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BioRxiv : the Preprint Server For... Dec 2023Genetic analyses of Schizophrenia (SCZ) patients have identified thousands of risk factors. In silico protein-protein interaction (PPI) network analysis has provided...
Genetic analyses of Schizophrenia (SCZ) patients have identified thousands of risk factors. In silico protein-protein interaction (PPI) network analysis has provided strong evidence that disrupted PPI networks underlie SCZ pathogenesis. In this study, we performed PPI analysis of several SCZ risk factors in the rodent brain. Using endogenous antibody immunoprecipitations coupled to mass spectrometry (MS) analysis, we constructed a SCZ network comprising 1612 unique PPI with a 5% FDR. Over 90% of the PPI were novel, reflecting the lack of previous PPI MS studies in brain tissue. Our SCZ PPI network was enriched with known SCZ risk factors, which supports the hypothesis that an accumulation of disturbances in selected PPI networks underlies SCZ. We used Stable Isotope Labeling in Mammals (SILAM) to quantitate phencyclidine (PCP) perturbations in the SCZ network and found that PCP weakened most PPI but also led to some enhanced or new PPI. These findings demonstrate that quantitating PPI in perturbed biological states can reveal alterations to network biology.
PubMed: 38168169
DOI: 10.1101/2023.12.12.571320 -
Neuropharmacology Aug 2024Pharmacological approaches to induce N-methyl-d-aspartate receptor (NMDAR) hypofunction have been intensively used to understand the aetiology and pathophysiology of...
Pharmacological approaches to induce N-methyl-d-aspartate receptor (NMDAR) hypofunction have been intensively used to understand the aetiology and pathophysiology of schizophrenia. Yet, the precise cellular and molecular mechanisms that relate to brain network dysfunction remain largely unknown. Here, we used a set of complementary approaches to assess the functional network abnormalities present in male mice that underwent a 7-day subchronic phencyclidine (PCP 10 mg/kg, subcutaneously, once daily) treatment. Our data revealed that pharmacological intervention with PCP affected cognitive performance and auditory evoked gamma oscillations in the prefrontal cortex (PFC) mimicking endophenotypes of some schizophrenia patients. We further assessed PFC cellular function and identified altered neuronal intrinsic membrane properties, reduced parvalbumin (PV) immunostaining and diminished inhibition onto L5 PFC pyramidal cells. A decrease in the strength of optogenetically-evoked glutamatergic current at the ventral hippocampus to PFC synapse was also demonstrated, along with a weaker shunt of excitatory transmission by local PFC interneurons. On a macrocircuit level, functional ultrasound measurements indicated compromised functional connectivity within several brain regions particularly involving PFC and frontostriatal circuits. Herein, we reproduced a panel of schizophrenia endophenotypes induced by subchronic PCP application in mice. We further recapitulated electrophysiological signatures associated with schizophrenia and provided an anatomical reference to critical elements in the brain circuitry. Together, our findings contribute to a better understanding of the physiological underpinnings of deficits induced by subchronic NMDAR antagonist regimes and provide a test system for characterization of pharmacological compounds.
Topics: Animals; Prefrontal Cortex; Male; Phencyclidine; Receptors, N-Methyl-D-Aspartate; Mice; Disease Models, Animal; Schizophrenia; Mice, Inbred C57BL; Parvalbumins; Adaptation, Physiological; Pyramidal Cells; Gamma Rhythm; Hippocampus; Excitatory Amino Acid Antagonists
PubMed: 38685343
DOI: 10.1016/j.neuropharm.2024.109970 -
Frontiers in Pharmacology 2023Ketamine acts primarily by blocking the N-methyl-D-aspartate (NMDA) receptor at the phencyclidine site. The rapid antidepressant properties of ketamine were demonstrated...
Ketamine acts primarily by blocking the N-methyl-D-aspartate (NMDA) receptor at the phencyclidine site. The rapid antidepressant properties of ketamine were demonstrated in the clinic and several behavioral models of depression in rodents. We hypothesized that the normalization of abnormal activity of monoamine neurons in Wistar Kyoto (WKY) rats contributes to the rapid antidepressant effects of ketamine. A single administration of ketamine (10 mg/kg, i. p) or saline was administered to anesthetized WKY rats before electrophysiological recordings of dorsal raphe nucleus (DRN) serotonin (5-HT), locus coeruleus (LC) norepinephrine (NE) and ventral tegmental area (VTA) dopamine (DA) neuronal activity. Pyramidal neurons from the medial prefrontal cortex (mPFC) were also recorded before and after a ketamine injection. In the VTA, ketamine elicited a significant increase in the population activity of DA neurons. This enhancement was consistent with findings in other depression-like models in which such a decreased population activity was observed. In the LC, ketamine normalized increased NE neuron burst activity found in WKY rats. In the DRN, ketamine did not significantly reverse 5-HT neuronal activity in WKY rats, which is dampened compared to Wistar rats. Ketamine did not significantly alter the neuronal activity of mPFC pyramidal neurons. These findings demonstrate that ketamine normalized NE neuronal activity and enhanced DA neuronal activity in WKY rats, which may contribute to its rapid antidepressant effect.
PubMed: 38026921
DOI: 10.3389/fphar.2023.1276309 -
Drug Testing and Analysis Jan 20243-Methoxyeticyclidine (3-MeO-PCE), a phencyclidine-type substance, has a higher N-methyl-D-aspartate receptor binding affinity than phencyclidine and an involvement in...
In vitro and in vivo metabolism of 3-Methoxyeticyclidine in human liver microsomes, a zebrafish model, and two human urine samples based on liquid chromatography-high-resolution mass spectrometry.
3-Methoxyeticyclidine (3-MeO-PCE), a phencyclidine-type substance, has a higher N-methyl-D-aspartate receptor binding affinity than phencyclidine and an involvement in fatal intoxication cases. The aim of this study was to identify new biomarkers and biotransformation pathways for 3-MeO-PCE. In vitro models were established using zebrafish and human liver microsomes for analysis of the phases I and II metabolites of 3-MeO-PCE by liquid chromatography-high-resolution mass spectrometry. Urine samples of known 3-MeO-PCE consumers in forensic cases were then subjected to analysis. Overall, 14 metabolites were identified in zebrafish and human liver microsomes, allowing postulation of the following metabolic pathways: hydroxylation, O-demethylation, N-dealkylation, dehydrogenation, combination, and glucuronidation or sulfation. 3-MeO-PCE and three metabolites (M2, M3, and M6) were detected in urine. We recommended M2 (the hydroxylation product) as a potential biomarker for documenting 3-MeO-PCE intake in clinical and forensic cases.
Topics: Animals; Humans; Microsomes, Liver; Zebrafish; Phencyclidine; Tandem Mass Spectrometry; Chromatography, Liquid; Ketamine
PubMed: 37125436
DOI: 10.1002/dta.3488 -
Methods in Molecular Biology (Clifton,... 2024Phencyclidine (PCP), a dissociative anesthetic, is a commonly abused recreational drug. In the 1950s, initially tested as an intravenous anesthetic, PCP was discontinued...
Phencyclidine (PCP), a dissociative anesthetic, is a commonly abused recreational drug. In the 1950s, initially tested as an intravenous anesthetic, PCP was discontinued for clinical use due to its severe adverse effects. Since then, it has gained popularity as a recreational drug due to its ability to induce hallucinations and alter perception. PCP can be detected in urine, serum, or plasma by immunoassays and quantified and its presence confirmed by gas or liquid chromatography-mass spectrometry. In the method described here, a deuterated internal standard is added to the sample and the drug is extracted under alkaline conditions. Analysis is conducted using gas chromatography-mass spectrometry (GC-MS). Selected ion monitoring is used for quantitation of PCP.
Topics: Humans; Phencyclidine; Gas Chromatography-Mass Spectrometry; Mass Spectrometry; Substance-Related Disorders; Illicit Drugs
PubMed: 38036841
DOI: 10.1007/978-1-0716-3541-4_37 -
Journal of Analytical Toxicology Mar 2024Due to the increase in the use of novel psychoactive substances (NPS) and their overall prevalence, it is important to have effective and reliable screening technologies...
Due to the increase in the use of novel psychoactive substances (NPS) and their overall prevalence, it is important to have effective and reliable screening technologies to detect NPS in biological matrices. Enzyme-linked immunosorbent assays (ELISA) are among the most popular screening methods. To evaluate the effectiveness of ELISA for NPS detection, five subclasses of NPS (novel synthetic opioids, fentanyl analogs, stimulants, benzodiazepines and hallucinogens) were evaluated in whole blood for their cross-reactivity on commercially available ELISA kits. A variety of novel synthetic opioids were tested at concentrations of 1-80 ng/mL and 50-2000 ng/mL and demonstrated no cross-reactivity to a morphine ELISA plate at either concentration range. Fentanyl analogs were tested at concentrations ranging from 0.01 to 1 ng/mL and had cross-reactivities ranging from 8% to 178% on the fentanyl ELISA kit used. Both para-chloro fentanyl (178%) and acryl fentanyl (164%) showed cross-reactivities well above that of fentanyl. Novel stimulants were tested at concentrations of 0.5-40 ng/mL and 20-2,000 ng/mL. 4-Fluoroamphetamine was the only novel stimulant with cross-reactivity (3,354%) to the amphetamine ELISA plate. Novel benzodiazepines were tested at concentrations of 1-40 ng/mL on a benzodiazepine plate. Cross-reactivities ranged from 36.1% to 263%, with desalkylflurazepam having the highest cross-reactivity. Finally, novel hallucinogens were tested at concentrations of 0.5-10 ng/mL on a phencyclidine (PCP) ELISA plate, which produced no cross-reactivity and then with 10-1,000 ng/mL, which gave results from 56.6% to 151%. Both hydroxy-PCP (151%) and chloro-PCP (137%) showed cross-reactivities above that of PCP. This research has demonstrated the utility of using ELISA-based screening for novel benzodiazepines, hallucinogens and for fentanyl analogs; however, there is limited application and risk of false-negative results for the other drug classes due to low or non-existent cross-reactivities.
Topics: Humans; Hallucinogens; Enzyme-Linked Immunosorbent Assay; Analgesics, Opioid; Fentanyl; Amphetamine; Central Nervous System Stimulants; Benzodiazepines; Substance Abuse Detection
PubMed: 38456706
DOI: 10.1093/jat/bkae017 -
Experimental Brain Research May 2024Hypofunctioning of NMDA receptors, and the resulting shift in the balance between excitation and inhibition, is considered a key process in the pathophysiology of...
Hypofunctioning of NMDA receptors, and the resulting shift in the balance between excitation and inhibition, is considered a key process in the pathophysiology of schizophrenia. One important manifestation of this phenomenon is changes in neural oscillations, those above 30 Hz (i.e., gamma-band oscillations), in particular. Although both preclinical and clinical studies observed increased gamma activity following acute administration of NMDA receptor antagonists, the relevance of this phenomenon has been recently questioned given the reduced gamma oscillations typically observed during sensory and cognitive tasks in schizophrenia. However, there is emerging, yet contradictory, evidence for increased spontaneous gamma-band activity (i.e., at rest or under baseline conditions). Here, we use the sub-chronic phencyclidine (PCP) rat model for schizophrenia, which has been argued to model the pathophysiology of schizophrenia more closely than acute NMDA antagonism, to investigate gamma oscillations (30-100 Hz) in the medial prefrontal cortex of anesthetized animals. While baseline gamma oscillations were not affected, oscillations induced by train stimulation of the posterior dorsal CA1 (pdCA1) field of the hippocampus were enhanced in PCP-treated animals (5 mg/kg, twice daily for 7 days, followed by a 7-day washout period). This effect was reversed by pharmacological enhancement of endocannabinoid levels via systemic administration of URB597 (0.3 mg/kg), an inhibitor of the catabolic enzyme of the endocannabinoid anandamide. Intriguingly, the pharmacological blockade of CB receptors by AM251 unmasked a reduced gamma oscillatory activity in PCP-treated animals. The findings are consistent with the observed effects of URB597 and AM251 on behavioral deficits reminiscent of the symptoms of schizophrenia and further validate the potential for cannabinoid-based drugs as a treatment for schizophrenia.
Topics: Animals; Male; Rats; Amidohydrolases; Arachidonic Acids; Benzamides; Carbamates; Disease Models, Animal; Endocannabinoids; Excitatory Amino Acid Antagonists; Gamma Rhythm; Phencyclidine; Piperidines; Polyunsaturated Alkamides; Prefrontal Cortex; Pyrazoles; Rats, Sprague-Dawley; Schizophrenia
PubMed: 38489023
DOI: 10.1007/s00221-024-06801-2 -
CNS Neuroscience & Therapeutics Apr 2024We aimed to evaluate the potential of a novel selective α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor (AMPAR) potentiator, LT-102, in treating...
Novel α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor (AMPAR) potentiator LT-102: A promising therapeutic agent for treating cognitive impairment associated with schizophrenia.
AIMS
We aimed to evaluate the potential of a novel selective α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor (AMPAR) potentiator, LT-102, in treating cognitive impairments associated with schizophrenia (CIAS) and elucidating its mechanism of action.
METHODS
The activity of LT-102 was examined by Ca influx assays and patch-clamp in rat primary hippocampal neurons. The structure of the complex was determined by X-ray crystallography. The selectivity of LT-102 was evaluated by hERG tail current recording and kinase-inhibition assays. The electrophysiological characterization of LT-102 was characterized by patch-clamp recording in mouse hippocampal slices. The expression and phosphorylation levels of proteins were examined by Western blotting. Cognitive function was assessed using the Morris water maze and novel object recognition tests.
RESULTS
LT-102 is a novel and selective AMPAR potentiator with little agonistic effect, which binds to the allosteric site formed by the intradimer interface of AMPAR's GluA2 subunit. Treatment with LT-102 facilitated long-term potentiation in mouse hippocampal slices and reversed cognitive deficits in a phencyclidine-induced mouse model. Additionally, LT-102 treatment increased the protein level of brain-derived neurotrophic factor and the phosphorylation of GluA1 in primary neurons and hippocampal tissues.
CONCLUSION
We conclude that LT-102 ameliorates cognitive impairments in a phencyclidine-induced model of schizophrenia by enhancing synaptic function, which could make it a potential therapeutic candidate for CIAS.
Topics: Animals; Mice; Rats; Phencyclidine; Schizophrenia; Cognitive Dysfunction; Isoxazoles; Propionates
PubMed: 38615362
DOI: 10.1111/cns.14713 -
Journal of Analytical Toxicology Mar 2024Current guidelines recommend universal screening for substance use disorders in obstetric patients, and neonatal drug testing is also frequently performed. Meconium is...
Current guidelines recommend universal screening for substance use disorders in obstetric patients, and neonatal drug testing is also frequently performed. Meconium is often the preferred specimen type to detect neonatal drug exposure due to a longer window of detection compared to urine, but most laboratories send out meconium testing to specialized reference laboratories, which can delay results for several days or more. Here, we evaluate a rapid and definitive liquid chromatography-tandem mass spectrometry method for neonatal urine drug testing and compare results obtained using this method to paired meconium drug testing in 1,424 neonates for amphetamines, cocaine, cannabinoids, opiates, oxycodone and phencyclidine. Urine testing showed equivalent sensitivity to current meconium methods for detecting in utero exposure to amphetamines and cocaine.
Topics: Infant, Newborn; Female; Pregnancy; Humans; Methamphetamine; Meconium; Substance Abuse Detection; Cocaine; Body Fluids
PubMed: 37952088
DOI: 10.1093/jat/bkad085 -
Molecular Neurobiology Feb 2024The pathogenesis of schizophrenia begins in early neurodevelopment and leads to excitatory-inhibitory imbalance. It is therefore essential that preclinical models used...
The pathogenesis of schizophrenia begins in early neurodevelopment and leads to excitatory-inhibitory imbalance. It is therefore essential that preclinical models used to understand disease, select drug targets and evaluate novel therapeutics encompass similar neurochemical deficits. One approach to improved preclinical modelling incorporates dual-hit neurodevelopmental insults, like neonatal administration of phencyclidine (PCP, to disrupt development of glutamatergic circuitry) then post-weaning isolation (Iso, to mimic adolescent social stress). We recently showed that male Lister-hooded rats exposed to PCP-Iso exhibit reduced hippocampal expression of the GABA interneuron marker calbindin. The current study expanded on this by investigating changes to additional populations of GABAergic interneurons in frontal cortical and hippocampal tissue from the same animals (by immunohistochemistry) as well as levels of GABA itself (via ELISA). Because inflammatory changes are also implicated in schizophrenia, we performed additional immunohistochemical evaluations of Iba-1 positive microglia as well as ELISA analysis of IL-6 in the same brain regions. Single-hit isolation-reared and dual-hit PCP-Iso rats both showed reduced parvalbumin immunoreactivity in the prelimbic/infralimbic region of the frontal cortex. However, this was more widespread in PCP-Iso, extending to the medial/ventral and lateral/dorsolateral orbitofrontal cortices. Loss of GABAergic markers was accompanied by increased microglial activation in the medial/ventral orbitofrontal cortices of PCP-Iso, together with frontal cortical IL-6 elevations not seen following single-hit isolation rearing. These findings enhance the face validity of PCP-Iso, and we advocate the use of this preclinical model for future evaluation of novel therapeutics-especially those designed to normalise excitatory-inhibitory imbalance or reduce neuroinflammation.
PubMed: 38363536
DOI: 10.1007/s12035-024-03987-y