Control of contextual memory through interneuronal α5-GABAA receptors.

γ-Aminobutyric acid type A receptors that incorporate α5 subunits (α5-GABAARs) are highly enriched in the hippocampus and are strongly implicated in control of learning and memory. Receptors located on pyramidal neuron dendrites have long been considered responsible, but here we report that mice in which α5-GABAARs have been eliminated from pyramidal neurons (α5-pyr-KO) continue to form strong spatial engrams and that they remain as sensitive as their pseudo-wild-type (p-WT) littermates to etomidate-induced suppression of place cells and spatial engrams. By contrast, mice with selective knockout in interneurons (α5-i-KO) no longer exhibit etomidate-induced suppression of place cells. In addition, the strength of spatial engrams is lower in α5-i-KO mice than p-WT littermates under control conditions. Consistent with the established role of the hippocampus in contextual fear conditioning, α5-i-KO mice resisted etomidate's suppression of freezing to context, but so too did α5-pyr-KO mice, supporting a role for extra-hippocampal regions in the development of contextual fear memory. Overall, our results indicate that interneuronal α5-GABAARs serve a physiological role in promoting spatial learning and that they mediate suppression of hippocampus-dependent contextual memory by etomidate.

Understanding the Effects of General Anesthetics on Cortical Network Activity Using Ex Vivo Preparations.

General anesthetics have been used to ablate consciousness during surgery for more than 150 yr. Despite significant advances in our understanding of their molecular-level pharmacologic effects, comparatively little is known about how anesthetics alter brain dynamics to cause unconsciousness. Consequently, while anesthesia practice is now routine and safe, there are many vagaries that remain unexplained. In this paper, the authors review the evidence that cortical network activity is particularly sensitive to general anesthetics, and suggest that disruption to communication in, and/or among, cortical brain regions is a common mechanism of anesthesia that ultimately produces loss of consciousness. The authors review data from acute brain slices and organotypic cultures showing that anesthetics with differing molecular mechanisms of action share in common the ability to impair neurophysiologic communication. While many questions remain, together, ex vivo and in vivo investigations suggest that a unified understanding of both clinical anesthesia and the neural basis of consciousness is attainable.

Zolpidem Activation of Alpha 1-Containing GABAA Receptors Selectively Inhibits High Frequency Action Potential Firing of Cortical Neurons.

Introduction: High frequency neuronal activity in the cerebral cortex can be induced by noxious stimulation during surgery, brain injury or poisoning. In this scenario, it is essential to block cortical hyperactivity to protect the brain against damage, e.g., by using drugs that act as positive allosteric modulators at GABAA receptors. Yet, cortical neurons express multiple, functionally distinct GABAA receptor subtypes. Currently there is a lack of knowledge which GABAA receptor subtypes would be a good pharmacological target to reduce extensive cortical activity. Methods: Spontaneous action potential activity was monitored by performing extracellular recordings from organotypic neocortical slice cultures of wild type and GABAAR-α1(H101R) mutant mice. Phases of high neuronal activity were characterized using peri-event time histograms. Drug effects on within-up state firing rates were quantified via Hedges' g. Results: We quantified the effects of zolpidem, a positive modulator of GABAA receptors harboring α1-subunits, and the experimental benzodiazepine SH-053-2'F-S-CH3, which preferably acts at α2/3/5- but spares α1-subunits. Both agents decreased spontaneous action potential activity but altered the firing patterns in different ways. Zolpidem reduced action potential firing during highly active network states. This action was abolished by flumazenil, suggesting that it was mediated by benzodiazepine-sensitive GABAA receptors. SH-053-2'F-S-CH3 also attenuated neuronal activity, but unlike zolpidem, failed to reduce high frequency firing. To confirm that zolpidem actions were indeed mediated via α1-dependent actions, it was evaluated in slices from wild type and α(H101R) knock-in mice. Inhibition of high frequency action potential firing was observed in slices from wild type but not mutant mice. Conclusion: Our results suggest that during episodes of scarce and high neuronal activity action potential firing of cortical neurons is controlled by different GABAA receptor subtypes. Exaggerated firing of cortical neurons is reduced by positive modulation of α1-, but not α2/3/5-subunit containing GABAA receptors.

Differential depression of neuronal network activity by midazolam and its main metabolite 1-hydroxymidazolam in cultured neocortical slices.

The benzodiazepine midazolam is widely used in critical care medicine. Midazolam has a clinically active metabolite, 1-hydroxymidazolam. The contribution of 1-hydroxymidazolam to the effects of midazolam is controversial. The aim of the current study was to compare the actions of midazolam and 1-hydroxymidazolam on network activity of cortical neurons. Midazolam depressed neuronal activity at a low concentration of 5 nM. When midazolam concentration was increased, it depressed neuronal discharge rates in a biphasic manner. In comparison, 1-hydroxymidazolam did not depress the cortical network activity at low nanomolar concentrations. Higher concentrations of 1-hydroxymidazolam consistently inhibited neuronal activity. Moreover, midazolam shortened cortical up states at low, but not at high concentrations, while the opposite effect was observed with 1-hydroxymidazolam. The network depressant action of midazolam at low concentrations was absent in slices from GABAA receptor α1(H101R)mutant mice. The α1(H101R)mutation renders α1-subunit containing GABAA receptors insensitive towards benzodiazepines. This GABAA receptor subtype is thought to mediate sedation. As midazolam is more potent than its metabolite 1-hydroxymidazolam, the major clinical effects are thus likely caused by midazolam itself. However, 1-hydroxymidazolam could add to the effects of midazolam, especially after the application of high doses of midazolam, and in case of impaired drug metabolism.

MDMA modulates spontaneous firing of subthalamic nucleus neurons in vitro.

3,4-Methylene-dioxy-N-methylamphetamine (MDMA, 'ecstasy') has a broad spectrum of molecular targets in the brain, among them receptors and transporters of the serotonergic (5-hydroxytryptamine, 5-HT) and noradrenergic systems. Its action on the serotonergic system modulates motor systems in rodents and humans. Although parts of the basal ganglia could be identified as mediators of the motor effects of MDMA, very little is known about the role of the subthalamic nucleus (STN). Therefore, this study investigated the modulation of spontaneous action potential activity of the STN by MDMA (2.5-20 µM) in vitro. MDMA had very heterogeneous effects, ranging from a complete but reversible inhibition to a more than twofold increase in firing at 5 µM. On average, MDMA excited STN neurons moderately, but lost its excitatory effect in the presence of the 5-HT(2A) antagonist MDL 11,939. 5-HT(1A) receptors did not appear to play a major role. Effects of MDMA on transporters for serotonin (SERT) and norepinephrine (NET) were investigated by coapplication of the reuptake inhibitors citalopram and desipramine, respectively. Similar to the effects of 5-HT(2A) receptor blockade, antagonism of SERT and NET bestowed an inhibitory effect on MDMA. From these results, we conclude that both the 5-HT and the noradrenergic system mediate MDMA-induced effects on STN neurons.

Impairment of GABA transporter GAT-1 terminates cortical recurrent network activity via enhanced phasic inhibition.

In the central nervous system, GABA transporters (GATs) very efficiently clear synaptically released GABA from the extracellular space, and thus exert a tight control on GABAergic inhibition. In neocortex, GABAergic inhibition is heavily recruited during recurrent phases of spontaneous action potential activity which alternate with neuronally quiet periods. Therefore, such activity should be quite sensitive to minute alterations of GAT function. Here, we explored the effects of a gradual impairment of GAT-1 and GAT-2/3 on spontaneous recurrent network activity--termed network bursts and silent periods--in organotypic slice cultures of rat neocortex. The GAT-1 specific antagonist NO-711 depressed activity already at nanomolar concentrations (IC50 for depression of spontaneous multiunit firing rate of 42 nM), reaching a level of 80% at 500-1000 nM. By contrast, the GAT-2/3 preferring antagonist SNAP-5114 had weaker and less consistent effects. Several lines of evidence pointed toward an enhancement of phasic GABAergic inhibition as the dominant activity-depressing mechanism: network bursts were drastically shortened, phasic GABAergic currents decayed slower, and neuronal excitability during ongoing activity was diminished. In silent periods, NO-711 had little effect on neuronal excitability or membrane resistance, quite in contrast to the effects of muscimol, a GABA mimetic which activates GABAA receptors tonically. Our results suggest that an enhancement of phasic GABAergic inhibition efficiently curtails cortical recurrent activity and may mediate antiepileptic effects of therapeutically relevant concentrations of GAT-1 antagonists.

Computation of measures of effect size for neuroscience data sets.

The overwhelming majority of research in the neurosciences employs P-values stemming from tests of statistical significance to decide on the presence or absence of an effect of some treatment variable. Although a continuous variable, the P-value is commonly used to reach a dichotomous decision about the presence of an effect around an arbitrary criterion of 0.05. This analysis strategy is widely used, but has been heavily criticized in the past decades. To counter frequent misinterpretations of P-values, it has been advocated to complement or replace P-values with measures of effect size (MES). Many psychological, biological and medical journals now recommend reporting appropriate MES. One hindrance to the more frequent use of MES may be their scarcity in standard statistical software packages. Also, the arguably most widespread data analysis software in neuroscience, matlab, does not provide MES beyond correlation and receiver-operating characteristic analysis. Here we review the most common criticisms of significance testing and provide several examples from neuroscience where use of MES conveys insights not amenable through the use of P-values alone. We introduce an open-access matlab toolbox providing a wide range of MES to complement the frequently used types of hypothesis tests, such as t-tests and analysis of variance. The accompanying documentation provides calculation formulae, intuitive explanations and example calculations for each measure. The toolbox described is usable without sophisticated statistical knowledge and should be useful to neuroscientists wishing to enhance their repertoire of statistical reporting.

Diazepam decreases action potential firing of neocortical neurons via two distinct mechanisms.

BACKGROUND: Benzodiazepines are widely used in clinical anesthesia as premedication, but also to induce general anesthesia. Recent in vitro studies suggest that γ-aminobutyric acid type A receptors, harboring a classical high-affinity benzodiazepine binding site, possess another "nonclassical" binding site for benzodiazepines. At present, it is unclear if, and to what extent, this novel nonclassical binding site is of relevance for the actions of benzodiazepines in the central nervous system. METHODS: Because neocortex is involved in mediating the sedative and hypnotic properties of general anesthetics, we quantified the actions of diazepam over a wide range of concentrations (from 10 nM up to 100 µM) in organotypic slice cultures using extracellular multiunit recordings of spontaneous action potential activity. RESULTS: Up to a concentration of 6.25 µM, diazepam reduced the activity of neocortical neurons, approaching a maximum of approximately 20%. This action was nullified by the benzodiazepine antagonist flumazenil. At concentrations >12.5 µM, diazepam evoked a second concentration-dependent dampening of network activity. Unlike the low concentration effect, this high concentration component was resistant to flumazenil. CONCLUSIONS: Diazepam induced a biphasic attenuation of spontaneous action potential firing of neocortical neurons. Low to moderate concentrations caused a monotonic, mild depression that is mediated via the classical binding site as it is antagonized by flumazenil. However, the effects of diazepam observed at high concentrations were not affected by flumazenil. Hence, these findings support the concept of at least 2 different binding sites for benzodiazepines on γ-aminobutyric acid type A receptors. Furthermore, our results are consistent with the hypothesis that the classical high-affinity binding site mediates low-dose diazepam actions, such as amnesia, anxiolysis, and sedation, while a second, nonclassical and independent site contributes to the anesthetic effects of diazepam, such as hypnosis and immobility.

The head-fixed behaving rat--procedures and pitfalls.

This paper describes experimental techniques with head-fixed, operantly conditioned rodents that allow the control of stimulus presentation and tracking of motor output at hitherto unprecedented levels of spatio-temporal precision. Experimental procedures for the surgery and behavioral training are presented. We place particular emphasis on potential pitfalls using these procedures in order to assist investigators who intend to engage in this type of experiment. We argue that head-fixed rodent models, by allowing the combination of methodologies from molecular manipulations, intracellular electrophysiology, and imaging to behavioral measurements, will be instrumental in combining insights into the functional neuronal organization at different levels of observation. Provided viable behavioral methods are implemented, model systems based on rodents will be complementary to current primate models--the latter providing highest comparability with the human brain, while the former offer hugely advanced methodologies on the lower levels of organization, for example, genetic alterations, intracellular electrophysiology, and imaging.

Cross-approximate entropy of cortical local field potentials quantifies effects of anesthesia--a pilot study in rats.

BACKGROUND: Anesthetics dose-dependently shift electroencephalographic (EEG) activity towards high-amplitude, slow rhythms, indicative of a synchronization of neuronal activity in thalamocortical networks. Additionally, they uncouple brain areas in higher (gamma) frequency ranges possibly underlying conscious perception. It is currently thought that both effects may impair brain function by impeding proper information exchange between cortical areas. But what happens at the local network level? Local networks with strong excitatory interconnections may be more resilient towards global changes in brain rhythms, but depend heavily on locally projecting, inhibitory interneurons. As anesthetics bias cortical networks towards inhibition, we hypothesized that they may cause excessive synchrony and compromise information processing already on a small spatial scale. Using a recently introduced measure of signal independence, cross-approximate entropy (XApEn), we investigated to what degree anesthetics synchronized local cortical network activity. We recorded local field potentials (LFP) from the somatosensory cortex of three rats chronically implanted with multielectrode arrays and compared activity patterns under control (awake state) with those at increasing concentrations of isoflurane, enflurane and halothane. RESULTS: Cortical LFP signals were more synchronous, as expressed by XApEn, in the presence of anesthetics. Specifically, XApEn was a monotonously declining function of anesthetic concentration. Isoflurane and enflurane were indistinguishable; at a concentration of 1 MAC (the minimum alveolar concentration required to suppress movement in response to noxious stimuli in 50% of subjects) both volatile agents reduced XApEn by about 70%, whereas halothane was less potent (50% reduction). CONCLUSIONS: The results suggest that anesthetics strongly diminish the independence of operation of local cortical neuronal populations, and that the quantification of these effects in terms of XApEn has a similar discriminatory power as changes of spontaneous action potential rates. Thus, XApEn of field potentials recorded from local cortical networks provides valuable information on the anesthetic state of the brain.

Altered GABAA,slow inhibition and network oscillations in mice lacking the GABAA receptor beta3 subunit.

Phasic GABAergic inhibition in hippocampus and neocortex falls into two kinetically distinct categories, GABA(A,fast) and GABA(A,slow). In hippocampal area CA1, GABA(A,fast) is generally believed to underlie gamma oscillations, whereas the contribution of GABA(A,slow) to hippocampal rhythms has been speculative. Hypothesizing that GABA(A) receptors containing the beta(3) subunit contribute to GABA(A,slow) inhibition and that slow inhibitory synapses control excitability as well as contribute to network rhythms, we investigated the consequences of this subunit's absence on synaptic inhibition and network function. In pyramidal neurons of GABA(A) receptor beta(3) subunit-deficient (beta(3)(-/-)) mice, spontaneous GABA(A,slow) inhibitory postsynaptic currents (IPSCs) were much less frequent, and evoked GABA(A,slow) currents were much smaller than in wild-type mice. Fittingly, long-lasting recurrent inhibition of population spikes was less powerful in the mutant, indicating that receptors containing beta(3) subunits contribute substantially to GABA(A,slow) currents in pyramidal neurons. By contrast, slow inhibitory control of GABA(A,fast)-producing interneurons was unaffected in beta(3)(-/-) mice. In vivo hippocampal network activity was markedly different in the two genotypes. In beta(3)(-/-) mice, epileptiform activity was observed, and theta oscillations were weaker, slower, less regular and less well coordinated across laminae compared with wild-type mice, whereas gamma oscillations were weaker and faster. The amplitude modulation of gamma oscillations at theta frequency ("nesting") was preserved but was less well coordinated with theta oscillations. With the caveat that seizure-induced changes in inhibitory circuits might have contributed to the changes observed in the mutant animals, our results point to a strong contribution of beta(3) subunits to slow GABAergic inhibition onto pyramidal neurons but not onto GABA(A,fast) -producing interneurons and support different roles for these slow inhibitory synapses in the generation and coordination of hippocampal network rhythms.

Muscarinic blockade weakens interaction of gamma with theta rhythms in mouse hippocampus.

theta (4-12 Hz) and gamma (40-90) oscillations are prominent rhythms in the mammalian brain. A striking feature of these rhythms, possibly vital to memory encoding, is their specific coordination in a manner that has been termed 'nesting', i.e. the preferred occurrence of bouts of gamma activity during specific phases of theta. Both rhythms are shaped by the neuromodulator acetylcholine, but it is unknown to what degree their coordination is influenced by cholinergic neuromodulation. Here, we investigated the effects of a blockade of muscarinic acetylcholine receptors by atropine on theta and gamma oscillations, and their interaction, in mouse hippocampus in vivo. Multi-site recordings from area CA1 of freely moving mice showed that under control conditions gamma activity was amplitude-modulated at theta frequencies. This coordination of theta and gamma oscillations, as assessed by cross-correlation of theta with the gamma envelope, was prominent in basal and apical dendritic laminae but not in intermediate laminae. It was stronger during active exploration than during awake immobility. Atropine (50 mg/kg intraperitoneal) altered several aspects of the individual and nested rhythms. It rendered theta activity irregular, decreased theta oscillation frequency and reduced gamma power. Atropine also reduced the amplitude-modulation of gamma oscillations at theta frequencies, in part by perturbing the coordination of the rhythms on a short time scale. Thus, our findings demonstrate that phase locking of the amplitude of gamma oscillations to theta in hippocampal area CA1 is partially governed by neuronal elements harbouring muscarinic receptors.

Cholinergic modulation of sevoflurane potency in cortical and spinal networks in vitro.

BACKGROUND: Victims of organophosphate intoxication with cholinergic crisis may have need for sedation and anesthesia, but little is known about how anesthetics work in these patients. Recent studies suggest that cholinergic stimulation impairs gamma-aminobutyric acid type A (GABAA) receptor function. Because GABAA receptors are major targets of general anesthetics, the authors investigated interactions between acetylcholine and sevoflurane in spinal and cortical networks. METHODS: Cultured spinal and cortical tissue slices were obtained from embryonic and newborn mice. Drug effects were assessed by extracellular voltage recordings of spontaneous action potential activity. RESULTS: Sevoflurane caused a concentration-dependent decrease in spontaneous action potential firing in spinal (EC50=0.17+/-0.02 mM) and cortical (EC50=0.29+/-0.01 mM) slices. Acetylcholine elevated neuronal excitation in both preparations and diminished the potency of sevoflurane in reducing action potential firing in cortical but not in spinal slices. This brain region-specific decrease in sevoflurane potency was mimicked by the specific GABAA receptor antagonist bicuculline, suggesting that (1) GABAA receptors are major molecular targets for sevoflurane in the cortex but not in the spinal cord and (2) acetylcholine impairs the efficacy of GABAA receptor-mediated inhibition. The latter hypothesis was supported by the finding that acetylcholine reduced the potency of etomidate in depressing cortical and spinal neurons. CONCLUSIONS: The authors raise the question whether cholinergic overstimulation decreases the efficacy of GABAA receptor function in patients with organophosphate intoxication, thereby compromising anesthetic effects that are mediated predominantly via these receptors such as sedation and hypnosis.

Amnesic concentrations of the nonimmobilizer 1,2-dichlorohexafluorocyclobutane (F6, 2N) and isoflurane alter hippocampal theta oscillations in vivo.

BACKGROUND: Drug-induced temporary amnesia is one of the principal goals of general anesthesia. The nonimmobilizer 1,2-dichlorohexafluorocyclobutane (F6, also termed 2N) impairs hippocampus-dependent learning at relative, i.e., lipophilicity-corrected, concentrations similar to isoflurane. Hippocampal theta oscillations facilitate mnemonic processes in vivo and synaptic plasticity (a cellular model of memory) in vitro and are thought to represent a circuit level phenomenon that supports memory encoding. Therefore, the authors investigated the effects of F6 and isoflurane on theta oscillations (4-12 Hz). METHODS: Thirteen adult rats were implanted with multichannel depth electrodes to measure the microelectroencephalogram and were exposed to a range of concentrations of isoflurane and F6 spanning the concentrations that produce amnesia. Five of these animals also underwent control experiments without drug injection. The authors recorded the behavioral state and hippocampal field potentials. They confirmed the electrode location postmortem by histology. RESULTS: The tested concentrations for isoflurane and F6 ranged from 0.035% to 0.77% and from 0.5% to 3.6%, respectively. Isoflurane increased the fraction of time that the animals remained immobile, consistent with sedation, whereas F6 had the opposite effect. Electroencephalographic power in the theta band was less when the animals were immobile than when they explored their environment. F6 suppressed the power of oscillations in the theta band. Isoflurane slowed theta oscillations without reducing total power in the theta band. CONCLUSIONS: Drug-induced changes in theta oscillations may be a common basis for amnesia produced by F6 and isoflurane. The different patterns suggest that these drugs alter network activity by acting on different molecular and/or cellular targets.

Central signals rapidly switch tactile processing in rat barrel cortex during whisker movements.

Palpatory movements ('active' touch) are an integral part of tactile sensing. It is known that tactile signals can be modulated in certain behavioral contexts, but it is still unresolved to what degree this modulation is related to movement kinematics and whether it stems from tactile receptors or from central sources. Using awake, head-fixed rats, trained to contact an object, we measured trajectories of muscle-propelled whisker movement precisely and compared tactile responses to contacts thus accomplished with 'passive' contacts (motionless whisker contacted by object). Multielectrode extracellular recordings in deep layers of barrel cortex revealed that when the animals moved their whiskers actively, tactile processing switched from high response amplitudes, wide cortical representation and low background firing, to low response amplitudes, narrow spatial representation and elevated background firing. Switching was fast (<100 ms) and unrelated to the degree of alertness as assessed by spectral analysis of pre-contact field potentials. Switching persisted when information about whisker kinematics was interrupted by transection of the infraorbital nerve and contacts were mimicked by peripheral electrical stimulation. Taken together, these characteristics render central signals derived from the motor system a likely contributor to the processing of active touch.

Neocortex is the major target of sedative concentrations of volatile anaesthetics: strong depression of firing rates and increase of GABAA receptor-mediated inhibition.

General anaesthetics cause sedation, amnesia and hypnosis. Although these clinically desired actions are indicative of an impairment of neocortical information processing, it is widely held that they are to a large part mediated by subcortical neural networks. Anaesthetic action on brain stem, basal forebrain and thalamus, all of which are known to modulate cortical excitability, would thus ultimately converge on neocortex, perturbing and reducing action potential activity therein. However, as neocortex harbours molecular targets of anaesthetics in high densities, notably GABA(A) receptors, neocortex itself should be very sensitive to anaesthetics. Here, we performed experiments to reveal the extent to which neocortex proper is a relevant target of the low concentrations of volatile anaesthetics causing sedation and hypnosis. We compared the effects of isoflurane, enflurane and halothane on spontaneous action potential activity of rat neocortical neurons in vivo and in isolated cortical networks in vitro, i.e. in the presence and absence of subcortical arousal systems. We observed that the anaesthetics decreased spontaneous firing of neurons via intracortical mechanisms; concentrations inducing hypnosis in humans reduced discharge rates both in vivo and in vitro to the same extent, approximately 50%. This decrease in neuronal activity was paralleled by a significant enhancement of neocortical GABA(A) receptor-mediated inhibition. These findings challenge the notion of predominantly subcortical effects of volatile anaesthetics and suggest that intracortical targets, among them neocortical GABA(A) receptors, mediate the sedative and hypnotic properties of volatile anaesthetics.