiological conditions. The phenotypic similarity between mACT transection and Gad1 knockdown in Mz699+ neurons also suggests that Mz699+ neurons provide the major inhibitory input through the mACT to the lateral horn in our experimental context.