Danko Georgiev

Lower Gene Expression for KCNS3 Potassium Channel Subunit in Parvalbumin-Containing Neurons in the Prefrontal Cortex in Schizophrenia

OBJECTIVE In schizophrenia, alterations in markers of cortical GABA neurotransmission are prominent in parvalbumin-containing neurons. Parvalbumin neurons selectively express KCNS3, the gene encoding the Kv9.3 potassium channel α-subunit. Kv9.3 subunits are present in voltage-gated potassium channels that contribute to the precise detection of coincident excitatory synaptic inputs to parvalbumin neurons. This distinctive feature of parvalbumin neurons appears important for the synchronization of cortical neural networks in γ-oscillations. Because impaired prefrontal cortical γ-oscillations are thought to underlie the cognitive impairments in schizophrenia, the authors investigated whether KCNS3 mRNA levels are altered in the prefrontal cortex of schizophrenia subjects. METHOD KCNS3 mRNA expression was evaluated by in situ hybridization in 22 matched pairs of schizophrenia and comparison subjects and by microarray analyses of pooled samples of individually dissected neurons that were labeled with Vicia villosa agglutinin (VVA), a parvalbumin neuron-selective marker, in a separate cohort of 14 pairs. Effects of chronic antipsychotic treatments on KCNS3 expression were tested in the prefrontal cortex of antipsychotic-exposed monkeys. RESULTS By in situ hybridization, KCNS3 mRNA levels were 23% lower in schizophrenia subjects. At the cellular level, both KCNS3 mRNA-expressing neuron density and KCNS3 mRNA level per neuron were significantly lower. By microarray, KCNS3 mRNA levels were lower by 40% in VVA-labeled neurons from schizophrenia subjects. KCNS3 mRNA levels were not altered in antipsychotic-exposed monkeys. CONCLUSIONS These findings reveal lower KCNS3 expression in prefrontal cortical parvalbumin neurons in schizophrenia, providing a molecular basis for compromised detection of coincident synaptic inputs to parvalbumin neurons that could contribute to altered γ-oscillations and impaired cognition in schizophrenia.

Insensitivity to glutamate neurotoxicity mediated by NMDA receptors in association with delayed mitochondrial membrane potential disruption in cultured rat cortical neurons

We have attempted to elucidate mechanisms underlying differential vulnerability to glutamate (Glu) using cultured neurons prepared from discrete structures of embryonic rat brains. Brief exposure to Glu led to a significant decrease in the mitochondrial activity in hippocampal neurons cultured for 9 or 12 days at 10 μM to 1 mM with an apoptosis‐like profile, without markedly affecting that in cortical neurons. Brief exposure to Glu also increased lactate dehydrogenase release along with a marked decrease in the number of cells immunoreactive for a neuronal marker protein in hippocampal, but not cortical, neurons. Similar insensitivity was seen to the cytotoxicity by NMDA, but not to that by tunicamycin, 2,4‐dinitrophenol, hydrogen peroxide or A23187, in cortical neurons. However, NMDA was more efficient in increasing intracellular free Ca2+ levels in cortical neurons than in hippocampal neurons. Antagonists for neuroprotective metabotropic Glu receptors failed to significantly affect the insensitivity to Glu, while NMDA was more effective in disrupting mitochondrial membrane potentials in hippocampal than cortical neurons. These results suggest that cortical neurons would be insensitive to the apoptotic neurotoxicity mediated by NMDA receptors through a mechanism related to mitochondrial membrane potentials, rather than intracellular free Ca2+ levels, in the rat brain.

A critical importance of polyamine site in NMDA receptors for neurite outgrowth and fasciculation at early stages of P19 neuronal differentiation

We have investigated the role of N-methyl-d-aspartate receptors (NMDARs) and gamma-aminobutyric acid receptors type A (GABA(A)Rs) at an early stage of P19 neuronal differentiation. The subunit expression was profiled in 24-hour intervals with RT-PCR and functionality of the receptors was verified via fluo-3 imaging of Ca(2+) dynamics in the immature P19 neurons showing that both NMDA and GABA excite neuronal bodies, but only polyamine-site sensitive NMDAR stimulation leads to enhanced Ca(2+) signaling in the growth cones. Inhibition of NR1/NR2B NMDARs by 1 muM ifenprodil severely impaired P19 neurite extension and fasciculation, and this negative effect was fully reversible by polyamine addition. In contrast, GABA(A)R antagonism by a high dose of 200 microM bicuculline had no observable effect on P19 neuronal differentiation and fasciculation. Except for the differential NMDAR and GABA(A)R profiles of Ca(2+) signaling within the immature P19 neurons, we have also shown that inhibition of NR1/NR2B NMDARs strongly decreased mRNA level of NCAM-180, which has been previously implicated as a regulator of neuronal growth cone protrusion and neurite extension. Our data thus suggest a critical role of NR1/NR2B NMDARs during the process of neuritogenesis and fasciculation of P19 neurons via differential control of local growth cone Ca(2+) surges and NCAM-180 signaling.

Selective Expression of KCNS3 Potassium Channel α-Subunit in Parvalbumin-Containing GABA Neurons in the Human Prefrontal Cortex

The cognitive deficits of schizophrenia appear to be associated with altered cortical GABA neurotransmission in the subsets of inhibitory neurons that express either parvalbumin (PV) or somatostatin (SST). Identification of molecular mechanisms that operate selectively in these neurons is essential for developing targeted therapeutic strategies that do not influence other cell types. Consequently, we sought to identify, in the human cortex, gene products that are expressed selectively by PV and/or SST neurons, and that might contribute to their distinctive functional properties. Based on previously reported expression patterns in the cortex of mice and humans, we selected four genes: KCNS3, LHX6, KCNAB1, and PPP1R2, encoding K+ channel Kv9.3 modulatory α-subunit, LIM homeobox protein 6, K+ channel Kvβ1 subunit, and protein phosphatase 1 regulatory subunit 2, respectively, and examined their colocalization with PV or SST mRNAs in the human prefrontal cortex using dual-label in situ hybridization with 35S- and digoxigenin-labeled antisense riboprobes. KCNS3 mRNA was detected in almost all PV neurons, but not in SST neurons, and PV mRNA was detected in >90% of KCNS3 mRNA-expressing neurons. LHX6 mRNA was detected in almost all PV and >90% of SST neurons, while among all LHX6 mRNA-expressing neurons 50% expressed PV mRNA and >44% expressed SST mRNA. KCNAB1 and PPP1R2 mRNAs were detected in much larger populations of cortical neurons than PV or SST neurons. These findings indicate that KCNS3 is a selective marker of PV neurons, whereas LHX6 is expressed by both PV and SST neurons. KCNS3 and LHX6 might be useful for characterizing cell-type specific molecular alterations of cortical GABA neurotransmission and for the development of novel treatments targeting PV and/or SST neurons in schizophrenia.

Bexarotene-Activated Retinoid X Receptors Regulate Neuronal Differentiation and Dendritic Complexity

Bexarotene-activated retinoid X receptors (RXRs) ameliorate memory deficits in Alzheimers disease mouse models, including mice expressing human apolipoprotein E (APOE) isoforms. The goal of this study was to gain further insight into molecular mechanisms whereby ligand-activated RXR can affect or restore cognitive functions. We used an unbiased approach to discover genome-wide changes in RXR cistrome (ChIP-Seq) and gene expression profile (RNA-Seq) in response to bexarotene in the cortex of APOE4 mice. Functional categories enriched in both datasets revealed that bexarotene-liganded RXR affected signaling pathways associated with neurogenesis and neuron projection development. To further validate the significance of RXR for these functions, we used mouse embryonic stem (ES) cells, primary neurons, and APOE3 and APOE4 mice treated with bexarotene. In vitro data from ES cells confirmed that bexarotene-activated RXR affected neuronal development at different levels, including proliferation of neural progenitors and neuronal differentiation, and stimulated neurite outgrowth. This effect was validated in vivo by demonstrating an increased number of neuronal progenitors after bexarotene treatment in the dentate gyrus of APOE3 and APOE4 mice. In primary neurons, bexarotene enhanced the dendritic complexity characterized by increased branching, intersections, and bifurcations. This effect was confirmed by in vivo studies demonstrating that bexarotene significantly improved the compromised dendritic structure in the hippocampus of APOE4 mice. We conclude that bexarotene-activated RXRs promote genetic programs involved in the neurogenesis and development of neuronal projections and these results have significance for the improvement of cognitive deficits. SIGNIFICANCE STATEMENT Bexarotene-activated retinoid X receptors (RXRs) ameliorate memory deficits in Alzheimers disease mouse models, including mice expressing human apolipoprotein E (APOE) isoforms. The goal of this study was to gain further insight into molecular mechanisms whereby ligand-activated RXR can affect or restore cognitive functions. We used an unbiased approach to discover genome-wide changes in RXR cistrome (ChIP-Seq) and gene expression profile (RNA-Seq) in response to bexarotene in the cortex of APOE4 mice. Functional categories enriched in both datasets revealed that liganded RXR affected signaling pathways associated with neurogenesis and neuron projection development. The significance of RXR for these functions was validated in mouse embryonic stem cells, primary neurons, and APOE3 and APOE4 mice treated with bexarotene.

Promotion of neuronal differentiation through activation of N-methyl-D-aspartate receptors transiently expressed by undifferentiated neural progenitor cells in fetal rat neocortex

Neural progenitor cell is a generic term for undifferentiated cell populations composed of neural stem, neuronal progenitor, and glial progenitor cells with abilities for self‐renewal and multipotentiality. In this study, we have attempted to evaluate the possible functional expression of N‐methyl‐D‐aspartate (NMDA) receptors by neural progenitor cells prepared from neocortex of 18‐day‐old embryonic rats. Cells were cultured in the presence of basic fibroblast growth factor (bFGF) for different periods up to 12 days under floating conditions. Reverse transcription‐polymerase chain reaction and fluorescence imaging analyses revealed transient expression of functional NMDA receptors in neurospheres formed by clustered progenitors during the culture with bFGF. A similarly potent increase was seen in the fluorescence intensity after brief exposure to NMDA in cells differentiated after the removal of bFGF under adherent conditions, and an NMDA receptor antagonist invariably prevented these increases by NMDA. Moreover, sustained exposure to NMDA not only inhibited the formation of neurospheres when exposed for 10 days from day 2 to day 12 but also promoted spontaneous and induced differentiation of neurospheres to cells immunoreactive for a neuronal marker protein on immunocytochemistry and Western blotting analyses. These results suggest that functional NMDA receptors may be transiently expressed to play a role in mechanisms underlying the modulation of proliferation along with the determination of subsequent differentiation fate toward a neuronal lineage in neural progenitor cells of developing rat neocortex.

Photons Do Collapse In the Retina Not in the Brain Cortex: Evidence from Visual Illusions

While looking for evidence of quantum coherent states within the brain, many quantum mind advocates proposed experiments based on the assumption that the coherent state of a photon entering the visual system could somehow be preserved through the neural processing, or in other words they suppose that photons collapse not in the retina, but in the brain cortex. In this paper we show that photons do collapse within the retina and subsequent processing of information at the level of neural membranes proceeds. Moreover, we explicitly stress on the fact that due to existent amplification of the signal produced by each photon, a basic quantum mechanical theorem forbids the photon state to be teleported from the retina to the brain cortex. The changes of the membrane potential of the neurons in the primary visual cortex are shown to be relevant to inputting visual sensory information that is already processed and is not identical to the visual image entering in the retina. A striking evidence for the existent processing of the incoming visual information by the retina is provided by visual illusions resulting from the lateral inhibition mechanism.

Cortical Gene Expression After a Conditional Knockout of 67 kDa Glutamic Acid Decarboxylase in Parvalbumin Neurons

In the cortex of subjects with schizophrenia, expression of glutamic acid decarboxylase 67 (GAD67), the enzyme primarily responsible for cortical GABA synthesis, is reduced in the subset of GABA neurons that express parvalbumin (PV). This GAD67 deficit is accompanied by lower cortical levels of other GABA-associated transcripts, including GABA transporter-1, PV, brain-derived neurotrophic factor (BDNF), tropomyosin receptor kinase B, somatostatin, GABAA receptor α1 subunit, and KCNS3 potassium channel subunit mRNAs. In contrast, messenger RNA (mRNA) levels for glutamic acid decarboxylase 65 (GAD65), another enzyme for GABA synthesis, are not altered. We tested the hypothesis that this pattern of GABA-associated transcript levels is secondary to the GAD67 deficit in PV neurons by analyzing cortical levels of these GABA-associated mRNAs in mice with a PV neuron-specific GAD67 knockout. Using in situ hybridization, we found that none of the examined GABA-associated transcripts had lower cortical expression in the knockout mice. In contrast, PV, BDNF, KCNS3, and GAD65 mRNA levels were higher in the homozygous mice. In addition, our behavioral test battery failed to detect a change in sensorimotor gating or working memory, although the homozygous mice exhibited increased spontaneous activities. These findings suggest that reduced GAD67 expression in PV neurons is not an upstream cause of the lower levels of GABA-associated transcripts, or of the characteristic behaviors, in schizophrenia. In PV neuron-specific GAD67 knockout mice, increased levels of PV, BDNF, and KCNS3 mRNAs might be the consequence of increased neuronal activity secondary to lower GABA synthesis, whereas increased GAD65 mRNA might represent a compensatory response to increase GABA synthesis.

Monte Carlo simulation of quantum Zeno effect in the brain

Environmental decoherence appears to be the biggest obstacle for successful construction of quantum mind theories. Nevertheless, the quantum physicist Henry Stapp promoted the view that the mind could utilize quantum Zeno effect to influence brain dynamics and that the efficacy of such mental efforts would not be undermined by environmental decoherence of the brain. To address the physical plausibility of Stapps claim, we modeled the brain using quantum tunneling of an electron in a multiple-well structure such as the voltage sensor in neuronal ion channels and performed Monte Carlo simulations of quantum Zeno effect exerted by the mind upon the brain in the presence or absence of environmental decoherence. The simulations unambiguously showed that the quantum Zeno effect breaks down for timescales greater than the brain decoherence time. To generalize the Monte Carlo simulation results for any n-level quantum system, we further analyzed the change of brain entropy due to the mind probing actions and proved a theorem according to which local projections cannot decrease the von Neumann entropy of the unconditional brain density matrix. The latter theorem establishes that Stapps model is physically implausible but leaves a door open for future development of quantum mind theories provided the brain has a decoherence-free subspace.

The quantum physics of synaptic communication via the SNARE protein complex

Twenty five years ago, Sir John Carew Eccles together with Friedrich Beck proposed a quantum mechanical model of neurotransmitter release at synapses in the human cerebral cortex. The model endorsed causal influence of human consciousness upon the functioning of synapses in the brain through quantum tunneling of unidentified quasiparticles that trigger the exocytosis of synaptic vesicles, thereby initiating the transmission of information from the presynaptic towards the postsynaptic neuron. Here, we provide a molecular upgrade of the Beck and Eccles model by identifying the quantum quasiparticles as Davydov solitons that twist the protein α-helices and trigger exocytosis of synaptic vesicles through helical zipping of the SNARE protein complex. We also calculate the observable probabilities for exocytosis based on the mass of this quasiparticle, along with the characteristics of the potential energy barrier through which tunneling is necessary. We further review the current experimental evidence in support of this novel bio-molecular model as presented.