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Neuronal activity of LMO4-deficient Sim1 neurons is reduced. A, GFP-positive neurons of the PVH (dashed oval, area recorded). Scale bar, 100 m. B, Representative traces showing spontaneous firing. C, Lmo4 KO neurons have a lower spontaneous firing frequency than their littermate controls (WT). D, Representative traces show low-threshold spiking (LTS) in Lmo4 KO and littermate control (WT) neurons of the caudal PVH. LTS, containing 2– 4 action potentials at 60 Hz, were elicited by injecting negative current (1 s) to hyperpolarize the membrane from 50 mV (holding potential) to 90 mV. Enlarged (200 ms) traces (inset) are shown. E, Percentage of the Sim1 neurons (WT, n 19 from 7 mice; KO, n 26 from 9 mice) showing LTS. F, Representative traces showing neuronal activity in response to depolarizing currents (20, 40, and 60 pA) from 50 mV of holding potential. G, Tonic spike frequency in response to various depolarizing currents presented as mean SEM, F (1,9) 64.90; two-way ANOVA: *p 0.05; **p 0.01; ***p 0.001.
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The dramatic increase in the prevalence of obesity reflects a lack of progress in combating one of the most serious health problems of this century. Recent studies have improved our understanding of the appetitive network by focusing on the paraventricular hypothalamus (PVH), a key region responsible for the homeostatic balance of food intake. Here...
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... patch-clamp recording, we compared neuronal activity in Sim1 neurons of the caudal PVH in Lmo4 KO and littermate control mice. To visualize Sim1 neurons, Sim1Cre/Lmo4 flox/flox mice were bred with ROSA26-EGFP mice allowing enhanced GFP expression in Sim1 neurons that express Cre-recom- binase (Fig. 3A). Spontaneous firing was much reduced in Lmo4 KO mice compared with littermate controls (Fig. 3 B, C), despite similar resting membrane potentials (Table 1). Re- duced spontaneous firing could result from increased inhibi- tory inputs, reduced excitatory inputs, or a cell-intrinsic reduction in excitability resulting from altered ...
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... activity in Sim1 neurons of the caudal PVH in Lmo4 KO and littermate control mice. To visualize Sim1 neurons, Sim1Cre/Lmo4 flox/flox mice were bred with ROSA26-EGFP mice allowing enhanced GFP expression in Sim1 neurons that express Cre-recom- binase (Fig. 3A). Spontaneous firing was much reduced in Lmo4 KO mice compared with littermate controls (Fig. 3 B, C), despite similar resting membrane potentials (Table 1). Re- duced spontaneous firing could result from increased inhibi- tory inputs, reduced excitatory inputs, or a cell-intrinsic reduction in excitability resulting from altered expression of membrane channels. Membrane resistance was significantly ele- vated (by 50%) in Lmo4-ablated ...
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... determine whether calcium currents are affected in Suzuki and Rogawski, 1989). LTS was detected in 78.5% of littermate control PVH neurons (n 15 of 19), whereas 21% (n 4 of 19) of the cells exhibited no LTS (Fig. 3D,E), as reported previously for parvocellular neurons of the caudal PVH ( Luther et al., 2002). In contrast, only 12% of GFP-positive LMO4-deficient neurons (n 3 of 26) exhibited LTS (Fig. ...
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... Suzuki and Rogawski, 1989). LTS was detected in 78.5% of littermate control PVH neurons (n 15 of 19), whereas 21% (n 4 of 19) of the cells exhibited no LTS (Fig. 3D,E), as reported previously for parvocellular neurons of the caudal PVH ( Luther et al., 2002). In contrast, only 12% of GFP-positive LMO4-deficient neurons (n 3 of 26) exhibited LTS (Fig. ...
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... firing was induced by injecting depolarizing currents in 10 pA stepwise increments (10 steps, 1 s duration) from a holding potential of 50 mV. Increased tonic spike frequency was observed with each increment of injected current. How- ever, a much lower response was observed in Lmo4 KO neurons (Fig. 3 F, G). Thus, ablation of Lmo4 in PVH neurons likely decreases the expression of both low-and high-voltage-gated calcium channels, and these would reduce spontaneous firing and cell excitability as we observed in Lmo4-ablated PVH ...
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... 2 channels play a critical role in neuronal excitability ( Perez-Reyes, 2003). The markedly reduced LTS we observed in LMO4-deficient PVH neurons (Fig. 3 D, E) led us to exam- ine the status of LVA Ca 2 currents. We measured LVA cur- rents in Cs -based whole-cell patch clamping as described previously ( Sun et al., 2001). Neurons were held at 100 mV (1 s) and depolarized to 40 mV, below the activation thresh- old for HVA Ca 2 channels. This depolarization step induces a fast-inactivating ...
Citations
... Functionally, LMO4 is involved in the regulation of Ca 2+ fluxes as it activates ryanodine receptor 2 (RyR2) expression [187]. Other Ca 2+ channels in the hypothalamus are also under the control of LMO4, thereby regulating neuronal excitability [188]. On the other hand, LMO4 can itself be regulated by Ca 2+ , indicating a circular relationship [189]. ...
... Interestingly, LMO4 has also been associated with dopamine D2 receptor signaling in the amygdala, where it contributes to cue-reward learning [192]. LMO4 s generally protective or homeostatic nature in the CNS is underlined by its reduced expression in Alzheimer's brains [193] and its obesity-offsetting role through the maintenance of central leptin sensitivity and food intake control via the regulation of Ca 2+ channels in the hypothalamus [188,194]. LMO4 expression is developmentally regulated in murine brains and is expressed early on during CNS development [184,193,195]. Mice with germline ablation of LMO4 die before birth and show defects in neural tube closure and skeletal patterning [183]. ...
Mast cells are evolutionarily old cells and the principal effectors in allergic responses and inflammation. They are seeded from the yolk sac during embryogenesis or are derived from hematopoietic progenitors and are therefore related to other leukocyte subsets, even though they form a separate clade in the hematopoietic system. Herein, we systematically bundle information from several recent high-throughput endeavors, especially those comparing MCs with other cell types, and combine such information with knowledge on the genes’ functions to reveal groups of neuronal markers specifically expressed by MCs. We focus on recent advances made regarding human tissue MCs, but also refer to studies in mice. In broad terms, genes hyper-expressed in MCs, but largely inactive in other myelocytes, can be classified into subcategories such as traffic/lysosomes (MLPH and RAB27B), the dopamine system (MAOB, DRD2, SLC6A3, and SLC18A2), Ca2+-related entities (CALB2), adhesion molecules (L1CAM and NTM) and, as an overall principle, the transcription factors and modulators of transcriptional activity (LMO4, PBX1, MEIS2, and EHMT2). Their function in MCs is generally unknown but may tentatively be deduced by comparison with other systems. MCs share functions with the nervous system, as they express typical neurotransmitters (histamine and serotonin) and a degranulation machinery that shares features with the neuronal apparatus at the synapse. Therefore, selective overlaps are plausible, and they further highlight the uniqueness of MCs within the myeloid system, as well as when compared with basophils. Apart from investigating their functional implications in MCs, a key question is whether their expression in the lineage is due to the specific reactivation of genes normally silenced in leukocytes or whether the genes are not switched off during mastocytic development from early progenitors.
... Our results are consistent with physiological observations that LMO4 knockdown has differential effects on neuronal firing in different brain regions. For example, knockdown of LMO4 in singleminded-one neurons in the hypothalamus reduces excitability [40], whereas knockdown in BLA pyramidal neurons increases excitability [18]. How increased excitability in BLA pyramidal neurons reduces drinking and whether LMO4 affects neuronal excitability in the NAc should be determined in future studies. ...
Repeated alcohol exposure leads to changes in gene expression that are thought to underlie the transition from moderate to excessive drinking. However, the mechanisms by which these changes are integrated into a maladaptive response that leads to alcohol dependence are not well understood. One mechanism could involve the recruitment of transcriptional co-regulators that bind and modulate the activity of transcription factors. Our results indicate that the transcriptional regulator LMO4 is one such candidate regulator. Lmo4-deficient mice (Lmo4gt/+) consumed significantly more and showed enhanced preference for alcohol in a 24 h intermittent access drinking procedure. shRNA-mediated knockdown of Lmo4 in the nucleus accumbens enhanced alcohol consumption, whereas knockdown in the basolateral amygdala (BLA) decreased alcohol consumption and reduced conditioned place preference for alcohol. To ascertain the molecular mechanisms that underlie these contrasting phenotypes, we carried out unbiased transcriptome profiling of these two brain regions in wild type and Lmo4gt/+ mice. Our results revealed that the transcriptional targets of LMO4 are vastly different between the two brain regions, which may explain the divergent phenotypes observed upon Lmo4 knockdown. Bioinformatic analyses revealed that Oprk1 and genes related to the extracellular matrix (ECM) are important transcriptional targets of LMO4 in the BLA. Chromatin immunoprecipitation revealed that LMO4 bound Oprk1 promoter elements. Consistent with these results, disruption of the ECM or infusion of norbinaltorphimine, a selective kappa opioid receptor antagonist, in the BLA reduced alcohol consumption. Hence our results indicate that an LMO4-regulated transcriptional network regulates alcohol consumption in the BLA.
... Based on Trodusquemine's effect to improve cognitive function (Qin et al., 2020;Qin et al., 2015;Ricke et al., 2020), a sample size of 11 mice/group provides 90% power to detect a 30% difference at p < 0.05 in a 2-tailed t-test. For patch-clamp recording studies, ~n = 8 cells/ group from 4 to 8 mice/group provide similar power (Qin et al., 2020;Qin et al., 2015;Zaman et al., 2014;Zhang et al., 2020). ...
Mutations in the beta-amyloid protein (APP) cause familial Alzheimer's disease. In hAPP-J20 mice expressing mutant APP, pharmacological inhibition or genetic ablation of the tyrosine phosphatase PTP1B prevents CA3 hippocampus neuron loss and cognitive decline. However, how targeting PTP1B affects the cellular mechanisms underlying these cognitive deficits remains unknown. Changes in synaptic strength at the hippocampus can affect information processing for learning and memory. While prior studies have focused on post-synaptic mechanisms to account for synaptic deficits in Alzheimer's disease models, presynaptic mechanisms may also be affected. Here, using whole cell patch-clamp recording, coefficient of variation (CV) analysis suggested a profound presynaptic deficit in long-term potentiation (LTP) of CA3:CA1 synapses in hAPP-J20 mice. While the membrane-impermeable ionotropic NMDA receptor (NMDAR) blocker norketamine in the post-synaptic recording electrode had no effect on LTP, additional bath application of the ionotropic NMDAR blockers MK801 could replicate the deficit in LTP in wild type mice. In contrast to LTP, the paired-pulse ratio and short-term facilitation (STF) were aberrantly increased in hAPP-J20 mice. These synaptic deficits in hAPP-J20 mice were associated with reduced phosphorylation of NMDAR GluN2B and the synaptic vesicle recycling protein NSF (N-ethylmaleimide sensitive factor). Phosphorylation of both proteins, together with synaptic plasticity and cognitive function, were restored by PTP1B ablation or inhibition by the PTP1B-selective inhibitor Trodusquemine. Taken together, our results indicate that PTP1B impairs presynaptic NMDAR-mediated synaptic plasticity required for spatial learning in a mouse model of Alzheimer's disease. Since Trodusquemine has undergone phase 1/2 clinical trials to treat obesity, it could be repurposed to treat Alzheimer's disease.
... Coronal brain sections (300 μm) containing PFC were prepared from 5 to 7-week-old mice as described (Qin et al., 2012;Qin et al., 2015b;Zaman et al., 2014). Mice were sacrificed 30 min after a single intraperitoneal administration of ketamine (50 mg/kg) and brain slices were prepared in ACSF and acclimated at room temperature prior to patchclamp recording over a 2-h window. ...
Subanesthetic doses of ketamine induce schizophrenia-like behaviors in mice including hyperlocomotion and deficits in working memory and sensorimotor gating. Here, we examined the effect of in vivo ketamine administration on neuronal properties and endocannabinoid (eCB)-dependent modulation of synaptic transmission onto layer 2/3 pyramidal neurons in brain slices of the prefrontal cortex, a region tied to the schizophrenia-like behavioral phenotypes of ketamine. Since deficits in working memory and sensorimotor gating are tied to activation of the tyrosine phosphatase PTP1B in glutamatergic neurons, we asked whether PTP1B contributes to these effects of ketamine. Ketamine increased membrane resistance and excitability of pyramidal neurons. Systemic pharmacological inhibition of PTP1B by Trodusquemine restored these neuronal properties and prevented each of the three main ketamine-induced behavior deficits. Ketamine also reduced mobilization of eCB by pyramidal neurons, while unexpectedly reducing their inhibitory inputs, and these effects of ketamine were blocked or occluded by PTP1B ablation in glutamatergic neurons. While ablation of PTP1B in glutamatergic neurons prevented ketamine-induced deficits in memory and sensorimotor gating, it failed to prevent hyperlocomotion (a psychosis-like phenotype). Taken together, these results suggest that PTP1B in glutamatergic neurons mediates ketamine-induced deficits in eCB mobilization, memory and sensorimotor gating whereas PTP1B in other cell types contributes to hyperlocomotion. Our study suggests that the PTP1B inhibitor Trodusquemine may represent a new class of fast-acting antipsychotic drugs to treat schizophrenia-like symptoms.
... PTP1B protein levels were quantified using ImageJ software. Three days after the last behavior test, mice were sacrificed and whole brain cryostat sections (20 μm) were subjected to immunofluorescence, as described (Chen et al., 2007a;Zaman et al., 2014). Immunofluorescence images were acquired on a Zeiss Z1 fluorescent microscope (Zeiss, North York, ON, Canada). ...
Ischemic brain injury causes neuronal death and inflammation. Inflammation activates protein-tyrosine phosphatase 1B (PTP1B). Here, we tested the significance of PTP1B activation in glutamatergic projection neurons on functional recovery in two models of stroke: by photothrombosis, focal ischemic lesions were induced in the sensorimotor cortex (SM stroke) or in the peri-prefrontal cortex (peri-PFC stroke). Elevated PTP1B expression was detected at 4 days and up to 6 weeks after stroke. While ablation of PTP1B in neurons of neuronal knockout (NKO) mice had no effect on the volume or resorption of ischemic lesions, markedly different effects on functional recovery were observed. SM stroke caused severe sensory and motor deficits (adhesive removal test) in wild type and NKO mice at 4 days, but NKO mice showed drastically improved sensory and motor functional recovery at 8 days. In addition, peri-PFC stroke caused anxiety-like behaviors (elevated plus maze and open field tests), and depression-like behaviors (forced swimming and tail suspension tests) in wild type mice 9 and 28 days after stroke, respectively, with minimal effect on sensory and motor function. Peri-PFC stroke-induced affective disorders were associated with fewer active (FosB +) neurons in the PFC and nucleus accumbens but more FosB + neurons in the basolateral amygdala, compared to sham-operated mice. In contrast, mice with neuronal ablation of PTP1B were protected from anxiety-like and depression-like behaviors and showed no change in FosB + neurons after peri-PFC stroke. Taken together, our study identifies neuronal PTP1B as a key component that hinders sensory and motor functional recovery and also contributes to the development of anxiety-like and depression-like behaviors after stroke. Thus, PTP1B may represent a novel therapeutic target to improve stroke recovery. All procedures for animal use were approved by the Animal Care and Use
... PTP1B protein levels were quantified using ImageJ software. Three days after the last behavior test, mice were sacrificed and whole brain cryostat sections (20 μm) were subjected to immunofluorescence, as described (Chen et al., 2007a;Zaman et al., 2014). Immunofluorescence images were acquired on a Zeiss Z1 fluorescent microscope (Zeiss, North York, ON, Canada). ...
Ischemic brain injury causes neuronal death and inflammation. Inflammation activates protein-tyrosine phosphatase 1B (PTP1B). Here, we tested the significance of PTP1B activation in glutamatergic projection neurons on functional recovery in two models of stroke: by photothrombosis, focal ischemic lesions were induced in the sensorimotor cortex (SM stroke) or in the peri-prefrontal cortex (peri-PFC stroke). Elevated PTP1B expression was detected at 4 days and up to 6 weeks after stroke. While ablation of PTP1B in neurons of neuronal knockout (NKO) mice had no effect on the volume or resorption of ischemic lesions, markedly different effects on functional recovery were observed. SM stroke caused severe sensory and motor deficits (adhesive removal test) in wild type and NKO mice at 4 days, but NKO mice showed drastically improved sensory and motor functional recovery at 8 days. In addition, peri-PFC stroke caused anxiety-like behaviors (elevated plus maze and open field tests), and depression-like behaviors (forced swimming and tail suspension tests) in wild type mice 9 and 28 days after stroke, respectively, with minimal effect on sensory and motor function. Peri-PFC stroke-induced affective disorders were associated with fewer active (FosB +) neurons in the PFC and nucleus accumbens but more FosB + neurons in the basolateral amygdala, compared to sham-operated mice. In contrast, mice with neuronal ablation of PTP1B were protected from anxiety-like and depression-like behaviors and showed no change in FosB + neurons after peri-PFC stroke. Taken together, our study identifies neuronal PTP1B as a key component that hinders sensory and motor functional recovery and also contributes to the development of anxiety-like and depression-like behaviors after stroke. Thus, PTP1B may represent a novel therapeutic target to improve stroke recovery. All procedures for animal use were approved by the Animal Care and Use
... Our neuroanatomical data are consistent with those of previous studies that show that metabolic signals are sensed by interoceptive neurons of the ARC and ventromedial nuclei of the hypothalamus that send synaptic projections to the PVN. As a major hypothalamic nucleus of autonomic regulation, the PVN is the site at which metabolic signals are integrated to control feeding behavior, via projections to the rostral ventrolateral medulla and spinal cord (Dampney et al., 2002;Pyner, 2009;Cassaglia et al., 2014;Zaman et al., 2014). ...
The most common side effects of cisplatin chemotherapy are nausea and vomiting, and the overwhelming majority of research studies on the mechanism of cisplatin-induced nausea have been focused on the “vomiting center.” As a modulatory center of gastric motility, the roles of the hypothalamus in nausea and vomiting remain unclear. In the present study, we investigated the effects of exogenous orexin-A injected into the arcuate nucleus (ARC) on cisplatin-induced nausea and vomiting, and the possible underlying mechanism. Kaolin intake was calculated daily in cisplatin-treated and saline-treated rats. Gastric motility recording, injections into the ARC, and lesions of the paraventricular nucleus (PVN) were used to study the effects of orexin-A and the hypothalamic nucleus on disorders of gastrointestinal function in cisplatin-treated rats. The pathway from the ARC to the PVN was observed through Fluoro-Gold retrograde tracing. Furthermore, an NPY Y1 receptor antagonist was administered to explore the possible mechanisms involved in the effects of orexin-A in the ARC. We illustrated that exogenous orexin-A injected into the ARC reduced kaolin intake and promoted gastric motility in cisplatin-treated rats, and these effects could have been blocked by an ipsilateral PVN lesion or co-injected antagonist of orexin-A-SB334867. Additional results showed that orexin-A-activated neurons in the ARC communicated directly with other neurons in the PVN that express neuropeptide Y (NPY). Furthermore, activation of the downstream NPY pathway was required for the observed effects of orexin in the ARC on cisplatin-induced nausea and vomiting. These findings reveal a novel neurobiological circuit from the ARC to the PVN that might provide a potential target for the prevention and treatment of cisplatin-induced nausea and vomiting.
... 16,17 It is also essential for development of the central nervous system, mediates calcium dependent transcription in cortical neurons, and regulates calcium release and synoptic plasticity in neurons of hippocampus. 18 The role of LMO4 in either renal development or function is largely unknown. Our previous studies indicated that cisplatin-induced nitration of cochlear LMO4 is associated with a decrease in LMO4 protein levels 5 and downregulation of signal transducer and activator of transcription 3, 19 a downstream target of LMO4, and suggested that these changes facilitate ototoxicity in Wistar rats. ...
Cytotoxic effects of cisplatin occur primarily through apoptosis. Though several pro- and anti-apoptotic signaling molecules have been identified to play an important role in mediating the ototoxic, nephrotoxic, and neurotoxic side effects of cisplatin, the underlying mechanism is yet to be fully characterized. We reported that nitration of LIM domain-only 4 (LMO4), a transcriptional regulator, facilitates cochlear apoptosis in cisplatin-induced ototoxicity. However, its role in cisplatin-mediated nephrotoxicity and neurotoxicity is poorly understood. Therefore, HK2 and SH-SY5Y cells were used along with UBOC1 cells, to investigate the perturbations of LMO4 in cisplatin-induced cytotoxicity, in renal, neuronal, and auditory cells, respectively. Cisplatin induced an increase in the expression of active caspase-3, indicating cellular apoptosis, and increased the nitration of proteins, 24 h post treatment. Immunostaining with anti-nitrotyrosine and anti-LMO4 indicated that nitrotyrosine co-localized with LMO4 protein in cisplatin-treated cells. Immunoblotting with anti-LMO4 indicated that cisplatin induced a decrease in LMO4 protein levels. However, a corresponding decrease in LMO4 gene levels was not observed. Inhibition of protein nitration with SRI110, a peroxynitrite decomposition catalyst, attenuated cisplatin-induced downregulation of LMO4. More importantly, overexpression of LMO4 mitigated the cytotoxic effects of cisplatin in UBOC1 cells while a dose-dependent decrease in LMO4 protein strongly correlated with cell viability in UBOC1, HK2, and SH-SY5Y cells. Collectively, these findings suggested a potential role of LMO4 in facilitating the cytotoxic effects of cisplatin in auditory, renal, and neuronal cells.
... Brain sections (300 mm) containing amygdala were prepared from 3-to 5week-old mice as described previously Zaman et al., 2014) and in detail in Supplemental Experimental Procedures. ...
Highlights
•Mice with ablation of an endogenous PTP1B inhibitor LMO4 have anxiety
•PTP1B dephosphorylates mGluR5 and impairs mGluR5-mediated eCB production
•Glucocorticoids impair LMO4 palmitoylation and increase PTP1B activity
•PTP1B inhibition in the amygdala restores eCBs and relieves stress-induced anxiety
Summary
Collapse of endocannabinoid (eCB) signaling in the amygdala contributes to stress-induced anxiety, but the mechanisms of this effect remain unclear. eCB production is tied to the function of the glutamate receptor mGluR5, itself dependent on tyrosine phosphorylation. Herein, we identify a novel pathway linking eCB regulation of anxiety through phosphorylation of mGluR5. Mice lacking LMO4, an endogenous inhibitor of the tyrosine phosphatase PTP1B, display reduced mGluR5 phosphorylation, eCB signaling, and profound anxiety that is reversed by genetic or pharmacological suppression of amygdalar PTP1B. Chronically stressed mice exhibited elevated plasma corticosterone, decreased LMO4 palmitoylation, elevated PTP1B activity, reduced amygdalar eCB levels, and anxiety behaviors that were restored by PTP1B inhibition or by glucocorticoid receptor antagonism. Consistently, corticosterone decreased palmitoylation of LMO4 and its inhibition of PTP1B in neuronal cells. Collectively, these data reveal a stress-responsive corticosterone-LMO4-PTP1B-mGluR5 cascade that impairs amygdalar eCB signaling and contributes to the development of anxiety.
... Brain sections (300 mm) containing amygdala were prepared from 3-to 5week-old mice as described previously Zaman et al., 2014) and in detail in Supplemental Experimental Procedures. ...
Collapse of endocannabinoid (eCB) signaling in the amygdala contributes to stress-induced anxiety, but the mechanisms of this effect remain unclear. eCB production is tied to the function of the glutamate receptor mGluR5, itself dependent on tyrosine phosphorylation. Herein, we identify a novel pathway linking eCB regulation of anxiety through phosphorylation of mGluR5. Mice lacking LMO4, an endogenous inhibitor of the tyrosine phosphatase PTP1B, display reduced mGluR5 phosphorylation, eCB signaling, and profound anxiety that is reversed by genetic or pharmacological suppression of amygdalar PTP1B. Chronically stressed mice exhibited elevated plasma corticosterone, decreased LMO4 palmitoylation, elevated PTP1B activity, reduced amygdalar eCB levels, and anxiety behaviors that were restored by PTP1B inhibition or by glucocorticoid receptor antagonism. Consistently, corticosterone decreased palmitoylation of LMO4 and its inhibition of PTP1B in neuronal cells. Collectively, these data reveal a stress-responsive corticosterone-LMO4-PTP1B-mGluR5 cascade that impairs amygdalar eCB signaling and contributes to the development of anxiety. VIDEO ABSTRACT:
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