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.

Changes in Memory, Sedation, and Receptor Kinetics Imparted by the ß2-N265M and ß3-N265M GABAA Receptor Point Mutations.

Point mutations in the ß2 (N265S) and ß3 (N265M) subunits of γ-amino butyric acid type A receptors (GABAARs) that render them insensitive to the general anesthetics etomidate and propofol have been used to link modulation of ß2-GABAARs to sedation and ß3-GABAARs to surgical immobility. These mutations also alter GABA sensitivity, and mice carrying the ß3-N265M mutation have been reported to have impaired baseline memory. Here, we tested the effects of the ß2-N265M and ß3-N265M mutations on memory, movement, hotplate sensitivity, anxiety, etomidate-induced sedation, and intrinsic kinetics. We found that both ß2-N265M and ß3-N265M mice exhibited baseline deficits in the Context Preexposure Facilitation Effect learning paradigm. Exploratory activity was slightly greater in ß2-N265M mice, but there were no changes in either genotype in anxiety or hotplate sensitivity. ß2-N265M mice were highly resistant to etomidate-induced sedation, and heterozygous mice were partially resistant. In rapid solution exchange experiments, both mutations accelerated deactivation two- to three-fold compared to wild type receptors and prevented modulation by etomidate. This degree of change in the receptor deactivation rate is comparable to that produced by an amnestic dose of etomidate but in the opposite direction, indicating that intrinsic characteristics of GABAARs are optimally tuned under baseline conditions to support mnemonic function.

Dose-dependent suppression of hippocampal contextual memory formation, place cells, and spatial engrams by the NMDAR antagonist (R)-CPP.

We recently reported that the competitive NMDAR antagonist (R,S)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) does not suppress NMDAR-mediated field EPSPs (fEPSPNMDA) or long-term potentiation (LTP) in vitro at concentrations that block contextual conditioning in vivo. Here we tested one possible explanation for the mismatch - that the hippocampus is relatively resistant to CPP compared to other brain structures engaged in contextual fear conditioning. Using the context pre-exposure facilitation effect (CPFE) paradigm to separate the hippocampal and extra-hippocampal components of contextual learning, we found that the active enantiomer (R)-CPP suppressed the hippocampal component with an IC50 of 3.1 mg/kg, a dose that produces brain concentrations below those required to block fEPSPNMDA or LTP. Moreover, using in-vivo calcium imaging of place cells and spatial engrams to directly assess hippocampal spatial coding, we found that (R)-CPP dose-dependently reduced the development of place cells and interfered with the formation of stable spatial engrams when it was administered prior to exposing mice to a novel context. Both effects occurred at doses that interfered with freezing to context in CPFE experiments. We conclude that (R)-CPP blocks memory formation by interfering with hippocampal function, but that it does so by modulating NMDARs at sites that are not engaged in vitro in the same manner that they are in vivo - perhaps through interneuron circuits that do not contribute to fEPSPs and are not required to elicit LTP using standard induction protocols in vitro, but are essential for successful mnemonic function in vivo.

Contrasting Effects of the γ-Aminobutyric Acid Type A Receptor ß3 Subunit N265M Mutation on Loss of Righting Reflexes Induced by Etomidate and the Novel Anesthetic Barbiturate R-mTFD-MPAB.

BACKGROUND: Previous studies have shown that etomidate modulates γ-aminobutyric acid type A receptors by binding at the ß-α subunit interface within the transmembrane domain of receptors that incorporate ß2 or ß3 subunits. Introducing an asparagine-to-methionine (N265M) mutation at position 265 of the ß3 subunit, which sits within the etomidate-binding site, attenuates the hypnotic effect of etomidate in vivo. It was reported recently that the photoactivatable barbiturate R-mTFD-MPAB also acts on γ-aminobutyric acid type A receptors primarily by binding to a homologous site at the γ-ß interface. Given this difference in drug-binding sites established by the in vitro experiments, we hypothesized that the ß3-N265M-mutant mice would not be resistant to the anesthetic effects of R-mTFD-MPAB in vivo, whereas the same mutant mice would be resistant to the anesthetic effects of R-etomidate. METHODS: We measured the effects of IV injection of etomidate and R-mTFD-MPAB on loss and recovery of righting reflex in wild-type mice and in mice carrying the ß3-N265M mutation. RESULTS: Etomidate-induced hypnosis, as measured by the duration of loss of righting reflex, was attenuated in the N265M knock-in mice, confirming prior results. By contrast, recovery of balance and coordinated movement, as measured by the ability to maintain all 4 paws on the ground, was unaffected by the mutation. Neither hypnosis nor impairment of coordinated movement produced by the barbiturate R-mTFD-MPAB was affected by the mutation. CONCLUSIONS: The findings confirmed our hypothesis that mutating the etomidate-binding site would not alter the response to the barbiturate R-mTFD-MPAB. Furthermore, we confirmed previous studies indicating that etomidate-induced hypnosis is mediated in part by ß3-containing receptors. We also extended previous findings by showing that etomidate-impaired balance and coordinated movement are not mediated by ß3-containing receptors, thus implicating ß2-containing receptors in this end point.

Exceptionally stable fluorous emulsions for the intravenous delivery of volatile general anesthetics.

BACKGROUND: IV delivery of volatile fluorinated anesthetics has a number of potential advantages when compared with the current inhalation method of administration. We reported previously that the IV delivery of sevoflurane can be achieved through an emulsion composed of a linear fluorinated diblock copolymer, a stabilizer, and the anesthetic. However, this original emulsion was subject to particle size growth that would limit its potential clinical utility. We hypothesized that the use of bulkier fluorous groups and smaller polyethylene glycol moieties in the polymer design would result in improved emulsion stability while maintaining anesthetic functionality. METHODS: The authors prepared emulsions incorporating sevoflurane, perfluorooctyl bromide as a stabilizing agent, and combinations of linear fluorinated diblock copolymer and a novel dibranched fluorinated diblock copolymer. Emulsion stability was assessed using dynamic light scattering. The ability of the emulsions to induce anesthesia was tested in vivo by administering them intravenously to 15 male Sprague-Dawley rats and measuring loss of the forepaw righting reflex. RESULTS: 20% (volume/volume) sevoflurane emulsions incorporating mixtures of dibranched and linear diblock copolymers had improved stability, with those containing an excess of the dibranched polymers displaying stability of particle size for more than 1 yr. The ED50s for loss of forepaw-righting reflex were all similar, and ranged between 0.55- 0.60 ml/kg body weight. CONCLUSIONS: Hemifluorinated dibranched polymers can be used to generate exceptionally stable sevoflurane nanoemulsions, as required of formulations intended for clinical use. IV delivery of the emulsion in rats resulted in induction of anesthesia with rapid onset and smooth and rapid recovery.

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.

Fluoropolymer-based emulsions for the intravenous delivery of sevoflurane.

BACKGROUND: The intravenous delivery of halogenated volatile anesthetics has been previously achieved using phospholipid-stabilized emulsions, e.g., Intralipid. However, fluorinated volatile anesthetics, such as sevoflurane, are partially fluorophilic and do not mix well with classic nonfluorinated lipids. This effect limits the maximum amount of sevoflurane that can be stably emulsified in Intralipid to 3.5% vol/vol. This is a significant limitation to the potential clinical use of Intralipid-based emulsions. METHODS: The authors prepared a 20% vol/vol sevoflurane emulsion using a novel fluorinated surfactant and tested its effectiveness and therapeutic index by administering it to male Sprague-Dawley rats via intravenous injection into the jugular vein. The median effective dose to induce anesthesia (ED50), the median lethal dose (LD50), and the therapeutic index (LD50/ED50) were determined. Anesthesia was measured by loss of the forepaw righting reflex. RESULTS: The ED50 and LD50 values were found to be 0.41 and 1.05 ml emulsion/kg body weight, respectively. These lead to a therapeutic index of 2.6, which compares favorably with previously determined values of emulsified isoflurane, as well as values for propofol and thiopental. CONCLUSIONS: A novel semifluorinated surfactant was able to considerably increase the maximum amount of stably emulsified sevoflurane compared with Intralipid. These formulations can be used to rapidly induce anesthesia with bolus dosing from which recovery is smooth and rapid.

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.