Application of natural resource valuation concepts for development of sustainable remediation plans for groundwater.

This paper explores the application of natural resource assessment and valuation procedures as a tool for developing groundwater remediation strategies that achieve the objectives for health and environmental protection, in balance with considerations of economic viability and conservation of natural resources. The natural resource assessment process, as applied under U.S. and international guidelines, entails characterization of groundwater contamination in terms of the pre-existing beneficial services of the impacted resource, the loss of these services caused by the contamination, and the measures and associated costs necessary to restore or replace the lost services. Under many regulatory programs, groundwater remediation objectives assume that the impacted groundwater may be used as a primary source of drinking water in the future, even if not presently in use. In combination with a regulatory preference for removal or treatment technologies, this assumed exposure, while protective of human health, can drive the remedy selection process toward remedies that may not be protective of the groundwater resource itself or of the other natural resources (energy, materials, chemicals, etc.) that may be consumed in the remediation effort. To achieve the same health and environmental protection goals under a sustainable remediation framework, natural resource assessment methods can be applied to restore the lost services and preserve the intact services of the groundwater so as to protect both current and future users of that resource. In this paper, we provide practical guidelines for use of natural resource assessment procedures in the remedy selection process and present a case study demonstrating the use of these protocols for development of sustainable remediation strategies.

Astrocyte activation and wound healing in intact-skull mouse after focal brain injury.

Localised brain tissue damage activates surrounding astrocytes, which significantly influences subsequent long-term pathological processes. Most existing focal brain injury models in rodents employ craniotomy to localise mechanical insults. However, the craniotomy procedure itself induces gliosis. To investigate perilesional astrocyte activation under conditions in which the skull is intact, we created focal brain injuries using light exposure through a cranial window made by thinning the skull without inducing gliosis. The lesion size was maximal at ~ 12 h and showed substantial recovery over the subsequent 30 days. Two distinct types of perilesional reactive astrocyte, identified by GFAP upregulation and hypertrophy, were found. In proximal regions the reactive astrocytes proliferated and expressed nestin, whereas in regions distal to the injury core the astrocytes showed increased GFAP expression but did not proliferate, lacked nestin expression, and displayed different morphology. Simply making the window did not induce any of these changes. There were also significant numbers of neurons in the recovering cortical tissue. In the recovery region, reactive astrocytes radially extended processes which appeared to influence the shapes of neuronal nuclei. The proximal reactive astrocytes also formed a cell layer which appeared to serve as a protective barrier, blocking the spread of IgG deposition and migration of microglia from the lesion core to surrounding tissue. The recovery was preceded by perilesional accumulation of leukocytes expressing vascular endothelial growth factor. These results suggest that, under intact skull conditions, focal brain injury is followed by perilesional reactive astrocyte activities that foster cortical tissue protection and recovery.

A review of physical, chemical, and hydrogeologic characteristics of stray gas migration: Implications for investigation and remediation.

Releases of natural gas into groundwater from oil and gas exploration, production, or storage (i.e., "stray gas") can pose a risk to groundwater users and landowners in the form of a fire or explosive hazard. The acute nature of stray gas risk differs from the long-term health risks posed by the ingestion or inhalation of other petroleum hydrocarbons (e.g., benzene). Stray gas also exhibits different fate and transport behaviors in the environment from other hydrocarbon contaminants, including the potential for rapid and extensive transport of free-phase gas through preferential pathways, and the resulting variable and discontinuous spatial distribution of free and dissolved gas phases. While there is extensive guidance on response actions for releases of other hydrocarbons such as benzene, there are relatively few examples available in the technical literature that discuss appropriate response measures for the investigation and remediation of stray gas impacts. This paper describes key considerations in the physical, chemical, and hydrogeological characteristics of stray gas releases and implications for the improved investigation and mitigation of associated risks.

NR2B-NMDA receptor-mediated increases in intracellular Ca2+ concentration regulate the tyrosine phosphatase, STEP, and ERK MAP kinase signaling.

NMDA receptors regulate both the activation and inactivation of the extracellular signal-regulated kinase (ERK) signaling cascade, a key pathway involved in neuronal plasticity and survival. This bi-directional regulation of ERK activity by NMDA receptors has been attributed to opposing actions of NR2A- versus NR2B-containing NMDA receptors, but how this is implemented is not understood. Here, we show that glutamate-mediated intracellular Ca(2+) increases occur in two phases, a rapid initial increase followed by a delayed larger increase. Both phases of the Ca(2+) increase were blocked by MK-801, a non-selective NMDA receptor inhibitor. On the other hand, selective inhibition of NR2B-NMDA receptors by Ifenprodil or Ro 25-6981 blocked the delayed larger phase but had only a small effect on the rapid initial increase. The rapid initial increase in Ca(2+), presumably because of NR2A-NMDAR activation, was sufficient to activate ERK, whereas the large delayed increases in Ca(2+) mediated by NR2B-NMDARs were necessary for dephosphorylation and subsequent activation of striatal-enriched phosphatase, a neuron-specific tyrosine phosphatase that in turn mediates the dephosphorylation and inactivation of ERK. We conclude that the magnitude of Ca(2+) increases mediated through NR2B-NMDA receptors plays a critical role in the regulation of the serine/threonine and tyrosine kinases and phosphatases that are involved in the regulation of ERK activity.

Localized loss of Ca2+ homeostasis in neuronal dendrites is a downstream consequence of metabolic compromise during extended NMDA exposures.

Excessive Ca(2+) loading is central to most hypotheses of excitotoxic neuronal damage. We examined dendritic Ca(2+) signals in single CA1 neurons, injected with fluorescent indicators, after extended exposures to a low concentration of NMDA (5 microM). As shown previously, NMDA produces an initial transient Ca(2+) elevation of several micromolar, followed by recovery to submicromolar levels. Then after a delay of approximately 20-40 min, a large Ca(2+) elevation appears in apical dendrites and propagates to the soma. We show here that this large delayed Ca(2+) increase is required for ultimate loss of membrane integrity. However, transient removal of extracellular Ca(2+) for varying epochs before and after NMDA exposure does not delay the propagation of these events. In contrast to compound Ca(2+) elevations, intracellular Na(+) elevations are monophasic and were promptly reversed by the NMDA receptor antagonist MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate]. MK-801 applied after the transient Ca(2+) elevations blocked the delayed propagating Ca(2+) increase. Even if applied after the propagating response was visualized, MK-801 restored resting Ca(2+) levels. Propagating Ca(2+) increases in dendrites were delayed or prevented by (1) reducing extracellular Na(+), (2) injecting ATP together with the Ca(2+) indicator, or (3) provision of exogenous pyruvate. These results show that extended NMDA exposure initiates degenerative signaling generally in apical dendrites. Although very high Ca(2+) levels can report the progression of these responses, Ca(2+) itself may not be required for the propagation of degenerative signaling along dendrites. In contrast, metabolic consequences of sustained Na(+) elevations may lead to failure of ionic homeostasis in dendrites and precede Ca(2+)-dependent cellular compromise.

Development of epileptiform excitability in the deep entorhinal cortex after status epilepticus.

Epileptiform neuronal activity during seizures is observed in many brain areas, but its origins following status epilepticus (SE) are unclear. We have used the Li low-dose pilocarpine rat model of temporal lobe epilepsy to examine early development of epileptiform activity in the deep entorhinal cortex (EC). We show that during the 3-week latent period that follows SE, an increasing percentage of neurons in EC layer 5 respond to a single synaptic stimulus with polysynaptic burst depolarizations. This change is paralleled by a progressive depolarizing shift of the inhibitory postsynaptic potential reversal potential in layer 5 neurons, apparently caused by upregulation of the Cl(-) inward transporter NKCC1 and concurrent downregulation of the Cl(-) outward transporter KCC2, both changes favoring intracellular Cl(-) accumulation. Inhibiting Cl(-) uptake in the latent period restored more negative GABAergic reversal potentials and eliminated polysynaptic bursts. The changes in the Cl(-) transporters were highly specific to the deep EC. They did not occur in layers 1-3, perirhinal cortex, subiculum or dentate gyrus during this period. We propose that the changes in Cl(-) homeostasis facilitate hyperexcitability in the deep entorhinal cortex leading to epileptiform discharge there, which subsequently affects downstream cortical regions.

An epigenetic induction of a right-shift in hippocampal asymmetry: selectivity for short- and long-term potentiation but not post-tetanic potentiation.

In humans, it is well established that major psychological functions are asymmetrically represented between the left and right cerebral cortices. The developmental origin of such functional lateralization remains unknown. Using the rat as a model system, we examined whether exposing neonates briefly to a novel environment can differentially affect synaptic plasticity in the left and right hippocampi during adulthood. During the first 3 weeks of life, one half of the pups from a litter spent 3 min daily away from their familiar home environment (Novel) while their littermates remained in that familiar environment (Home). At adulthood (7-months old), post-tetanic potentiation (PTP) of excitatory post-synaptic potentials (EPSPs), a very short-lasting form of plasticity, was greater among the Novel than the Home rats in both left and right hippocampi. In contrast, the novelty-induced increases in short- and long-term potentiation (STP, LTP), two relatively longer-lasting forms of plasticity, were found only in the right hippocampus. These findings demonstrate that a phase-selective asymmetry in hippocampal synaptic plasticity can be induced epigenetically by seemingly small systematic differences in early life environment. The selectivity of this asymmetry for the longer-lasting forms of synaptic plasticity suggests that the observed asymmetry in plasticity may contribute specifically to an asymmetric learning process which, in turn, may contribute to a functional asymmetry in the neocortex.

Purging and other sampling variables affecting dissolved methane concentration in water supply wells.

Determining whether changes in groundwater methane concentration are naturally occurring or related to oil and gas operations can be complicated by numerous sources of variability. This study of 10 residential water supply wells in Northeastern Pennsylvania evaluates how i) sampling from different points within the water well system, ii) purging different water volumes prior to sampling, and ii) natural variation over time, affects concentrations of naturally occurring dissolved methane and other water quality parameters. Among the population of wells, all had dissolved methane concentrations >1mg/L. Regardless of the volume of water purged or the timing between events, the maximum change in methane concentration (ratio of maximum to minimum concentration) among samples from a single well was 3.2, with eight out of ten wells exhibiting a maximum change less than a factor of two (i.e., <±100%). Among water wells where methane concentration changed by ±50% or more, there was a strong correlation with changes in the concentrations of sodium, chloride, and other salinity indicators such as specific conductivity and TDS. This suggests that significant variability in methane concentration is predominantly related to changes in the relative volumes of sodium-rich fluids feeding the wellbore at any given time. Among study well locations with bladder and diaphragm pressure tanks, there was no significant difference in dissolved methane concentrations between samples collected either upstream or downstream of a pressure tank. There appears to be little benefit to purging multiple casing volumes of water from a well prior to sampling because such volumes tend to be much larger than those representative of normal residential use. We recommend purging a volume sufficient to remove standing water in the pressure tank and lines above the pump intake. This article culminates with additional recommendations for improving sample collection methods and interpreting sampling data.

Emergence of a spermine-sensitive, non-inactivating conductance in mature hippocampal CA1 pyramidal neurons upon reduction of extracellular Ca2+: dependence on intracellular Mg2+ and ATP.

Large and protracted elevations of intracellular [Ca(2+)] and [Na(+)] play a crucial role in neuronal injury in ischemic conditions. In addition to excessive glutamate receptor activation, other ion channels may contribute to disruption of intracellular ionic homeostasis. During episodes of ischemia, extracellular [Ca(2+)] falls significantly. Here we report the emergence of an inward current in hippocampal CA1 pyramidal neurons in acute brain slices from adult mice upon reduction/removal of [Ca(2+)](e). The magnitude of the current was 100-300pA at -65mV holding potential, depending on intracellular constituents. The current was accompanied by intense neuronal discharge, observed in both whole-cell and cell-attached patch configurations. Sustained currents and increased neuronal firing rates were both reversed by restoration of physiological levels of [Ca(2+)](e), or by application of spermine (1mM). The amplitudes of the sustained currents were strongly reduced by raising intracellular [Mg(2+)], but not by extracellular [Mg(2+)] increases. Elevated intracellular ATP also reduced the current. This conductance is similar in several respects to the "calcium-sensing, non-selective cation current" (csNSC), previously described in cultured mouse hippocampal neurons of embryonic origin. The dependence on intracellular [ATP] and [Mg(2+)] shown here, suggests a possible role for this current in disruption of ionic homeostasis during metabolic stress that accompanies excessive neuronal stimulation.

A Tiered Approach to Evaluating Salinity Sources in Water at Oil and Gas Production Sites.

A suspected increase in the salinity of fresh water resources can trigger a site investigation to identify the source(s) of salinity and the extent of any impacts. These investigations can be complicated by the presence of naturally elevated total dissolved solids or chlorides concentrations, multiple potential sources of salinity, and incomplete data and information on both naturally occurring conditions and the characteristics of potential sources. As a result, data evaluation techniques that are effective at one site may not be effective at another. In order to match the complexity of the evaluation effort to the complexity of the specific site, this paper presents a strategic tiered approach that utilizes established techniques for evaluating and identifying the source(s) of salinity in an efficient step-by-step manner. The tiered approach includes: (1) a simple screening process to evaluate whether an impact has occurred and if the source is readily apparent; (2) basic geochemical characterization of the impacted water resource(s) and potential salinity sources coupled with simple visual and statistical data evaluation methods to determine the source(s); and (3) advanced laboratory analyses (e.g., isotopes) and data evaluation methods to identify the source(s) and the extent of salinity impacts where it was not otherwise conclusive. A case study from the U.S. Gulf Coast is presented to illustrate the application of this tiered approach.

1,4-Dioxane drinking water occurrence data from the third unregulated contaminant monitoring rule.

This study examined data collected from U.S. public drinking water supplies in support of the recently-completed third round of the Unregulated Contaminant Monitoring Rule (UCMR3) to better understand the nature and occurrence of 1,4-dioxane and the basis for establishing drinking water standards. The purpose was to evaluate whether the occurrence data for this emerging but federally-unregulated contaminant fit with common conceptual models, including its persistence and the importance of groundwater contamination for potential exposure. 1,4-Dioxane was detected in samples from 21% of 4864 PWSs, and was in exceedance of the health-based reference concentration (0.35µg/L) at 6.9% of these systems. In both measures, it ranked second among the 28 UCMR3 contaminants. Although much of the focus on 1,4-dioxane has been its role as a groundwater contaminant, the detection frequency for 1,4-dioxane in surface water was only marginally lower than in groundwater (by a factor of 1.25; p<0.0001). However, groundwater concentrations were higher than those in surface water (p<0.0001) and contributed to a higher frequency of exceeding the reference concentration (by a factor of 1.8, p<0.0001), indicating that surface water sources tend to be more dilute. Sampling from large systems increased the likelihood that 1,4-dioxane was detected by a factor of 2.18 times relative to small systems (p<0.0001). 1,4-Dioxane detections in drinking water were highly associated with detections of other chlorinated compounds particularly 1,1-dichlorethane (odds ratio=47; p<0.0001), which is associated with the release of 1,4-dioxane as a chlorinated solvent stabilizer. Based on aggregated nationwide data, 1,4-dioxane showed evidence of a decreasing trend in concentration and detection frequency over time. These data suggest that the loading to drinking water supplies may be decreasing. However, in the interim, some water supply systems may need to consider improving their treatment capabilities in response to further regulatory review of this compound.

NAD(P)H fluorescence transients after synaptic activity in brain slices: predominant role of mitochondrial function.

Excitatory stimulation in hippocampal slices results in biphasic NAD(P)H fluorescence transients. Previous studies using differing stimulus protocols agreed that the oxidation phase is a consequence of mitochondrial metabolism, but the reduction phase has been attributed to (1) mitochondrial nicotinamide adenine dinucleotide (NADH) generation or (2) astrocytic glycolysis triggered by glutamate uptake. In an attempt to reconcile these two views, the present study examined NAD(P)H signals evoked by a wide range of stimulus durations (40 ms to 20 secs). A combination of ionotropic glutamate receptor (iGluR) antagonists (6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 2-amino-5-phosphonopentanoic acid (APV)) virtually abolished responses to brief stimuli (40 to 200 ms, 50 Hz), but a significant fraction of the signal elicited by extended stimulation (20 secs, 32 Hz) was resistant to CNQX/APV. Glycolysis was inhibited by removal of glucose and addition of 2-deoxyglucose (2DG) (10 mmol/L) or iodoacetic acid (IAA, 1 mmol/L). Pyruvate was provided as an alternative substrate for oxidative phosphorylation and the A1 receptor antagonist 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX) included to prevent decreases in synaptic efficacy. If sufficient pyruvate was supplied, responses to brief and extended stimuli were unaffected by glycolytic inhibition and not significantly reduced by an inhibitor of glucose uptake (3-O-methyl glucose, 3 mmol/L). When timed to arrive at the peak of overshoots generated by extended synaptic stimulation, brief pyruvate applications (10 mmol/L, 2 mins) had little effect on evoked NAD(P)H increases. Flavoprotein autofluorescence transients after extended stimuli matched (with inverted sign) NAD(P)H responses. Responses to extended stimuli were not reduced by a nonselective inhibitor of glutamate uptake DL-Threo-beta-benzyloxyaspartic acid (TBOA). These results suggest that NAD(P)H transients report mitochondrial dynamics, rather than recruitment of glycolytic metabolism, over a wide range of stimulus intensities.

Environmental Factors Associated With Natural Methane Occurrence in the Appalachian Basin.

The recent boom in shale gas development in the Marcellus Shale has increased interest in the methods to distinguish between naturally occurring methane in groundwater and stray methane associated with drilling and production operations. This study evaluates the relationship between natural methane occurrence and three principal environmental factors (groundwater redox state, water type, and topography) using two pre-drill datasets of 132 samples from western Pennsylvania, Ohio, and West Virginia and 1417 samples from northeastern Pennsylvania. Higher natural methane concentrations in residential wells are strongly associated with reducing conditions characterized by low nitrate and low sulfate ([NO3- ] < 0.5 mg/L; [SO42- ] < 2.5 mg/L). However, no significant relationship exists between methane and iron [Fe(II)], which is traditionally considered an indicator of conditions that have progressed through iron reduction. As shown in previous studies, water type is significantly correlated with natural methane concentrations, where sodium (Na) -rich waters exhibit significantly higher (p<0.001) natural methane concentrations than calcium (Ca)-rich waters. For water wells exhibiting Na-rich waters and/or low nitrate and low sulfate conditions, valley locations are associated with higher methane concentrations than upland topography. Consequently, we identify three factors ("Low NO3- & SO42- " redox condition, Na-rich water type, and valley location), which, in combination, offer strong predictive power regarding the natural occurrence of high methane concentrations. Samples exhibiting these three factors have a median methane concentration of 10,000 µg/L. These heuristic relationships may facilitate the design of pre-drill monitoring programs and the subsequent evaluation of post-drill monitoring results to help distinguish between naturally occurring methane and methane originating from anthropogenic sources or migration pathways.

Effect of Different Sampling Methodologies on Measured Methane Concentrations in Groundwater Samples.

Analysis of dissolved light hydrocarbon gas concentrations (primarily methane and ethane) in water supply wells is commonly used to establish conditions before and after drilling in areas of shale gas and oil extraction. Several methods are currently used to collect samples for dissolved gas analysis from water supply wells; however, the reliability of results obtained from these methods has not been quantified. This study compares dissolved methane and ethane concentrations measured in groundwater samples collected using three sampling methods employed in pre- and post-drill sampling programs in the Appalachian Basin. These include an open-system collection method where 40 mL volatile organic analysis (VOA) vials are filled directly while in contact with the atmosphere (Direct-Fill VOA) and two alternative methods: (1) a semi-closed system method whereby 40 mL VOA vials are filled while inverted under a head of water (Inverted VOA) and (2) a relatively new (2013) closed system method in which the sample is collected without direct contact with purge water or the atmosphere (IsoFlask® ). This study reveals that, in the absence of effervescence, the difference in methane concentrations between the three sampling methods was relatively small. However, when methane concentrations equaled or exceeded 20 mg/L (the approximate concentration at which effervescence occurs in the study area), IsoFlask® (closed system) samples yielded significantly higher methane concentrations than Direct-Fill VOA (open system) samples, and Inverted VOA (semi-closed system) samples yielded lower concentrations. These results suggest that open and semi-closed system sample collection methods are adequate for non-effervescing samples. However, the use of a closed system collection method provides the most accurate means for the measurement of dissolved hydrocarbon gases under all conditions.

NAD(P)H fluorescence imaging of postsynaptic neuronal activation in murine hippocampal slices.

We examined mechanisms contributing to stimulus-evoked changes in NAD(P)H fluorescence as a marker of neuronal activation in area CA1 of murine hippocampal slices. Three types of stimuli (electrical, glutamate iontophoresis, bath-applied kainate) produced biphasic fluorescence changes composed of an initial transient decrease ("initial component," 1-3%), followed by a longer-lasting transient increase ("overshoot," 3-8%). These responses were matched by inverted biphasic flavin adenine dinucleotide (FAD) fluorescence transients, suggesting that these transients reflect mitochondrial function rather than optical artifacts. Both components of NAD(P)H transients were abolished by ionotropic glutamate receptor block, implicating postsynaptic neuronal activation as the primary event involved in generating the signals, and not presynaptic activity or reuptake of synaptically released glutamate. Spatial analysis of the evoked signals indicated that the peak of each component could arise in different locations in the slice, suggesting that there is not always obligatory coupling between the two components. The initial NAD(P)H response showed a strong temporal correspondence to intracellular Ca+ increases and mitochondrial depolarization. However, despite the fact that removal of extracellular Ca2+ abolished neuronal cytosolic Ca2+ transients to exogenous glutamate or kainate, this procedure did not reduce slice NAD(P)H responses evoked by either of these agonists, implying that mechanisms other than neuronal mitochondrial Ca2+ loading underlie slice NAD(P)H transients. These data show that, in contrast to previous proposals, slice NAD(P)H transients in mature slices do not reflect neuronal Ca2+ dynamics and demonstrate that these signals are sensitive indicators of both the spatial and temporal characteristics of postsynaptic neuronal activation in these preparations.

Review of quantitative surveys of the length and stability of MTBE, TBA, and benzene plumes in groundwater at UST sites.

Quantitative information regarding the length and stability condition of groundwater plumes of benzene, methyl tert-butyl ether (MTBE), and tert-butyl alcohol (TBA) has been compiled from thousands of underground storage tank (UST) sites in the United States where gasoline fuel releases have occurred. This paper presents a review and summary of 13 published scientific surveys, of which 10 address benzene and/or MTBE plumes only, and 3 address benzene, MTBE, and TBA plumes. These data show the observed lengths of benzene and MTBE plumes to be relatively consistent among various regions and hydrogeologic settings, with median lengths at a delineation limit of 10 µg/L falling into relatively narrow ranges from 101 to 185 feet for benzene and 110 to 178 feet for MTBE. The observed statistical distributions of MTBE and benzene plumes show the two plume types to be of comparable lengths, with 90th percentile MTBE plume lengths moderately exceeding benzene plume lengths by 16% at a 10-µg/L delineation limit (400 feet vs. 345 feet) and 25% at a 5-µg/L delineation limit (530 feet vs. 425 feet). Stability analyses for benzene and MTBE plumes found 94 and 93% of these plumes, respectively, to be in a nonexpanding condition, and over 91% of individual monitoring wells to exhibit nonincreasing concentration trends. Three published studies addressing TBA found TBA plumes to be of comparable length to MTBE and benzene plumes, with 86% of wells in one study showing nonincreasing concentration trends.

Exceptionally Long MTBE Plumes of the Past Have Greatly Diminished.

Studies published in the late 1990s and early 2000s identified the presence of exceptionally long methyl tert-butyl ether (MTBE) plumes (more than 600 m or 2000 feet) in groundwater and have been cited in technical literature as characteristic of MTBE plumes. However, the scientific literature is incomplete in regard to the subsequent behavior and fate of these MTBE plumes over the past decade. To address this gap, this issue paper compiles recent groundwater monitoring records for nine exceptional plumes that were identified in prior studies. These nine sites exhibited maximum historical MTBE groundwater plume lengths ranging from 820 m (2700 feet) to 3200 m (10,500 feet) in length, exceeding the lengths of 99% of MTBE plumes, as characterized in multiple surveys at underground storage tank sites across the United States. Groundwater monitoring data compiled in our review demonstrate that these MTBE plumes have decreased in length over the past decade, with five of the nine plumes exhibiting decreases of 75% or more compared to their historical maximum lengths. MTBE concentrations within these plumes have decreased by 93% to 100%, with two of the nine sites showing significant decreases (98% and 99%) such that the regulatory authority has subsequently designated the site as requiring no further action.

Long-term Depression of Glutamate Currents in Cultured Cerebellar Purkinje Neurons Does Not Require Nitric Oxide Signalling.

Cerebellar long-term depression (LTD) is produced when conjunctive stimulation of parallel fibre (PF) and climbing fibre (CF) inputs to a Purkinje neuron (PN) results in a prolonged decrease in the strength of the PF - PN synapse. In cultured PNs, LTD may be induced by substituting depolarization of the PN and iontophoretic glutamate pulses for CF and PF stimulation, respectively, allowing an unambiguous analysis of post-synaptic processes (Linden et al., Neuron, 7, 81 - 89, 1991). Recent studies have suggested that release of the newly described second messenger, nitric oxide, in the cerebellar molecular layer, is necessary for LTD produced in the slice preparation by PF/CF conjunctive stimulation (Shibuki and Okada, Nature, 349, 326 - 328, 1991) or PF/depolarization conjunctive stimulation (Crepel and Jaillard, NeuroReport, 1, 133 - 136, 1990). We report that LTD of glutamate currents produced without synaptic stimulation in cultured PNs is unaffected by reagents that stimulate (sodium nitroprusside) or inhibit (haemoglobin, NG-nitro-l-arginine) nitric oxide signalling.