Oxysterols have emerged as important biomarkers in disease and as signaling molecules. We recently showed that the oxysterol 24(S)-hydroxycholesterol, the major brain cholesterol metabolite, potently and selectively enhances NMDA receptor function at a site distinct from other modulators. Here we further characterize the pharmacological mechanisms of 24(S)-hydroxycholesterol and its synthetic analogue SGE201. We describe an oxysterol antagonist of this positive allosteric modulation, 25-hydroxycholesterol. We found that 24(S)-hydroxycholesterol and SGE201 primarily increased the efficacy of NMDAR agonists but did not directly gate the channel or increase functional receptor number. Rather than binding to a direct aqueous-accessible site, oxysterols may partition into the plasma membrane to access the NMDAR, likely explaining slow onset and offset kinetics of modulation. Interestingly, oxysterols were ineffective when applied to the cytosolic face of inside-out membrane patches or through a whole-cell pipette solution, suggesting a non-intracellular site. We also found that another natural oxysterol, 25-hydroxycholesterol, although exhibiting slight potentiation on its own, non-competitively and enantioselectively antagonized the effects of 24(S)-hydroxycholesterol analogues. In summary, we suggest two novel allosteric sites on NMDARs that separately modulate channel gating, but together oppose each other.
The major cholesterol metabolite in brain, 24S-hydroxycholesterol (24S-HC), serves as a vehicle for cholesterol removal. Its effects on neuronal function, however, have only recently begun to be investigated. Here we review that nascent work. Our own studies have demonstrated that 24S-HC has potent positive modulatory effects on NMDA receptor (NMDAR) function. This could have implications for brain plasticity but also for pathological NMDAR overuse. Other work has demonstrated effects of 24S-HC on neuronal survival and as a possible biomarker of neurodegenerative disease. Depending on circumstances, both upregulation/mimicry of 24S-HC signaling and downregulation/antagonism may have therapeutic potential. We are interested in the possibility that synthetic analogues of 24S-HC with positive effects at NMDARs may hold neurotherapeutic promise, given the role of NMDA receptor hypofunction in certain neuropsychiatric disorders.
Memantine and ketamine, voltage-and activation-dependent channel blockers of N-methyl-D-aspartate (NMDA) receptors (NMDARs), have enjoyed a recent resurgence in clinical interest. Steady-state pharmacodynamic differences between these blockers have been reported, but it is unclear whether the compounds differentially affect dynamic physiologic signaling. In this study, we explored nonequilibrium conditions relevant to synaptic transmission in hippocampal networks in dissociated culture and hippocampal slices. Equimolar memantine and ketamine had indistinguishable effects on the following measures: steadystate NMDA currents, NMDAR excitatory postsynaptic current (EPSC) decay kinetics, progressive EPSC inhibition during repetitive stimulation, and extrasynaptic NMDAR inhibition. Therapeutic drug efficacy and tolerability of memantine have been attributed to fast kinetics and strong voltage dependence. However, pulse depolarization in drug presence revealed a surprisingly slow and similar time course of equilibration for the two compounds, although memantine produced a more prominent fast component (62% versus 48%) of re-equilibration. Simulations predicted that low gating efficacy underlies the slow voltage-dependent relief from block. This prediction was empirically supported by faster voltage-dependent blocker re-equilibration with several experimental manipulations of gating efficacy. Excitatory postsynaptic potential-like voltage commands produced drug differences only with large, prolonged depolarizations unlikely to be attained physiologically. In fact, we found no difference between drugs on measures of spontaneous network activity or acute effects on plasticity in hippocampal slices. Despite indistinguishable synaptic pharmacodynamics, ketamine provided significantly greater neuroprotection from damage induced by oxygen glucose deprivation, consistent with the idea that under extreme depolarizing conditions, the biophysical difference between drugs becomes detectable. We conclude that despite subtle differences in voltage dependence, during physiologic activity, blocker pharmacodynamics are largely indistinguishable and largely voltage independent.
BACKGROUND AND PURPOSEMemantine and ketamine are clinically used, open-channel blockers of NMDA receptors exhibiting remarkable pharmacodynamic similarities despite strikingly different clinical profiles. Although NMDA channel gating constitutes an important difference between memantine and ketamine, it is unclear how positive allosteric modulators (PAMs) might affect the pharmacodynamics of these NMDA blockers. EXPERIMENTAL APPROACHWe used two different PAMs: SGE-201, an analogue of an endogenous oxysterol, 24S-hydroxycholesterol, along with pregnenolone sulphate (PS), to test on memantine and ketamine responses in single cells (oocytes and cultured neurons) and networks (hippocampal slices), using standard electrophysiological techniques. KEY RESULTSSGE-201 and PS had no effect on steady-state block or voltage dependence of a channel blocker. However, both PAMs increased the actions of memantine and ketamine on phasic excitatory post-synaptic currents, but neither revealed underlying pharmacodynamic differences. SGE-201 accelerated the re-equilibration of blockers during voltage jumps. SGE-201 also unmasked differences among the blockers in neuronal networks -measured either by suppression of activity in multi-electrode arrays or by neuroprotection against a mild excitotoxic insult. Either potentiating NMDA receptors while maintaining the basal activity level or increasing activity/depolarization without potentiating NMDA receptor function is sufficient to expose pharmacodynamic blocker differences in suppressing network function and in neuroprotection. CONCLUSIONS AND IMPLICATIONSPositive modulation revealed no pharmacodynamic differences between NMDA receptor blockers at a constant voltage, but did expose differences during spontaneous network activity. Endogenous modulator tone of NMDA receptors in different brain regions may underlie differences in the effects of NMDA receptor blockers on behaviour.
It is widely appreciated that neuronal networks exhibit patterns of bursting and synchrony that are not captured by simple measures such as average spike rate. These patterns can encode information or represent pathological behavior such as seizures. However, methods for quantifying bursting and synchrony are not agreed upon and can be confounded with spike rate measures. Previous validation has largely relied on in silico networks and single experimental conditions. How published measures of bursting and synchrony perform when applied to biological networks of varied average spike rate and subjected to varied experimental challenges is unclear. In multielectrode array recordings of network activity, we found that two mechanistically distinct drugs, cyclothiazide and bicuculline, produced equivalent increases in average spike rate but differed in bursting and synchrony. We applied several measures of bursting to the recordings (2 threshold interval methods and a surprise-based method) and found that a measure based on an average critical interval, adjusted for the array-wide spike rate, performed best in quantifying differential drug effects. To quantify synchrony, we compared a coefficient of variation-based measure, the recently proposed spike time tiling coefficient, the SPIKE-distance measure, and a global synchrony index. The spike time tiling coefficient, the SPIKE-distance measure, and the global synchrony index all captured a difference between drugs with the best performance exhibited by the global synchrony index. In summary, our exploration should aid other investigators by highlighting strengths and limitations of current methods.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.