The expression and activity of ionotropic glutamate receptors control signal transduction at the excitatory synapses in the CNS. The NMDAR comprises two obligatory GluN1 subunits and two GluN2 or GluN3 subunits in different combinations. Each GluN subunit consists of four domains: the extracellular amino-terminal and agonist-binding domains, the transmembrane domain, and the intracellular C-terminal domain (CTD). The CTD interaction with various classes of intracellular proteins is critical for trafficking and synaptic localization of NMDARs. Amino acid mutations or the inclusion of premature stop codons in the CTD could contribute to the emergence of neurodevelopmental and neuropsychiatric disorders. Here, we describe the method of preparing primary hippocampal neurons and lentiviral particles expressing GluN subunits that can be used as a model to study cell surface expression and synaptic localization of NMDARs. We also show a simple method of fluorescence immunostaining of eGFP-tagged GluN2 subunits and subsequent microscopy technique and image analysis to study the effects of disease-associated mutations in the CTDs of GluN2A and GluN2B subunits.

• Klíčová slova
• Colocalization, Fluorescence immunostaining, Fluorescence microscopy, Glutamate receptor, ImageJ analysis, Lentivirus, Primary hippocampal neurons, Surface expression,
• MeSH
• exprese genu MeSH
• hipokampus * metabolismus   cytologie   MeSH
• krysa rodu Rattus MeSH
• kultivované buňky MeSH
• Lentivirus genetika   MeSH
• lidé MeSH
• neurony * metabolismus   MeSH
• podjednotky proteinů metabolismus   genetika   MeSH
• primární buněčná kultura metody   MeSH
• receptory N-methyl-D-aspartátu * metabolismus   genetika   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• N-methyl D-aspartate receptor subtype 2A MeSH Prohlížeč
• podjednotky proteinů MeSH
• receptory N-methyl-D-aspartátu * MeSH

Evidence from clinical and preclinical studies implicates dysfunction of N-methyl-D-aspartate receptors (NMDARs) in schizophrenia progression and symptoms. We investigated the antipsychotic effect of two neuroactive steroids in an animal model of schizophrenia induced by systemic application of MK-801. The neuroactive steroids differ in their mechanism of action at NMDARs. MS-249 is positive, while PA-Glu is a negative allosteric NMDAR modulator. We hypothesized that the positive NMDA receptor modulator would attenuate deficits caused by MK-801 co-application more effectively than PA-Glu. The rats were tested in a battery of tests assessing spontaneous locomotion, anxiety and cognition. Contrary to our expectations, PA-Glu exhibited a superior antipsychotic effect to MS-249. The performance of MS-249-treated rats in cognitive tests differed depending on the level of stress the rats were exposed to during test sessions. In particular, with the increasing severity of stress exposure, the performance of animals worsened. Our results demonstrate that enhancement of NMDAR function may result in unspecific behavioral responses. Positive NMDAR modulation can influence other neurobiological processes besides memory formation, such as anxiety and response to stress.

• Klíčová slova
• MK-801, anxiety, cognition, neurosteroids, schizophrenia, stress,
• MeSH
• antipsychotika farmakologie   MeSH
• bicyklické sloučeniny heterocyklické metabolismus   MeSH
• chování zvířat účinky léků   MeSH
• dizocilpinmaleát farmakologie   MeSH
• HEK293 buňky MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• modely nemocí na zvířatech MeSH
• potkani Long-Evans MeSH
• potkani Wistar MeSH
• pregnenolon metabolismus   farmakologie   MeSH
• receptory N-methyl-D-aspartátu antagonisté a inhibitory   metabolismus   MeSH
• schizofrenie farmakoterapie   metabolismus   MeSH
• steroidy farmakologie   MeSH
• test vyvýšeného křížového bludiště MeSH
• úleková reakce účinky léků   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antipsychotika MeSH
• bicyklické sloučeniny heterocyklické MeSH
• dizocilpinmaleát MeSH
• pregnenolon MeSH
• pregnenolone sulfate MeSH Prohlížeč
• receptory N-methyl-D-aspartátu MeSH
• steroidy MeSH
• tert-butylbicyclophosphorothionate MeSH Prohlížeč

Ageing is accompanied by deterioration in physical condition and a number of physiological processes and thus a higher risk of a range of diseases and disorders. In particular, we focused on the changes associated with aging, especially the role of small molecules, their role in physiological and pathophysiological processes and potential treatment options. Our previously published results and data from other authors lead to the conclusion that these unwanted changes are mainly linked to the hypothalamic-pituitary-adrenal axis can be slowed down, stopped, or in some cases even reversed by an appropriate treatment, but especially by a life-management adjustment.

• MeSH
• hormony metabolismus   MeSH
• knihovny malých molekul MeSH
• lidé MeSH
• receptory buněčného povrchu metabolismus   MeSH
• stárnutí účinky léků   metabolismus   patologie   MeSH
• systém hypofýza - nadledviny účinky léků   fyziologie   MeSH
• systém hypotalamus-hypofýza účinky léků   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• hormony MeSH
• knihovny malých molekul MeSH
• receptory buněčného povrchu MeSH

UNLABELLED: Postsynaptic N-methyl-d-aspartate receptors (NMDARs) phasically activated by presynaptically released glutamate are critical for synaptic transmission and plasticity. However, under pathological conditions, excessive activation of NMDARs by tonically increased ambient glutamate contributes to excitotoxicity associated with various acute and chronic neurological disorders. Here, using heterologously expressed GluN1/GluN2A and GluN1/GluN2B receptors and rat autaptic hippocampal microisland cultures, we show that pregnanolone sulfate inhibits NMDAR currents induced by a prolonged glutamate application with a higher potency than the NMDAR component of EPSCs. For synthetic pregnanolone derivatives substituted with a carboxylic acid moiety at the end of an aliphatic chain of varying length and attached to the steroid skeleton at C3, the difference in potency between tonic and phasic inhibition increased with the length of the residue. The steroid with the longest substituent, pregnanolone hemipimelate, had no effect on phasically activated receptors while inhibiting tonically activated receptors. In behavioral tests, pregnanolone hemipimelate showed neuroprotective activity without psychomimetic symptoms. These results provide insight into the influence of steroids on neuronal function and stress their potential use in the development of novel therapeutics with neuroprotective action. SIGNIFICANCE STATEMENT: Synaptic activation of N-methyl-d-aspartate receptors (NMDARs) plays a key role in synaptic plasticity, but excessive tonic NMDAR activation mediates excitotoxicity associated with many neurological disorders. Therefore, there is much interest in pharmacological agents capable of selectively blocking tonically activated NMDARs while leaving synaptically activated NMDARs intact. Here, we show that an endogenous neurosteroid pregnanolone sulfate is more potent at inhibiting tonically than synaptically activated NMDARs. Further, we report that a novel synthetic analog of pregnanolone sulfate, pregnanolone hemipimelate, inhibits tonic NMDAR currents without inhibiting the NMDAR component of the EPSC and shows neuroprotective activity in vivo without inducing psychomimetic side effects. These results suggest steroids may have a clinical advantage over other known classes of NMDAR inhibitors.

• Klíčová slova
• NMDA receptor, memantine, neuroprotection, neurosteroid, pregnanolone sulfate, synaptic transmission,
• MeSH
• excitační postsynaptické potenciály účinky léků   MeSH
• HEK293 buňky MeSH
• hipokampus metabolismus   MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• metoda terčíkového zámku MeSH
• nervový přenos účinky léků   MeSH
• neuroprotektivní látky farmakologie   MeSH
• pohybová aktivita účinky léků   MeSH
• potkani Wistar MeSH
• pregnanolon chemie   farmakologie   MeSH
• pregnany chemie   farmakologie   MeSH
• receptory N-methyl-D-aspartátu antagonisté a inhibitory   účinky léků   genetika   MeSH
• učení vyhýbat se účinky léků   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• N-methyl D-aspartate receptor subtype 2A MeSH Prohlížeč
• neuroprotektivní látky MeSH
• NR2B NMDA receptor MeSH Prohlížeč
• pregnanolon MeSH
• pregnany MeSH
• receptory N-methyl-D-aspartátu MeSH