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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Neuropsychopharmacology. Author manuscript; available in PMC 2010 March 1.
Published in final edited form as:
PMCID: PMC2726900
NIHMSID: NIHMS114953

Abused inhalants enhance GABA-mediated synaptic inhibition

M. Bruce MacIver, MSc PhD, Associate Professor

Abstract

Abused inhalants are widely used, especially among school age children and teenagers, and are ‘gateway’ drugs leading to the abuse of alcohol and other addictive substances. In spite of this widespread use, little is known about the effects produced by inhalants on the central nervous system. The similarity in behavioral effects produced by inhalants and inhaled anesthetics, together with their common chemical features, prompted the present study of inhalant actions on a well characterized anesthetic target, GABA synapses. Whole cell patch clamp recordings were conducted on CA1 pyramidal neurons in rat hippocampal brain slices to measure effects on resting membrane properties, action potential discharge and GABA-mediated inhibitory responses. Toluene, 1,1,1-trichloroethane and trichloroethylene depressed CA1 excitability in a concentration-dependent and reversible manner. This depression appeared to involve enhanced GABA-mediated inhibition, evident in its reversal by a GABA receptor antagonist. Consistent with this, the abused inhalants increased inhibitory postsynaptic potentials produced using minimal stimulation of stratum radiatum inputs to CA1 neurons, in the presence of CNQX and APV to block excitatory synaptic responses and GGP to block GABAB responses. The enhanced inhibition appeared to come about by a presynaptic action on GABA nerve terminals, since spontaneous inhibitory postsynaptic current (IPSC) frequency was increased with no change in the amplitude of postsynaptic currents, both in the presence and absence of tetrodotoxin used to block interneuron action potentials and cadmium used to block calcium influx into nerve terminals. The toluene-induced increase in mIPSC frequency was blocked by dantrolene or ryanodine, indicating that the abused inhalant acted to increase the release of calcium from intracellular nerve terminal stores. This presynaptic action produced by abused inhalants is shared by inhaled anesthetics and would contribute to the altered behavorial effects produced by both classes of drugs, and could be especially important in the context of a disruption of learning and memory by abused inhalants.

Keywords: Neuropharmacology, Abuse, Hippocampus, Synapse, Inhibition, Calcium, Dantrolene, Ryanodine, Intracellular, Endoplasmic reticulum

Introduction

Over the past ten years, solvent abuse has increased among grade school children, adolescents and some adults. The Monitoring the Future Survey (2000) found that 8th graders reported a high rate of current (4.5 percent), past year (10.2 percent) and lifetime (17.9 percent) inhalant abuse. The National Household Survey on Drug Abuse (2001) indicates that abuse of inhalants outpaced both crack cocaine and heroin use among young adults. In addition, abused inhalants are a major ‘gateway’ drug – leading to abuse of alcohol and other drugs. Recent data from the National Survey on Drug Use (2006) reported that over 783,000 initiates aged 12 or older have tried inhalants. Despite this widespread usage, there is little known about the effects of inhaled solvents on the nervous system (Balster, 1998; Del Re et al, 2006; Lubman et al, 2008).

Abused inhalant solvents could share similar actions with inhalational anesthetics, since both types of chemicals are halogenated or aromatic hydrocarbons, and because volatile anesthetics also have a history of abuse potential (Balster, 1998; Beckstead et al, 2000; Bowen et al, 1999a). Also like anesthetics, abused inhalants exhibit non-selective actions on a number of neurotransmitter and voltage gated ion channels (Bale et al, 2005; Cruz et al, 2000; Del Re et al, 2006; Shafer et al, 2005; Smothers and Woodward, 2007). Synaptic sites of action play a major role for inhalational anesthetic effects on the central nervous system (Pittson et al, 2004; Pocock and Richards, 1991). Anesthetics enhance inhibitory synaptic responses (Nicoll et al, 1975; Nishikawa and MacIver, 2000, 2001). Early research indicated that amino acid transmitters in hippocampal cortex were altered by abused inhalant exposure in animals (Bjornaes and Naalsund, 1988; Briving et al, 1986) and more recent work demonstrates effects on isolated GABA and glutamate receptors occur (Bale et al, 2005; Bowen et al, 1999b; Cruz et al, 2000), and chronic exposure alters GABA synapses (Liu et al, 2007), and these effects could alter inhibitory synaptic transmission. Toluene can directly excite ventral tegmental area dopaminergic neurons recorded in brain slices (Riegel et al, 2007). Toluene also stimulated non-dopaminergic cells in vitro (Riegel et al, 2007), but not in vivo (Riegel and French, 1999), suggesting additional depressant effects to enhance GABAergic inhibition could be involved (Riegel et al, 2004; Riegel et al, 2007).

A previous study using field potential recordings in hippocampal slices found that toluene could inhibit synaptically evoked CA1 neuron population spikes (Ikeuchi and Hirai, 1994), suggesting that enhanced inhibition could occur. CA 1 neuron GABA-mediated synaptic inhibition is enhanced during anesthesia in vivo (Ma and Leung, 2006; MacIver et al, 1996; Nicoll et al, 1975; Pearce et al, 1989) and abused inhalants could produce similar alterations, since abused inhalants can produce anesthesia and inhaled anesthetics produce some behavioral changes similar to abused inhalants in mice (Balster, 1998). The present study compared effects produced by abused inhalants on GABA-mediated inhibitory synapses, using whole cell patch clamp recordings from CA 1 pyramidal cells in rat hippocampal brain slices. Three abused solvents were compared, two halogenated hydrocarbons (1,1,1-trichloroethane & trichloroethylene) as well as the aromatic agent, toluene, providing representation of inhalants with distinct chemical profiles.

Materials and Methods

Brain slice preparation methods have previously been described in detail (Bieda and MacIver, 2004). In short, standard transverse hippocampal slices (450□m) from adolescent Sprague-Dawley rats (P33−36) were prepared using a vibratome. All procedures conform to Society for Neuroscience and NIH guidelines and were approved by the Stanford University Animal Use Committee.

Electrophysiology

Standard visualized slice procedures were used (Nishikawa et al, 2000). All recordings were from CA1 pyramidal neurons in stratum pryamidale. Pyramidal neurons exhibited accommodating action potential trains in response to depolarizing current injection, but varied in resting membrane potential and threshold current required to produce spiking; there was no spontaneous spiking evident. Input resistances varied from 200 MOhm to 550 MOhm. There were no obvious differences between cells in response to toluene: every neuron displayed only a small decrease in spiking and input resistance. Throughout current clamp experiments, sets of current steps were applied repetitively at fixed intervals (typically, 3 steps/set: 1 hyperpolarizing, one at zero pA, and 1 depolarizing; 1 set/minute). The depolarizing current step was adjusted to produce 7 action potentials during the depolarization in control conditions for each neuron. In both current clamp and voltage clamp experiments cells were allowed to remain at rest, tonic currents were not applied to adjust resting potentials. All experiments were conducted at room temperature (22−23C) using a submersion chamber and perfusion vessels, valves and > 95 % of connectors and tubing were Teflon to minimize drug loss or binding. Using continuous perfusion of ACSF at 3 mL/minute complete bath replacement took < 30 seconds, as measured by dye exchange. Each slice was used for only a single experiment, and each animal provided 1 to 3 slices – limited by the long duration of each experiment (1 to 3 hours). The following external (ACSF) was used (in mM): 124 NaCl, 3.5 KCl, 1.25 NaH2PO4, 2 MgSO4, 2 CaCl2, 26 NaHCO3, 10 glucose; it was bubbled with 95% O2/5% CO2 to reach pH 7.4. To prevent loss of the abused inhalant from the perfusate, a known volume of inhalant was added to pre-gassed ACSF and stored in Teflon perfusion vessels. Preliminary experiments showed that this prevented loss of volatile agents (less than 5 % in five hours) measured using High Pressure Liquid Chromatography (HPLC; see below). A similar degree of loss was evident from samples taken next to brain slices in the experimental chamber used for recording neuronal responses.

We used standard whole-cell methods (pipette resistance 4−6 Mohm). For all current-clamp recordings a KGluconate-based internal solution was used (in mM): 100 KGluconate, 10 EGTA, 5 MgCl2, 40 HEPES, 2 Na2ATP, 0.3 NaGTP (pH 7.2 with KOH). For voltage-clamp experiments, a KCl internal based solution with QX-314 (1−5 mM) to block sodium currents was used, and, in experiments on evoked IPSCs, the concentration of MgCl2 was changed to 3 mM. KCl internal was:100 KCl, 10 EGTA, 5 MgCl2, 40 HEPES, 2 Na2ATP, 0.3 NaGTP, 1 QX-314 (pH 7.2 with KOH).

Slice viability was tested using population spike responses and only slices exhibiting response amplitudes > 10 mV were used for subsequent experiments. For recording of population spikes from CA1 pyramidal cells, an ACSF filled pipette (2 to 4 MOhm) was placed at the boarder of stratum pyramidale and stratum oriens. To stimulate synaptic inputs, a bipolar tungsten electrode was placed in stratum radiatum, as previously described.(Bieda et al, 2009) Recordings were established for > 15 minutes before recording baseline data, and preparations showing unstable response properties (> 5 % variability) were not used.

Compounds and Concentration Analysis

All compounds were reagent grade or better from Sigma/RBI (St Louis, MO), except for toluene, tricholorethane and trichloroethylene (all > 99.99 %), which were obtained from Aldrich (Milwaukee, WI). All solutions were made fresh daily using HPLC grade ‘OmniSolv’ water obtained from EMD – Merck (Gibbstown, NJ).

Due to the volatile nature of the abused inhalants, it was essential to measure bath concentrations to determine the extent of loss of drug from the perfusion vessels, tubing and brain slice chamber. Chemical analysis was performed on ACSF samples (1.5 ml) taken from the perfusate and run on an HP 1050 HPLC using a 40/60 % (water/acetonitrile) carrier solvent on a 25 cm spherisorb column. Major detection peaks for concentration analysis were found at 225 and 240 nm wavelengths with a retention time of approximately 8.5 min.

Data Collection and Analysis

Whole-cell and synaptically evoked responses were collected and analyzed online using Igor Pro 6.0 (Wavemetrics, Oswego, OR) and graphed on computer monitors to ensure that consistent baselines were established for each preparation. A two-tailed Student's t-test or repeated measures ANOVA with Tukey test were used to evaluate statistical significance (p < 0.05 or below) (GraphPad Prism software, Prism Inc., San Diego, CA, USA).

Results

Abused Inhalants Produce Little effect on Postsynaptic Excitability

Even at high concentrations, toluene produced only a small depression of CA1 neuron excitability (Fig 1), measured as an ability to generate action potentials in response to depolarizing current injection. There was no effect apparent on action potential peak amplitude, rise time, half-width, or decay phase. At a concentration of 1 mM, toluene depressed action potential discharge from 7 spikes in control to 6.2 ± 0.7 (mean ± SD) spikes in the presence of the inhalant (Fig 1A). Although this depression was statistically significant (p < 0.05; n = 10 experiments, each from different slices, from 8 rats), only a minor degree of inhibition was evident. The depression of spike discharge was not accompanied by a change in the resting membrane potential of CA1 neurons, although a small, statistically insignificant, decrease in membrane resistance was observed.

Figure 1
Toluene depressed excitability of CA1 pyramidal neurons, but produced very little effect on resting membrane responses. A, Direct current evoked discharge frequency was slowed by toluene (950 μM; top recordings), but there was no apparent change ...

To determine whether the depression of action potential discharge was due to an inhalant-induced enhancement of synaptic inhibition, the ability of a GABA receptor antagonist to reverse the depression was studied. The GABA receptor antagonist, gabazine (SR95531) at a saturating concentration (10 μM) completely reversed the inhalant-induced depression of discharge (8.1 ± 0.6 spikes), indicating that an increase in GABA-mediated inhibition could account for this depression. Gabazine also reversed the small change in membrane resistance that was observed, but the difference was only significant at the p < 0.05 level when gabzine data were compared to the normalized resistance in the presence of toluene (Fig 1B). Gabazine alone had no effect on action potential amplitude, rise time, duration, or on resting membrane potential.

Abused Inhalants Enhanced GABA-Mediated Synaptic Inhibition

To study the effects of abused inhalants on GABA-mediated inhibition, isolated, monosynaptic inhibitory postsynaptic potentials (IPSPs) were evoked with stimulating electrodes placed in stratum radiatum. Glutamate and GABA-B-mediated synaptic responses were blocked using receptor antagonists – CNQX (18 μM and APV 100 μM) and CGP (1.0 μM), respectively. Stimulus location, within stratum radiatum, as well as intensities and polarity were adjusted to produce minimal, stable response amplitudes; typically about 1.5 times threshold for the smallest amplitude responses seen. Using this approach, IPSPs exhibited amplitudes from 5 to 15 mV and failure rates of approximately 20 %. The abused inhalants increased IPSP amplitudes, but did not appear to alter the rise time, decay time, duration or failure rate of these synaptic responses (Fig 2).

Figure 2
Toluene enhanced GABA-mediated inhibition measured using monosynaptically-evoked IPSPs recorded from CA1 pyramidal neurons (top recordings) and this effect recovered following washout of the inhalant. IPSP response amplitude was measured from pre-stimulus ...

Toluene (TOL), trichloroethane (TCE) and trichloroethylene (TCY) appeared to be equally efficacious at increasing GABA-mediated inhibition, enhancing IPSP amplitudes to 129 ± 2.3, 128 ± 1.6 and 127 ± 0.9 % of control responses, respectively. The inhalants differed considerably, however, in potencies with a rank order of TCY > TOL > TCE over a two fold range of concentrations. The concentration producing a half maximal effect (EC50) was determined from Hill equations, fitting the data with a least squares approach. The TCY EC50 was 360 μM, and EC50s for TOL and TCE were 628 μM and 895 μM, respectively. Hill coefficients also varied considerably: 1.2 for TCE, 2.7 for TOL and 4.7 for TCY.

Abused Inhalants Enhanced GABA-Mediated Synaptic Inhibition by a Presynaptic Action

To determine whether the enhanced GABA-mediated inhibition resulted from pre- or post-synaptic mechanisms of action, spontaneous inhibitory postsynaptic currents (IPSCs) were recorded from voltage clamped CA1 neurons (Fig 3). In control conditions, IPSC amplitudes ranged from 2 pA, just detectable above noise, to 650 pA with an average amplitude of 75.8 ± 14.2 pA seen across 23,720 events recorded from 10 CA1 neurons. Frequency varied considerably, even within each cell, but averaged 4.3 ± 1.7 Hz. IPSC rise time (1.58 ± 0.21 ms), decay time (16.9 ± 5.2 ms) and duration (24.6 ± 7.8 ms) measurements were quite consistent across cells. These IPSCs were completely blocked with 20 μM gabazine (not shown), indicating that they were GABA-mediated synaptic currents.

Figure 3
Toluene enhanced GABA inhibition by a presynaptic mechanism, evident in spontaneous IPSC recordings from CA1 pyramidal neurons. Recordings on top show 1.0 second long consecutive traces in control or after exposure to 950 μM) toluene. Unlike inhaled ...

All three abused inhalants significantly increased IPSC frequency: TOL to 134 ± 5.6 % of control, TCE to 130 ± 6.3 % and TCY to 123 ± 7.1 % of control, for concentrations of 940 μM TOL, and 800 μM of both TCE and TCY (Fig 3). These frequency increases were not accompanied by any measurable effect on holding currents needed to maintain the resting membrane potential at the original control values for the CA1 neurons. No significant effects on IPSC amplitude, rise time, decay time or duration were observed in the presence of any abused inhalant. Thus, the abused inhalant-induced increase in inhibition appears to come about by a presynaptic action to increase the release of GABA from inhibitory nerve terminals.

Abused Inhalants Act Directly on GABA Nerve Terminals

The increased IPSC frequency produced by the inhalants could have come about by increased discharge of action potentials in inhibitory interneurons, or by a direct effect on interneuron nerve terminals. To test whether either or both of these effects contribute to the increased IPSC frequency, miniature IPSCs (mIPSCs) were recorded in the presence of TTX (1.0 μM) to block sodium channels, and, hence, abolish action potential discharge of inhibitory neurons. If the inhalant-induced increase in mIPSC frequency still occurred, then a direct action on GABA nerve terminals would be implicated.

In control conditions, mIPSC amplitudes were much smaller than IPSCs, averaging 27.4 ± 5.3 pA. Rise times and decay times were essentially the same as seen for control conditions in the absence of TTX, however, mIPSC frequency was reduced to 3.25 ± 1.89 Hz across the 9,189 events recorded from 10 CA1 neurons studied. In the neuron shown in Fig 4B, toluene (940 μM) produced a near doubling in mIPSC frequency (from 5.5 to 8.3 Hz), that reached steady state within 10 min and recovered following removal of toluene within 20 min. All of these synaptic currents were blocked by 20 μM gabazine applied at 50 min. Grouped data for all 10 experiments are shown in Fig 4C and the increased frequency of mIPSCs was statistically significant (p < 0.01), as was the gabazine block (p < 0.001), both compared to control frequencies measured before application of drug.

Figure 4
Toluene appeared to act directly at GABA nerve terminals, since an increase in miniature IPSC frequency was evident in the presence of tetrodotoxin used to block action potentials in the inhibitory interneurons. A, Consecutive five second long recordings ...

For the majority of experiments (7 of 10), neither mIPSC amplitude, rise time, decay time nor duration were altered by the inhalant. In the remaining three experiments, an apparent toluene-induced increase in rise time (from 1.78 ± 0.63 to 2.55 ± 0.98 ms), decay time (from 17.11 ± 11.23 to 29.87 ± 28.65 ms) and duration was seen in the initial analysis. On closer inspection, it was evident that these cells had prominent GABAA Slow mIPSCs mixed in amongst the typical GABAA Fast responses seen during control recordings (Fig 5). Toluene appeared to have a selective effect to enhance the frequency of GABAA Slow mIPSCs (to 120 % of control) in these cells and this produced an apparent increase in decay time and duration, since more slow events were contributing to the analysis in the presence of inhalant.

Figure 5
Toluene appeared to selectively enhance the frequency of GABAA slow synaptic currents. In some experiments (3 of 10), two kinds of miniature IPSC kinetics were evident: fast and slow (recording on top). In these experiments, toluene selectively enhanced ...

Previous studies have shown that volatile anesthetics increase mIPSC frequency by acting on intracellular calcium stores in GABA nerve terminals.(Doze and MacIver, 1998; Yamashita et al, 2001) To determine whether a similar mechanism contributed to the toluene-induced increase in mIPSC frequency, the effects of blocking calcium influx into nerve terminals was studied. The toluene-induced increase in mIPSC frequency persisted in the presence of 500 μM Cd++, used to block calcium entry into nerve terminals, thus, toluene appeared to share an action on intracellular stores with volatile anesthetics. Nerve terminals appear to have at least two intracellular calcium storage compartments: a thapsigargin-sensitive store that is associated with IP3 receptor-mediated regulation, and a caffeine-sensitive store that is associated with ryanodine receptor-mediated regulation. Neither thapsigargin, ryanodine nor dantrolene, when applied alone, appeared to alter mIPSC frequency, except for a transient increase (2 to 5 min) following dantrolene exposure. Pretreatment of preparations with thapsigargin did not alter the toluene-induced increase in mIPSC frequency (Figure 6), however, pretreatment with either dantrolene (a calcium store depletion agent) or with ryanodine (a caffeine store calcium channel blocker) blocked the effect produced by toluene.

Figure 6
Toluene increased IPSC frequency by releasing calcium from intracellular stores, since the effect persisted in the presence of Cd++, used to block calcium entry through nerve terminal membrane channels, but was blocked when calcium stores were depleted, ...

Discussion

Abused inhalants depressed CA1 pyramidal neurons by increasing inhibition at GABA synapses. All three inhalants appeared to increase inhibition over the concentration range thought to occur in vivo (Balster, 1987; Beckstead et al, 2000). This increased inhibition was evident in recordings of monosynaptic IPSPs and occurred with no apparent effect on resting membrane potential or action potential threshold produced by the inhalants. The increased inhibition appeared to come about by a presynaptic mechanism, since it was associated with an increase in spontaneous IPSC frequency with no effect on postsynaptic current amplitudes. The increased IPSC frequency appeared to involve actions directly on GABA nerve terminals, since the effect persisted after blockade of action potentials secondary to blocking sodium channels with tetrodotoxin. Similarly, the toluene effect persisted after blocking calcium influx into nerve terminals, indicating that the increased mIPSC frequency involved a toluene-induced release of calcium from intracellular stores. The increase in mIPSC frequency was blocked by pretreatment with either dantrolene or ryanodine, but not by thapsigargin, indicating that toluene caused a release of calcium from a caffeine-sensitive store, but not from an IP3-regulated store. In this respect, the abused inhalants appear to act via a mechanism that is similar to that used by inhaled anesthetics.

Enhanced GABA-mediated inhibition is a common effect produced by inhaled anesthetics, like halothane and isoflurane (Banks and Pearce, 1999; Bieda et al, 2009; Franks, 2008; Jones and Harrison, 1993; Nishikawa et al, 2001; Tanelian et al, 1993). As this is a prevalent effect seen in hippocampal neurons, it has been proposed to play a role in anesthetic-induced memory loss (Simon et al, 2001; Tanelian et al, 1993). Indeed, volatile anesthetics have been shown to block long-term potentiation (MacIver et al, 1989) and long-term depression at CA1 neuron synapses (Ishizeki et al, 2008; Simon et al, 2001), as do other anesthetics (Cheng et al, 2006). The selective enhancement of GABAA Slow mIPSCs observed could be particularly important in this regard because these synaptic currents have the ideal time course to modulate NMDA responses (Banks et al, 2000), which are critical for synaptic plasticity in several major synaptic pathways. These forms of synaptic plasticity are widely thought to contribute to hippocampal-dependent learning and memory, as well as to some long-term mechanisms of drug addiction (Hyman et al, 2006). Thus, the enhanced GABA-mediated inhibition seen in the present study would provide a mechanism for the disruption of learning and memory that is produced by abused inhalants (Bowen et al, 2006). It should be noted that good evidence exists for additional effects of abused solvents that could also impair learning mechanisms, in particular actions on NMDA receptors and other neurotransmitter systems, and brain regions, would be very likely to contribute (Beckstead et al, 2000; Riegel et al, 2007; Smothers et al, 2007; Woodward et al, 2004).

Although anesthetics and abused inhalants both increase GABA-mediated inhibition, important differences in the mechanism of action were seen. Anesthetics enhance inhibition by at least three distinct actions: 1) a presynaptic effect similar to that seen here (i.e. an increased frequency of IPSCs; (Banks et al, 1999; Nishikawa et al, 2000; Yamashita et al, 2001)), 2) a postsynaptic effect to prolong synaptic inhibition (Mody et al, 1991; Nicoll et al, 1975; Pittson et al, 2004), and 3) a postsynaptic effect to enhance tonic (extrasynaptic) GABA-gated currents (Bieda et al, 2004; Bieda et al, 2009; Caraiscos et al, 2004). Abused inhalants, in contrast, shared only the first (presynaptic) effect with inhaled anesthetics, and neither of the postsynaptic actions were evident. This enhanced presynaptic release of GABA has also been observed for ethanol (Weiner and Valenzuela, 2006). Prolongation of IPSCs, such as an increase in decay time or duration, was not produced. Neither was an increase in tonic, extrasynaptic currents evident in either current clamp or voltage clamp experiments. Perhaps it is only the presynaptic effects of the anesthetics and abused inhalants that are needed to disrupt learning and memory, while the postsynaptic actions contribute to sedation and loss of consciousness produced by the inhaled anesthetics. The relatively weak anesthetic properties of abused inhalants are consistent with the lack of postsynaptic actions seen in the present study, of course it might only reflect a more limited degree of enhanced inhibition too, regardless of pre- or postsynaptic sites of action.

In summary, abused inhalants increased synaptic inhibition in hippocampal neurons and this likely contributes to learning deficits associated with solvent abuse.

Acknowledgments

I thank Marin MacDonald and Patricia Turnquist for technical assistance with brain slice preparation and electrophysiology. I thank Eric Hu for technical assistance with HPLC analysis and Professor James R. Trudell for HPLC assistance, guidance and advice about the use and chemical properties of the abused inhalants throughout this project. Drs. J.J. Woodward, and R. A. Pearce and provided helpful comments and suggestions on portions of this work.

Research support was provided by NIH: NIDA R01DA017884 and I am thankful for the encouragement, and guidance provided by Drs. Charles Sharp and Jerry Frankenheim at NIDA.

Footnotes

Disclosure/Conflict of Interest: The author declares that no financial support or compensation has been received from any individual or corporate entity and there are no personal financial holdings that could be perceived as constituting a real or potential conflict of interest in this work.

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