Bioelectronic neural pixel: Chemical stimulation and electrical sensing at the same site.

Local control of neuronal activity is central to many therapeutic strategies aiming to treat neurological disorders. Arguably, the best solution would make use of endogenous highly localized and specialized regulatory mechanisms of neuronal activity, and an ideal therapeutic technology should sense activity and deliver endogenous molecules at the same site for the most efficient feedback regulation. Here, we address this challenge with an organic electronic multifunctional device that is capable of chemical stimulation and electrical sensing at the same site, at the single-cell scale. Conducting polymer electrodes recorded epileptiform discharges induced in mouse hippocampal preparation. The inhibitory neurotransmitter, γ-aminobutyric acid (GABA), was then actively delivered through the recording electrodes via organic electronic ion pump technology. GABA delivery stopped epileptiform activity, recorded simultaneously and colocally. This multifunctional "neural pixel" creates a range of opportunities, including implantable therapeutic devices with automated feedback, where locally recorded signals regulate local release of specific therapeutic agents.

Segmental exchanges define 4-aminopyridine binding and the inner mouth of K+ pores.

4-Aminopyridine (4AP) blocks the intracellular mouth of voltage-gated K+ channels. We identified critical regions for 4AP binding with chimeric channels in which segments of a low affinity clone (Kv2.1, IC50 = 18 mM) were replaced with those of a high affinity clone (Kv3.1, IC50 = 0.1 mM). 4AP sensitivity was not transferred with the S5-S6 linker (pore or P region). Instead, a chimera of the cytoplasmic half of S6 increased block 20-fold, without affecting gating. A double chimera of the cytoplasmic halves of S5 and S6 fully transferred 4AP sensitivity. Because 4AP block was inhibited by tetrapentylammonium, we conclude that determinants of 4AP binding lie in the S6 segment that forms the cytoplasmic vestibule of the pore and that this site may overlap a quaternary ammonium site.

A glue-based, screw-free method for implantation of intra-cranial electrodes in young mice.

Intra-cranial electroencephalographic recordings are increasingly employed in mice because of the availability of genetically manipulated mouse models. Currently, dental acrylic and anchoring screws are used to cement implanted electrodes. This technique works well for adult animals but often encounters difficulty when employed in young mice because their skulls are not strong enough to bear the anchoring screws. Here we describe a novel method favorable for implantation of intra-cranial electrodes in mice as young as postnatal 18 days and suitable for long-term intra-cranial electroencephalographic recordings. Our approach is to construct a multi-electrode assembly according to the desired stereotaxic coordinates of intra-cranial recordings and to secure the implanted electrode assembly to the skull via glue rather than dental acrylic/anchoring screws. The surgical operation for such electrode implantation is relatively quick and rarely associated with complications such as infection, bleeding, neurological deficits, spontaneous seizures or behavioral disturbances. The implanted electrodes are stable, allowing repeated monitoring for several months. Data obtained by simultaneous intra-hippocampal and intra-cortical recordings indicate that our method is suitable for the examination of behaviorally related electroencephalographic activities and experimentally induced seizures. Technical aspects of our methods are discussed, and the procedures for constructing the electrode assembly are presented in detail.

Heteromultimeric Kv1 channels contribute to myogenic control of arterial diameter.

Inhibition of vascular smooth muscle (VSM) delayed rectifier K+ channels (K(DR)) by 4-aminopyridine (4-AP; 200 micromol/L) or correolide (1 micromol/L), a selective inhibitor of Kv1 channels, enhanced myogenic contraction of rat mesenteric arteries (RMAs) in response to increases in intraluminal pressure. The molecular identity of K(DR) of RMA myocytes was characterized using RT-PCR, real-time PCR, and immunocytochemistry. Transcripts encoding the pore-forming Kvalpha subunits, Kv1.2, Kv1.4, Kv1.5, and Kv1.6, were identified and confirmed at the protein level with subunit-specific antibodies. Kvbeta transcript (beta1.1, beta1.2, beta1.3, and beta2.1) expression was also identified. Kv1.5 message was approximately 2-fold more abundant than that for Kv1.2 and Kv1.6. Transcripts encoding these three Kv1alpha subunits were approximately 2-fold more abundant in 1st/2nd order conduit compared with 4th order resistance RMAs, and Kvbeta1 was 8-fold higher than Kvbeta2 message. RMA K(DR) activated positive to -50 mV, exhibited incomplete inactivation, and were inhibited by 4-AP and correolide. However, neither alpha-dendrotoxin or kappa-dendrotoxin affected RMA K(DR), implicating the presence of Kv1.5 in all channels and the absence of Kv1.1, respectively. Currents mediated by channels because of coexpression of Kv1.2, Kv1.5, Kv1.6, and Kvbeta1.2 in human embryonic kidney 293 cells had biophysical and pharmacological properties similar to those of RMA K(DR). It is concluded that K(DR) channels composed of heteromultimers of Kv1 subunits play a critical role in myogenic control of arterial diameter.

A soft, transparent, freely accessible cranial window for chronic imaging and electrophysiology.

Chronic in vivo imaging and electrophysiology are important for better understanding of neural functions and circuits. We introduce the new cranial window using soft, penetrable, elastic, and transparent, silicone-based polydimethylsiloxane (PDMS) as a substitute for the skull and dura in both rats and mice. The PDMS can be readily tailored to any size and shape to cover large brain area. Clear and healthy cortical vasculatures were observed up to 15 weeks post-implantation. Real-time hemodynamic responses were successfully monitored during sensory stimulation. Furthermore, the PDMS window allowed for easy insertion of microelectrodes and micropipettes into the cortical tissue for electrophysiological recording and chemical injection at any location without causing any fluid leakage. Longitudinal two-photon microscopic imaging of Cx3Cr1(+/- GFP) transgenic mice was comparable with imaging via a conventional glass-type cranial window, even immediately following direct intracortical injection. This cranial window will facilitate direct probing and mapping for long-term brain studies.

Functional reconstitution of bacterially expressed human potassium channels in proteoliposomes: membrane potential measurements with JC-1 to assay ion channel activity.

Structure-function studies on ion channels have been greatly facilitated by the cloning of cDNAs from a variety of sources. However, obtaining detailed structural information on these proteins requires overexpression, purification and reconstitution in a functionally competent form. In this communication, we report on the functional reconstitution of a human potassium channel, Kv1.4, overexpressed in bacteria. We have assessed the activity of these channels using a spectroscopic assay with a potential-sensitive dye, JC-1. The presence of ion channels renders proteoliposomes selectively permeable to potassium ions as monitored by measurements of transmembrane electrical potential. We have optimised conditions wherein a 12% change in the fluorescence signal of the carbocyanine dye JC-1 per 10 mV change in membrane potential is obtained. Using this assay, we find that the reconstituted protein is potassium selective and its activity is blocked by 4-aminopyridine, a known potassium channel blocker.

On the mechanism by which 4-Aminopyridine occludes quinidine block of the cardiac K+ channel, hKv1.5.

4-Aminopyridine (4-AP) binds to potassium channels at a site or sites in the inner mouth of the pore and is thought to prevent channel opening. The return of hKv1.5 off-gating charge upon repolarization is accelerated by 4-AP and it has been suggested that 4-AP blocks slow conformational rearrangements during late closed states that are necessary for channel opening. On the other hand, quinidine, an open channel blocker, slows the return or immobilizes off-gating charge only at opening potentials (>-25 mV). The aim of this study was to use quinidine as a probe of open channels to test the kinetic state of 4-AP-blocked channels. In the presence of 0.2-1 mM 4-AP, quinidine slowed charge return and caused partial charge immobilization, corresponding to an increase in the Kd of approximately 20-fold. Peak off-gating currents were reduced and decay was slowed approximately 2- to 2.5-fold at potentials negative to the threshold of channel activation and during depolarizations shorter than normally required for channel activation. This demonstrated access of quinidine to 4-AP-blocked channels, a lack of competition between the two drugs, and implied allosteric modulation of the quinidine binding site by 4-AP resident within the channel. Single channel recordings also showed that quinidine could modulate the 4-AP-induced closure of the channels, with the result that frequent channel reopenings were observed when both drugs were present. We propose that 4-AP-blocked channels exist in a partially open, nonconducting state that allows access to quinidine, even at more negative potentials and during shorter depolarizations than those required for channel activation.

Gating-dependent mechanism of 4-aminopyridine block in two related potassium channels.

4-aminopyridine (4AP) is widely used as a selective blocker of voltage-activated K+ currents in excitable membranes, but its mechanism and site of action at the molecular level are not well understood. To address this problem we have analyzed 4AP block in Kv2.1 and Kv3.1, mammalian representatives of the Drosophila Shab and Shaw subfamilies of voltage-gated K+ channels, respectively. The two channels were expressed in Xenopus oocytes and analyzed at both the macroscopic and single channel levels. Whole cell analysis showed that 4AP sensitivity of Kv3.1 was approximately 150 times greater than that of Kv2.1. Patch clamp analysis revealed that the mechanism of 4AP block in both channels was qualitatively similar. 4AP reached its blocking site via the cytoplasmic side of the channels, the ON rate for block was strongly accelerated when channels opened and the drug was trapped in closed channels. Single channel analysis showed that 4AP decreased burst duration and increased the latency-to-first-opening. These effects were found to be related, respectively to drug ON and OFF rates in the activated channel. Kv3.1's high 4AP sensitivity relative to Kv2.1 was associated with both a slower OFF rate and therefore increased stability of the blocked state, as well as a faster ON rate and therefore increased access to the binding site. Our results indicate that in both channels 4AP enters the intracellular mouth to bind to a site that is guarded by the gating mechanism. Differences in channel gating as well as differences in the structure of the intracellular mouth may be important for specifying the 4AP sensitivity in related voltage-gated K+ channels.

What do the synaptic vesicles contain?

The intersynaptic membranes of the rat brain cortex were found to remain firmly attached to one another after perfusion of strongly anisotonic solutions. Brains perfused with depolarizing and excitotoxic agents showed abundant, apparent intermingling of mitochondria and synaptic vesicles. The results suggest (i) that the intersynaptic membranes are not separated from one another by an essentially fluid intersynaptic medium as it is commonly assumed, but rather firmly attached to one another by a layer of faintly osmiophilic yet remarkably stable, water-insoluble material; and (ii) that the synaptic vesicles may be involved in adenosine triphosphate carriage. Well established multidisciplinary data are presented which appear to be in line with both possibilities.

Enzymatic synthesis of dextran-containing hydrogels.

Dextran, a natural glucose-containing polysaccharide, has been acylated by Proleather FG-F and lipase AY, a protease and lipase from Bacillus sp. and Candida rugosa, respectively, in anhydrous dimethylsulfoxide in the presence of vinyl acrylate (VA). The efficiency of the reaction in the presence of Proleather FG-F and the isolated yields were ca. 71% and 45%, respectively. Dextran derivatized with VA (dexT70-VA) was characterized by gel permeation chromatography and its structure was established by NMR indicating two positional isomers at the 2 and 3 positions on the glucose moieties in equal amounts. Furthermore, the dextran glucopyranose residues were mono-substituted. The benefits of the biocatalytic synthesis of dextran acrylates was demonstrated using 4-dimethylaminopyridine as a chemical catalyst. Gels were prepared by free radical polymerization of aqueous solutions of dexT70-VA with different degrees of substitution and monomer concentrations. Intermolecular linkages and physical entanglements are predominantly formed by concentrated solutions, however, a part of the acrylate groups did not react. Gel pore sizes were calculated from swelling experiments and ranged from ca. 18 to 182 A.

Characteristics of a fast transient outward current in guinea pig trigeminal motoneurons.

A fast transient voltage dependent outward current (TOC) in trigeminal motoneurons (TMNs) was studied in guinea pig brainstem slices by use of sharp electrodes in combination with single electrode voltage clamp techniques. In solutions containing TTX, low Ca2+/Mn2+ and 20 mM TEA this current activated around -55 to -60 mV from holding potentials negative to resting potential, obtained its peak amplitude within 5 ms and decayed as a single exponential with a time constant of 6-8 ms. Half maximal values for inactivation and activation were -72 and -37 mV, respectively. Bath application of 5 mM 4-AP suppressed this current by approximately 90% and eliminated the early depolarizing transient membrane rectification observed in response to a constant depolarizing current pulse, prolonged the action potential duration, and reduced the threshold voltage and delay to onset of the action potential. It is suggested that this current resembles the typical A-current observed in many CNS neurons and, as a result of its voltage and time dependent properties, could contribute to control of motoneuronal discharge and timing of burst onset during rhythmical jaw movements. Therefore, any cellular models of masticatory activity should include the properties of this current.

Stress induces CRF release in the paraventricular nucleus, and both CRF and GABA release in the amygdala.

In the hypothalamus, corticotropin-releasing factor (CRF) initiates the hypothalamic-pituitary-adrenal (HPA) axis response to stress, resulting in the release of glucocorticoids, including cortisol. Extrahypothalamic CRF, particularly in the limbic system, also appears to play a role in the stress response. To further define brain CRF response to stress, immunosensor-based microdialysis probes were used to measure the extracellular levels of CRF in the paraventricular nucleus of the hypothalamus (PVN) and in the amygdala of sheep during a predator (dog) exposure stress. In addition, gamma amino butyric acid (GABA) was measured in the amygdala and cortisol was measured in venous blood. Exposure to the predator stress increased CRF in the PVN and both CRF and GABA in the amygdala. These were followed in time by a rise in venous cortisol. Application of a CRF antagonist to the amygdala, immediately prior to stress, had a small effect on the subsequent observed stress responses. This treatment, however, significantly reduced the responses to a repeat stress administered 2 days later, compared to nontreated animals. Application of a GABA antagonist to the amygdala prior to stress had no effect on the subsequent observed stress response but increased the response to the stress repeated 2 days later. Perfusion with 4-aminopyridine, a neuronal depolarising agent, into the PVN induced a release of CRF accompanied shortly thereafter by a small increase in CRF in the amygdala, and 5-10 min later by an increase in venous cortisol. Perfusion into the amygdala increased the levels of both CRF and GABA but had no effect on either PVN CRF or venous cortisol. These data support roles for both the PVN and amygdala in stress responsiveness. It suggests further that actions at the amygdala can strongly influence subsequent responsiveness to a further stress, mediated in part by both CRF and GABA actions.

Glucocorticoid feedback increases the sensitivity of the limbic system to stress.

In the hypothalamus, corticotropin-releasing hormone (CRH) has a well-described role initiating the hypothalamic-pituitary-adrenal (HPA) axis response to stress. Cortisol, released from the adrenal gland, exerts negative feedback on this axis. The role of extrahypothalamic CRH in stress responses is less well known. The purpose of this study was to measure the response of CRH in the amygdala to acute and repeated stress and to examine if cortisol had any effect on this response. Immunosensor-based microdialysis probes were used to measure CRH and cortisol in the amygdala and cortisol systemically in sheep exposed to a predator stress (a dog). Upon presentation of a dog, CRH increased in the amygdala of the sheep and then fell off. Cortisol levels rose both systemically and in the amygdala, and as they peaked, a second CRH response was observed. Repeated stress changed this response, with the magnitude of the first CRH peak decreasing while the second peak increased. Repeated stress also produced an exaggeration in both of the CRH peaks to presentation of a subsequent novel stress (a forelimb electric shock). Animals that had an escape route from the repeated dog stress did not show this exaggeration when faced subsequently with the novel stress. Administration of mifepristone, a glucocorticoid receptor antagonist, prior to the delivery of the repeat stress prevented subsequent changes in the CRH response. The data suggest that the amygdala shows a CRH response to presentation of a stressor acutely and repeatedly and that repeated stress can alter subsequent amygdala responsiveness to the same or a different stressor. This alteration appears dependent on circulatory glucocorticoids.

Acetylation of rice straw with or without catalysts and its characterization as a natural sorbent in oil spill cleanup.

An investigation of the acetylation of rice straw with acetic anhydride at 100 and 120 degrees C for 1-4 h with four tertiary amine catalysts (pyridine, 4-dimethylaminopyridine, N-methylpyrrolidine, and N-methylpyrrolidinone) or without catalyst in a solvent-free system was undertaken, and the extent of acetylation was measured by weight percent gain, which increased with the extent of reaction time and temperature and the amounts of catalyst used. 4-Dimethylaminopyridine was found to be the most effective catalyst of those studied. At a concentration of 7% of the catalyst in acetic anhydride, a weight percent gain of 15.4% was realized, compared with 11.2% for the noncatalyst reaction, after 0.5 h of exposure to the system at 120 degrees C. Characterization of acetylated straw was performed by FT-IR, CP MAS (13)C NMR, and thermal studies. Interestingly, the acetylated straw is significantly hydrophobic and does not get wet with water, thereby offering potential for the better utilization of cheap waste materials as natural sorbents in oil cleanup.

A voltage-dependent transient K(+) current in rat dental pulp cells.

We characterized a voltage-dependent transient K(+) current in dental pulp fibroblasts on dental pulp slice preparations by using a nystatin perforated-patch recording configuration. The mean resting membrane potential of dental pulp fibroblasts was -53 mV. Depolarizing voltage steps to +60 mV from a holding potential of -80 mV evoked transient outward currents that are activated rapidly and subsequently inactivated during pulses. The activation threshold of the transient outward current was -40 mV. The reversal potential of the current closely followed the K(+) equilibrium potential, indicating that the current was selective for K(+). The steady-state inactivation of the peak outward K(+) currents described by a Boltzmann function with half-inactivation occurred at -47 mV. The K(+) current exhibited rapid activation, and the time to peak amplitude of the current was dependent on the membrane potentials. The inactivation process of the current was well fitted with a single exponential function, and the current exhibited slow inactivating kinetics (the time constants of decay ranged from 353 ms at -20 mV to 217 ms at +60 mV). The K(+) current was sensitive to intracellular Cs(+) and to extracellular 4-aminopyridine in a concentration-dependent manner, but it was not sensitive to tetraethylammonium, mast cell degranulating peptide, and dendrotoxin-I. The blood depressing substance-I failed to block the K(+) current. These results indicated that dental pulp fibroblasts expressed a slow-inactivating transient K(+) current.

Vasodilatory effect of hydroxyethyl methacrylate and triethylene glycol dimethacrylate in rat aorta through calcium antagonistic action.

INTRODUCTION: Resin-based dental materials contain various diluent monomers that can interfere with vascular function by causing vasodilation. In this study, we evaluated the vasoactive potential of hydroxyethyl methacrylate (HEMA) and triethylene glycol dimethacrylate (TEGDMA) and the possible mechanism of their vascular action on isolated rat aorta. METHODS: Responses of thoracic aorta rings were recorded isometrically by using force displacement transducers. After precontracting aorta rings with phenylephrine, relaxations to HEMA and TEGDMA were recorded in the absence and presence of nitric oxide synthase inhibitor N(ω)-nitro-L-arginine methyl ester, cyclooxygenase inhibitor indomethacin, and K(+) channel inhibitors tetraethylammonium, glibenclamide, and 4-aminopyridine. To investigate the Ca(2+)-channel antagonistic effect of HEMA and TEGDMA in different aorta rings, concentration-response curves to CaCl(2) were obtained in the absence and presence of the test monomers. RESULTS: Both HEMA and TEGDMA elicited concentration-dependent relaxations. The vasorelaxant effect of HEMA and TEGDMA was not mediated via endothelium-dependent nitric oxide and prostanoid-dependent mechanisms or by K(+) efflux through K(+) channels. Both monomers significantly inhibited the contractions induced by CaCl(2). CONCLUSIONS: Our results showed that HEMA and TEGDMA induce vasodilation via Ca(2+)-antagonistic action, whereas nitric oxide and cyclooxgenase pathway and K(+) channels were not responsible for this vasoactive effect.

Vasorelaxant effect of a self-etch adhesive system through calcium antagonistic action.

Etch-and-rinse adhesives can cause vasorelaxation via mechanisms occurring in the endothelium and smooth muscle, including the release of nitric oxide (NO). This effect might promote or aggravate bleeding if such adhesives are placed inadvertently on iatrogenic pulp microexposures. The present study assessed the vasoactive potential of a newer generation self-etch adhesive, Clearfil Protect Bond (CPB), on isolated rat aorta. Cumulative concentrations of the primer, bond, and mixture of CPB elicited concentration-dependent relaxation of phenylephrine-induced active tonus in the rat aorta, demonstrating that the tested self-etch adhesive can lead to vasorelaxation of pulp vessels that is mediated by Ca(2+) antagonistic effect. The vasorelaxant effect of CPB or its components was mediated neither via endothelium-dependent NO and prostanoid-dependent mechanisms nor by K(+) efflux through K(+) channels. Mechanical removal of the endothelium did not significantly alter the relaxation induced by CPB. Assuming these data can be extrapolated to the clinical situation, CPB, either in mixed form or by its components, can lead to vasorelaxation of pulp vessels that is mediated by a Ca(2+) antagonistic effect. If CPB is placed inadvertently on pulp microexposures during direct pulp capping, this effect might promote bleeding that might impair healing and, via plasma exatravasation, might compromise resin infiltration and polymerization.

Acute repair of crushed guinea pig spinal cord by polyethylene glycol.

Acute repair of crushed guinea pig spinal cord by polyethylene glycol. We have studied the responses of adult guinea pig spinal cord white matter to a standardized compression within a sucrose gap recording chamber. This injury eliminated compound action potential (CAP) conduction through the lesion, followed by little or no recovery of conduction by 1 h postinjury. We tested the ability of polyethylene glycol (PEG) to repair the injured axons and restore physiological function. Local application of PEG (1,800 MW, 50% by weight in water) for approximately 2 min restored CAP conduction through the injury as early as 1 min post PEG application. The recovery of the CAP

Mutational analysis of ion conduction and drug binding sites in the inner mouth of voltage-gated K+ channels.

Pore properties that distinguish two cloned, voltage-gated K+ channels, Kv2.1 and Kv3.1, include single-channel conductance, block by external and internal tetraethylammonium, and block by 4-aminopyridine. To define the inner mouth of voltage-gated K+ channels, segmental exchanges and point mutations of nonconserved residues were used. Transplanting the cytoplasmic half of either transmembrane segments S5 or S6 from Kv3.1 into Kv2.1 reduced sensitivity to block by internal tetraethylammonium, increased sensitivity to 4-aminopyridine, and reduced single-channel conductance. In S6, changes in single-channel conductance and internal tetraethylammonium sensitivity were associated with point mutations V400T and L403 M, respectively. Although individual residues in both S5 and S6 were found to affect 4-aminopyridine blockade, the most effective change was L327F in S5. Thus, both S5 and S6 contribute to the inner mouth of the pore but different residues regulate ion conduction and blockade by internal tetraethylammonium and 4-aminopyridine.

Characterization of a 25-pS nonselective cation channel in a cultured secretory epithelial cell line.

We have studied a 25-pS nonselective cation channel from the apical membranes of cell line ST885, derived from neonatal mouse mandibular glands. Its Cl- permeability was not significantly different from zero. The permeabilities (relative to Na+) for inorganic cations were NH4+ (1.87) greater than K+ (1.12) greater than Li+ (1.02) greater than Na+ (1) greater than Rb+ (0.81) greater than Mg2+ (0.07) greater than Ca2+ (0.002), and for organic cations, guanidinium (1.61) greater than ethanolamine (0.70) greater than 4-aminopyridine (0.66) greater than diethylamine (0.54) greater than piperazine (0.25) greater than Tris (0.18) greater than N-methylglucamine (0.12). The Tris and N-methylglucamine permeabilities differed significantly from zero. Fitting the Renkin equation indicated that the channel had an equivalent pore radius of 0.49 nm. The channel was activated by Ca2+ on the cytosolic surface (greater than 0.1 mmol/liter) with a Hill coefficient of 1.2; it was also activated by depolarization. Open- and closed-time histograms indicated that it had at least two open and two closed states. The channel was blocked by cytosolic AMP or ATP (0.1 mmol/liter). It was also blocked by the Cl- channel blocker, diphenylamine-2-carboxylate (DPC; 0.1 mmol/liter), applied to the extracellular but not the cytosolic surface. 4-Amino-pyridine, which permeated the channel when applied to the extracellular surface, blocked it when applied in low concentrations (5 mmol/liter) to the cytosolic surface. Quinine (0.1 mmol/liter) blocked from both the extracellular and cytosolic surfaces, blockade from either side being enhanced by depolarization. The channel was held open by application of SITS (0.1 mmol/liter) to the cytosolic surface. The channel shows striking similarities to the nicotinic acetylcholine receptor channel, viz., both channel types are abnormally permeable to 4-aminopyridine applied externally, and their selectivity sequences for inorganic ions are similar and for organic cations are identical.