Cross Talk between α7 and α3β4 Nicotinic Receptors Prevents Their Desensitization in Human Chromaffin Cells

The physical interaction and functional cross talk among the different subtypes of neuronal nicotinic acetylcholine receptors (nAChRs) expressed in the various tissues is unknown. Here, we have investigated this issue between the only two nAChRs subtypes expressed, the α7 and α3β4 subtypes, in a human native neuroendocrine cell (the chromaffin cell) using electrophysiological patch-clamp, fluorescence, and Förster resonance energy transfer (FRET) techniques. Our data show that α7 and α3β4 receptor subtypes require their mutual and maximal efficacy of activation to increase their expression, to avoid their desensitization, and therefore, to increase their activity. In this way, after repetitive stimulation with acetylcholine (ACh), α7 and α3β4 receptor subtypes do not desensitize, but they do with choline. The nicotinic current increase associated with the α3β4 subtype is dependent on Ca²⁺. In addition, both receptor subtypes physically interact. Interaction and expression of both subtypes are reversibly reduced by tyrosine and serine/threonine phosphatases inhibition, not by Ca²⁺. In addition, expression is greater in human chromaffin cells from men compared to women, but FRET efficiency is not affected. Together, our findings indicate that human α7 and α3β4 subtypes mutually modulate their expression and activity, providing a promising line of research to pharmacologically regulate their activity.SIGNIFICANCE STATEMENT Desensitization of nicotinic receptors is accepted to occur with repetitive agonist stimulation. However, here we show that human native α3β4 and α7 nicotinic acetylcholine receptor (nAChR) subtypes do not desensitize, and instead, increase their activity when they are activated by the physiological agonist acetylcholine (ACh). An indispensable requirement is the activation of the other receptor subtype with maximal efficacy, and the presence of Ca²⁺ to cooperate in the case of the α3β4 current increase. Because choline is an α3β4 partial agonist, it will act as a limiting factor of nicotinic currents enhancement in the absence of ACh, but in its presence, it will further potentiate α7 currents.     

Meprin β: A novel regulator of blood–brain barrier integrity

The metalloprotease meprin β (Mep1b) is capable of cleaving cell-adhesion molecules in different tissues (e.g. skin, kidney and intestine) and is dysregulated in several diseases associated with barrier breakdown (Alzheimer´s disease, kidney disruption, inflammatory bowel disease). In this study, we demonstrate that Mep1b is a novel regulator of tight junction (TJ) composition and blood–brain barrier (BBB) integrity in brain endothelium. In Mep1b-transfected mouse brain endothelial cells (bEnd.3), we observed a reduction of the TJ protein claudin-5, decreased transendothelial electrical resistance (TEER) and an elevated permeability to paracellular diffusion marker [¹⁴C]-inulin. Analysis of global Mep1b knock-out (Mep1b^−/−) mice showed increased TJ protein expression (claudin-5, occludin, ZO-1) in cerebral microvessels and increased TEER in cultivated primary mouse brain endothelial compared to wild-type (wt) mice. Furthermore, we investigated the IgG levels in cerebrospinal fluid (CSF) and the brain water content as additional permeability markers and detected lower IgG levels and reduced brain water content in Mep1b^−/− mice compared to wt mice. Showing opposing features in overexpression and knock-out, we conclude that Mep1b plays a role in regulating brain endothelial TJ-proteins and therefore affecting BBB tightness in vitro and in vivo.     

One‐week escitalopram intake alters the excitation–inhibition balance in the healthy female brain

Neural health relies on cortical excitation–inhibition balance (EIB). Previous research suggests a link between increased cortical excitation and neuroplasticity induced by selective serotonin reuptake inhibitors (SSRIs). Whether there are modulations of EIB following SSRI‐administration in the healthy human brain, however, remains unclear. Thus, in a randomized double‐blind study, we administered a clinically relevant dose of 20 mg escitalopram for 7 days (time when steady state is achieved) in 59 healthy women (28 escitalopram, 31 placebo) on oral contraceptives. We acquired resting‐state electroencephalography data at baseline, after a single dose, and at steady state. We assessed 1/f slope of the power spectrum as a marker of EIB, compared individual trajectories of 1/f slope changes contrasting single dose and 1‐week drug intake, and tested the relationship of escitalopram plasma levels and cortical excitatory and inhibitory balance shifts. Escitalopram‐intake was associated with decreased 1/f slope, indicating an EIB shift in favor of excitation. Furthermore, 1/f slope at baseline and after a single dose of escitalopram was associated with 1/f slope at steady state. Higher plasma escitalopram levels at a single dose were associated with better maintenance of these EIB changes throughout the drug administration week. These findings demonstrate the potential for 1/f slope to predict individual cortical responsivity to SSRIs and widen the lens through which we map the human brain by testing an interventional psychopharmacological design in a clearly defined endocrinological state.     

Only the Fastest Corticospinal Fibers Contribute to β Corticomuscular Coherence

Human corticospinal transmission is commonly studied using brain stimulation. However, this approach is biased to activity in the fastest conducting axons. It is unclear whether conclusions obtained in this context are representative of volitional activity in mild-to-moderate contractions. An alternative to overcome this limitation may be to study the corticospinal transmission of endogenously generated brain activity. Here, we investigate in humans (N = 19; of either sex), the transmission speeds of cortical β rhythms (∼20 Hz) traveling to arm (first dorsal interosseous) and leg (tibialis anterior; TA) muscles during tonic mild contractions. For this purpose, we propose two improvements for the estimation of corticomuscular β transmission delays. First, we show that the cumulant density (cross-covariance) is more accurate than the commonly-used directed coherence to estimate transmission delays in bidirectional systems transmitting band-limited signals. Second, we show that when spiking motor unit activity is used instead of interference electromyography, corticomuscular transmission delay estimates are unaffected by the shapes of the motor unit action potentials (MUAPs). Applying these improvements, we show that descending corticomuscular β transmission is only 1–2 ms slower than expected from the fastest corticospinal pathways. In the last part of our work, we show results from simulations using estimated distributions of the conduction velocities for descending axons projecting to lower motoneurons (from macaque histologic measurements) to suggest two scenarios that can explain fast corticomuscular transmission: either only the fastest corticospinal axons selectively transmit β activity, or else the entire pool does. The implications of these two scenarios for our understanding of corticomuscular interactions are discussed.SIGNIFICANCE STATEMENT We present and validate an improved methodology to measure the delay in the transmission of cortical β activity to tonically-active muscles. The estimated corticomuscular β transmission delays obtained with this approach are remarkably similar to those expected from transmission in the fastest corticospinal axons. A simulation of β transmission along a pool of corticospinal axons using an estimated distribution of fiber diameters suggests two possible mechanisms by which fast corticomuscular transmission is achieved: either a very small fraction of the fastest descending axons transmits β activity to the muscles or, alternatively, the entire population does and natural cancellation of slow channels occurs because of the distribution of axon diameters in the corticospinal tract.     

Evidence of an interaction between FXR1 and GSK3β polymorphisms on levels of Negative Symptoms of Schizophrenia and their response to antipsychotics

BackgroundGenome-Wide Association Studies (GWASs) have identified several genes associated with Schizophrenia (SCZ) and exponentially increased knowledge on the genetic basis of the disease. In addition, products of GWAS genes interact with neuronal factors coded by genes lacking association, such that this interaction may confer risk for specific phenotypes of this brain disorder. In this regard, fragile X mental retardation syndrome-related 1 (FXR1) gene has been GWAS associated with SCZ. FXR1 protein is regulated by glycogen synthase kinase-3β (GSK3β), which has been implicated in pathophysiology of SCZ and response to antipsychotics (APs). rs496250 and rs12630592, two eQTLs (Expression Quantitative Trait Loci) of FXR1 and GSK3β, respectively, interact on emotion stability and amygdala/prefrontal cortex activity during emotion processing. These two phenotypes are associated with Negative Symptoms (NSs) of SCZ suggesting that the interaction between these SNPs may also affect NS severity and responsiveness to medication.MethodsTo test this hypothesis, in two independent samples of patients with SCZ, we investigated rs496250 by rs12630592 interaction on NS severity and response to APs. We also tested a putative link between APs administration and FXR1 expression, as already reported for GSK3β expression.ResultsWe found that rs496250 and rs12630592 interact on NS severity. We also found evidence suggesting interaction of these polymorphisms also on response to APs. This interaction was not present when looking at positive and general psychopathology scores. Furthermore, chronic olanzapine administration led to a reduction of FXR1 expression in mouse frontal cortex.DiscussionOur findings suggest that, like GSK3β, FXR1 is affected by APs while shedding new light on the role of the FXR1/GSK3β pathway for NSs of SCZ.     

Presenilin/γ-Secretase Activity Is Located in Acidic Compartments of Live Neurons

Presenilin (PSEN)/γ-secretase is a protease complex responsible for the proteolytic processing of numerous substrates. These substrates include the amyloid precursor protein (APP), the cleavage of which by γ-secretase results in the production of β-amyloid (Aβ) peptides. However, exactly where within the neuron γ-secretase processes APP C99 to generate Aβ and APP intracellular domain (AICD) is still not fully understood. Here, we employ novel Förster resonance energy transfer (FRET)-based multiplexed imaging assays to directly “visualize” the subcellular compartment(s) in which γ-secretase primarily cleaves C99 in mouse cortex primary neurons (from both male and female embryos). Our results demonstrate that γ-secretase processes C99 mainly in LysoTracker-positive low-pH compartments. Using a new immunostaining protocol which distinguishes Aβ from C99, we also show that intracellular Aβ is significantly accumulated in the same subcellular loci. Furthermore, we found functional correlation between the endo-lysosomal pH and cellular γ-secretase activity. Taken together, our findings are consistent with Aβ being produced from C99 by γ-secretase within acidic compartments such as lysosomes and late endosomes in living neurons.SIGNIFICANCE STATEMENT Alzheimer''s disease (AD) genetics and histopathology highlight the importance of amyloid precursor protein (APP) processing by γ-secretase in pathogenesis. For the first time, this study has enabled us to directly “visualize” that γ-secretase processes C99 mainly in acidic compartments such as late endosomes and lysosomes in live neurons. Furthermore, we uncovered that intracellular β-amyloid (Aβ) is significantly accumulated in the same subcellular loci. Emerging evidence proposes the great importance of the endo-lysosomal pathway in mechanisms of misfolded proteins propagation (e.g., Tau, α-Syn). Therefore, the predominant processing of C99 and enrichment of Aβ in late endosomes and lysosomes may be critical events in the molecular cascade leading to AD.     

Volume of β-Bursts,But Not Their Rate,Predicts Successful Response Inhibition

In humans, impaired response inhibition is characteristic of a wide range of psychiatric diseases and of normal aging. It is hypothesized that the right inferior frontal cortex (rIFC) plays a key role by inhibiting the motor cortex via the basal ganglia. The electroencephalography (EEG)-derived β-rhythm (15–29 Hz) is thought to reflect communication within this network, with increased right frontal β-power often observed before successful response inhibition. Recent literature suggests that averaging spectral power obscures the transient, burst-like nature of β-activity. There is evidence that the rate of β-bursts following a Stop signal is higher when a motor response is successfully inhibited. However, other characteristics of β-burst events, and their topographical properties, have not yet been examined. Here, we used a large human (male and female) EEG Stop Signal task (SST) dataset (n = 218) to examine averaged normalized β-power, β-burst rate, and β-burst “volume” (which we defined as burst duration × frequency span × amplitude). We first sought to optimize the β-burst detection method. In order to find predictors across the whole scalp, and with high temporal precision, we then used machine learning to (1) classify successful versus failed stopping and to (2) predict individual stop signal reaction time (SSRT). β-burst volume was significantly more predictive of successful and fast stopping than β-burst rate and normalized β-power. The classification model generalized to an external dataset (n = 201). We suggest β-burst volume is a sensitive and reliable measure for investigation of human response inhibition.SIGNIFICANCE STATEMENT The electroencephalography (EEG)-derived β-rhythm (15–29 Hz) is associated with the ability to inhibit ongoing actions. In this study, we sought to identify the specific characteristics of β-activity that contribute to successful and fast inhibition. In order to search for the most relevant features of β-activity, across the whole scalp and with high temporal precision, we employed machine learning on two large datasets. Spatial and temporal features of β-burst “volume” (duration × frequency span × amplitude) predicted response inhibition outcomes in our data significantly better than β-burst rate and normalized β-power. These findings suggest that multidimensional measures of β-bursts, such as burst volume, can add to our understanding of human response inhibition.     

GSK3β regulates oligodendrogenesis in the dorsal microdomain of the subventricular zone via Wnt‐β‐catenin signaling

Oligodendrocytes, the myelinating cells of the CNS, are derived postnatally from oligodendrocyte precursors (OPs) of the subventricular zone (SVZ). However, the mechanisms that regulate their generation from SVZ neural stem cells (NSC) are poorly understood. Here, we have examined the role of glycogen synthase kinase 3β (GSK3β), an effector of multiple converging signaling pathways in postnatal mice. The expression of GSK3β by rt‐qPCR was most prominent in the SVZ and in the developing white matter, around the first 1–2 weeks of postnatal life, coinciding with the peak periods of OP differentiation. Intraventricular infusion of the GSK3β inhibitor ARA‐014418 in mice aged postnatal day (P) 8–11 significantly increased generation of OPs in the dorsal microdomain of the SVZ, as shown by expression of cell specific markers using rt‐qPCR and immunolabelling. Analysis of stage specific markers revealed that the augmentation of OPs occurred via increased specification from earlier SVZ cell types. These effects of GSK3β inhibition on the dorsal SVZ were largely attributable to stimulation of the canonical Wnt/β‐catenin signaling pathway over other pathways. The results indicate GSK3β is a key endogenous factor for specifically regulating oligodendrogenesis from the dorsal SVZ microdomain under the control of Wnt‐signaling. GLIA 2014;62:778–789     

Is γ-aminobutyric acid the neurotransmitter of barnacle photoreceptors?

The hypothesis that γ-aminobutyric acid (GABA) is the neurotransmitter ofbarnacle photoreceptors was tested by studying the effect of GABA on the membrane of the cell directly postsynaptic to the photoreceptor, by testing the ability of GABA antagonists to block transmission at this synapse, and by estimating the free GABA content of the photoreceptor. The results of these experiments suggest that GABA is not the photoreceptor's neurotransmitter.     

Effect of transient cerebral ischemia on γ-aminobutyric acidA receptor α1-subunit–immunoreactive interneurons in the gerbil CA1 hippocampus

Following transient cerebral ischemia, pyramidal cells within area CA1 of the hippocampus exhibit delayed neuronal death. While interneurons within this sector continue to survive long-term, there is evidence that some interneurons in area CA1 are vulnerable to damage. To determine the nature of vulnerability in a neurochemically heterogeneous population of interneurons throughout area CA1, we examined the labeling of γ-aminobutyric acid (GABA)ergic interneurons with an antibody to the GABA_A receptor α₁-subunit 1–35 days following cerebral ischemia in the Mongolian gerbil. Unlike some other GABA interneuron markers, this antibody labels both the dendrites and soma of interneurons, allowing dendritic structure to be examined. Three to four days following ischemia, the pyramidal cells in area CA1 had degenerated, and the α₁-subunit–positive interneurons in all layers of area CA1 had developed severely beaded dendrites. At longer survival times (21–35 days), the α₁-subunit–immunolabeled dendrites of these interneurons had a fragmented appearance. In contrast, interneurons bordering str. oriens and alveus typically exhibited normal dendritic morphology. Despite the pathologic changes, there was no evidence of interneuron loss in area CA1 up to 35 days post-ischemia. Normal interneuron morphology was also observed in area CA3 and dentate gyrus, regions where neither pyramidal neurons nor granule cells, respectively, die following 5 min of cerebral ischemia. To determine if the ischemia-induced changes in interneuron morphology could be prevented, diazepam was administered 30 and 90 min following ischemia. Diazepam produces long-term neuroprotection of area CA1 pyramidal neurons. In gerbils sacrificed 35 days after ischemia, diazepam markedly attenuated the dendritic beading of the area CA1 interneurons. In addition, the dendrites did not display the fragmented labeling by the α₁-subunit antibody. Thus, despite their long-term survival, CA1 hippocampal interneurons in the gerbil can express severe structural abnormalities after transient cerebral ischemia coincident with pyramidal cell degeneration, and the injury to the dendrites can be prevented by the neuroprotectant diazepam. Hippocampus 1997; 7:511–523. © 1997 Wiley-Liss, Inc.     

Lithium attenuates blood–brain barrier damage and brain edema following intracerebral hemorrhage via an endothelial Wnt/β‐catenin signaling‐dependent mechanism in mice

BackgroundVasogenic cerebral edema resulting from blood–brain barrier (BBB) damage aggravates the devastating consequences of intracerebral hemorrhage (ICH). Although augmentation of endothelial Wnt/β‐catenin signaling substantially alleviates BBB breakdown in animals, no agents based on this mechanism are clinically available. Lithium is a medication used to treat bipolar mood disorders and can upregulate Wnt/β‐catenin signaling.MethodsWe evaluated the protective effect of lithium on the BBB in a mouse model of collagenase IV‐induced ICH. Furthermore, we assessed the effect and dependency of lithium on Wnt/β‐catenin signaling in mice with endothelial deletion of the Wnt7 coactivator Gpr124.ResultsLithium treatment (3 mmol/kg) significantly decreased the hematoma volume (11.15 ± 3.89 mm³ vs. 19.97 ± 3.20 mm³ in vehicle controls, p = 0.0016) and improved the neurological outcomes of mice following ICH. Importantly, lithium significantly increased the BBB integrity, as evidenced by reductions in the levels of brain edema (p = 0.0312), Evans blue leakage (p = 0.0261), and blood IgG extravasation (p = 0.0009) into brain tissue around the hematoma. Mechanistically, lithium upregulated the activity of endothelial Wnt/β‐catenin signaling in mice and increased the levels of tight junction proteins (occludin, claudin‐5 and ZO‐1). Furthermore, the protective effect of lithium on cerebral damage and BBB integrity was abolished in endothelial Gpr124 knockout mice, suggesting that its protective effect on BBB function was mainly dependent on Gpr124‐mediated endothelial Wnt/β‐catenin signaling.ConclusionOur findings indicate that lithium may serve as a therapeutic candidate for treating BBB breakdown and brain edema following ICH.     

Memory Destabilization and Reconsolidation Dynamically Regulate the PKMζ Maintenance Mechanism

Useful memory must balance between stability and malleability. This puts effective memory storage at odds with plasticity processes, such as reconsolidation. What becomes of memory maintenance processes during synaptic plasticity is unknown. Here we examined the fate of the memory maintenance protein PKMζ during memory destabilization and reconsolidation in male rats. We found that NMDAR activation and proteasome activity induced a transient reduction in PKMζ protein following retrieval. During reconsolidation, new PKMζ was synthesized to re-store the memory. Failure to synthesize new PKMζ during reconsolidation impaired memory but uninterrupted PKMζ translation was not necessary for maintenance itself. Finally, NMDAR activation was necessary to render memories vulnerable to the amnesic effect of PKMζ-antisense. These findings outline a transient disruption and renewal of the PKMζ memory maintenance mechanism during plasticity. We argue that dynamic changes in PKMζ protein levels can serve as an exemplary model of the molecular changes underlying memory destabilization and reconsolidation.SIGNIFICANCE STATEMENT Maintenance of long-term memory relies on the persistent activity of PKMζ. However, after retrieval, memories can become transiently destabilized and must be reconsolidated within a few hours to persist. During this period of plasticity, what happens to maintenance processes, such as those involving PKMζ, is unknown. Here we describe dynamic changes to PKMζ expression during both destabilization and reconsolidation of auditory fear memory in the amygdala. We show that destabilization induces a NMDAR- and proteasome-dependent loss of synaptic PKMζ and that reconsolidation requires synthesis of new PKMζ. This work provides clear evidence that memory destabilization disrupts ongoing synaptic maintenance processes which are restored during reconsolidation.     

β-Adrenoceptors regulate myoelectric activity in the small intestine of rats: stimulation by β2 and inhibition by β3 subtypes

Abstract Using β-adrenergic agonists and antagonists this study investigated the importance of three different adrenoceptor subtypes for the regulation of migrating myoelectric complexes (MMCs) of the upper small intestine in conscious, naive rats. After a control period of 60 min with four activity fronts, agonists were given as an intravenous infusion for another 60 min. The non-selective β-adrenoceptor agonist isoprenaline (1 μg kg^?1 min^?1) inhibited MMCs and induced irregular spiking during the infusion period. This effect was blocked by intravenous administration of a bolus dose of either the non-selective β-adrenoceptor antagonist propranolol (1 mg kg^?1), or the β₂-antagonist ICI 118 551 (1 mg kg^?1), both given prior to isoprenaline. However, acebutolol (1 mg kg^?1), a selective β₁-antagonist, failed to antagonize the effect of isoprenaline. Furthermore, prenalterol, a selective β₁-agonist (12.5–800.0 μg kg^?1 min^?1), had no effect on the MMC pattern, whereas the β₂-selective agonist ritodrine (25–100 μg kg^?1 min^?1) induced a myoelectric pattern similar to one induced by isoprenaline. The partial β₃-adrenoceptor agonist D7114 (50–100 μg kg^?1 min^?1), disrupted the MMCs and induced quiescence. Neither of the antagonists, i.e. propranolol (1 mg kg^?1), acebutolol (1 mg kg^?1) nor ICI 118 551 (1 mg kg^?1), given alone induced changes in the MMC pattern. In conclusion, β₂-adrenoceptors in particular but also β₃-adrenoceptors seem to be of importance in the regulation of small intestinal motility by disrupting the regular MMC pattern in rats.     

Reading and Modulating Cortical β Bursts from Motor Unit Spiking Activity

β Oscillations (13–30 Hz) are ubiquitous in the human motor nervous system. Yet, their origins and roles are unknown. Traditionally, β activity has been treated as a stationary signal. However, recent studies observed that cortical β occurs in “bursting events,” which are transmitted to muscles. This short-lived nature of β events makes it possible to study the main mechanism of β activity found in the muscles in relation to cortical β. Here, we assessed whether muscle β activity mainly results from cortical projections. We ran two experiments in healthy humans of both sexes (N = 15 and N = 13, respectively) to characterize β activity at the cortical and motor unit (MU) levels during isometric contractions of the tibialis anterior muscle. We found that β rhythms observed at the cortical and MU levels are indeed in bursts. These bursts appeared to be time-locked and had comparable average durations (40–80 ms) and rates (approximately three to four bursts per second). To further confirm that cortical and MU β have the same source, we used a novel operant conditioning framework to allow subjects to volitionally modulate MU β. We showed that volitional modulation of β activity at the MU level was possible with minimal subject learning and was paralleled by similar changes in cortical β activity. These results support the hypothesis that MU β mainly results from cortical projections. Moreover, they demonstrate the possibility to decode cortical β activity from MU recordings, with a potential translation to future neural interfaces that use peripheral information to identify and modulate activity in the central nervous system.SIGNIFICANCE STATEMENT We show for the first time that β activity in motor unit (MU) populations occurs in bursting events. These bursts observed in the output of the spinal cord appear to be time-locked and share similar characteristics of β activity at the cortical level, such as the duration and rate at which they occur. Moreover, when subjects were exposed to a novel operant conditioning paradigm and modulated MU β activity, cortical β activity changed in a similar way as peripheral β. These results provide evidence for a strong correspondence between cortical and peripheral β activity, demonstrating the cortical origin of peripheral β and opening the pathway for a new generation of neural interfaces.     

Nature of the 5′ residue in the M2 domain affects function of the human α1β1 GABAA receptor

The effects on the functional properties of the α₁β₁ GABA_A receptor when the 5′ (α₁ Val260; β₁ Ile255) hydrophobic amino acids in the second transmembrane (M2) region were changed to threonine were examined. In response to a saturating concentration of GABA, the current evoked in mutant receptors showed a decreased rate of desensitization and at equilibrium was a greater fraction of the peak current than in wild-type receptors. The half-saturation concentration of the peak current response to GABA in mutant receptors was comparable to that in wild-type receptors, but the Hill coefficient was reduced to less than one. It was concluded that the 5′ amino acids in the M2 region have a role in the conformational changes that occur within the α₁β₁ GABA_A receptor in response to GABA. Synapse 26:324–327, 1997. © 1997 Wiley-Liss, Inc.