Learning related modulation of functional retrieval networks in man.

The medial temporal lobe has been implicated in studies of episodic memory tasks involving spatio-temporal context and object-location conjunctions. We have previously demonstrated that an increased level of practice in a free-recall task parallels a decrease in the functional activity of several brain regions, including the medial temporal lobe, the prefrontal, the anterior cingulate, the anterior insular, and the posterior parietal cortices, that in concert demonstrate a move from elaborate controlled processing towards a higher degree of automaticity. Here we report data from two experiments that extend these initial observations. We used a similar experimental approach but probed for effects of retrieval paradigms and stimulus material. In the first experiment we investigated practice related changes during recognition of object-location conjunctions and in the second during free-recall of pseudo-words. Learning in a neural network is a dynamic consequence of information processing and network plasticity. The present and previous PET results indicate that practice can induce a learning related functional restructuring of information processing. Different adaptive processes likely subserve the functional re-organisation observed. These may in part be related to different demands for attentional and working memory processing. It appears that the role(s) of the prefrontal cortex and the medial temporal lobe in memory retrieval are complex, perhaps reflecting several different interacting processes or cognitive components. We suggest that an integrative interactive perspective on the role of the prefrontal and medial temporal lobe is necessary for an understanding of the processing significance of these regions in learning and memory. It appears necessary to develop elaborated and explicit computational models for prefrontal and medial temporal functions in order to derive detailed empirical predictions, and in combination with an efficient use and development of functional neuroimaging approaches, to further the understanding of the processing significance of these regions in memory.

Reactivation of motor brain areas during explicit memory for actions.

Recent functional brain imaging studies have shown that sensory-specific brain regions that are activated during perception/encoding of sensory-specific information are reactivated during memory retrieval of the same information. Here we used PET to examine whether verbal retrieval of action phrases is associated with reactivation of motor brain regions if the actions were overtly or covertly performed during encoding. Compared to a verbal condition, encoding by means of overt as well as covert activity was associated with differential activity in regions in contralateral somatosensory and motor cortex. Several of these regions were reactivated during retrieval. Common to both the overt and covert conditions was reactivation of regions in left ventral motor cortex and left inferior parietal cortex. A direct comparison of the overt and covert activity conditions showed that activation and reactivation of left dorsal parietal cortex and right cerebellum was specific to the overt condition. These results support the reactivation hypothesis by showing that verbal-explicit memory of actions involves areas that are engaged during overt and covert motor activity.

Voltage-dependent formation of gramicidin channels in lipid bilayers.

The formation kinetics of gramicidin A channels in lipid bilayer membranes has been characterized as a function of voltage for different solution conditions and membrane composition. The frequency of channel events was measured during the application of voltage ramps and counted in given intervals, a procedure that eliminated the effects of drift in gramicidin concentration. The formation rate was found to increase strongly with voltages up to approximately 50 mV and then to level off slightly. The shape of the voltage dependence was independent of lipid solvent and ramp speed but differed for different ions and different solution concentrations. This suggested an ion occupancy effect on the formation rate that was further supported by the fact that the minimum of the formation rate was shifted toward the equilibrium potential in asymmetric solution concentrations. The effects are explained in terms of a model that contains two contributions to the voltage dependence, a voltage-dependent ion binding to the monomers and a polarization of monomers by the applied electric field and by the occupied ions. The theory is found to give a good fit to experimental data.

Cytoplasmic Ca2+ oscillations in human leukemia T-cells are reduced by 50 Hz magnetic fields.

The effect of 50 Hz magnetic fields on the cytosolic calcium oscillator in Jurkat E6.1 cells was investigated for field strengths within the range from 0 to 0.40 mT root mean square. The intracellular Ca2+ concentration data were collected for single Jurkat cells that exhibited a sustained spiking for at least 1 h while repeatedly exposing them to an alternating magnetic field in 10-min intervals interposed with nonexposure intervals of the same length. The obtained data were analysed by computing spectral densities of the Ca2+ oscillating patterns for each of these 10-min intervals. For every single-cell experiment the spectra of all exposure as well as nonexposure periods were then averaged separately. A comparison between the resulting averages showed that the total spectral power of the cytosolic Ca2+ oscillator was reduced by exposure of the cells to an alternating magnetic field and that the effect increased in an explicit dose-response manner. The same relationship was observed within the 0-10 mHz (10 x 10(-3) Hz) subinterval of the Ca2+ oscillation spectrum. For subintervals at higher frequencies, the change caused by the exposure to the magnetic field was not significant.

Amplification of electromagnetic signals by ion channels.

Cells may respond to the exposure of low-frequency electromagnetic fields with changes in cell division, ion influx, chemical reaction rates, etc. The chain of events leading to such responses is difficult to study, mainly because of extremely small energies associated with low-frequency fields, usually much smaller than the thermal noise level. However, the presence of stochastic systems (for instance, ion channels) provides a basis for signal amplification, and could therefore, despite the low signal-to-noise ratio of the primary response, lead to the transmission of weak signals along the signaling pathways of cells. We have explored this possibility for an ion channel model, and we present a theory, based on the formalism of stochastically driven processes, that relates the time averages of the ion channel currents to the amplitude and frequency of the applied signal. It is concluded from this theory that the signal-to-noise ratio increases with the number of channels, the magnitude of the rate constants, and the frequency response of the intracellular sensing system (for instance, a calcium oscillator). The amplification properties of the stochastic system are further deduced from numerical simulations carried out on the model, which consists of multiple identical two-state channels, and the behavior for different parameters is examined. Numerical estimates of the parameters show that under optimum conditions, even very weak low-frequency electromagnetic signals (<100 Hz and down to 100 microT) may be detected in a cellular system with a large number of ion channels.

The influence of 50-Hz magnetic fields on cytoplasmic Ca2+ oscillations in human leukemia T-cells.

We have studied the effects of 50-Hz 100-microT rms magnetic fields on intracellular Ca2+ concentration in the Jurkat T lymphocyte variant E6.1 using fluorescent probes Indo-1 and Fura-2. We found, however, that the pattern of intracellular Ca2+ fluctuations also depended on the agent used for cell attachment, in our case the polypeptide poly-L-lysine. In order to isolate possible effects of magnetic field exposure from those of poly-L-lysine, the action of polypeptide on cytosolic Ca2+ was studied as well. It was found that a 10(-7)% concentration of polypeptide triggered prolonged Ca2+ spiking. Higher (10(-4)%) concentrations induced rapid increases in intracellular Ca2+ followed by high, unstable Ca2+ levels. The response of these cells to the monoclonal antibody anti-CD3 was also inhomogeneous, similar to one caused by poly-L-lysine. The effect of magnetic field exposure was studied on cells initially exhibiting (1) non-oscillating, low Ca2+ concentration and (2) prolonged Ca2+ concentration oscillations. In case (1) the result was negative. In case (2), statistically significant changes were found: the oscillation amplitude was reduced on average by 30%, and the frequency composition was shifted towards higher frequencies.

The frequency response of ion channel currents in a combined diffusion and barrier type model.

The dynamics of ion transport through channels has been calculated using a small signal analysis method. This is applied to the equations describing the rates of ion transport through channels represented by a pair of entrance barriers in series with an interior diffusion regime. The responses to combinations of applied DC and AC electric fields, assumed to be constant in space, have been calculated and are represented as complex admittances. On examining the admittance properties, the channel is found to behave electrically as a double reactance network, which can give rise to resonance effects. A further interesting property is the limiting slope of the current amplitude at high frequencies, which is found to exhibit an f-2/3 dependence for small entrance barriers and an f-1 dependence for large entrance barriers. This points to the possibility of using noise measurements for drawing conclusions about ion transport properties through channels.

Ca2+ ion transport through patch-clamped cells exposed to magnetic fields.

The total current of Ca2+ ions through patch-clamped cell membranes was measured while exposing clonal insulin-producing beta-cells (RINm5F) to a combination of DC and AC magnetic fields at so-called cyclotron resonance conditions. Previous experimental evidence supports the theory that a resonant interaction between magnetic fields and organisms can exist. This experiment was designed to test one possible site of interaction: channels in the cell membrane. The transport of Ca2+ ions through the protein channels of the plasma membrane did not show any resonant behavior in the frequency range studied.

Theoretical study of the resonant behaviour of an ion confined to a potential well in a combination of AC and DC magnetic fields.

Numerical solutions are presented to the equation of motion for an ion confined to a region of space by a restoring force and subject to DC and AC magnetic fields. We have expanded on the theoretical work of Durney et al. [1988] by including a potential well as a confining factor. This additional term in the equation of motion, being nondissipative, could allow for the buildup of stored energy within the system to a level necessary for a macroscopic resonant phenomenon. Resonant behaviour has been studied, including calculation of the trajectory and energy (kinetic and potential) of a confined ion, with emphases on the appearance of both amplitude and frequency windows. The results are discussed in relation to ion transport through transmembrane channels exposed to magnetic fields. When realistic values of the frictional and restoring-force coefficients are considered, all predicted resonant behaviour disappears, except at very high field strengths.

Experimental search for combined AC and DC magnetic field effects on ion channels.

The hypothesis that specific combinations of DC and low frequency AC magnetic fields at so-called cyclotron-resonance conditions could affect the transport of ions through ion channels, or alter the kinetics of ion channels (opening and closing rates), has been tested. As a model system, the ion channels formed by gramicidin A incorporated in lipid bilayer membranes were studied. No significant changes in channel conductance, average lifetime, or formation rate as a function of applied fields could be detected over a wide range of frequencies and field strengths. Experiments were carried out to measure the time-resolved single-channel events and the average conductances of many-channel events in the presence of K+ and H+ ions. The channel blocking effect of Ca++ was also studied.

The effect of microwave radiation on the stability and formation of gramicidin-A channels in lipid bilayer membranes.

The effects of microwaves on the single-channel kinetics of gramicidin-A channels in lipid bilayer membranes were examined. Attempts were made to separate thermal and athermal effects by accurate measurements of temperature at the site of the membrane and by relating the measured parameters to their previously characterized temperature dependence. It was found that microwave radiation does not affect single-channel conductance or channel life time to a degree that is significantly different from that expected of a purely thermal effect. On the other hand, the rate of channel formation is decreased during exposure, which is opposite to that expected of a purely thermal effect. The mechanism of this effect is discussed in terms of the dimerization process of channel formation.

Expression of voltage-gated K+ channels in insulin-producing cells. Analysis by polymerase chain reaction.

We have used the polymerase chain reaction (PCR) with primers against the S5 and S6 regions of voltage-gated K+ channels to identify 8 different specific amplification products using poly(A)+ RNA isolated from islets of Langerhans from obese hyperglycemic (ob/ob) mice and from the two insulin-producing cell lines HIT T15 and RINm5F. Sequence analysis suggests that they derive from mRNAs coding for a family of voltage-gated K+ channels; 5 of these have been recently identified in mammalian brain and 3 are novel. These hybridize in classes to different mRNAs which distribute differently to a number of tissues and cell lines including insulin-producing cells.

Evaluation of surface tension and ion occupancy effects on gramicidin A channel lifetime.

The surface tension of glycerylmonooleate-hexadecane lipid bilayer membranes and the lifetime of gramicidin A channels were measured at various concentrations of the surrounding solutions. For HCl the surface tension is essentially constant at approximately 5 mN/m up to approximately 1 M, whereas the average lifetime increases approximately 40-fold. At higher concentrations the surface tension decreases markedly. For CsCl the surface tension is constant up to about 1 M then increases with salt level. The average lifetime in this case increases about sixfold. In both cases the lifetime levels off and even decreases at higher salt levels. The increase in lifetime observed with ion activity is therefore qualitatively different from, and not explained by, the established dependence of lifetime on membrane properties (Elliot, J.R., D. Needham, J.P. Dilger, and D.A. Haydon. 1983. Biochim. Biophys. Acta. 735:95-103). We have previously proposed that ion occupancy is a determinant of channel stability, and to test this hypothesis the voltage dependence of channel lifetime was measured in asymmetrical solutions. For the case of a potassium chloride solution on one side of the membrane and a hydrogen chloride solution, on the other, the voltage dependence of the lifetime is asymmetrical. The asymmetry is such that when the electrical field is applied in the direction of the chemical gradient for each of the ions, the channel lifetime approaches, at increasing field strengths, that of a symmetrical solution of the respective ion. The voltage dependence of the surface tension, on the other hand, is negligible for the range of voltages used. These results, and the earlier findings that the order of the lifetimes for the alkali cations generally agree with the order of the permeability selectivity of the gramicidin A channel, support the hypothesis that ion occupancy is a major factor determining the lifetime of gramicidin A channels. The effects of multivalent blockers and osmotic agents were also tested. Ba2", La3+,and Mg2" decrease the lifetime and conductance markedly. Sucrose and urea increase the lifetime and decrease the conductance. The voltage dependence of the lifetime in symmetrical solutions was examined. Contrary to previous reports it was found that the lifetimes for K+, Cs', and H+ are voltage dependent. For 0.5 M HCI the lifetime decreases monotonically by .60% at 150 mV, and for 0.5 M KCI the lifetime increases by -60% at 200 mV. Below 10 mM there is no effect of voltage for H+, K+, and Cs+. These effects of blockers, osmotic agents, and voltage on the lifetime, as well as the lack of effect of voltage at low salt levels, are consistent with the occupancy hypothesis.

Modulation of gramicidin A open channel lifetime by ion occupancy.

The hypothesis that the gramicidin A channel stability depends on the level of ion occupancy of the channel was used to derive a mathematical model relating channel lifetime to channel occupancy. Eyring barrier permeation models were examined for their ability to fit the zero-voltage conductance, current-voltage, as well as lifetime data. The simplest permeation model required to explain the major features of the experimental data consists of three barriers and four sites (3B4S) with a maximum of two ions occupying the channel. The average lifetime of the channel was calculated from the barrier model by assuming the closing rate constant to be proportional to the probability of the internal channel sites being empty. The link between permeation and lifetime has as its single parameter the experimentally determined averaged lifetime of gramicidin A channels in the limit of infinitely dilute solutions and has therefore no adjustable parameters. This simple assumption that one or more ions inside the channel completely stabilize the dimer conformation is successful in explaining the experimental data considering the fact that this model for stabilization is independent of ion species and configurational occupancy. The model is used to examine, by comparison with experimental data, the asymmetrical voltage dependence of the lifetime in asymmetrical solutions, the effects of blockers, and the effects of elevated osmotic pressure.

Access permeability of ionic channels. Dependence on aqueous jump distance.

The access diffusion permeability of pores with diameters comparable to the aqueous jump distance is characterized using a rate theory analysis for the aqueous diffusion process. It is found that this process gives rise to two permeability terms, one associated with bulk diffusion and the other a jump from the aqueous solution into a position where it has access to the channel. The latter term dominates for small channel diameters and vice versa for large channel diameters. The properties of access diffusion with respect to concentration polarization is shown to be different in the two limits of large and small values of the channel radius. A necessary criterion for bulk access diffusion to be rate limiting is given in terms of measured channel conductance G, aqueous jump distance lambda and aqueous resistivity rho, G greater than pi lambda/rho, which does not require a knowledge of channel geometry.

Effect of hypoglycemic sulfonylureas on Ca2+ fluxes across lipid bilayers.

Black lipid membranes and liposomes loaded with Ca2+ or 5,6-carboxyfluorescein were used for exploring the mechanism of action of insulin-releasing sulfonylureas. Unlike the Ca2+/H+ exchanging ionophore A-23187, tolbutamide did not stimulate the net efflux of Ca2+ from the liposomes. Glibenclamide caused a sustained release of Ca2+, but this effect could be attributed to labilization of the liposomal membrane as indicated by a quantitatively similar loss of the stability marker 5,6-carboxyfluorescein. Unlike the neutral ionophore nonactin or the channel forming quasi-ionophore gramicidin A, the sulfonylureas did not alter the conductance of black lipid membranes in medium containing Na+, K+, Ca2+, Mg2+, and Cl-. It is concluded that the sulfonylureas tested lack ionophore properties but that glibenclamide can labilize membranes.

Multioccupancy models for single filing ionic channels: theoretical behavior of a four-site channel with three barriers separating the sites.

A procedure is developed for dealing with multioccupancy in single-filing channels having any number of sites internal to the barriers at the channel ends but having the outermost sites in equilibrium with the bathing solutions. Using this procedure, a general theory is developed for a single-filing channel having three barriers and four sites, the outermost of which are in equilibrium with the bathing solutions. By introducing a vectorial representation, it is shown that the four-site model can be reduced to an equivalent two-site model with respect to the number of possible transitions, thereby simplifying the algebraic steps required to solve transport equations for the system. The transport coefficients are derived and expressed in terms of the energy levels of the peaks and the wells for the different occupancy configurations. An explicit solution to the transport equations is given in a comprised form for a single permeable species. The solution allows some important properties for the system to be deduced, specifically with regard to the conductance at zero current, the correlation factor between electrical conductance and tracer flux, and the current-voltage relationship. Examples are given for the use of the present results in a physical interpretation of the data from the gramicidin A channel.