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1.
J Neurophysiol ; 131(3): 529-540, 2024 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-38323322

RESUMEN

Cortical electroencephalograms (EEGs) may help understanding of neuropsychiatric illness and new treatment mechanisms. The aperiodic component (1/f) of EEG power spectra is often treated as noise, but recent studies suggest that changes to the aperiodic exponent of power spectra may reflect changes in excitation/inhibition balance, a concept linked to antidepressant effects, epilepsy, autism, and other clinical conditions. One confound of previous studies is behavioral state, because factors associated with behavioral state other than excitation/inhibition ratio may alter EEG parameters. Thus, to test the robustness of the aperiodic exponent as a predictor of excitation/inhibition ratio, we analyzed video-EEG during active exploration in mice of both sexes during various pharmacological manipulations with the fitting oscillations and one over f (FOOOF) algorithm. We found that GABAA receptor (GABAAR)-positive allosteric modulators increased the aperiodic exponent, consistent with the hypothesis that an increased exponent signals enhanced cortical inhibition, but other drugs (ketamine and GABAAR antagonists at subconvulsive doses) did not follow the prediction. To tilt excitation/inhibition ratio more selectively toward excitation, we suppressed the activity of parvalbumin-positive interneurons with Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). Contrary to our expectations, circuit disinhibition with the DREADD increased the aperiodic exponent. We conclude that the aperiodic exponent of EEG power spectra does not yield a universally reliable marker of cortical excitation/inhibition ratio.NEW & NOTEWORTHY Neuropsychiatric illness may be associated with altered excitation/inhibition balance. A single electroencephalogram (EEG) parameter, the aperiodic exponent of power spectra, may predict the ratio between excitation and inhibition. Here, we use cortical EEGs in mice to evaluate this hypothesis, using pharmacological manipulations of known mechanism. We show that the aperiodic exponent of EEG power spectra is not a reliable marker of excitation/inhibition ratio. Thus, alternative markers of this ratio must be sought.


Asunto(s)
Electroencefalografía , Ketamina , Masculino , Femenino , Ratones , Animales , Receptores de GABA-A , Ketamina/farmacología , Ácido gamma-Aminobutírico
2.
Nature ; 540(7631): 74-79, 2016 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-27775718

RESUMEN

Mitochondria are dynamic organelles that exchange contents and undergo remodelling during cyclic fusion and fission. Genetic mutations in MFN2 (the gene encoding mitofusin 2) interrupt mitochondrial fusion and cause the untreatable neurodegenerative condition Charcot-Marie-Tooth disease type 2A (CMT2A). It has not yet been possible to directly modulate mitochondrial fusion, in part because the structural basis of mitofusin function is not completely understood. Here we show that mitofusins adopt either a fusion-constrained or a fusion-permissive molecular conformation, directed by specific intramolecular binding interactions, and demonstrate that mitofusin-dependent mitochondrial fusion can be regulated in mouse cells by targeting these conformational transitions. On the basis of this model, we engineered a cell-permeant minipeptide to destabilize the fusion-constrained conformation of mitofusin and promote the fusion-permissive conformation, reversing mitochondrial abnormalities in cultured fibroblasts and neurons that harbour CMT2A-associated genetic defects. The relationship between the conformational plasticity of mitofusin 2 and mitochondrial dynamism reveals a central mechanism that regulates mitochondrial fusion, the manipulation of which can correct mitochondrial pathology triggered by defective or imbalanced mitochondrial dynamics.


Asunto(s)
GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Dinámicas Mitocondriales/efectos de los fármacos , Péptidos/farmacología , Animales , Células Cultivadas , Enfermedad de Charcot-Marie-Tooth/genética , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , GTP Fosfohidrolasas/genética , Ratones , Mitocondrias/genética , Mitocondrias/patología , Dinámicas Mitocondriales/genética , Modelos Moleculares , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Péptidos/química , Permeabilidad , Conformación Proteica/efectos de los fármacos
3.
J Neurosci ; 38(38): 8128-8145, 2018 09 19.
Artículo en Inglés | MEDLINE | ID: mdl-30076210

RESUMEN

Two major GABAA receptor classes mediate ionotropic GABA signaling, those containing a δ subunit and those with a γ2 subunit. The classical viewpoint equates γ2-containing receptors with IPSCs and δ-containing receptors with tonic inhibition because of differences in receptor localization, but significant questions remain because the populations cannot be pharmacologically separated. We removed this barrier using gene editing to confer a point mutation on the δ subunit in mice, rendering receptors containing the subunit picrotoxin resistant. By pharmacologically isolating δ-containing receptors, our results demonstrate their contribution to IPSCs in dentate granule neurons and weaker contributions to thalamocortical IPSCs. Despite documented extrasynaptic localization, we found that receptor localization does not preclude participation in isolated IPSCs, including mIPSCs. Further, phasic inhibition from δ subunit-containing receptors strongly inhibited summation of EPSPs, whereas tonic activity had little impact. In addition to any role that δ-containing receptors may play in canonical tonic inhibition, our results highlight a previously underestimated contribution of δ-containing receptors to phasic inhibition.SIGNIFICANCE STATEMENT GABAA receptors play key roles in transient and tonic inhibition. The prevailing view suggests that synaptic γ2-containing GABAA receptors drive phasic inhibition, whereas extrasynaptic δ-containing receptors mediate tonic inhibition. To re-evaluate the impact of δ receptors, we took a chemogenetic approach that offers a sensitive method to probe the synaptic contribution of δ-containing receptors. Our results reveal that localization does not strongly limit the contribution of δ receptors to IPSCs and that δ receptors make an unanticipated robust contribution to phasic inhibition.


Asunto(s)
Giro Dentado/metabolismo , Neuronas/metabolismo , Receptores de GABA-A/metabolismo , Sinapsis/metabolismo , Animales , Giro Dentado/citología , Potenciales Postsinápticos Excitadores/fisiología , Edición Génica , Potenciales Postsinápticos Inhibidores/fisiología , Ratones , Inhibición Neural/fisiología , Neuronas/citología , Receptores de GABA-A/genética , Transmisión Sináptica/fisiología
4.
J Neurosci ; 38(13): 3218-3229, 2018 03 28.
Artículo en Inglés | MEDLINE | ID: mdl-29476014

RESUMEN

N-methyl-d-aspartate receptors (NMDARs) are ionotropic glutamate receptors important for synaptic plasticity, memory, and neuropsychiatric health. NMDAR hypofunction contributes to multiple disorders, including anti-NMDAR encephalitis (NMDARE), an autoimmune disease of the CNS associated with GluN1 antibody-mediated NMDAR internalization. Here we characterize the functional/pharmacological consequences of exposure to CSF from female human NMDARE patients on NMDAR function, and we characterize the effects of intervention with recently described positive allosteric modulators (PAMs) of NMDARs. Incubation (48 h) of rat hippocampal neurons of both sexes in confirmed NMDARE patient CSF, but not control CSF, attenuated NMDA-induced current. Residual NMDAR function was characterized by lack of change in channel open probability, indiscriminate loss of synaptic and extrasynaptic NMDARs, and indiscriminate loss of GluN2B-containing and GluN2B-lacking NMDARs. NMDARs tagged with N-terminal pHluorin fluorescence demonstrated loss of surface receptors. Thus, function of residual NMDARs following CSF exposure was indistinguishable from baseline, and deficits appear wholly accounted for by receptor loss. Coapplication of CSF and PAMs of NMDARs (SGE-301 or SGE-550, oxysterol-mimetic) for 24 h restored NMDAR function following 24 h incubation in patient CSF. Curiously, restoration of NMDAR function was observed despite washout of PAMs before electrophysiological recordings. Subsequent experiments suggested that residual allosteric potentiation of NMDAR function explained the persistent rescue. Further studies of the pathogenesis of NMDARE and intervention with PAMs may inform new treatments for NMDARE and other disorders associated with NMDAR hypofunction.SIGNIFICANCE STATEMENT Anti-N-methyl-d-aspartate receptor encephalitis (NMDARE) is increasingly recognized as an important cause of sudden-onset psychosis and other neuropsychiatric symptoms. Current treatment leaves unmet medical need. Here we demonstrate cellular evidence that newly identified positive allosteric modulators of NMDAR function may be a viable therapeutic strategy.


Asunto(s)
Encefalitis/líquido cefalorraquídeo , Enfermedad de Hashimoto/líquido cefalorraquídeo , Neurotransmisores/farmacología , Receptores de N-Metil-D-Aspartato/metabolismo , Potenciales Sinápticos/efectos de los fármacos , Adulto , Regulación Alostérica , Animales , Línea Celular , Células Cultivadas , Encefalitis/tratamiento farmacológico , Encefalitis/inmunología , Femenino , Enfermedad de Hashimoto/tratamiento farmacológico , Enfermedad de Hashimoto/inmunología , Humanos , Masculino , Ratones , Neurotransmisores/líquido cefalorraquídeo , Neurotransmisores/inmunología , Neurotransmisores/uso terapéutico , Transporte de Proteínas , Ratas , Receptores de N-Metil-D-Aspartato/inmunología
5.
J Neurophysiol ; 115(3): 1263-72, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26745248

RESUMEN

N-methyl-D-aspartate receptors (NMDARs), a major subtype of glutamate receptors mediating excitatory transmission throughout the central nervous system (CNS), play critical roles in governing brain function and cognition. Because NMDAR dysfunction contributes to the etiology of neurological and psychiatric disorders including stroke and schizophrenia, NMDAR modulators are potential drug candidates. Our group recently demonstrated that the major brain cholesterol metabolite, 24S-hydroxycholesterol (24S-HC), positively modulates NMDARs when exogenously administered. Here, we studied whether endogenous 24S-HC regulates NMDAR activity in hippocampal slices. In CYP46A1(-/-) (knockout; KO) slices where endogenous 24S-HC is greatly reduced, NMDAR tone, measured as NMDAR-to-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) excitatory postsynaptic current (EPSC) ratio, was reduced. This difference translated into more NMDAR-driven spiking in wild-type (WT) slices compared with KO slices. Application of SGE-301, a 24S-HC analog, had comparable potentiating effects on NMDAR EPSCs in both WT and KO slices, suggesting that endogenous 24S-HC does not saturate its NMDAR modulatory site in ex vivo slices. KO slices did not differ from WT slices in either spontaneous neurotransmission or in neuronal intrinsic excitability, and exhibited LTP indistinguishable from WT slices. However, KO slices exhibited higher resistance to persistent NMDAR-dependent depression of synaptic transmission induced by oxygen-glucose deprivation (OGD), an effect restored by SGE-301. Together, our results suggest that loss of positive NMDAR tone does not elicit compensatory changes in excitability or transmission, but it protects transmission against NMDAR-mediated dysfunction. We expect that manipulating this endogenous NMDAR modulator may offer new treatment strategies for neuropsychiatric dysfunction.


Asunto(s)
Potenciales Postsinápticos Excitadores , Hipocampo/metabolismo , Hidroxicolesteroles/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Colesterol 24-Hidroxilasa/genética , Colesterol 24-Hidroxilasa/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/fisiología , Hidroxicolesteroles/farmacología , Potenciación a Largo Plazo , Ratones , Ratones Endogámicos C57BL , Oxígeno/metabolismo
6.
J Neurosci ; 34(35): 11792-802, 2014 Aug 27.
Artículo en Inglés | MEDLINE | ID: mdl-25164674

RESUMEN

Few other neurotransmitters are of as intense interest to neuropsychiatry and neurology as dopamine, yet existing techniques to monitor dopamine release leave an important spatiotemporal gap in our understanding. Electrochemistry and fluorescence imaging tools have been developed to fill the gap, but these methods have important limitations. We circumvent these limitations by introducing a dopamine-gated chloride channel into rat dorsal striatal medium spiny neurons, targets of strong dopamine innervation, thereby transforming dopamine from a slow transmitter into a fast transmitter and revealing new opportunities for studying moment-to-moment regulation of dopamine release. We demonstrate pharmacological and biophysical properties of the channel that make it suitable for fast, local dopamine measurements, and we demonstrate for the first time spontaneous and evoked responses to vesicular dopamine release in the dorsal striatum. Evoked dopamine currents were separated into a fast, monosynaptic component and a slower-rising and decaying disynaptic component mediated by nicotinic receptor activation. In summary, LGC-53 represents a dopamine biosensor with properties suitable for temporal separation of distinct dopamine signals in targets of dopamine innervation.


Asunto(s)
Técnicas Biosensibles/métodos , Canales de Cloruro/metabolismo , Cuerpo Estriado/metabolismo , Dopamina/metabolismo , Canales Iónicos Activados por Ligandos/metabolismo , Neuronas/metabolismo , Animales , Caenorhabditis elegans , Línea Celular , Humanos , Técnicas de Placa-Clamp , Ratas , Xenopus
7.
bioRxiv ; 2024 Jun 14.
Artículo en Inglés | MEDLINE | ID: mdl-38915641

RESUMEN

In medial prefrontal cortex (mPFC), fast-spiking parvalbumin (PV) interneurons regulate excitability and microcircuit oscillatory activity important for cognition. Although PV interneurons inhibit pyramidal neurons, they themselves express δ subunits of GABAA receptors important for slow inhibition. However, the specific contribution of δ-containing GABAA receptors to the function of PV interneurons in mPFC is unclear. We explored cellular, synaptic, and local-circuit activity in PV interneurons and pyramidal neurons in mouse mPFC after selectively deleting δ subunits in PV interneurons (cKO mice). In current-clamp recordings, cKO PV interneurons exhibited a higher frequency of action potentials and higher input resistance than wild type (WT) PV interneurons. Picrotoxin increased firing and GABA decreased firing in WT PV interneurons but not in cKO PV interneurons. The δ-preferring agonist THIP reduced spontaneous inhibitory postsynaptic currents in WT pyramidal neurons but not in cKO pyramidal neurons. In WT slices, depolarizing the network with 400 nM kainate increased firing of pyramidal neurons but had little effect on PV interneuron firing. By contrast, in cKO slices kainate recruited PV interneurons at the expense of pyramidal neurons. At the population level, kainate induced broadband increases in local field potentials in WT but not cKO slices. These results on cells and the network can be understood through increased excitability of cKO PV interneurons. In summary, our study demonstrates that δ-containing GABAA receptors in mPFC PV interneurons play a crucial role in regulating their excitability and the phasic inhibition of pyramidal neurons, elucidating intricate mechanisms governing cortical circuitry. Significance statement: By selectively deleting δ-containing GABAA receptors in PV interneurons, we demonstrate the importance of these receptors on PV interneuron excitability, synaptic inhibition of pyramidal neurons, and circuit function.

8.
Sci Rep ; 14(1): 1042, 2024 01 10.
Artículo en Inglés | MEDLINE | ID: mdl-38200205

RESUMEN

Neuropsychiatric and neurodegenerative disorders are correlated with cellular stress. Macroautophagy (autophagy) may represent an important protective pathway to maintain cellular homeostasis and functionality, as it targets cytoplasmic components to lysosomes for degradation and recycling. Given recent evidence that some novel psychiatric treatments, such as the neuroactive steroid (NAS) allopregnanolone (AlloP, brexanolone), may induce autophagy, we stably transfected human embryonic kidney 293 (HEK) cells with a ratiometric fluorescent probe to assay NAS effects on autophagy. We hypothesized that NAS may modulate autophagy in part by the ability of uncharged NAS to readily permeate membranes. Microscopy revealed a weak effect of AlloP on autophagic flux compared with the positive control treatment of Torin1. In high-throughput microplate experiments, we found that autophagy induction was more robust in early passages of HEK cells. Despite limiting studies to early passages for maximum sensitivity, a range of NAS structures failed to reliably induce autophagy or interact with Torin1 or starvation effects. To probe NAS in a system where AlloP effects have been shown previously, we surveyed astrocytes and again saw minimal autophagy induction by AlloP. Combined with other published results, our results suggest that NAS may modulate autophagy in a cell-specific or context-specific manner. Although there is merit to cell lines as a screening tool, future studies may require assaying NAS in cells from brain regions involved in neuropsychiatric disorders.


Asunto(s)
Neuroesteroides , Humanos , Autofagia , Macroautofagia , Riñón , Lisosomas
9.
bioRxiv ; 2024 Mar 29.
Artículo en Inglés | MEDLINE | ID: mdl-38585911

RESUMEN

GABA A receptors containing δ subunits have been shown to mediate tonic/slow inhibition in the CNS. These receptors are typically found extrasynaptically and are activated by relatively low levels of ambient GABA in the extracellular space. In the mouse neocortex, δ subunits are expressed on the surface of some pyramidal cells as well as on parvalbumin positive (PV+) interneurons. An important function of PV+ interneurons is the organization of coordinated network activity that can be measured by EEG; however, it remains unclear what role tonic/slow inhibitory control of PV+ neurons may play in shaping oscillatory activity. After confirming a loss of functional δ mediated tonic currents in PV cells in cortical slices from mice lacking Gabrd in PV+ neurons (PV δcKO), we performed EEG recordings to survey network activity across wake and sleep states. PV δcKO mice showed altered spectral content of EEG during NREM and REM sleep that was a result of increased oscillatory activity in NREM and the emergence of transient high amplitude bursts of theta frequency activity during REM. Viral reintroduction of Gabrd to PV+ interneurons in PV δcKO mice rescued REM EEG phenotypes, supporting an important role for δ subunit mediated inhibition of PV+ interneurons for maintaining normal REM cortical oscillations. Significance statement: The impact on cortical EEG of inhibition on PV+ neurons was studied by deleting a GABA A receptor subunit selectively from these neurons. We discovered unexpected changes at low frequencies during sleep that were rescued by viral reintroduction.

10.
Biomolecules ; 14(3)2024 Feb 21.
Artículo en Inglés | MEDLINE | ID: mdl-38540675

RESUMEN

Brain cholesterol metabolic products include neurosteroids and oxysterols, which play important roles in cellular physiology. In neurons, the cholesterol oxidation product, 24S-hydroxycholesterol (24S-HC), is a regulator of signaling and transcription. Here, we examined the behavioral effects of 24S-HC loss, using global and cell-selective genetic deletion of the synthetic enzyme CYP46A1. Mice that are globally deficient in CYP46A1 exhibited hypoactivity at young ages and unexpected increases in conditioned fear memory. Despite strong reductions in hippocampal 24S-HC in mice with selective loss of CYP46A1 in VGLUT1-positive cells, behavioral effects were not recapitulated in these conditional knockout mice. Global knockout produced strong, developmentally dependent transcriptional effects on select cholesterol metabolism genes. These included paradoxical changes in Liver X Receptor targets. Again, conditional knockout was insufficient to recapitulate most changes. Overall, our results highlight the complex effects of 24S-HC in an in vivo setting that are not fully predicted by known mechanisms. The results also demonstrate that the complete inhibition of enzymatic activity may be needed for a detectable, therapeutically relevant impact on gene expression and behavior.


Asunto(s)
Colesterol , Hidroxicolesteroles , Ratones , Animales , Colesterol 24-Hidroxilasa/metabolismo , Hidroxicolesteroles/metabolismo , Colesterol/metabolismo , Hipocampo/metabolismo
11.
eNeuro ; 10(3)2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36725341

RESUMEN

Allopregnanolone (AlloP) is a neurosteroid that potentiates ionotropic GABAergic (GABAA) inhibition and is approved for treating postpartum depression in women. Although the antidepressant mechanism of AlloP is largely unknown, it could involve selective action at GABAA receptors containing the δ subunit. Despite previous evidence for selective effects of AlloP on α4/δ-containing receptors of hippocampal dentate granule cells (DGCs), other recent results failed to demonstrate selectivity at these receptors (Lu et al., 2020). In contrast to DGCs, hippocampal fast-spiking parvalbumin (PV) interneurons express an unusual variant partnership of δ subunits with α1 subunits. Here, we hypothesized that native α1/δ receptors in hippocampal fast-spiking interneurons may provide a preferred substrate for AlloP. Contrary to the hypothesis, electrophysiology from genetically tagged PV interneurons in hippocampal slices from male mice showed that 100 nm AlloP promoted phasic inhibition by increasing the sIPSC decay, but tonic inhibition was not detectably altered. Co-application of AlloP with 5 µm GABA did augment tonic current, which was not primarily through δ-containing receptors. Furthermore, AlloP decreased the membrane resistance and the number of action potentials of DGCs, but the impact on PV interneurons was weaker than on DGCs. Thus, our results indicate that hippocampal PV interneurons possess low sensitivity to AlloP and suggest they are unlikely contributors to mood-altering effects of neurosteroids through GABA effects.


Asunto(s)
Parvalbúminas , Pregnanolona , Ratones , Masculino , Femenino , Animales , Pregnanolona/farmacología , Parvalbúminas/metabolismo , Interneuronas/fisiología , Receptores de GABA-A/metabolismo , Hipocampo/metabolismo , Ácido gamma-Aminobutírico/farmacología
12.
bioRxiv ; 2023 Sep 22.
Artículo en Inglés | MEDLINE | ID: mdl-37790570

RESUMEN

Cortical electroencephalograms (EEG) may help understanding of neuropsychiatric illness and new treatment mechanisms. The aperiodic component (1/ f ) of EEG power spectra is often treated as noise, but recent studies suggest that changes to the aperiodic exponent of power spectra may reflect changes in excitation/inhibition (E/I) balance, a concept linked to antidepressant effects, epilepsy, autism, and other clinical conditions. One confound of previous studies is behavioral state, because factors associated with behavioral state other than E/I ratio may alter EEG parameters. Thus, to test the robustness of the aperiodic exponent as a predictor of E/I ratio, we analyzed active exploration in mice using video EEG following various pharmacological manipulations with the Fitting Oscillations & One Over F (FOOOF) algorithm. We found that GABA A receptor (GABA A R) positive allosteric modulators increased the aperiodic exponent, consistent with the hypothesis that an increased exponent signals enhanced cortical inhibition, but other drugs (ketamine and GABA A R antagonists at sub-convulsive doses) did not follow the prediction. To tilt E/I ratio more selectively toward excitation, we suppressed the activity of parvalbumin (PV) interneurons with Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). Contrary to our expectations and studies demonstrating increased cortical activity following PV suppression, circuit disinhibition with the DREADD increased the aperiodic exponent. We conclude that the aperiodic exponent of EEG power spectra does not yield a universally reliable marker of E/I ratio. Alternatively, the concept of E/I state may be sufficiently oversimplified that it cannot be mapped readily onto an EEG parameter. Significance StateBment: Neuropsychiatric illness is widely prevalent and debilitating. Causes are not well understood, but some hypotheses point toward altered excitation/inhibition (E/I) balance. Here, we use cortical electroencephalograms (EEG) in mice, given applicability of cortical EEG across species, and evaluate the impact of validated drugs, including anxiolytics (pentobarbital and diazepam), along with novel rapid-acting antidepressants (ketamine and allopregnanolone). We focus on analyzing the aperiodic component of EEG power spectra, which may be associated with changes in E/I ratio. We show that aperiodic exponent of EEG power spectra is not a reliable marker of E/I ratio. Moreover, the concept of E/I ratio may be too broad and complex to be defined by an EEG parameter.

13.
Neuropsychopharmacology ; 48(2): 371-379, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-36168047

RESUMEN

Neurosteroids that positively modulate GABAA receptors are among a growing list of rapidly acting antidepressants, including ketamine and psychedelics. To develop increasingly specific treatments with fewer side effects, we explored the possibility of EEG signatures in mice, which could serve as a cross-species screening tool. There are few studies of the impact of non-sedative doses of rapid antidepressants on EEG in either rodents or humans. Here we hypothesize that EEG features may separate a rapid antidepressant neurosteroid, allopregnanolone, from other GABAA positive modulators, pentobarbital and diazepam. Further, we compared the actions GABA modulators with those of ketamine, an NMDA antagonist and prototype rapid antidepressant. We examined EEG spectra during active exploration at two cortical locations and examined cross-regional and cross-frequency interactions. We found that at comparable doses, the effects of allopregnanolone, despite purported selectivity for certain GABAAR subtypes, was indistinguishable from pentobarbital during active waking exploration. The actions of diazepam had recognizable common features with allopregnanolone and pentobarbital but was also distinct, consistent with subunit selectivity of benzodiazepines. Finally, ketamine exhibited no distinguishing overlap with allopregnanolone in the parameters examined. Our results suggest that rapid antidepressants with different molecular substrates may remain separated at the level of large-scale ensemble activity, but the studies leave open the possibility of commonalities in more discrete circuits and/or in the context of a dysfunctional brain.


Asunto(s)
Ketamina , Neuroesteroides , Humanos , Ratones , Animales , Pregnanolona/farmacología , Ketamina/farmacología , Pentobarbital/farmacología , Receptores de GABA-A/fisiología , Diazepam/farmacología , Antidepresivos/farmacología , Ácido gamma-Aminobutírico , Electroencefalografía
14.
Biofactors ; 48(4): 897-907, 2022 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-35170815

RESUMEN

Uptake into intestinal cells and intracellular distribution into metabolically competent organelles, such as the endoplasmic reticulum, are important processes potentially limiting the bioavailability of xenobiotics. The incorporation of curcumin into polysorbate 80 micelles improves its naturally low oral bioavailability in humans. Here, we investigated uptake and time-dependent localization of curcumin in intestinal cells when administered as native or micellar formulation. Differentiated Caco-2 cells were incubated with 200 µmol/L native or micellar curcumin for up to 180 min and cellular uptake was quantified. Intracellular curcumin was detected already after 30 min and did not differ significantly between formulations or over time. Subcellular localization of native and micellar curcumin in Caco-2 cells was studied by density gradient centrifugation. After 30 min, curcumin from both formulations was mainly associated with mitochondria and lysosomes, after 180 min native curcumin was associated with mitochondria and peroxisomes, micellar curcumin with peroxisomes only. Uptake and localization of native and micellar curcumin in intestinal cells do not differ significantly and consequently do not explain differences in bioavailability in humans. The temporary co-localization with lysosomes is in agreement with the previously proposed role of endocytosis in cellular uptake of curcumin and warrants further investigation.


Asunto(s)
Curcumina , Micelas , Disponibilidad Biológica , Transporte Biológico , Células CACO-2 , Curcumina/metabolismo , Curcumina/farmacología , Humanos
15.
Bioorg Med Chem Lett ; 20(22): 6680-4, 2010 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-20875742

RESUMEN

Alphaxalone, a neuroactive steroid containing a 17ß-acetyl group, has potent anesthetic activity in humans. This pharmacological activity is attributed to this steroid's enhancement of γ-amino butyric acid-mediated chloride currents at γ-amino butyric acid type A receptors. The conversion of alphaxalone into Δ(16)-alphaxalone produces an analogue that lacks anesthetic activity in humans and that has greatly diminished receptor actions. By contrast, the corresponding 17ß-carbonitrile analogue of alphaxalone and the Δ(16)-17-carbonitrile analogue both have potent anesthetic and receptor actions. The differential effect of the Δ(16)-double bond on the actions of alphaxalone and the 17ß-carbonitrile analogue is accounted for by a differential effect on the orientation of the 17-acetyl and 17-carbonitrile substituents.


Asunto(s)
Anestésicos/farmacología , Nitrilos/química , Pregnanodionas/farmacología , Ácido gamma-Aminobutírico/efectos de los fármacos , Animales , Cristalografía por Rayos X , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Pregnanodionas/química , Ratas , Espectrofotometría Infrarroja
16.
J Neurosci ; 28(20): 5159-68, 2008 May 14.
Artículo en Inglés | MEDLINE | ID: mdl-18480272

RESUMEN

Glutamate generates fast postsynaptic depolarization throughout the CNS. The positive-feedback nature of glutamate signaling likely necessitates flexible adaptive mechanisms that help prevent runaway excitation. We have previously explored presynaptic adaptive silencing, a form of synaptic plasticity produced by ongoing neuronal activity and by strong depolarization. Unsilencing mechanisms that maintain active synapses and restore normal function after adaptation are also important, but mechanisms underlying such presynaptic reactivation remain unexplored. Here we investigate the involvement of the cAMP pathway in the basal balance between silenced and active synapses, as well as the recovery of baseline function after depolarization-induced presynaptic silencing. Activation of the cAMP pathway activates synapses that are silent at rest, and pharmacological inhibition of cAMP signaling silences basally active synapses. Adenylyl cyclase (AC) 1 and AC8, the major Ca2+-sensitive AC isoforms, are not crucial for the baseline balance between silent and active synapses. In cells from mice doubly deficient in AC1 and AC8, the baseline percentage of active synapses was only modestly reduced compared with wild-type synapses, and forskolin unsilencing was similar in the two genotypes. Nevertheless, after strong presynaptic silencing, recovery of normal function was strongly inhibited in AC1/AC8-deficient synapses. The entire recovery phenotype of the double null was reproduced in AC8-deficient but not AC1-deficient cells. We conclude that, under normal conditions, redundant cyclase activity maintains the balance between presynaptically silent and active synapses, but AC8 plays a particularly important role in rapidly resetting the balance of active to silent synapses after adaptation to strong activity.


Asunto(s)
Adaptación Fisiológica/fisiología , Adenilil Ciclasas/metabolismo , Señalización del Calcio/fisiología , Inhibición Neural/fisiología , Terminales Presinápticos/enzimología , Transmisión Sináptica/fisiología , Potenciales de Acción/fisiología , Adenilil Ciclasas/genética , Animales , Células Cultivadas , Sistema Nervioso Central/enzimología , Sistema Nervioso Central/fisiología , AMP Cíclico/biosíntesis , Metabolismo Energético/fisiología , Exocitosis/fisiología , Retroalimentación Fisiológica/fisiología , Ácido Glutámico/metabolismo , Homeostasis/fisiología , Ratones , Ratones Noqueados , Ratas
17.
Mol Pharmacol ; 76(4): 754-65, 2009 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-19596835

RESUMEN

We have shown that fluorescent, 7-nitro-2,1,3-benzoxadiazol-4-yl amino (NBD)-conjugated neurosteroid analogs photopotentiate GABA(A) receptor function. These compounds seem to photosensitize a modification of receptor function, resulting in long-lived increases in responses to exogenous or synaptic GABA. Here we extend this work to examine the effectiveness of different fluorophore positions, conjugations, steroid structures, and fluorophores. Our results are generally in agreement with the idea that steroids with activity at GABA(A) receptors are the most potent photopotentiators. In particular, we find that an unnatural enantiomer of an effective photopotentiating steroid is relatively weak, excluding the idea that membrane solubility alone, which is identical for enantiomer pairs, is solely responsible for potent photopotentiation. Furthermore, there is a significant correlation between baseline GABA(A) receptor activity and photopotentiation. Curiously, both sulfated steroids, which bind a presumed external neurosteroid antagonist site, and hydroxysteroids, which bind an independent site, are effective. We also find that a rhodamine dye conjugated to a 5beta-reduced 3alpha-hydroxy steroid is a particularly potent and effective photopotentiator, with minimal baseline receptor activity up to 10 muM. Steroid conjugated fluorescein and Alexa Fluor 546 also supported photopotentiation, although the Alexa Fluor conjugate was weaker and required 10-fold higher concentration to achieve similar potentiation to the best NBD and rhodamine conjugates. Filling cells with steroid-conjugated or free fluorophores via whole-cell patch pipette did not support photopotentiation. FM1-43, another membrane-targeted, structurally unrelated fluorophore, also produced photopotentiation at micromolar concentrations. We conclude that further optimization of fluorophore and carrier could produce an effective, selective, light-sensitive GABA(A) receptor modulator.


Asunto(s)
Colorantes Fluorescentes/química , Receptores de GABA-A/química , Animales , Femenino , Técnicas de Placa-Clamp , Ratas , Xenopus laevis
18.
Neuropharmacology ; 144: 91-103, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30332607

RESUMEN

Positive modulators of NMDA receptors are important candidates for therapeutic development to treat psychiatric disorders including autism and schizophrenia. Sulfated neurosteroids have been studied as positive allosteric modulators of NMDA receptors for years, but we understand little about the cellular fate of these compounds, an important consideration for drug development. Here we focus on a visualizable sulfated neurosteroid analogue, KK-169. As expected of a pregnenolone sulfate analogue, the compound strongly potentiates NMDA receptor function, is an antagonist of GABAA receptors, exhibits occlusion with pregnenolone sulfate potentiation, and requires receptor domains important for pregnenolone sulfate potentiation. KK-169 exhibits somewhat higher potency than the natural parent, pregnenolone sulfate. The analogue contains a side-chain alkyne group, which we exploited for retrospective click labeling of neurons. Although the anionic sulfate group is expected to hinder cell entry, we detected significant accumulation of KK-169 in neurons with even brief incubations. Adding a photolabile diazirine group revealed that the expected plasma membrane localization of KK-169 is likely lost during fixation. Overall, our studies reveal new facets of the structure-activity relationship of neurosteroids at NMDA receptors, and their intracellular distribution suggests that sulfated neurosteroids could have unappreciated targets in addition to plasma membrane receptors.


Asunto(s)
Membrana Celular/efectos de los fármacos , Citoplasma/efectos de los fármacos , Agonistas de Aminoácidos Excitadores/farmacología , Receptores de N-Metil-D-Aspartato/agonistas , Regulación Alostérica , Animales , Membrana Celular/metabolismo , Células Cultivadas , Química Clic , Citoplasma/metabolismo , Agonistas de Aminoácidos Excitadores/química , Ácido Glutámico/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Humanos , Ratones , Oocitos , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismo , Relación Estructura-Actividad , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Xenopus laevis
19.
Sci Rep ; 9(1): 16431, 2019 11 11.
Artículo en Inglés | MEDLINE | ID: mdl-31712706

RESUMEN

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


Asunto(s)
Hipocampo/metabolismo , Hipocampo/fisiopatología , Inhibición Neural , Animales , Biomarcadores , Línea Celular , Diazepam/farmacología , Susceptibilidad a Enfermedades , Electroencefalografía , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Humanos , Inmunohistoquímica , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Ratones , Ratones Noqueados , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Ácido gamma-Aminobutírico/metabolismo
20.
J Med Chem ; 51(5): 1309-18, 2008 Mar 13.
Artículo en Inglés | MEDLINE | ID: mdl-18275132

RESUMEN

Although the structural features of binding sites for neuroactive steroids on gamma-aminobutryic acid type A (GABA A) receptors are still largely unknown, structure-activity studies have established a pharmacophore for potent enhancement of GABA A receptor function by neuroactive steroids. This pharmacophore emphasizes the importance of the position and stereochemistry of hydrogen-bonding groups on the steroid. However, the importance of the steroid ring system in mediating hydrophobic interactions with the GABA A receptor is unclear. We have taken the cyclopenta[ b]phenanthrene (tetracyclic compounds with a nonlinear ring system different from that of steroids) and cyclopenta[ b]anthracene (tetracyclic molecules with a linear 6-6-6-5 carbocyclic ring system) ring systems and properly substituted them to satisfy the pharmacophore requirements of the critical hydrogen-bond donor and acceptor groups found in neuroactive steroids. We have found these cyclopenta[ b]phenanthrene and cyclopenta[ b]anthracene analogues to have potent activity at the GABA A receptor, rivaling that of the most potent steroid modulators. Single-channel analysis of electrophysiological data indicates that similarly substituted analogues in the different ring systems affect the kinetic components of macroscopic currents in different ways. Mutations to the hydrogen bonding amino acids at the putative steroid binding site (alpha1Q241L mutation and alpha1N407A/Y410F double mutation) produce similar effects on macroscopic current amplitude by the different ring system analogues suggesting that the different kinetic effects are explained by the precise interactions of each analogue with the same binding site(s).


Asunto(s)
Anestésicos/síntesis química , Antracenos/síntesis química , Moduladores del GABA/síntesis química , Fenantrenos/síntesis química , Receptores de GABA-A/fisiología , Anestésicos/química , Anestésicos/farmacología , Animales , Antracenos/química , Antracenos/farmacología , Sitios de Unión , Encéfalo/metabolismo , Línea Celular , Moduladores del GABA/química , Moduladores del GABA/farmacología , Humanos , Técnicas In Vitro , Larva , Modelos Moleculares , Mutación , Oocitos/efectos de los fármacos , Oocitos/fisiología , Técnicas de Placa-Clamp , Fenantrenos/química , Fenantrenos/farmacología , Ensayo de Unión Radioligante , Ratas , Receptores de GABA-A/genética , Estereoisomerismo , Esteroides/farmacología , Relación Estructura-Actividad , Xenopus laevis
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