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NEUROPHARMACOLOGY
Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, Pennsylvania
Received for publication
April 2, 2008
Accepted
July 2, 2008.
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Abstract |
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). Thus, MDMA has a powerful impact on sensory processing; yet, the neural processes underlying this effect are unknown. It is important to gain insights into such mechanisms if we are to create rational arguments regarding the abuse liability of MDMA. In addition, because clinical studies are underway to investigate the utility of MDMA in treating post-traumatic stress disorder (Doblin, 2002), new knowledge regarding the drug effects on sensory physiology is critical for establishing the drug's therapeutic potential.
MDMA is a releaser and reuptake inhibitor of serotonin (5-HT), norepinephrine (NE) (Gough et al., 1991; White et al., 1994; Wichems et al., 1995; Gudelsky and Nash, 1996), and, to a lesser extent, dopamine (Rothman et al., 2001). Thus, MDMA increases extracellular levels of these neurotransmitters within efferent targets of serotonergic and catecholaminergic pathways, including circuits along the primary sensory pathways.
In the present report, we chose the rat trigeminal somatosensory pathway as a model to investigate the short-term effects of MDMA on 5-HT and NE efflux and sensory-evoked discharge. This system transmits tactile information from the facial whiskers of rat to the contralateral somatosensory cortex via the brainstem trigeminal nucleus of V and the ventral posterior medial thalamus (VPM). All regions in this network receive serotonergic and noradrenergic projections and, thus, are vulnerable to the pharmacological effects of MDMA (Simpson et al., 1997; Kirifides et al., 2001).
It has been demonstrated in several species that 5-HT depresses thalamocortical transmission (Pape and McCormick, 1989; McCormick and Pape, 1990; Monckton and McCormick, 2002). Furthermore, in vivo iontophoretic 5-HT elevates spontaneous discharge and suppresses sensory-evoked discharge of rat somatosensory (Waterhouse et al., 1986) and visual cortical neurons (Waterhouse et al., 1990). Thus, both in vitro and in vivo studies demonstrate that 5-HT dampens signal transmission in the somatosensory pathway.
Investigations in rat somatosensory cortex indicate that iontophoretic NE (Waterhouse and Woodward, 1980; Waterhouse et al., 1980) or activation of the noradrenergic pathway from LC (Waterhouse et al., 1998) enhances neuronal responsiveness to excitatory and inhibitory synaptic stimuli. Furthermore, in vivo local application of NE decreases spontaneous discharge and sharpens receptive fields of VPM thalamic neurons (Castro-Alamancos et al., 2006). Thus, NE enhances signal transmission at multiple sites along the somatosensory pathway.
The goal of the present study was to determine the effects of short-term, recreationally relevant doses of MDMA (determined by plasma blood level analysis) on 5-HT and NE efflux in the VPM thalamus of conscious rats. Next, we used an anesthetized preparation to determine the electrophysiological effects of acute MDMA on transmission of sensory signals through the VPM thalamus. As predicted, the results indicate that low-dose MDMA increases serotonin efflux in the VPM thalamus. However, although human MDMA users report "enhanced" tactile awareness after consuming the drug, we found that, in the rat, MDMA suppresses tactile-evoked responses in the VPM thalamus. Although the net effects of MDMA on tactile sensory processing are likely to involve the interaction of multiple brain regions, both within and outside the trigeminal somatosensory network, this study clearly implicates the VPM thalamus as at least one sensory relay circuit involved in producing this effect.
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Materials and Methods |
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Plasma Level Experiments
After short-term injection of 3 mg/kg i.p. MDMA, animals were decapitated at one of seven different time points (10, 20, 40, 60, 80, 100, and 200 min). Four samples per time point were collected, using a total of 28 rats. Trunk blood (10 ml) was collected from each rat and placed into heparinized culture tubes and kept on ice. Analyses were performed by National Medical Services (Willow Grove, PA) according to the following protocol. Internal standard (propylamphetamine) was added to a 1.0-ml blood aliquot that was made basic with sodium hydroxide and extracted with a mixed solvent containing isopropanol, methylene chloride, and petroleum ether. Samples were back-extracted into dilute hydrochloric acid, which were then made basic, and samples were extracted into 200 µl of methylene chloride. The amines were then derivatized by adding butyric anhydride containing a trace of 4-dimethylaminopyridine as a catalyst and heating to dryness. Note that the butyric anhydride prevents evaporative losses of the analytes of interest. Extractions were reconstituted with toluene. Instrumental analysis was by gas chromatography and mass spectrometry. Quantitative analyses were performed by gas chromatography with a nitrogen selective detector. Extractions were injected on both an Agilent Technologies 6890 Gas Chromatograph equipped with a DB-17 capillary column and a nitrogen selective detector (Agilent Technologies, Santa Clara, CA). In addition, each sample was injected on a gas spectrograph with the same analytical column but with a mass selective detector (Agilent Technologies 6890 GC with a 5970 mass selective detector) that was run in a selective ion-monitoring mode. The purpose of the mass spectrometry analysis was to verify identity, whereas the nitrogen-selective detector was used for quantification.
Amounts of MDMA and MDA, its primary metabolite, were quantified on the gas spectrograph using calibrators from 10 to 500 ng/ml. The method demonstrated between run coefficients of variation of 6.8 and 5.5% at 20 and 300 ng/ml MDMA and 10.5 and 6.0% at 20 and 300 ng/ml MDA, respectively.
Dialysis Probe Construction and Calibration
Vertical concentric microdialysis probes were utilized (Abercrombie et al., 1988). In brief, a piece of fused silica (Polymicro Technologies, Phoenix, AZ) was inserted through PE10 tubing (Clay Adams, Parsippany, NJ), and a semi-permeable membrane of hollow cuprammonium rayon fibers with a 224-µm outer diameter and a molecular weight cutoff of 35,000 (C series; Terumo Corp., Somerset, NJ) was fixed over the fused silica into the PE10 tubing with epoxy. The open end of the dialysis fiber was sealed with a 0.5-mm epoxy plug, and a region was coated with epoxy, leaving an active area of 1 mm for exchange across the membrane. The in vitro recovery rate was determined by placing the probe in a beaker filled with artificial cerebrospinal fluid containing a known concentration of NE or 5-HT standard. The concentration of NE or 5-HT in the perfusate was compared with the amount in the bath. Probes that did not correspond to the typical range of recovery (11–21%) were identified and eliminated. Because the diffusion properties of neurochemicals in brain tissue are probably different from in vitro conditions, diasylate values reported were not corrected for the recovery of the probe.
Surgeries
Microdialysis Studies. Animals were allowed to acclimate to the animal facility for at least one week before surgery. The day before an experiment, rats were anesthetized with sodium pentobarbital (17.5 mg/kg i.p.) and choral hydrate (400 mg/kg i.p.), positioned in a stereotaxic apparatus with the skull flat, and allowed to breathe spontaneously. The body temperature of the animal was monitored using a rectal probe and maintained at 37°C by a heating pad. Anesthesia was maintained throughout the surgery, such that animals were not responsive to foot pinch, the blinking reflex was absent, and the breathing was slow and regular. Supplemental doses of sodium pentobarbital and choral hydrate were alternately administered as needed. Under anesthesia, microdialysis probes were surgically implanted into the VPM (anteroposterior, 3.5; mediolateral, 2.9; dorsoventral, 5.5) thalamus. The rats were then placed in cylindrical Plexiglas containers lined with bedding material, connected to a liquid swivel by a spring tether, and allowed to recover overnight.
Electrophysiology Studies. The surgical procedure used for electrode implant was the same as described above for the microdialysis probe implants, with the exception of the following modifications. Four stainless steel screws were fixed to the skull to serve as electrical grounds and anchors for affixing the electrodes to the skull. Microelectrode bundles (eight microwires per bundle) were implanted in the VPM thalamus. The microelectrode bundles were placed initially using stereotaxic coordinates (anteroposterior, 3.5; mediolateral, 2.9; dorsoventral, 5.5); however, the final position of the bundle was determined using electrophysiological verification that the site contained neurons that met established criteria for VPM thalamic neurons. Microelectrode bundles were permanently attached to the head using a connector and dental cement. The wound was sutured, and the animal was administered topical antibiotics to prevent infection. All animals were allowed to recover for 1 week before the beginning of an experiment.
Microdialysis Experiments
Microdialysis probes were continuously perfused with artificial cerebrospinal fluid (147 mM NaCl, 1.7 mM CaCl2, 0.9 mM MgCl2, and 4 mM KCl) at a flow rate of 1.5 µl/min. Baseline sample collection began the next morning. Rats received an injection of MDMA (either 1, 3, or 10 mg/kg i.p.) or vehicle (volume equivalent injections of 0.9% saline i.p.) after 2 h of baseline sampling. Animals in the 10 mg/kg group served as a comparison group to contrast the effects of lower doses of MDMA (1 and 3 mg/kg), as determined by the principals of "effects scaling" with a high dose that is based on the principals of "interspecies scaling". Diasylate samples were collected every 20 min for 7 h and frozen at -80°C for subsequent HPLC analysis.
HPLC
5-HT Detection and Quantification. Samples of 15 µl (each sample was divided in half to analyze both 5-HT and NE) were injected directly onto the HPLC column using an autosampler (model 542; ESA, Chelmsford, MA). The HPLC consisted of an ESA solvent delivery system and an ESA column (model MD-150; 150 x 2 mm). The mobile phase consisted of 32 mM NaH2PO4, 0.67 µM EDTA, 0.43 mM octyl sulfate, and 19% methanol adjusted to a pH of 5.6. The detection system consisted of an ESA Coulochem II electrochemical detector with two electrodes in series—the guard cell set at +150 mV and the compounds of interest quantified at the analytical cell (model 5041; ESA), which was set at +500 mV. Peak heights were measured and compared with the peak height of a 10-8 M standard calibrated daily. The detection limit, defined as the sample amount producing a peak height that was twice the height of background noise, was approximately 0.5 pg of 5-HT.
NE Detection and Quantification
Samples of 15-µl were injected directly onto the HPLC column using an autosampler (model 542; ESA). The HPLC consisted of an ESA solvent delivery system and an ESA column (model MD-150; 150 x 2 mm). The mobile phase consisted of 60 mM sodium phosphate buffer, pH 4.2, with 100 µm EDTA, 1.5 mM sodium octyl-sulfate, and 3.5% (v/v) methanol. The detection system consisted of an ESA Coulochem II electrochemical detector with two electrodes in series—the applied potential of the guard cell set at -150 mV and the compounds of interest quantified at the analytical cell (model 5041; ESA), which was set at +220 mV. Peak heights were measured and compared with the peak height of a 10-8 M standard calibrated daily. The detection limit, defined as the sample amount producing a peak height that was twice the height of background noise, was approximately 0.5 pg of NE.
Extracellular Unit Recordings. Microelectrode bundles were used to record the extracellular activity from ensembles of single neurons in the VPM thalamus of the halothane-anesthetized rat. Although conscious rats were used for microdialysis studies, we deliberately chose the anesthetized preparation to assess MDMA effect on sensory neuronal responsiveness to maximize our control of whisker stimulation parameters and minimize confounds arising from drug effects on behavioral state. In particular, MDMA at doses of 3 mg/kg and higher produces increased locomotion. Output from the electrodes was amplified and displayed on an oscilloscope and sent to a window discriminator for spike isolation. Neuronal activity was recorded from the eight microelectrodes simultaneously using the multichannel acquisition processor hardware and real-time acquisition system programs for unit timing in neuroscience (RASPUTIN) from Plexon Inc (Dallas, TX). Data acquisition parameters (amplification, filtering, and threshold of detection) were set independently for each channel. Multiple methods for real-time spike sorting online were employed, such as time-voltage window discrimination and template-based discrimination. Alternatively, we stored all of the waveforms that met detection criteria and sorted them later using an off-line sorting routine. Output from the window discriminator was sent to a digital computer to build real-time poststimulus time histograms (PSTHs) and raster records of neuronal activity.
Single-unit recordings in our data were determined off-line by analyzing waveforms, as well as interspike interval histograms. A recording was identified as arising from a single-unit if it met all three of the following criteria: i) the presence of reproducible waveforms; ii) interspike interval histograms with <5% of the interspike intervals shorter than 1.2 ms; and iii) waveform amplitude at least three times the average background noise levels.
We generated PSTHs in NeuroEXplorer (Plexon, Dallas, TX), using the whisker pad stimulation as the reference event and a bin width of 1 ms. Two hundred 2-s trials (20 min total) were used to build PSTHs. Each recording session consisted of one 20-min control period, one 20-min period immediately after a saline (0.09% i.p.) injection, and eight 20-min periods after MDMA (3 mg/kg i.p.) injection. The duration of the recordings and limits were determined from the results of our microdialysis studies. We chose recording periods based on the time course of 5-HT and NE elevation after MDMA administration.
The response to the whisker pad stimulation usually consisted of a short latency excitation (E1). Numerical data from PSTHs were exported to MatLab (The Mathworks, Inc., Natick, MA) for determination of the amplitude of the whisker-evoked response and the spontaneous firing rate (SFR) within 400 ms preceding whisker stimulation. The amplitude of E1 (PeakE1) was defined as the maximal firing rate during the first 25 ms following whisker stimulation minus the SFR. The onset, offset, latency, and duration of whisker-evoked responses were determined using a Gaussian 95% confidence interval. The onset (OnsetE1) and offset (OffsetE1) of the whisker-evoked response was defined as the time where the firing rate increased and decreased above and below the 95% confidence interval limit, respectively. The duration of E1 (DurE1) was determined by subtracting the OnsetE1 from OffsetE1. The latency of E1 (LatE1) was defined as the amount of time in milliseconds (beginning 2 ms after whisker stimulation to account for any potential artifact from the whisker stimulator) that it took to reach to the peak amplitude of the E1 response. The onset of E1 occurred between 3 and 9 ms following whisker stimulation, and the latency of E1 was between 4 and 20 ms. Figure 1 illustrates the response of a typical VPM thalamic unit response to mechanical stimulation of the primary whisker.
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Vibrissae Stimulation. Somatosensory afferent pathways were activated by mechanical displacement of single whiskers on the side of the face contralateral to recording sites in the VPM thalamus. Initially, a cell's receptive field was characterized by deflecting individual whiskers using a hand-held probe while listening to spike discharges on an audio monitor. After initial characterization of a cell's receptive field, a multiangular piezoelectric stimulator was used to produce uniform repetitive displacement of individual whiskers. Activation of the piezoelectric stimulator also triggered computer collection of spike train data. This device has the ability to reliably generate a range of small whisker deflections (1–1000 µm) in any direction (360°) or velocity (1–1440 deg/s) of motion as a linear function of input voltage. To determine whether MDMA administration differentially affects responsiveness of VPM thalamic neurons to whisker inputs of different intensity, we deflected the primary whisker using two randomly presented stimulus intensities, which we termed "medium" and "high". Medium and high intensity whisker stimulation deflected the whisker 
200 and 500 µm, respectively. Medium intensity produced a less robust response in VPM thalamic neurons compared with high intensity whisker stimulation (see Fig. 1B). It is important to note that whisker deflection was achieved with this method without disturbance to the surrounding whiskers. For detailed information on this device, see Armstrong-James and Fox (1987).
Behavioral Observations
All animals were videotaped throughout the microdialysis experiments. After each experiment, videotapes were scored for locomotor activity. At the onset of locomotion, the observer activated a stop-watch and then deactivated the timer once locomotion had ceased. Locomotor activity was operationally defined as forward movement of all four paws (see Ball et al., 2003). Measurements were taken during the first 10 min of every hour throughout the experiment. The data are presented here as the amount of time spent in locomotion per 10-min observation period (10 min, being the maximal amount of time the animal could be engaged in locomotion.) (Fig. 2).
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Electrophysiology Experiments. Computer-generated cumulative rasters and PSTHs were used to characterize stimulus-evoked responses and to quantitate evoked activity as both spikes/stimulus (excitations only) and percentage of baseline spontaneous firing rate. Rasters and histograms were generated preceding, during, and after intraperitoneal administration of MDMA. Equal numbers of stimuli were used to compute each histogram.
Changes in evoked and spontaneous activity were calculated for each cell by comparing discharges in identical portions of histograms computed during saline and MDMA interaction periods. Spontaneous and evoked discharge period rates were calculated as described above. These rates were computed from control and MDMA interaction histograms and compared, and the difference was expressed as percentage suppression or potentiation from control response. Spikes per stimulus comparisons were made between histograms using a similar method. Changes in stimulus-bound activity between saline and MDMA postinjection periods were statistically compared using one-way ANOVA. Such analytical procedures have been used previously in our laboratory to characterize 5-HT and NE actions on rodent somatosensory and visual cortical neurons.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). Given that the 1 and 3 mg/kg doses used in the current study were significantly lower than what are used typically in this field, we measured the effect of each dose on locomotion to determine whether the low doses used in our studies were relevant in terms of this outcome and to establish a baseline for future comparisons of the locomotor effects of short-term versus long-term MDMA administration. As illustrated in Fig. 2, short-term MDMA administration (1, 3, and 10 mg/kg i.p.) produced significant dose-dependent increases in locomotor activity [F(4,5) = 6.172; p < 0.0021], [F(4,5) = 16.48; p < 0.0001 and F(4,5) = 12.35; p < 0.0001; one-way ANOVA], respectively. Locomotor activity peaked 20 min after injection of either 1 or 3 mg/kg MDMA and returned to baseline levels within 60 min (Dunnett's test; p < 0.01). However, injection of 10 mg/kg produced the greatest increase in locomotion, an effect that peaked 60 min after injection and returned to baseline levels within 150 min (Dunnett's test; p < 0.05).
Effects of Short-Term MDMA Administration on Plasma Levels of MDMA and MDA. Short-term administration of 3 mg/kg i.p. MDMA increased plasma levels of MDMA and its major metabolite MDA (see Fig. 3). Peak plasma MDMA levels of 322.5 ± 19.738 ng/ml were reached within 20 min after injection. Peak plasma MDA levels of 55.5 ± 3.279 ng/ml were reached within 80 min after injection. Plasma levels of MDMA and MDA remained elevated for 100 min and returned to near zero levels by 200 min after injection. Plasma levels of MDMA and MDA are expressed as the mean nanogram/milliliter ± S.E.M for each time point that samples were collected (10, 20, 40, 60, 80, 100, and 200 min). It is important to note that intraperitoneal injection of 3 mg/kg MDMA results in peak plasma levels of MDMA (322.5 ± 19.738) that are similar to those seen in humans (223 + 48 ng/ml human) after an oral dose of 100 mg (Pizarro et al., 2002).
Effect of Short-Term MDMA Administration on 5-HT Efflux in VPM Thalamus. In the current study, short-term systemic administration of MDMA at 1, 3, or 10 mg/kg i.p. led to a rapid dose-dependent increase in 5-HT efflux in the VPM thalamus (see Fig. 4B). An overall ANOVA revealed a significant effect of treatment [Fdose(3,20) = 25.72; p < 0.0001], time [Ftime(4,80) = 16.25; p < 0.0001], and a significant time by treatment interaction [Fint(12,80) = 6.668; p < 0.0001]. All three doses of MDMA caused significant increases in 5-HT efflux [F(6,4) = 2.814; p = 0.0478; one-way ANOVA], [F(5,4) = 10.84; p < 0.0001; one-way ANOVA], and [F(6,7) = 16.10; p < 0.0001; one-way ANOVA], respectively. MDMA (1 mg/kg) increased the concentration of extracellular 5-HT in diasylates from 0.789 ± 0.191 to 2.356 ± 2.24 pg/15 µl. The 3 and 10 mg/kg doses increased extracellular 5-HT from 0.922 ± 0.451 to 3.145 ± 0.696 pg/15 µl and from 1.31 ± 0.345 to 9.709 ± 4.18 pg/15 µl, respectively. These effects were evident within 20 min of drug administration. 5-HT levels peaked 40 min after MDMA administration at all of the doses tested, with the exception of the 1 mg/kg dose in which 5-HT levels peaked 20 min after administration. Serotonin levels returned to baseline within 40 min after injection of 1 mg/kg (Dunnett's; p < 0.05), within 80 min after injection of 3 mg/kg (Dunnett's; p < 0.05), and within 100 min after injection of 10 mg/kg MDMA (Dunnett's; p < 0.05). Extracellular levels of 5-HT did not change significantly after saline injection (0.566 ± 0.058 to 0.908 ± 0.221 pg/15 µl) (see Fig. 4B).
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MDMA Effects on VPM Thalamic Unit Responsiveness (Peak E1) to Whisker Deflection. MDMA administration (3 mg/kg) led to decreased responsiveness of VPM thalamic neurons to both medium-and high-intensity whisker stimulation: [F(54,10) = 11.25; p < 0.0001; one-way ANOVA]; [F(65, 10) = 11.65; p < 0.0001; one-way ANOVA] and [F(66,12) = 4.783; p < 0.0001; one-way ANOVA]; [F(66,12) = 3.844; p < 0.0001; one-way ANOVA], respectively (see Fig. 5, A and B). There was no evidence of a differential effect of MDMA on responses to medium versus high stimulation intensities (Fig. 5B). It is interesting that animals treated short-term display a significant overall increase in responsiveness to medium- (n = 8; 47 cells; 55 responses) and high-intensity (n = 8; 49 cells; 66 responses) whisker stimulation at 160 to 180 min after drug administration (Dunnett's; p < 0.05). Peak E1 was defined as the maximal firing rate that occurs from 2 to 25 ms after stimulation onset minus the spontaneous firing rate.
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MDMA Effects on the SFR of Neurons in the VPM Thalamus. In addition to the direct suppression of evoked discharge, short-term administration of MDMA increased the spontaneous firing rate of individual neurons during medium and high intensity whisker stimulation, followed by a return to control levels: [F(46,10) = 4.005; p < 0.0001; one-way ANOVA]; [F(48,10) = 4.177; p < 0.0001; one-way ANOVA] and [F(37,12) = 2.088; p < 0.01; one-way ANOVA]; [F(44,12) = 2.43; p < 0.004; one-way ANOVA], respectively (see Fig. 5, A and C). After short-term administration recovery to control levels of spontaneous firing were observed by 140 to 160 min postdrug, respectively. SFR was defined as the average firing rate of 400 ms before stimulation onset. As a result of MDMA suppression of evoked discharge and elevation of SFR, the signal-to-noise ratio was markedly reduced. Scatter plots were generated to illustrate percentage change in Peak E1 and SFR for all cells studied. Percentage change in Peak E1 and SFR were calculated using the following formulas: percentage change Peak E1 = control Peak E1 - Peak E1 (during peak drug effect) x 100, and percentage change SFR = control SFR - Peak E1 (during peak drug effect) x 100, respectively. As indicated in Fig. 6, the majority of points lie to the left of the 45° line, indicating instances where MDMA suppressed evoked firing and elevated spontaneous discharge.
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Effect of MDMA on Onset Latency of the Whisker-Evoked Response. Short-term MDMA administration had no significant effect on the onset latency of whisker-evoked responses (see Fig. 5E). Onset latency was defined as the time in milliseconds from stimulation onset to when the firing rate first crosses the 95% Gaussian confidence interval - 2 ms to control for stimulation-related artifact.
Effects of MDMA on the Offset Latency of the Whisker-Evoked Response. MDMA administration led to an initial decrease in the offset latency during medium- and high-intensity whisker stimulation [F(51,10) = 5.988; p < 0.0001; one-way ANOVA]; [F(56,10) = 3.71; p < 0.0001; one-way ANOVA], respectively (see Fig. 5F). Offset latency was defined as the time in milliseconds from stimulation onset to when the firing rate first falls below the 95% Gaussian confidence interval (after the peak response) - 2msto control for stimulation-related artifact.
Effects of MDMA on the Duration of the Whisker-Evoked Response. MDMA administration decreased the duration of the whisker-evoked response during medium- and high-intensity whisker stimulation [F(51,10) = 3.684; p < 0.0001; one-way ANOVA]; [F(56,10) = 3.005; p < 0.0009; one-way ANOVA]; and [F(66,12) = 5.439; p < 0.0001; one-way ANOVA]; [F(66,12) = 10.44; p < 0.0001; one-way ANOVA], respectively (see Fig. 4D). In order for a decrease in duration of the whisker-evoked response to occur, either the onset latency alone must increase, the offset latency alone must decrease, or the onset latency must increase combined with a decrease in the offset latency. It appears that short-term MDMA administration decreases the duration of the whisker-evoked response by causing a decrease in the offset latency. Duration was defined as offset latency - onset latency in milliseconds.
Effects of MDMA on Thalamic Field Potential Activity. Power spectral density (PSD) analysis was performed to determine the effects of short-term MDMA administration on thalamic field potential activity. Short-term MDMA administration led to decreases in the mean PSD values (see Fig. 7, A and B). In other words, MDMA administration caused a reduction in high-frequency field potential activity compared with control periods. This suggests that administration of MDMA to an anesthetized animal causes the animal to transition to a "lighter" level of anesthesia. Time had no effect on the mean PSD values in the thalamus.
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Effects of Anesthesia Level on Responsiveness of VPM Thalamic Neurons to Whisker Stimulation. To determine whether depth of anesthesia affected neuronal responsiveness to mechanical stimulation of the primary whisker, animals (n = 2) were anesthetized with halothane and spike train data were collected for 3 h, 20 min. The principle whisker was stimulated continuously throughout the recording session. We found that, as the concentration of halothane was increased, there was a corresponding decrease in both spontaneous firing rate and responsiveness to whisker stimulation (see Fig. 8, C and D). In contrast, at lower concentrations of halothane, spontaneous firing and responsiveness to whisker stimulation increased. By contrast, MDMA produced a decrease in VPM thalamic unit responsiveness to whisker stimulation combined with an increase in spontaneous firing rate. Thus, MDMA-induced transition to a lighter plane of anesthesia (see above) cannot account for MDMA-associated decreases in sensory-driven discharges.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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) have shown that MDMA displays similar potency at the norepinephrine transporter and serotonin transporter sites in vitro. However, it is unknown whether there is equal expression of 5-HT and norepinephrine transporter proteins in the VPM thalamus. An imbalance in transporter expression for these two transmitters could help explain our result. In addition, we found that short-term (3 mg/kg) MDMA administration led to decreased responsiveness of individual VPM thalamic neurons to whisker stimulation. This effect resulted from an increased level of spontaneous firing in conjunction with a reduction in stimulus evoked discharge. A significant increase or "rebound" in responsiveness to whisker stimulation was observed 160 to 180 min after drug administration. One possible explanation for this effect could be that 5-HT becomes depleted over time. If the role of 5-HT in the VPM thalamus is to suppress sensory responsiveness, reduced 5-HT levels could lead to less sensory-evoked inhibition. Although, data from the microdialysis studies did not show a decrease below baseline 5-HT levels at this time point, it is possible that the technique is not sensitive enough to measure slight drops in neurotransmitter levels that are nonetheless capable of producing electrophysiological effects.
An important consideration in evaluating this work is the fact that microdialysis studies were carried out in waking animals, whereas electrophysiology experiments were completed under anesthetized conditions. An advantage of performing in vivo microdialysis in the awake animal is that neurochemical measurements are made under circumstances that closely approximate normal physiology. However, we chose to conduct multiunit recording studies in the anesthetized preparation to characterize the effects of MDMA under conditions that allow for maximal control over stimulus parameters and a fixed level of arousal. Until the effects of MDMA on sensory-evoked responsiveness are described in this manner, it is impossible to evaluate the effect of the drug under more dynamic conditions where the analysis must contend with locomotion and behavioral arousal. For example, MDMA administration to the waking animal causes dose-dependent increases in locomotion (see Fig. 2), a behavioral state that has effects on sensory signal processing (Fanselow and Nicolelis, 1999). Anesthesia eliminates confounds introduced by locomotion while still providing for systemic drug delivery.
Because of its psychostimulant properties, MDMA also has the ability to increase arousal, an effect that by itself could affect sensory neuronal responsiveness. However, when the anesthesia level was alternated between "low" and "high" levels while monitoring spike train activity, we found that spontaneous and stimulus-evoked discharge increased as depth of anesthesia was reduced. Thus, arousal from anesthesia produced a pattern of discharge, unlike that observed after MDMA administration. Likewise, increased locomotion alters sensory neuronal responsiveness (Fanselow and Nicolelis, 1999), but results in our anesthetized preparation demonstrate that MDMA alters sensory signal processing in the absence of locomotion.
Kalén et al. (1988) reported baseline levels of hippocampal NE to be lower in the halothane-anesthetized rat, which could potentially make correlation between NE efflux in the waking animal and electrophysiological effects in the anesthetized preparation difficult. However, we demonstrated that the dose used in the electrophysiological recordings (3 mg/kg) has no significant effect on NE efflux in the awake animal. In addition, Silva et al. (2008) recently demonstrated that halothane induces 5-HT release from rat brain cortical slices. However, because MDMA also causes 5-HT release, it is likely that the potential effect of halothane on our results is most likely a matter of degree rather than direction.
The current findings are consistent with the results from in vitro studies that suggest that 5-HT suppresses thalamocortical transmission (Pape and McCormick, 1989; McCormick and Pape, 1990; Monckton and McCormick, 2002). Our results are also consistent with the findings that iontophoretic 5-HT elevates spontaneous ring and suppresses stimulusevoked discharges of rat somatosensory (Waterhouse et al., 1986) and visual cortical neurons (Waterhouse et al., 1990).
Support for Effects Scaling. Until very recently, the doses of MDMA used in most animal studies have been based upon the model of "interspecies scaling" (Ricaurte et al., 2000). This model is based upon various underlying biological similarities among mammals and states that smaller animals require higher dosages of drug to achieve equivalent effects (see Ricaurte et al., 2000 for a detailed explanation).
Based on these assumptions, some have argued that high doses used in laboratory animal studies are equivalent to what a human consumes recreationally. As a result, MDMA doses as high as 100 mg/kg have been administered to rats (Jensen et al., 1993). The limitations of this dosing method have been reviewed (de la Torre and Farre, 2004), and an alternative approach, "effects scaling," has been suggested (Baumann et al., 2007). This approach involves selection and study of doses that produce similar behavioral and pharmacological effects in rats versus humans. Recent data indicate that humans self-administer on average 1.5 mg/kg MDMA in one night (Cole and Sumnall, 2003), a level of consumption that is significantly lower than the doses typically administered to experimental animals. Our microdialysis data are consistent with Kankaanpää et al. (1998), who found that a short-term subcutaneous injection of 1 and 3 mg/kg MDMA led to a 300 and 350% increase in 5-HT efflux, respectively, in rat nucleus accumbens. Thus, we consider our 1 and 3 mg/kg doses to be within the relevant range for eliciting behavioral and neurochemical effects. This contention is further supported by the observation that doses in this range elicit hyperactivity (see Fig. 2), an effect that is evident in human users. In addition, data from our plasma level measurements show that intraperitoneal injection of 3 mg/kg MDMA results in peak plasma levels of MDMA (322 ± 20 ng/m) that are similar to those seen in humans (223 ± 48 ng/ml human) after an oral dose of 100 mg (Pizarro et al., 2002). Thus, current and previous findings provide a compelling argument for the implementation of effects scaling for MDMA dose selection rather than interspecies scaling in animal studies aimed at elucidating the behavioral, neurochemical, and physiological outcomes of drug administration.
Functional Consequences of MDMA Actions on Sensory Neuronal Responsiveness. The observation of consistent decreases in the magnitude and duration of the sensory-evoked response in thalamic neurons accompanied by increases in spontaneous firing suggests an alteration in the transmission of sensory information to the cortex after short-term MDMA administration. A combination of these effects may produce a net delay or distortion of sensory signal transfer from the periphery to the cortex and, thus, may begin to provide an electrophysiological explanation for the ability of MDMA to alter somatosensory experiences.
Significance of the Current Findings. This study is the first to assess the impact of short-term MDMA administration on transmitter levels of 5-HT and NE in the VPM thalamus, an area important for processing tactile sensory information. Although previous studies have shown that MDMA blocks reuptake of 5-HT and NE in various brain regions, no studies have quantified the effects of MDMA on monoamine transmission within the thalamus. The results of the present studies provide an explicit description of the time course and the degree to which NE and 5-HT efflux in thalamus are affected by short-term low-dose MDMA administration. These findings are consistent with other work where the effects of MDMA on neurotransmitter efflux have been shown to be time- and dose-dependent and can also vary across brain regions (Gartside et al., 1996).
Taken together, these data indicate that short-term low-dose (1 and 3 mg/kg) MDMA selectively increases 5-HT efflux in sensory thalamus. In contrast, an injection of high-dose MDMA (10 mg/kg) elicits increases in both 5-HT and NE in the VPM thalamus. Because low-dose (3 mg/kg) MDMA administration produces peak plasma levels of drug that are similar to those seen in a human after a recreational dose of MDMA, we conclude that the 5-HT efflux is likely responsible for mediating the recreationally desirable effects of Ecstasy in humans. This conclusion is supported by the studies of Liechti et al. (2000) and, more recently, Tancer and Johanson (2007), who have shown that certain subjective effects produced by MDMA can be antagonized with selective serotonin reuptake inhibitor pretreatment. It is unclear how high-dose MDMA-mediated release of both 5-HT and NE affect physiological response properties of VPM thalamic neurons or other central nervous system functions.
The current study is also the first to examine the effects of short-term MDMA administration on sensory-evoked responsiveness in any brain region of intact animals. Our data are inconsistent with human reports of "enhanced" tactile sensory awareness after MDMA self-administration. These findings suggest that more controlled physiological studies are needed to determine whether recreational doses of MDMA do indeed increase tactile sensitivity in humans.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: MDMA, 3,4-methylenedioxymethamphetamine; MDA, methylenedioxyamphetamine; NE, norepinephrine; PSTH, poststimulus time histogram; 5-HT, 5-hydroxytryptamine, serotonin; SFR, spontaneous firing rate; VPM, ventral posterior medial; ANOVA, analysis of variance; HPLC, high-performance liquid chromatography; PSD, power spectral density.
Address correspondence to: Dr. Melanie A. Starr, Department of Neurobiology and Anatomy, Drexel University College of Medicine, 2900 Queen Lane, Philadelphia, PA 19129. E-mail: mla29{at}drexel.edu
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Abercrombie ED, Keller RW Jr, and Zigmond MJ (1988) Characterization of hippocampal norepinephrine release as measured by microdialysis perfusion: pharmacological and behavioral studies. Neuroscience 27: 897-904.[CrossRef][Medline]
Armstrong-James M and Fox K (1987) Spatiotemporal convergence and divergence in the rat SI "barrel" cortex. J Comp Neurol 263: 265-281.[CrossRef][Medline]
Ball KT, Budreau D, and Rebec GV (2003) Acute effects of 3,4-methylenedioxymethamphetamine on striatal single unit activity and behavior in freely moving rats: differential involvement of dopamine D1 and D2 receptors. Brain Res 994: 203-215.[CrossRef][Medline]
Bankson MG and Cunningham KA (2001) 3,4-Methylenedioxymethamphetamine (MDMA) as a unique model of serotonin receptor function and serotonin-dopamine interactions. J Pharmacol Exp Ther 297: 846-852.
Baumann MH, Wang X, and Rothman RB (2007) 3,4-Methylenedioxymethamphetamine (MDMA) neurotoxicity in rats: a reappraisal of the past and present findings. Psychopharmacologia 189: 407-424.[CrossRef][Medline]
Cole JC and Sumnall HR (2003) The pre-clinical behavioural pharmacology of 3,4-methylenedioxymethamphetamine (MDMA). Neurosci Biobehav Rev 27: 199-217.[CrossRef][Medline]
de la Torre R and Farré M (2004) Neurotoxicity of MDMA (Ecstasy): the limitations of scaling from animals to humans. Trends Pharmacol Sci 25: 505-508.[CrossRef][Medline]
Doblin R (2002) A clinical plan for MDMA (Ecstasy) in the treatment of posttraumatic stress disorder (PTSD): partnering with the FDA. J Psychoactive Drugs 34: 185-194.[Medline]
Fanselow EE and Nicolelis MA (1999) Behavioral modulation of tactile responses in the rat somatosensory system. J Neurosci 19: 7603-7616.
Gartside SE, McQuade R, and Sharp T (1996) Effects of repeated administration of 3,4-methylenedioxymethamphetamine on 5-hydroxytryptamine neuronal activity and release in the rat brain in vivo. J Pharmacol Exp Ther 279: 277-283.
Gough B, Ali SF, Slikker W Jr, and Holson RR (1991) Acute effects of 3,4-methylenedioxymethamphetamine (MDMA) on monoamines in rat caudate. Pharmacol Biochem Behav 39: 619-623.[CrossRef][Medline]
Gudelsky GA and Nash JF (1996) Carrier-mediated release of serotonin by 3,4-methylenedioxymethamphetamine: implications for serotonin-dopamine interactions. J Neurochem 66: 243-249.[Medline]
Hirata A and Castro-Alamanos MA (2006) Noradrenergic activation amplifies bottom-up and top-down signal-to-noise ratios in sensory thalamus. J Neurosci 26: 4426-4436.
Jensen KF, Olin J, Haykal-Coates N, O'Callaghan J, Miller DB, and de Olmos JS (1993) Mapping toxicant-induced nervous system damage with cupric silver stain: a quantitative analysis of neural degeneration induced by 3,4-methylenedioxymethamphetamine. NIDA Res Monogr 136: 133-149.[Medline]
Kalén P, Kokaia M, Lindvall O, and Björklund A (1988) Basic characteristics of noradrenaline release in the hippocampus of intact and 6-hydroxydopamine-lesioned rats as studied by in vivo microdialysis. Brain Res 474: 374-379.[CrossRef][Medline]
Kankaanpää A, Meririnne E, Lillsunde P, and Seppälä T (1998) The acute effects of amphetamine derivatives on extracellular serotonin and dopamine levels in rat nucleus accumbens. Pharmacol Biochem Behav 59: 1003-1009.[CrossRef][Medline]
Kirifides ML, Simpson KL, Lin RC, and Waterhouse BD (2001) Topographic organization and neurochemical identity of dorsal raphe neurons that project to the trigeminal somatosensory pathway in the rat. J Comp Neurol 435: 325-340.[CrossRef][Medline]
Liechti ME, Baumann C, Gamma A, and Vollenweider FX (2000) Acute psychological effects of 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") are attenuated by the serotonin uptake inhibitor citalopram. Neuropsychopharmacology 22: 513-521.[CrossRef][Medline]
McCormick DA and Pape HC (1990) Noradrenergic and serotonergic modulation of a hyperpolarization-activated cation current in thalamic relay neurons. J Physiol 431: 319-342.
Monckton JE and McCormick DA (2002) Neuromodulatory role of serotonin in the ferret thalamus. J Neurophysiol 87: 2124-2136.
Pape HC and McCormick DA (1989) Noradrenalin and serotonin selectively modulate thalamic burst firing by enhancing a hyperpolarization-activated cation current. Nature 340: 715-718.[CrossRef][Medline]
Pizarro N, Ortuno J, Farre M, Hernandez-Lopez C, Pujadas M, Llebaria A, Joglar J, Roset PN, Mas M, Segura J, et al. (2002) Determination of MDMA and its metabolites in blood and urine by gas chromatography-mass spectrometry and analysis of enantiomers by capillary electrophoresis. J Anal Toxicol 26: 157-165.[Medline]
Ricaurte GA, Yuan J, and McCann UD (2000) (+/-)3,4-Methylenedioxymethamphetamine ("Ecstasy")-induced serotonin neurotoxicity: studies on animals. Neuropsychobiology 42: 5-10.[CrossRef][Medline]
Rothman RB, Baumann MH, Dersch CM, Romero DV, Rice KC, Carroll FI, and Partilla JS (2001) Amphetamine-type central nervous system stimulants release norepinephrine more potently than they release dopamine and serotonin. Synapse 39: 32-41.[CrossRef][Medline]
Saunders N and Doblin R (1996) Personal accounts. In: Ecstasy: Dance, Trance, and Transformations, p 207, Quick American Archive, Oakland, CA.
Silva JH, Gomez MV, Silva JH, Guatimosim C, and Gomez RS (2008) Halothane induces vesicular and carrier-mediated release of [3H]serotonin from rat brain cortical slices. Neurochem Int 52: 1240-1246.[CrossRef][Medline]
Simpson KL, Altman DW, Wang L, Kirifides ML, Lin RC, and Waterhouse BD (1997) Lateralization and functional organization of the locus coeruleus projection to the trigeminal somatosensory pathway in rat. J Comp Neurol 385: 135-147.[CrossRef][Medline]
Tancer M and Johanson CE (2007) The effects of fluoxetine on the subjective and physiological effects of 3,4-methylenedioxymethamphetamine (MDMA) in humans. Psychopharmacology (Berl) 189: 565-573.[CrossRef][Medline]
Waterhouse BD, Azizi SA, Burne RA, and Woodward DJ (1990) Modulation of area 17 simple and complex cell responses to moving visual stimuli during norepinephrine and serotonin microiontophoresis. Brain Res 514: 276-292.[CrossRef][Medline]
Waterhouse BD, Moises HC, and Woodward DJ (1986) Interaction of serotonin with somatosensory cortical neuronal responses to afferent synaptic inputs and putative neurotransmitters. Brain Res Bull 17: 507-518.[CrossRef][Medline]
Waterhouse BD, Moises HC, and Woodward DJ (1980) Noradrenergic modulation of somatosensory cortical neuronal responses to iontophoretically applied putative neurotransmitters. Exp Neurol 69: 30-49.[CrossRef][Medline]
Waterhouse BD, Moises HC, and Woodward DJ (1998) Phasic activation of the locus coeruleus enhances responses of primary sensory cortical neurons to peripheral receptive field stimulation. Brain Res 790: 33-44.[CrossRef][Medline]
Waterhouse BD and Woodward DJ (1980) Interactions of norepinephrine with cerebro-cortical activity evoked by stimulation of somatosensory afferent pathways in the rat. Exp Neurol 67: 11-34.[CrossRef][Medline]
White SR, Duffy P, and Kalivas PW (1994) Methylenedioxymethamphetamine depresses glutamate-evoked neuronal firing and increases extracellular levels of dopamine and serotonin in the nucleus accumbens in vivo. Neuroscience 62: 41-50.[CrossRef][Medline]
Wichems CH, Hollingsworth CK, and Bennett BA (1995) Release of serotonin induced by 3,4-methylenedioxymethamphetamine (MDMA) and other substituted amphetamines in cultured fetal raphe neurons: further evidence for calcium-independent mechanisms of release. Brain Res 695: 10-18.[CrossRef][Medline]
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