Hypervulnerability to Sound Exposure through Impaired Adaptive Proliferation of Peroxisomes.

A deficiency in pejvakin, a protein of unknown function, causes a strikingly heterogeneous form of human deafness. Pejvakin-deficient (Pjvk(-/-)) mice also exhibit variable auditory phenotypes. Correlation between their hearing thresholds and the number of pups per cage suggest a possible harmful effect of pup vocalizations. Direct sound or electrical stimulation show that the cochlear sensory hair cells and auditory pathway neurons of Pjvk(-/-) mice and patients are exceptionally vulnerable to sound. Subcellular analysis revealed that pejvakin is associated with peroxisomes and required for their oxidative-stress-induced proliferation. Pjvk(-/-) cochleas display features of marked oxidative stress and impaired antioxidant defenses, and peroxisomes in Pjvk(-/-) hair cells show structural abnormalities after the onset of hearing. Noise exposure rapidly upregulates Pjvk cochlear transcription in wild-type mice and triggers peroxisome proliferation in hair cells and primary auditory neurons. Our results reveal that the antioxidant activity of peroxisomes protects the auditory system against noise-induced damage.

The Pharmacological Effects of Phenylephrine are Indirect, Mediated by Noradrenaline Release from the Cytoplasm.

Phenylephrine (PE) is a canonical α1-adrenoceptor-selective agonist. However, unexpected effects of PE have been observed in preclinical and clinical studies, that cannot be easily explained by its actions on α1-adrenoceptors. The probability of the involvement of α2- and ß-adrenoceptors in the effect of PE has been raised. In addition, our earlier study observed that PE released noradrenaline (NA) in a [Ca2+]o-independent manner. To elucidate this issue, we have investigated the effects of PE on [3H]NA release and α1-mediated smooth muscle contractions in the mouse vas deferens (MVD) as ex vivo preparation. The release experiments were designed to assess the effects of PE at the presynaptic terminal, whereas smooth muscle isometric contractions in response to electrical field stimulation were used to measure PE effect postsynaptically. Our results show that PE at concentrations between 0.3 and 30 µM significantly enhanced the resting release of [3H]NA in a [Ca2+]o-independent manner. In addition, prazosin did not affect the release of NA evoked by PE. On the contrary, PE-evoked smooth muscle contractions were inhibited by prazosin administration indicating the α1-adrenoceptor-mediated effect. When the function of the NA transporter (NAT) was attenuated with nisoxetine, PE failed to release NA and the contractions were reduced by approximately 88%. The remaining part proved to be prazosin-sensitive. The present work supports the substantial indirect effect of PE which relays on the cytoplasmic release of NA, which might explain the reported side effects for PE.

Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes.

The understanding of brain computations requires methods that read out neural activity on different spatial and temporal scales. Following signal propagation and integration across a neuron and recording the concerted activity of hundreds of neurons pose distinct challenges, and the design of imaging systems has been mostly focused on tackling one of the two operations. We developed a high-resolution, acousto-optic two-photon microscope with continuous three-dimensional (3D) trajectory and random-access scanning modes that reaches near-cubic-millimeter scan range and can be adapted to imaging different spatial scales. We performed 3D calcium imaging of action potential backpropagation and dendritic spike forward propagation at sub-millisecond temporal resolution in mouse brain slices. We also performed volumetric random-access scanning calcium imaging of spontaneous and visual stimulation-evoked activity in hundreds of neurons of the mouse visual cortex in vivo. These experiments demonstrate the subcellular and network-scale imaging capabilities of our system.

Roller Coaster Scanning reveals spontaneous triggering of dendritic spikes in CA1 interneurons.

Inhibitory interneurons are considered to be the controlling units of neural networks, despite their sparse number and unique morphological characteristics compared with excitatory pyramidal cells. Although pyramidal cell dendrites have been shown to display local regenerative events--dendritic spikes (dSpikes)--evoked by artificially patterned stimulation of synaptic inputs, no such studies exist for interneurons or for spontaneous events. In addition, imaging techniques have yet to attain the required spatial and temporal resolution for the detection of spontaneously occurring events that trigger dSpikes. Here we describe a high-resolution 3D two-photon laser scanning method (Roller Coaster Scanning) capable of imaging long dendritic segments resolving individual spines and inputs with a temporal resolution of a few milliseconds. By using this technique, we found that local, NMDA receptor-dependent dSpikes can be observed in hippocampal CA1 stratum radiatum interneurons during spontaneous network activities in vitro. These NMDA spikes appear when approximately 10 spatially clustered inputs arrive synchronously and trigger supralinear integration in dynamic interaction zones. In contrast to the one-to-one relationship between computational subunits and dendritic branches described in pyramidal cells, here we show that interneurons have relatively small (∼14 µm) sliding interaction zones. Our data suggest a unique principle as to how interneurons integrate synaptic information by local dSpikes.

Presynaptic nanoscale components of retrograde synaptic signaling.

While our understanding of the nanoscale architecture of anterograde synaptic transmission is rapidly expanding, the qualitative and quantitative molecular principles underlying distinct mechanisms of retrograde synaptic communication remain elusive. We show that a particular form of tonic cannabinoid signaling is essential for setting target cell-dependent synaptic variability. It does not require the activity of the two major endocannabinoid-producing enzymes. Instead, by developing a workflow for physiological, anatomical, and molecular measurements at the same unitary synapse, we demonstrate that the nanoscale stoichiometric ratio of type 1 cannabinoid receptors (CB1Rs) to the release machinery is sufficient to predict synapse-specific release probability. Accordingly, selective decrease of extrasynaptic CB1Rs does not affect synaptic transmission, whereas in vivo exposure to the phytocannabinoid Δ9-tetrahydrocannabinol disrupts the intrasynaptic nanoscale stoichiometry and reduces synaptic variability. These findings imply that synapses leverage the nanoscale stoichiometry of presynaptic receptor coupling to the release machinery to establish synaptic strength in a target cell-dependent manner.

Comparison of Ca2+ transients and [Ca2+]i in the dendrites and boutons of non-fast-spiking GABAergic hippocampal interneurons using two-photon laser microscopy and high- and low-affinity dyes.

Using two-photon laser microscopy, high- and low-affinity dyes and patch clamp electrophysiology, we successfully measured somatic stimulation-evoked Ca(2+) transients simultaneously in the dendrites and axonal boutons of the same non-fast-spiking GABAergic interneurons in acute slice preparations obtained from hippocampal area CA1. The advantage of the acute preparation is that both neuronal connections and anatomy are maintained. Calculated as unperturbed values, the amplitudes of Ca(2+) transients and changes in [Ca(2+)]i in response to somatic single or burst stimulation were much higher in boutons (428 nM/AP) than in dendrites (49 nM/AP), leading to the conclusion that the much greater influx of Ca(2+) observed in terminals might be due to a higher density of N-type voltage-sensitive Ca(2+) channels compared to the L-type channels present in dendrites. Whereas the decay of Ca(2+) transients recorded in dendrites was primarily mono-exponential, the decay in boutons was bi-exponential, as indicated by an initial fast phase, followed by a much slower reduction in fluorescence intensity. The extrusion of Ca(2+) was much faster in boutons than in dendrites. To avoid saturation effects and the flawed conversion of fluorescence measures of [Ca(2+)]i, we assessed the limits of [Ca(2+)] measurements (which ranged between 6 and 82% of the applied dye saturation) when high- and low-affinity dyes were applied at different concentrations. When two APs were delivered at a high frequency (>3 Hz) of stimulation, the low-affinity indicators OGB-6F (KD = 3.0 µM) and OGB-5N (KD = 20 µM) were able to accurately reflect the changes in ΔF/F produced by the consecutive APs. There was no difference in the endogenous buffer capacity (κE), which can shape Ca(2+) signals, calculated in dendrites (κE = 354) or boutons (κE = 458).

P2X7 purinergic receptor modulates dentate gyrus excitatory neurotransmission and alleviates schizophrenia-like symptoms in mouse.

ATP-gated P2X7 receptors (P2X7Rs) play a crucial role in brain disorders. However, how they affect normal and pathological synaptic transmission is still largely unclear. Here, by using whole-cell patch-clamp technique to record AMPA- and NMDA receptor-mediated excitatory postsynaptic currents (s/mEPSCs) in dentate gyrus granule cells (DG GCs), we revealed a modulation by P2X7Rs of presynaptic sites, especially originated from entorhinal cortex (EC)-GC path but not the mossy cell (MC)-GC path. The involvement of P2X7Rs was confirmed using a pharmacological approach. Additionally, the acute activation of P2X7Rs directly elevated calcium influx from EC-GC terminals. In postnatal phencyclidine (PCP)-induced mouse model of schizophrenia, we observed that P2X7R deficiency restored the EC-GC synapse alteration and alleviated PCP-induced symptoms. To summarize, P2X7Rs participate in the modulation of GC excitatory neurotransmission in the DG via EC-GC pathway, contributing to pathological alterations of neuronal functions leading to neurodevelopmental disorders.

A(2B) adenosine receptors in immunity and inflammation.

A(2B) adenosine receptors are increasingly recognized as important orchestrators of inflammation. A(2B) receptor activation promotes the inflammatory response of mast cells, epithelial cells, smooth muscle cells and fibroblasts, thereby contributing to the pathophysiology of asthma and colitis. A(2B) receptor stimulation limits endothelial cell inflammatory responses and permeability and suppresses macrophage activation thereby preventing tissue injury after episodes of hypoxia and ischemia. A(2B) receptor stimulation also promotes the production of angiogenic cytokines by endothelial cells, mast cells and dendritic cells, aiding granuloma tissue formation and inflammatory resolution, but can also contribute to tumor growth. A(2B) receptors are, thus, potentially important pharmacological targets in treating immune system dysfunction and inflammation.

A2B adenosine receptors protect against sepsis-induced mortality by dampening excessive inflammation.

Despite intensive research, efforts to reduce the mortality of septic patients have failed. Adenosine is a potent extracellular signaling molecule, and its levels are elevated in sepsis. Adenosine signals through G-protein-coupled receptors and can regulate the host's response to sepsis. In this study, we studied the role of A(2B) adenosine receptors in regulating the mortality and inflammatory response of mice following polymicrobial sepsis. Genetic deficiency of A(2B) receptors increased the mortality of mice suffering from cecal ligation and puncture-induced sepsis. The increased mortality of A(2B) knockout mice was associated with increased levels of inflammatory cytokines and chemokines and augmented NF-kappaB and p38 activation in the spleen, heart, and plasma in comparison with wild-type animals. In addition, A(2B) receptor knockout mice showed increased splenic apoptosis and phosphatase and tensin homolog activation and decreased Akt activation. Experiments using bone-marrow chimeras revealed that it is the lack of A(2B) receptors on nonhematopoietic cells that is primarily responsible for the increased inflammation of septic A(2B) receptor-deficient mice. These results indicate that A(2B) receptor activation may offer a new therapeutic approach for the management of sepsis.

Convergent cross-species pro-cognitive effects of RGH-235, a new potent and selective histamine H3 receptor antagonist/inverse agonist.

The histamine H3 receptor is a favourable target for the treatment of cognitive deficits. Here we report the in vitro and in vivo profile of RGH-235, a new potent, selective, and orally active H3 receptor antagonist/inverse agonist developed by Gedeon Richter Plc. Radioligand binding and functional assays were used for in vitro profiling. Procognitive efficacy was investigated in rodent cognitive tests, in models of attention deficit hyperactive disorder (ADHD) and in cognitive tests of high translational value (rat touch screen visual discrimination test, primate fixed-foreperiod visual reaction time task). Results were supported by pharmacokinetic studies, neurotransmitter release, sleep EEG and dipsogenia. RGH-235 displayed high affinity to H3 receptors (Ki = 3.0-9.2 nM, depending on species), without affinity to H1, H2 or H4 receptors and >100 other targets. RGH-235 was an inverse agonist ([35S] GTPγS binding) and antagonist (pERK1/2 ELISA), showing favourable kinetics, inhibition of the imetit-induced dipsogenia and moderate effects on sleep-wake EEG. RGH-235 stimulated neurotransmitter release both in vitro and in vivo. RGH-235 was active in spontaneously hypertensive rats (SHR), generally considered as a model of ADHD, and revealed a robust pro-cognitive profile both in rodent and primate tests (in 0.3-1 mg/kg) and in models of high translational value (e.g. in a rodent touch screen test and in non-human primates). The multiple and convergent procognitive effects of RGH-235 support the view that beneficial cognitive effects can be linked to antagonism/inverse agonism of H3 receptors.

Adenosine A2A receptor activation protects CD4+ T lymphocytes against activation-induced cell death.

Activation-induced cell death (AICD) is initiated by T-cell receptor (TCR) restimulation of already activated and expanded peripheral T cells and is mediated through Fas/Fas ligand (FasL) interactions. Adenosine is a purine nucleoside signaling molecule, and its immunomodulatory effects are mediated by 4 G-protein-coupled receptors: A(1), A(2A), A(2B), and A(3). In this study, we investigated the role of A(2A) receptors in regulating CD4(+) T lymphocyte AICD. Our results showed that the selective A(2A) receptor agonist CGS21680 (EC(50)=15.2-32.6 nM) rescued mouse CD4(+) hybridomas and human Jurkat cells from AICD and that this effect was reversed by the selective A(2A) receptor antagonist ZM241385 (EC(50)=2.3 nM). CGS21680 decreased phosphatidylserine exposure on the membrane, as well as the cleavage of caspase-3, caspase-8 and poly(ADP-ribose) polymerase indicating that A(2A) receptor stimulation blocks the extrinsic apoptotic pathway. In addition, CGS21680 attenuated both Fas and FasL mRNA expression. This decrease in FasL expression was associated with decreased activation of the transcription factor systems NF-kappaB, NF-ATp, early growth response (Egr)-1, and Egr-3. The antiapoptotic effect of A(2A) receptor stimulation was mediated by protein kinase A. Together, these results demonstrate that A(2A) receptor activation suppresses the AICD of peripheral T cells.

Fast- or slow-inactivated state preference of Na+ channel inhibitors: a simulation and experimental study.

Sodium channels are one of the most intensively studied drug targets. Sodium channel inhibitors (e.g., local anesthetics, anticonvulsants, antiarrhythmics and analgesics) exert their effect by stabilizing an inactivated conformation of the channels. Besides the fast-inactivated conformation, sodium channels have several distinct slow-inactivated conformational states. Stabilization of a slow-inactivated state has been proposed to be advantageous for certain therapeutic applications. Special voltage protocols are used to evoke slow inactivation of sodium channels. It is assumed that efficacy of a drug in these protocols indicates slow-inactivated state preference. We tested this assumption in simulations using four prototypical drug inhibitory mechanisms (fast or slow-inactivated state preference, with either fast or slow binding kinetics) and a kinetic model for sodium channels. Unexpectedly, we found that efficacy in these protocols (e.g., a shift of the "steady-state slow inactivation curve"), was not a reliable indicator of slow-inactivated state preference. Slowly associating fast-inactivated state-preferring drugs were indistinguishable from slow-inactivated state-preferring drugs. On the other hand, fast- and slow-inactivated state-preferring drugs tended to preferentially affect onset and recovery, respectively. The robustness of these observations was verified: i) by performing a Monte Carlo study on the effects of randomly modifying model parameters, ii) by testing the same drugs in a fundamentally different model and iii) by an analysis of the effect of systematically changing drug-specific parameters. In patch clamp electrophysiology experiments we tested five sodium channel inhibitor drugs on native sodium channels of cultured hippocampal neurons. For lidocaine, phenytoin and carbamazepine our data indicate a preference for the fast-inactivated state, while the results for fluoxetine and desipramine are inconclusive. We suggest that conclusions based on voltage protocols that are used to detect slow-inactivated state preference are unreliable and should be re-evaluated.

Contribution of analog signaling to neurotransmitter interactions and behavior: Role of transporter-mediated nonquantal dopamine release.

Neuronal networks cause changes in behaviorally important information processing through the vesicular release of neurotransmitters governed by the rate and timing of action potentials (APs). Herein, we provide evidence that dopamine (DA), nonquantally released from the cytoplasm, may exert similar effects in vivo. In mouse slice preparations, (+/-)-3,4-methylenedioxy-methamphetamine (MDMA, or ecstasy) and ß-phenylethylamine (ß-PEA)-induced DA release in the striatum and nucleus accumbens (NAc), two regions of the brain involved in reward-driven and social behavior and inhibited the axonal stimulation-induced release of tritiated acetylcholine ([3 H]ACh) in the striatum. The DA transporter (DAT) inhibitor (GBR-12909) prevented MDMA and ß-PEA from causing DA release. GBR-12909 could also restore some of the stimulated acetylcholine release reduced by MDMA or ß-PEA in the striatum confirming the fundamental role of DAT. In addition, hypothermia could prevent the ß-PEA-induced release in the striatum and in the NAc. Sulpiride, a D2 receptor antagonist, also prevented the inhibitory effects of MDMA or ß-PEA on stimulated ACh release, suggesting they act indirectly via binding of DA. Reflecting the neurochemical interactions in brain slices at higher system level, MDMA altered the social behavior of rats by preferentially enhancing passive social behavior. Similar to the in vitro effects, GBR-12909 treatment reversed specific elements of the MDMA-induced changes in behavior, such as passive social behavior, while left others including social play unchanged. The changes in behavior by the high level of extracellular DA-- a significant amount originating from cytoplasmic release--suggest that in addition to digital computation through synapses, the brain also uses analog communication, such as DA signaling, to mediate some elements of complex behaviors, but in a much longer time scale.

Enhanced dendritic action potential backpropagation in parvalbumin-positive basket cells during sharp wave activity.

In this study two-photon imaging and single cell electrophysiological measurements were carried out in PV+ hippocampal interneurons to compare the dendritic calcium dynamics of somatically evoked backpropagating action potentials (BAPs) and in vitro sharp wave oscillation (SPW) activated BAPs at different distances from the soma. In the case of 300 µm thick, non-oscillating slices, the BAP-evoked Ca(2+) (BAP-Ca(2+)) influx propagated along the dendritic tree in a non-uniform manner and its amplitude gradually reduced when measured at more distal regions. In contrast to the evoked BAP-Ca(2+)s, the spontaneous SPW-induced Ca(2+) influx had only a small distance-dependent decrement. Our results suggest that similarly to nicotinic acetylcholine receptor activation, synaptic activity during hippocampal SPWs increases AP backpropagation into distant dendritic segments. Bath application of Nimodipine, a specific Ca(2+) channel blocker and tetrodotoxine decreased the amplitude of the somatically evoked Ca(2+) influx, which suggests that L-type Ca(2+) channels play an important role both during somatically evoked and SPW-induced BAPs.

P2X7 Receptor-Dependent Layer-Specific Changes in Neuron-Microglia Reactivity in the Prefrontal Cortex of a Phencyclidine Induced Mouse Model of Schizophrenia.

Background: It has been consistently reported that the deficiency of the adenosine triphosphate (ATP) sensitive purinergic receptor P2X7 (P2X7R) ameliorates symptoms in animal models of brain diseases. Objective: This study aimed to investigate the role of P2X7R in rodent models of acute and subchronic schizophrenia based on phencyclidine (PCP) delivery in animals lacking or overexpressing P2X7R, and to identify the underlying mechanisms involved. Methods: The psychotomimetic effects of acute i.p. PCP administration in C57Bl/6J wild-type, P2X7R knockout (P2rx7-/-) and overexpressing (P2X7-EGFP) young adult mice were quantified. The medial prefrontal cortex (mPFC) of P2rx7-/- and heterozygous P2X7-EGFP acutely treated animals was characterized through immunohistochemical staining. The prefrontal cortices of young adult P2rx7-/- and P2rx7tg/+ mice were examined with tritiated dopamine release experiments and the functional properties of the mPFC pyramidal neurons in layer V from P2rx7-/- mice were assessed by patch-clamp recordings. P2rx7-/- animals were subjected to a 7 days subchronic systemic PCP treatment. The animals working memory performance and PFC cytokine levels were assessed. Results: Our data strengthen the hypothesis that P2X7R modulates schizophrenia-like positive and cognitive symptoms in NMDA receptor antagonist models in a receptor expression level-dependent manner. P2X7R expression leads to higher medial PFC susceptibility to PCP-induced circuit hyperactivity. The mPFC of P2X7R knockout animals displayed distinct alterations in the neuronal activation pattern, microglial organization, specifically around hyperactive neurons, and were associated with lower intrinsic excitability of mPFC neurons. Conclusions: P2X7R expression exacerbated PCP-related effects in C57Bl/6J mice. Our findings suggest a pleiotropic role of P2X7R in the mPFC, consistent with the observed behavioral phenotype, regulating basal dopamine concentration, layer-specific neuronal activation, intrinsic excitability of neurons in the mPFC, and the interaction of microglia with hyperactive neurons. Direct measurements of P2X7R activity concerning microglial ramifications and dynamics could help to further elucidate the molecular mechanisms involved.

Adenosine A2A receptor activation inhibits T helper 1 and T helper 2 cell development and effector function.

Adenosine is an immunosuppressive nucleoside, and adenosine A(2A) receptors inhibit T-cell activation. We investigated the role of A(2A) receptors in regulating T helper (Th)1- and Th2-cell development and effector function. A(2A)-receptor stimulation suppressed the development of T-cell receptor (TCR) -stimulated naive T cells into both Th1 and Th2 cells, as indicated by decreased IFN-gamma production by cells developed under Th1-skewing conditions and decreased interleukin (IL) -4, IL-5, and IL-10 production by cells developed under Th2-skewing conditions. Using A(2A) receptor-deficient mice, we demonstrate that A(2A) receptor activation inhibits Th1- and Th2-cell development by decreasing the proliferation and IL-2 production of naive T cells, irrespective of whether the cells are expanded under Th1- or Th2-skewing environment. Using in vivo established Th1 and Th2 cells, we further demonstrate the nonselective nature of A(2A) receptor-mediated immunosuppressive effects, because A(2A) receptor activation decreased IFN-gamma and IL-4 secretion and mRNA level of TCR-stimulated effector Th1 and Th2 cells, respectively. A(2A) receptor mRNA expression in both Th1 and Th2 effector cells increased following TCR stimulation. In summary, these data demonstrate that A(2A) receptor activation has strong inhibitory actions during early developmental, as well as late effector, stages of Th1- and Th2-cell responses.

Modulation of dopaminergic neurotransmission in rat striatum upon in vitro and in vivo diclofenac treatment.

Diclofenac (DCF) is a widely used non-steroidal anti-inflammatory drug, which also act as a mitochondrial toxin. As it is known that selective mitochondrial complex I inhibition combined with mild oxidative stress causes striatal dopaminergic dysfunction, we tested whether DCF also compromise dopaminergic function in the striatum. [3H]Dopamine ([3H]DA) release was measured from rat striatal slices after in vitro (2 h, 10-25 micromol/L) or in vivo (3 mg/kg i.v. for 28 days) DCF treatment. In vitro treatment significantly decreased [3H]DA uptake and dopamine (DA) content of the slices. H2O2 (0.1 mmol/L)-evoked DA release was enhanced. Intracellular reactive oxygen species production was not significantly changed in the presence of DCF. After in vivo DCF treatment no apparent decrease in striatal DA content was observed and the uptake of [3H]DA into slices was increased. The intensity of tyrosine hydroxylase immunoreactivity in the striatum was highly variable, and both decrease and increase were observed in individual rats. The H2O2-evoked [3H]DA release was significantly decreased and the effluent contained a significant amount of [3H]octopamine, [3H]tyramine, and [3H]beta-phenylethylamine. The ATP content and adenylate energy charge were decreased. In conclusion, whereas in vitro DCF pre-treatment resembles the effect of the mitochondrial toxin rotenone, in vivo it rather counteracts than aggravates dopaminergic dysfunction.

The effect of nicotine on spiking activity and Ca2+ dynamics of dendritic spines in rat CA1 pyramidal neurons.

Nicotinic acetylcholine receptors (nAChRs) of the hippocampus have been thought to contribute to cognitive enhancement by cigarette smoking. Although positive modulation on cognitive functions is linked to the smoked, low-dose nicotine, the cellular correlate behind this modulation is unknown. It has been accepted that cellular mechanisms underlying plastic effects on memory involve the association of backpropagating action potentials (bAPs) with synaptic activity in the hippocampus. Here, we show the effects of low-dose (1 microM) nicotine on bAP-evoked Ca2+ transients in basal dendrites and spines of pyramidal neurons in rat hippocampal slices. Although nicotine application failed to have any direct effect in low concentration, it could significantly enhance bAP-evoked Ca2+ transients through presynaptic nAChRs located on axon terminals innervating pyramidal cells. The activation of these receptors is known to release neurotransmitters and induce postsynaptic currents. High-dose (250-500 microM) nicotine could induce firing and Ca2+ accumulation in spines. Large amplitude currents were observed occasionally (8 out of 18 cells) in voltage clamp recordings in response to pressure application of high-dose nicotine. This may explain the relatively low incidence of nicotine-induced firing (7 out of 27 cells) under current clamp. These data indicate that (i) activation of presynaptic nAChRs can modulate backreporting in dendrites of pyramidal neurons and (ii) there is a group of pyramidal neurons with higher nicotine-sensitivity, producing firing at strong stimulations. Our data revealed a subcellular effect of nicotine through regulation of Ca2+ levels in the computational units of pyramidal neurons.

Layer-specific differences in reactive oxygen species levels after oxygen-glucose deprivation in acute hippocampal slices.

The major role of reactive oxygen species (ROS) in the pathomechanism of ischemia have been widely recognized. Still, measurements of the precise time course and regional distribution of ischemia-induced ROS level changes in acute brain slices have been missing. By using acute hippocampal slices and the fluorescent dye CM-H2DCFDA, we showed that reoxygenation after in vitro ischemia (oxygen-glucose deprivation; OGD) increased ROS levels in the hippocampal CA1 layers vulnerable to ischemia but did not have significant effects in the resistant stratum granulosum in the dentate gyrus (DG). Production of ROS started during OGD, but, contrary to reoxygenation, it manifested as a ROS level increase exclusively in the presence of catalase and glutathione peroxidase inhibition. The mechanism of ROS production involves the activation of NMDA receptors and nitric oxide synthases. The inhibition of ROS response by either AP-5 or L-NAME together with the ROS sensitivity profile of the dye suggest that peroxynitrite, the reaction product of superoxide and nitric oxide, plays a role in the response. Direct visualization of layer-specific effects of ROS production and its scavenging, shown for the first time in acute hippocampal slices, suggests that distinct ROS homeostasis may underlie the different ischemic vulnerability of CA1 and DG.

Dendritic nicotinic receptors modulate backpropagating action potentials and long-term plasticity of interneurons.

Stratum radiatum interneurons, unlike pyramidal cells, are rich in nicotinic acetylcholine receptors (nAChRs); however, the role of these receptors in plasticity has remained elusive. As opposed to previous physiological studies, we found that functional alpha7-subunit-containing nAChRs (alpha7-nAChRs) are abundant on interneuron dendrites of rats. Moreover, dendritic Ca2+ transients induced by activation of alpha7-nAChRs increase as a function of distance from soma. The activation of these extrasynaptic alpha7-nAChRs by cholinergic agonists either facilitated or depressed backpropagating action potentials, depending on the timing of alpha7-nAChR activation. We have previously shown that dendritic alpha7-nAChRs are involved in the regulation of synaptic transmission, suggesting that alpha7-nAChRs may play an important role in the regulation of the spike timing-dependent plasticity. Here we provide evidence that long-term potentiation is indeed boosted by stimulation of dendritic alpha7-nAChRs. Our results suggest a new mechanism for a cholinergic switch in memory encoding and retrieval.