Validation of a suite of ERP and QEEG biomarkers in a pre-competitive, industry-led study in subjects with schizophrenia and healthy volunteers.

OBJECTIVE: Complexity and lack of standardization have mostly limited the use of event-related potentials (ERPs) and quantitative EEG (QEEG) biomarkers in drug development to small early phase trials. We present results from a clinical study on healthy volunteers (HV) and patients with schizophrenia (SZ) that assessed test-retest, group differences, variance, and correlation with functional assessments for ERP and QEEG measures collected at clinical and commercial trial sites with standardized instrumentation and methods, and analyzed through an automated data analysis pipeline. METHODS: 81 HV and 80 SZ were tested at one of four study sites. Subjects were administered two ERP/EEG testing sessions on separate visits. Sessions included a mismatch negativity paradigm, a 40 Hz auditory steady-state response paradigm, an eyes-closed resting state EEG, and an active auditory oddball paradigm. SZ subjects were also tested on the Brief Assessment of Cognition (BAC), Positive and Negative Syndrome Scale (PANSS), and Virtual Reality Functional Capacity Assessment Tool (VRFCAT). RESULTS: Standardized ERP/EEG instrumentation and methods ensured few test failures. The automated data analysis pipeline allowed for near real-time analysis with no human intervention. Test-retest reliability was fair-to-excellent for most of the outcome measures. SZ subjects showed significant deficits in ERP and QEEG measures consistent with published academic literature. A subset of ERP and QEEG measures correlated with functional assessments administered to the SZ subjects. CONCLUSIONS: With standardized instrumentation and methods, complex ERP/EEG testing sessions can be reliably performed at clinical and commercial trial sites to produce high-quality data in near real-time.

Cloning and characterization of two K+ inward rectifier (Kir) 1.1 potassium channel homologs from human kidney (Kir1.2 and Kir1.3).

The DNA sequence encoding the rat brain inward rectifier-10 K+ channel was amplified from rat brain RNA using reverse transcription-polymerase chain reaction and used to clone the human homolog. Low stringency screening of a human kidney cDNA library and subsequent DNA sequence analysis identified two related K+ inward rectifier cDNAs, referred to as Kir1.2 and Kir1.3, which were derived from transcription of distinct human genes. Kir1.2 represents the human homolog of the rat BIRK-10 sequence, whereas Kir1.3 was unique compared with all available sequence data bases. The genes that encode Kir1.2 and Kir1.3 were mapped to human chromosomes 1 and 21, respectively. Both genes showed tissue-specific expression when analyzed by Northern blots. Kir1.2 was only detected in brain >> kidney and was detected at high levels in all brain regions examined. Kir1.3 was most readily detected in kidney and was also expressed in pancreas > lung. Comparative analysis of the predicted amino acid sequences for Kir1.2 and Kir1.3 revealed they were 62% identical. The most remarkable difference between the two polypeptides is that the Walker Type A consensus binding motif present in both Kir1.1 and Kir1.2 was not conserved in the Kir1.3 sequence. Expression of the Kir1.2 polypeptide in Xenopus oocytes resulted in the synthesis of a K+-selective channel that exhibited an inwardly rectifying current-voltage relationship and was inhibited by external Ba2+ and Cs+. Kir1.2 current amplitude was reduced by >85% when the pH was decreased from pH 7.4 to 5.9 using the membrane-permeant buffer acetate but was relatively unaffected when pH was similarly lowered using membrane-impermeant biphthalate. The inhibition by intracellular protons was voltage-independent with an IC50 of pH 6.2 and a Hill coefficient of 1.9, suggesting the cooperative binding of 2 protons to the intracellular face of the channel. In contrast, Kir1.3 expression in Xenopus oocytes was not detectable despite the fact that the cRNA efficiently directed the synthesis of a polypeptide of the expected Mr in an in vitro translation system. Co-expression of Kir1.3 with either Kir1.1 or Kir1.2 reduced currents resulting from expression of these inward-rectifier subunits alone, consistent with a dominant negative influence on Kir1.1 and Kir1.2 expression.

Cannabinoid receptor agonists inhibit glutamatergic synaptic transmission in rat hippocampal cultures.

Activation of cannabinoid receptors inhibits voltage-gated Ca2+ channels and activates K+ channels, reminiscent of other G-protein-coupled signaling pathways that produce presynaptic inhibition. We tested cannabinoid receptor agonists for effects on excitatory neurotransmission between cultured rat hippocampal neurons. Reducing the extracellular Mg2+ concentration to 0.1 mM elicited repetitive, transient increases in intracellular Ca2+ concentration ([Ca2+]i spikes) that resulted from bursts of action potentials, as measured by combined whole-cell current clamp and indo-1-based microfluorimetry. Pharmacological characterization indicated that the [Ca2+]i spikes required glutamatergic synaptic transmission. Cannabinoid receptor ligands inhibited stereoselectively the frequency of [Ca2+]i spiking in the rank order of potency: CP 54,939 > CP 55,940 > Win 55,212-2 > anandamide, with EC50 values of 0.36, 1.2, 2.7, and 71 nM, respectively. CP 55,940 was potent, but not efficacious, and reversed the inhibition produced by Win 55,212-2, indicating that it is a partial agonist. Inhibition of [Ca2+]i spiking by Win 55,212-2 was prevented by treatment of cultures with active, but not heat-treated, pertussis toxin. Win 55,212-2 (100 nM) inhibited stereoselectively CNQX-sensitive excitatory postsynaptic currents (EPSCs) elicited by presynaptic stimulation with an extracellular electrode, but did not affect the presynaptic action potential or currents elicited by direct application of kainate. Consistent with a presynaptic site of action, Win 55,212-2 increased both the number of response failures and the coefficient of variation of the evoked EPSCs. In contrast, cannabimimetics did not affect bicuculline-sensitive inhibitory postsynaptic currents. Thus, activation of cannabinoid receptors inhibits the presynaptic release of glutamate via an inhibitory G-protein.

Complete and reversible block by omega-grammotoxin SIA of glutamatergic synaptic transmission between cultured rat hippocampal neurons.

omega-Grammotoxin SIA (omega-GsTx SIA), a peptide isolated from tarantula venom, inhibits synaptosomal Ca2+ influx and neurotransmitter release, and blocks N-, P-, and Q-type voltage-gated Ca2+ channels. The whole-cell patch-clamp was used to record glutamatergic excitatory post-synaptic currents (EPSCs) evoked by extracellular stimulation of presynaptic neurons in primary rat hippocampal cultures. EPSCs displayed rapid kinetics and were blocked by CNQX. omega-Conotoxin (1 microM) GVIA inhibited EPSCs by 46%, while 30 nM and 1 microM omega-agatoxin IVA produced 12% and 69% inhibition, respectively, consistent with coupling of N-, P- and Q-type Ca2+ channels to glutamatergic synaptic transmission. omega-GsTx SIA (1 microM) rapidly, completely, and reversibly blocked glutamatergic EPSCs, but did not affect currents evoked by bath application of kainate. Thus, omega-GsTx SIA blocks glutamatergic synaptic transmission by blocking presynaptic voltage-gated Ca2+ channels. omega-GsTx SIA is the only agent that blocks selectively and reversibly the Ca2+ channels coupled to glutamate release. omega-GsTx SIA provides a unique and powerful tool for experiments requiring recovery of function following presynaptic block of synaptic transmission.

Omega-grammotoxin SIA blocks multiple, voltage-gated, Ca2+ channel subtypes in cultured rat hippocampal neurons.

Omega-Grammotoxin SIA is a peptide isolated from tarantula venom on the basis of its ability to block the voltage-gated Ca2+ channels that mediate glutamate release. To determine the Ca2+ channel subtype selectivity of omega-grammotoxin SIA, whole-cell Ba2+ current (IBa) was measured in cultured rat hippocampal neurons. Selective Ca2+ channel blockers were used to identify components of IBa mediated by Ca2+ channel subtypes. omega-Agatoxin IVA at 30 nM, 1 microM omega-conotoxin GVIA, and 3 microM omega-contoxin MVIIC, applied consecutively, each elicited a fractional increase in the cumulative block of IBa, identifying components of IBa mediated by P-, N-, and Q-type calcium channels. omega-Grammotoxin at 1 microM, a maximally effective concentration, blocked 52% of IBa. omega-Conotoxin MVIIC and the combination of omega-conotoxin GVIA and micromolar omega-agatoxin IVA blocked 52% and 54% of IBa, respectively, and block of IBa by omega-grammotoxin SIA was mutually occlusive of block of IBa by either treatment, both of which block N-, P-, and Q-type Ca2+ channels. The L channel blocker nimodipine produced identical block of IBa in the presence and absence of omega-grammotoxin SIA. These results indicate that omega-grammotoxin SIA blocks N-, P-, and Q-type but not L-type voltage-gated calcium channels. Block of IBa by omega-grammotoxin SIA was faster in onset and less sensitive to external divalent cation concentrations than was block by omega-conotoxin MVIIC, and it was rapidly and substantially reversible. Rapid onset, relative insensitivity to divalent cation concentrations, and reversibility render omega-grammotoxin SIA a useful tool for inhibition of neuronal voltage-gated Ca2+ channels.

omega-Grammotoxin blocks action-potential-induced Ca2+ influx and whole-cell Ca2+ current in rat dorsal-root ganglion neurons.

Field-potential stimulation of rat dorsal-root ganglion (DRG) neurons evoked action-potential-mediated transient increases in intracellular free calcium concentration ([Ca2+]i) as measured by indo-1-based microfluorimetry. Field-potential-evoked [Ca2+]i transients were abolished by tetrodotoxin, and their dependence on stimulus intensity exhibited an abrupt threshold. omega-Conotoxin GVIA (omega-CgTx, 100 nM) inhibited action-potential-mediated Ca2+ influx by 79%, while nitrendipine (1 microM) had little effect. omega-Grammotoxin SIA (omega-GsTx, 267 nM), a peptide toxin purified from the venom of the tarantula spider, Grammostola spatulata, blocked action-potential-mediated Ca2+ influx as effectively as did omega-CgTx, suggesting that omega-GsTx blocks N-type Ca2+ channels. In contrast to block by omega-CgTx, the block produced by omega-GsTx reversed upon washout of the peptide. omega-GsTx (270 nM) blocked 80%, and omega-CgTx (1 microM) blocked 64%, of whole-cell Ca2+ current (ICa) elicited by step depolarization to 0 mV from a holding potential of -80 mV. omega-GsTx completely occluded inhibition of ICa by omega-CgTx. However, when applied after omega-CgTx, omega-GsTx produced an additional inhibition of 27%, indicating that omega-GsTx also blocked a non-N-type Ca2+ channel. BayK8644 (1 microM) elicited an increase in ICa in the presence of maximally effective concentrations of omega-GsTx, suggesting that omega-GsTx does not block L-type channels. Thus, omega-GsTx displays a selectivity for Ca2+ channel subtypes which should prove useful for studying Ca2+ channels and Ca(2+)-channel-mediated processes.

Effects of stimulus intensity on the inhibition by omega-conotoxin GVIA and neomycin of K(+_-evoked [3H]norepinephrine release from hippocampal brain slices and synaptosomal calcium influx.

The effects of various K+ concentrations on the inhibition of [3H]norepinephrine release from rat hippocampal brain slices and evoked synaptosomal 45Ca2+ influx by omega-conotoxin GVIA (omega-CgTx) and neomycin were examined. K+ (15-75 mM) caused a concentration-dependent release of [3H]norepinephrine that was greater than 90% dependent on extracellular calcium. The ability of omega-CgTx to inhibit [3H]norepinephrine release was optimal at 25 mM K+ and was reduced substantially at higher concentrations of K+. omega-CgTx maximally inhibited [3H]norepinephrine release by 49% (15 mM K+), 58% (25 mM K+), 22% (50 mM K+), and 12% (75 mM K+). In contrast, neomycin caused a concentration-dependent and virtually complete inhibition of [3H]norepinephrine release at all concentrations of K+, with IC50 values of 210 microM (15 mM K+), 150 microM (25 mM K+), 450 microM (50 mM K+), and 1500 microM (75 mM K+). omega-CgTx (1 microM) had little effect (10% or less inhibition) on hippocampal synaptosomal 45Ca2+ influx at any concentration of K+, whereas 3 mM neomycin caused at least 75% inhibition of 45Ca2+ influx, with the largest inhibition (96%) occurring at 25 mM K+. The results suggest that increasing stimulus intensity decreases the contribution of N-type voltage-sensitive calcium channels (VSCC) in mediating K(+)-evoked release of [3H]norepinephrine. The comparative absence of omega-CgTx-sensitive synaptosomal 45Ca(2+)-influx sites suggests that N-type calcium channels are a small subset of channels in rat hippocampal synaptosomes. The demonstration that neomycin can inhibit omega-CgTx-sensitive and -insensitive neurotransmitter release and calcium influx suggests that neomycin may block N-type VSCC as well as non-N-type VSCC.

HIV-1 envelope protein evokes intracellular calcium oscillations in rat hippocampal neurons.

Treatment of single rat hippocampal neurons with 200 pM recombinant HIV-1 envelope glycoprotein, gp120, resulted in large increases in the intracellular free calcium concentration ([Ca2+]i) as measured with indo-1-based microfluorimetry. Three patterns of [Ca2+]i increases were observed: in one pattern the [Ca2+]i rose rapidly and transiently as a single peak, in a second pattern gp120 induced [Ca2+]i oscillations that subsided when the protein was removed, and in a third pattern the oscillations continued long after washout of gp120. Both single peak and oscillatory [Ca2+]i increases were completely blocked by the Ca2+ channel blocker nitrendipine (1 microM). The sustained oscillatory responses were also blocked completely and reversibly by the N-methyl-D-aspartate (NMDA) receptor antagonist CGS19755 (10 microM) and the Na+ channel blocker tetrodotoxin (1 microM). Complete block by antagonists of Ca2+, Na+, and NMDA-gated ion channels suggests that at least two cells are required to maintain the [Ca2+]i oscillations. We hypothesize that gp120 acts as an excitotoxin by increasing synaptic activity in the network of neurons established in primary culture.