Glutamate iontophoresis induces long-term potentiation in the absence of evoked presynaptic activity.

Protocols that induce long-term potentiation (LTP) typically involve afferent stimulation. We tested the hypothesis that LTP induction does not require presynaptic activity. The significance of this hypothesis is underscored by results suggesting that LTP expression may involve activity-dependent presynaptic changes. An induction protocol using glutamate iontophoresis was developed that reliably induced LTP in hippocampal slices without afferent stimulation. Iontophoresis LTP was Ca2+ dependent, was blocked by MK-801, and occluded tetanus-induced LTP. Iontophoresis LTP was induced when excitatory postsynaptic potentials were completely blocked by adenosine plus tetrodotoxin. Our results suggest constraints on the involvement of presynaptic mechanisms and putative retrograde messengers in LTP induction and expression; namely, these processes must function without many forms of activity-dependent presynaptic processes.

Shear deformation of polymer melt observed via proton NMR: theory and experiment.

We here develop a theory for the effect of shearing flow on residual proton dipole-dipole interactions for polymer melts. The model is based on the use of correlation functions which derive from the return to origin probability for polymers reptating in the tube of surrounding constraints. Using Doi-Edwards theory we calculate the spin-echo response under equilibrium conditions and then consider the effect of a shearing flow which deforms the tube, finding that there exists a strong dependence of transverse relaxation on Weissenberg number. The results are compared with NMR measurements of shear-perturbed proton T2 relaxation in 494kDa poly (dimethylsiloxane).

Expression of fructose sensitive glucose transporter in the brains of fructose-fed rats.

Glucose transporters play a critical role in mammalian brain energy metabolism because glucose is the principal brain energy source and these transporters promote glucose movement into neural cells. When glucose is unavailable, fructose can serve as an alternative energy source. Using real-time polymerase chain reaction and actin as a reference mRNA, we investigated the impact of fructose feeding on rat brain and other tissue mRNA expression of glucose transporter 5 which has high affinity for fructose. Brain mRNA levels of glucose transporter 5 increased 1.5-fold in 35-day old rats after 7 days of fructose feeding compared with controls, whereas it increased 2.5-fold in jejunum. Semi-quantitative analysis of protein expression by immunofluorescence of glucose transporter 5 in rat hippocampi indicated a 2.4-fold increase. We demonstrated the specificity of fructose feeding on glucose transporter 5 expression by showing that the expression of the neuronal glucose transporter 3 and insulin-regulated glucose transporter 4 were unaffected. In addition, the expression of glucose transporter 5 increased in fructose fed older adult rats (8-months and 12-months old) when compared with controls. These results suggest that short-term fructose feeding increases the expression of glucose transporter 5 in both young and aging adult rats. Increased brain expression of glucose transporter 5 is likely to be important in the role of fructose as an alternative energy source.

Astrocytes exert a pro-apoptotic effect on neurons in postnatal hippocampal cultures.

We investigated conditions that promote basal and activity-dependent neuronal apoptosis in postnatal rat hippocampal cultures. Low-density mixed cultures of astrocytes and neurons exhibited lower sensitivity than high-density cultures to basal neuronal death and activity-sensitive neuronal death, induced with glutamate receptor blockers, sodium channel blockers, or calcium channel blockers. Although elevations of [Ca(2+)](i) protect neurons from apoptosis, low-density microcultures and mass cultures exhibited only minor differences in resting [Ca(2+)](i) and Ca(2+) current density, suggesting that these variables are unlikely to explain differences in susceptibility. Astrocytes, rather than neurons, were implicated in the neuronal loss. Several candidate molecules implicated in other astrocyte-dependent neurotoxicity models were excluded, but heat inactivation experiments suggested that a heat-labile factor is critically involved. In sum, our results suggest the surprising result that astrocytes can be negative modulators of neuronal survival during development and when the immature nervous system is challenged with drugs that dampen electrical excitability.

Time-dependence of nuclear magnetic resonance quadrupole interactions for polymers under shear.

We consider the problem of performing NMR spectroscopy under conditions of flow, a central issue in Rheo-NMR. By way of example, the effects of rotational motion on the deuterium NMR spectrum are considered for Couette cell experiments involving deformation of polymers under shearing conditions. The polymer was modelled as a power law fluid and for each streamline, the spin Hamiltonian evolved to allow for flow reorientation. The gap-integral spectra are compared with the 'ideal' spectra for a polymer under shear, but without reorientation. It is found that flow does affect the shape of the deuterium spectra, as well as slightly perturbing the splittings.

Biaxial deformation of a polymer under shear: NMR test of the Doi-Edwards model with convected constraint release.

2H NMR quadrupole interaction spectroscopy has been used to measure the deformation of a 670 kD poly(dimethylsiloxane) melt under shear in a Couette cell. The signals were acquired from a per deuterated benzene probe molecule which provides a motionally averaged sampling of the entire segmental ensemble. We have measured the dependence on shear rate of the S(XX) (velocity), S(YY) (velocity gradient), S(ZZ) (vorticity), and S(XY) (shear) elements of the segmental alignment tensor, as well as the angular dependence of the deuterium quadrupole splitting at fixed shear rate. We show that the data agree quite well with the Doi-Edwards theory but significantly better when convected constraint release effects are included. These fits return a value for the tube disengagement time of 100 ms.

Cumulative effects of glutamate microstimulation on Ca(2+) responses of CA1 hippocampal pyramidal neurons in slice.

Glutamate stimulation of hippocampal CA1 neurons in slice was delivered via iontophoresis from a microelectrode. Five pulses (approximately 5 muA, 10 s duration, repeated at 1 min intervals) were applied with the electrode tip positioned in the stratum radiatum near the dendrites of a neuron filled with the Ca(2+) indicator fura-2. A single stimulus set produced Ca(2+) elevations that ranged from several hundred nM to several microM and that, in all but a few neurons, recovered within 1 min of stimulus termination. Subsequent identical stimulation produced Ca(2+) elevations that outlasted the local glutamate elevations by several minutes as judged by response recoveries in neighboring cells or in other parts of the same neuron. These long responses ultimately recovered but persisted for up to 10 min and were most prominent in the mid and distal dendrites. Recovery was not observed for responses that spread to the soma. The elevated Ca(2+) levels were accompanied by membrane depolarization but did not appear to depend on the depolarization. High-resolution images demonstrated responsive areas that involved only a few mu(m) of dendrite. Our results confirm the previous general findings from isolated and cell culture neurons that glutamate stimulation, if carried beyond a certain range, results in long-lasting Ca(2+) elevation. The response characterized here in mature in situ neurons was significantly different in terms of time course and reversibility. We suggest that the extended Ca(2+) elevations might serve not only as a trigger for delayed neuron death but, where more spatially restricted, as a signal for local remodeling in dendrites.

Glutamate-induced long-term potentiation enhances spontaneous EPSC amplitude but not frequency.

1. Many examples of long-term potentiation (LPT) are induced by repetitive electrical stimulation of presynaptic axons. LTP also is induced by direct glutamate iontophoresis (1 M, 1-2 microA, 10 s) onto postsynaptic neurons in hippocampal slices without evoked presynaptic stimulation; this form of LTP is called "ionto-LTP". The studies herein test the hypothesis that ionto-LTP is expressed primarily through postsynaptic mechanisms. 2. Whole cell recordings were used to examine the amplitude and frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in CA1 pyramidal neurons. sEPSCs were composed of an equal mixture of tetrodotoxin (TTX)-insensitive miniature EPSCs and EPSCs that appeared to result from spontaneous action potentials (i.e., TTX-sensitive EPSCs). The detection of all sEPSCs was virtually eliminated by 6-cyano-7-nitroquinoxaline-2,3-dione (20 microM), suggesting that sEPSCs were glutamate-mediated synaptic events. 3. Changes in the amplitude and frequency of sEPSCs were examined during the expression of ionto-LTP to obtain new information about the cellular location of mechanisms involved in synaptic plasticity. Our findings show that ionto-LTP expression results in increased sEPSC amplitude in the absence of lasting increases in sEPSC frequency. 4. Potentiation of sEPSC amplitude without changes in sEPSC frequency has been previously interpreted to be due to postsynaptic mechanisms. Although this interpretation is supported by findings from peripheral synapses, its application to the central nervous system is unclear. We have considered alternative mechanisms. Models based on increased release probability for action potential dependent transmitter release appeared insufficient to explain our results. The most straightforward interpretation of our results is that LTP induced by glutamate iontophoresis on dendrites of CA1 pyramidal neurons is mediated largely by postsynaptic changes.

Bidirectional synaptic plasticity correlated with the magnitude of dendritic calcium transients above a threshold.

The magnitude of postsynaptic Ca(2+) transients is thought to affect activity-dependent synaptic plasticity associated with learning and memory. Large Ca(2+) transients have been implicated in the induction of long-term potentiation (LTP), while smaller Ca(2+) transients have been associated with long-term depression (LTD). However, a direct relationship has not been demonstrated between Ca(2+) measurements and direction of synaptic plasticity in the same cells, using one induction protocol. Here, we used glutamate iontophoresis to induce Ca(2+) transients in hippocampal CA1 neurons injected with the Ca(2+)-indicator fura-2. Test stimulation of one or two synaptic pathways before and after iontophoresis showed that the direction of synaptic plasticity correlated with glutamate-induced Ca(2+) levels above a threshold, below which no plasticity occurred (approximately 180 nM). Relatively low Ca(2+) levels (180-500 nM) typically led to LTD of synaptic transmission and higher levels (>500 nM) often led to LTP. Failure to show plasticity correlated with Ca(2+) levels in two distinct ranges: <180 nM and approximately 450-600 nM, while only LTD occurred between these ranges. Our data support a class of models in which failure of Ca(2+) transients to affect transmission may arise either from insufficient Ca(2+) to affect Ca(2+)-sensitive proteins regulating synaptic strength through opposing activities or from higher Ca(2+) levels that reset activities of such proteins without affecting the net balance of activities. Our estimates of the threshold Ca(2+) level for LTD (approximately 180 nM) and for the transition from LTD to LTP (approximately 540 nM) may assist in constraining the molecular details of such models.

Basal levels of adenosine modulate mGluR5 on rat hippocampal astrocytes.

Neuronal activity elicits increases in intracellular Ca2+ in astrocytes, which in turn can elevate neuronal Ca2+ and potentiate the efficacy of excitatory synaptic transmission. Therefore, understanding the modulation of astrocyte Ca2+ elevations by neurotransmitters should aid in understanding astrocyte-neuronal interactions. On cultured hippocampal microislands containing only astrocytes, activation of metabotropic glutamate receptors (mGluRs) with the specific agonist 1S,3R-ACPD triggers Ca2+ elevations that are potentiated by adenosine A1 receptor activation. A1 receptor modulation of mGluR-induced Ca2+ elevations is blocked by pertussis toxin and is mimicked by the wasp venom peptide mastoparan, suggesting that potentiation occurs by means of a G(i/o) mechanism. Surprisingly, on microislands containing only astrocytes, A1 receptor antagonism or adenosine degradation suppresses mGluR-triggered Ca2+ elevations, strongly suggesting that astrocytes are a source of physiologically relevant concentrations of adenosine.

Reduced voltage-dependent Ca2+ signaling in CA1 neurons after brief ischemia in gerbils.

An initial overload of intracellular Ca2+ plays a critical role in the delayed death of hippocampal CA1 neurons that die a few days after transient ischemia. Without direct evidence, the prevailing hypothesis has been that Ca2+ overload may recur until cell death. Here, we report the first measurements of intracellular Ca2+ in living CA1 neurons within brain slices prepared 1, 2, and 3 days after transient (5 min) ischemia. With no sign of ongoing Ca2+ overload, voltage-dependent Ca2+ transients were actually reduced after 2-3 days of reperfusion. Resting Ca2+ levels and recovery rate after loading were similar to neurons receiving no ischemic insult. The tetrodotoxin-insensitive Ca spike, normally generated by these neurons, was absent at 2 days postischemia, as was a large fraction of Ca2+-dependent spike train adaptation. These surprising findings may lead to a new perspective on delayed neuronal death and intervention.