* Neurotoxicology Division, MD B105-05, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
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ABSTRACT |
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Key Words: carbon disulfide (CS2); flash-evoked potential (FEP); norepinephrine (NE).
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INTRODUCTION |
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Two FEP peaks that may be differentially altered by solvents are a negative peak occurring at approximately 3036 ms (N36), and a negative peak at about 166 ms latency (N166) following flash stimulation. Dichloromethane reduces the amplitude of FEP peak N36 (0.25 h after treatment), 1,3-dichloropropane reduces the amplitudes of both N36 (0.5 and 1 h) and N166 (at 0.5, 1, and 4 h after treatment), and 1,2-dichlorobenzene only reduces the amplitude of N166 (at 0.5, 1, 2, and 4 h after treatment) (Herr and Boyes, 1997). Treatment with toluene (Dyer et al., 1988
; Rebert et al., 1989b
,c
) or p-xylene (Dyer et al., 1988
) has also been shown to decrease the latter portions of FEPs. Interestingly, the decreases in peak N166 amplitude produced by these solvents were observed in the absence of decrements in peak N36 amplitude. Little is known regarding the possible neurochemical basis for these changes in FEPs. Carbon disulfide (CS2) is another solvent which is known to alter the amplitude of both peak N36 and N166, but with a distinctly different time-course after exposure (Herr et al., 1992
). Additionally, treatment with CS2 is known to decrease levels of brain norepinephrine (NE), supposedly mediated via inhibition of dopamine ß-hydroxylase (Bus, 1985
; McKenna and DiStefano, 1977
), suggesting one possible neurochemical mechanism for the alterations observed in FEPs.
The function of the noradrenergic system has been associated with modulating a subjects level of arousal and cortical responsiveness to external stimuli (Aston-Jones et al., 1984, 1999
; Foote et al., 1983
; Grant et al., 1988
; McCormick, 1989
). The NE-containing neurons of the locus coeruleus (LC) discharge slowly during slow-wave sleep, not at all during REM sleep, and rapidly during times of increased vigilance such as during orientation toward an unexpected or preferred stimulus (Foote et al., 1983
). Neuroanatomical tracing studies have shown fibers from the LC infiltrating the visual cortex (Aston-Jones et al., 1984
; Foote et al., 1983
; Parnavelas and Papadopoulos, 1989
). Both excitatory and inhibitory responses of cortical cells to NE have been reported (Devilbiss and Waterhouse, 2000
; Waterhouse et al., 1990
, 2000
). One interpretation of such data is that NE may enhance the relative responsiveness to external stimuli by producing an increased signal-to-noise ratio (Aston-Jones et al., 1984
; Foote et al., 1983
; Hasselmo et al., 1997
; Waterhouse et al., 1990
), and may also be involved in synaptic plasticity of the visual cortex (Kirkwood et al., 1999
). The increased signal-to-noise ratio is achieved via inhibition of spontaneous discharges and a resulting net increase above background of the visually evoked response (Aston-Jones and Bloom, 1981
; Aston-Jones et al., 1999
; Waterhouse et al., 1990
). Changes in the cortical processing of the "salience" of the flash stimulus could be involved in the increases in FEP peak N166 amplitude (and/or the photic after-discharge (PhAD)) observed with repeated testing (Dyer, 1989
; Herr et al., 1991
, 1994
).
Arousal levels have been implicated in modulating the amplitude of the late portions of FEPs. While stimulus intensity is very important in influencing the amplitude of peak N36 (Herr et al., 1991, 1994
), additional factors such as the subjects state of arousal are believed to modulate the amplitude of peak N166 and the PhAD. For these portions of the FEP, maximum amplitudes are obtained when the subject is believed to be in a state of relaxed awareness, neither inattentive nor anxious (Bigler, 1977
; Bigler and Fleming, 1976a
; Bigler et al., 1976
; Joseph et al., 1981
; Shearer and Creel, 1978
). Behavioral and/or parametric test manipulations have been used to alter arousal levels and modulate the amplitude of the late FEP peaks (Bigler et al., 1976
; Dyer, 1989
; Schaefer et al., 1974
; Standage and Fleming, 1978
). Thus, the late portions of FEPs appear to be modified by the subjects arousal level, which in turn may be influenced by the function of the NE system.
In this study, we have utilized CS2 as a means to examine the relationship between altered brain levels of NE and the amplitude of FEP peaks N36 and N166. This study replicated our previously reported differential effects of CS2 on peak N36 and N166 amplitudes (Herr et al., 1992). Additionally, we examined the dose- and time-course of CS2-induced depression of NE levels (as well as other catecholamine and indolamine neurotransmitters) in four brain regions. The purpose of the experiment was to increase our knowledge of the role of cortical NE in modulating FEP peaks, especially peak N166, which is believed to be influenced by psychological constructs such as arousal level, which may be modulated by NE.
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MATERIALS AND METHODS |
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Flash-evoked potential testing and CS2 treatment.
Animals were randomly assigned to treatment groups, which were counterbalanced across testing chambers. Flash-evoked potential testing took place in sound and light attenuating Faraday cages (Hamm et al., 2000). Unanesthetized rats were restrained in cones made of plastic film (decapicones; Braintree Scientific, Inc., Braintree, MA), their head and pinnae exposed, placed on a platform inside the test chambers, and allowed to acclimate for at least 1 min before stimulation. The rats eyes were located approximately 37 cm below a photic stimulator (Model PS22, Grass Instrument Division, Astro-Med, West Warwick, RI) that was encased in a foam-lined and electrically shielded box. Acoustic white noise (80 dB SPL; calibrated at the animals ear level) was presented using a 10-cm cone speaker located about 57 cm above the subjects ear level (Hamm et al., 2000
). The white noise was present during testing to mask auditory potentials produced by the strobe discharge (Herr et al., 1996
; Shaw, 1992
). An overhead DC-powered light bulb produced an ambient illumination of about 20.3 lux. A 10 µs flash stimulus was delivered at 203.1 lux-s (strobe setting 16) for 75 trials (collection time: 50 ms prestimulus baseline + 500 ms data). The EEG signals were amplified (10,000x; filter bandpass: 0.11000 Hz; Model 12A5 Neurodata Acquisition System, Grass Instrument Division, Astro-Med, Inc.), digitized at 4000 Hz, and averaged using custom written software (Hamm et al., 2000
). Prior to any testing, the amplitude and latency response factors of the amplifiers and computer were calibrated using sine waves of 178 µV RMS at 3, 100, and 300 kHz. Flash intensity and ambient light were calibrated using a photometer (Model 450 with Model 5503 Pulse Integration Module, Gamma Scientific, Inc., San Diego, CA). Masking noise, auditory calibration was performed at the rats ear level in the test chamber using a 1.27 cm microphone (Model 4166, Brüel & Kjær) and a measuring amplifier (Model 2636, Brüel & Kjær).
Animals had FEPs collected for two consecutive days prior to dosing with CS2, in order to develop FEP peak N166 (Dyer, 1989; Herr et al., 1991
, 1994
). Colonic temperature was monitored during testing using a rectal probe (Model RET-1; Thermalert Thermometer Model TH-8, Physitemp Instruments, Clifton, NJ) inserted approximately 8 cm and was recorded at the conclusion of FEP testing. On the third day, FEPs were recorded as a pretreatment baseline. One group of animals (10 rats/dose) was immediately sacrificed following pretreatment baseline FEP testing, to serve as non-injected controls (NIC) for the biogenic amine assays. The remaining animals were injected with 0 (corn oil vehicle, Mazola), 100, 200, or 400 mg/kg CS2 (ip, 2 ml/kg dosing volume). All treatment groups used 10 animals/dose/time point. The doses of CS2 were based on previous work, which showed a different time-course for CS2-induced depression of FEP peak N36 and N166 amplitudes (Herr et al., 1992
). The animals were then retested 1, 4, 8, or 24 h posttreatment. Separate groups of animals were used at each time point.
Tissue collection, biogenic amine assay, and protein assay.
Immediately following posttreatment FEP testing, on the third day, the animals were sacrificed by decapitation, and their brains removed and dissected following a modification of Glowinski and Iversen (1966). One hemisphere of cortical tissue (posterior to the optic chiasm and with the hippocampus removed), cerebellum, brain stem, and striatum were collected, weighed, immediately frozen on dry ice, and stored at -80°C until analysis.
The brain regions used for high performance liquid chromatography (HPLC) analysis were chosen based on their NE and DA innervation, the generator sites for FEPs, and predicted differences in neurochemical alterations produced by CS2. The visual cortex contains the neurons responsible for the generation of FEPs (Brancak et al., 1990
; Kenan-Vaknin and Teyler, 1994
; Kraut et al., 1985
) and has NE and 5-HT terminal innervation (Baumgarten and Lachenmayer, 1985
; Lindvall and Björklund, 1983
). The brain stem contains the cell bodies of NE and 5-HT neurons (Baumgarten and Lachenmayer, 1985
; Lindvall and Björklund, 1983
). The cerebellum receives both NE and 5-HT innervation (Baumgarten and Lachenmayer, 1985
; Bishop and Ho, 1985
; Lindvall and Björklund, 1983
), and the striatum has a rich DA innervation and also contains 5-HT terminals (Li et al., 2001
; Lindvall and Björklund, 1983
; Vertes, 1991
). The striatum was chosen as a negative control area (e.g., changes in NE synthesis should produce none-to-minimal changes in striatal neurotransmitter levels).
Frozen brain samples were sonicated on ice at 15 watts for 515 s in 1 ml of 0.1 N perchloric acid (PCA) containing 12.83 µM ethylenediaminetetraacetic acid (EDTA) and the internal standard NT-methyl-5-hydroxytryptamine (5-MHT). The samples were then centrifuged at 4°C for 10 min (12,500 x g) and the supernatants removed. The striatum was diluted 1:4 (v:v) and the brain stem was diluted 2:1 (v:v) with 0.1 N PCA. For all samples, 50 µl of the final supernatant (maintained at 4°C) were injected into the HPLC systems using a refrigerated autosampler. The final injected concentration of 5-MHT was 0.25 ng/µl (or 0.5 ng/µl for striatal samples using System 1). Samples were maintained on ice under light-shielded conditions at all times. The sample size for all groups for the HPLC analysis was 10, with the exceptions of the NIC striatum for the 100 mg/kg group (n = 9) and the cortex for the 0 mg/kg group at the 24-h time (n = 9).
HPLC analysis of brain catecholamines, NE and dopamine (DA), and its metabolites 3,4-dihyroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and the indolamine serotonin (5-HT) and its metabolite 5-hydroxyindole acetic acid (5-HIAA) were performed using two HPLC systems. The first system (System 1) consisted of a Waters 510 pump, a WISP 712 autosampler with a cooling unit (Waters Corp., Milford, MA), an Eppendorf CH-30 column heater and TC-50 controller (Eppendorf, Madison, WI), a pulse dampener (Model LP-21, Scientific Systems, Inc., State College, PA), and an ESA Coulochem electrochemical detector (Model 5100A, ESA, Inc., Chelmsford, MA). The second HPLC system (System 2) consisted of an Alliance 2690 Separations Module with column heater module (Waters), and an ESA Coulochem II electrochemical detector (Model 5200A', ESA, Inc.). All chromatographic separations were performed using a Guard-Pak precolumn holder and 10µ µBondpak C18 insert (Waters), and a 5µ Microsorb C18 column (250 x 4.6 mm; Varian Associates, Walnut Creek, CA). The columns were thermostated to 35°C (System 1) or 30°C (System 2). Both systems had a Guard Cell (Model 5020, ESA, Inc.) set at +450 mV, and an Analytical Cell (Model 5011, ESA Inc.) with detector 1 set at +50 mV, and the monitored detector 2 set at +420 mV. The gain setting for detector 2 was 500 nA/V for all tissues using System 2, and 10 x 15 (666.7 nA/V) for striatum, 100 x 3 (333.3 nA/V) for brain stem and cerebellum, and 100 x 6 (166.7 nA/V) for cortex using System 1. Standards for NE, DOPAC, DA, 5-HIAA, HVA, 5-HT, and 5-MHT were purchased from Sigma Chemical Co. (St. Louis, MO). The mobile phase contained 80 mM H3PO4 (Fischer Scientific, Fair Lawn, NJ), 2.59 mM heptanesulfonic acid (Sigma), and 12.83 µM EDTA (Sigma), at apparent pH of 2.7. The absolute concentration of MeOH varied from 911%, based on the age of the columns. The mobile phase was filtered (0.22 µ GVWP filters, Milipore, Bedford, MA), sparged with helium, and held under a helium blanket in a reservoir with UV protective coating (Kontes Glass Co., Vineland, NJ). Isocratic elutions were performed with a mobile phase flow rate of 1.2 ml/min for 60 min. Data was collected and analyzed using either Maxima or Millennium32 chromatography software (Waters), using area ratios to determine sample concentrations.
The tissue pellets remaining from the PCA extraction were analyzed for total protein content by the Pierce BCA assay (Pierce, Rockford, IL) against a bovine serum albumin (RIA Grade, Sigma) standard in a Thermomax microplate reader with SOFTmax® PRO software (Molecular Devices, Sunnyvale, CA). Previously frozen pellets were thawed and sonicated on ice in 1 ml of 0.1 N NaOH (Sigma), digested overnight, resonicated, and diluted to a final concentration of 0.01 N NaOH.
Statistical analysis.
Peak amplitudes and latencies were measured from each animals average waveform. Peak amplitudes (in µV) were measured from baseline (defined as the average voltage over the prestimulus period). Peak latencies (in ms) were calculated from stimulus onset. Peaks were identified by their polarity and latency according to the average waveform from each treatment group. Based on our previous results (Herr et al., 1992), statistical power was focused on changes in the amplitude of FEP peaks N36 and N166. Other peak amplitudes and latencies were examined in an exploratory manner using Bonferroni-corrected
levels (critical
= 0.05/[number of peak amplitudes and latencies]) (Abt, 1981
; Muller et al., 1983
). Only effects related to CS2 treatment are discussed in the manuscript. Significant effects were followed by step-down analysis, with further Bonferroni corrections to maintain the familywise
0.05. Within-subject effects had a Greenhouse-Geisser correction factor (
) applied to their degrees of freedom (Geisser and Greenhouse, 1958
; Greenhouse and Greisser, 1959
; Keselman and Rogan, 1980
). Due to the potentially decreased statistical power produced by these adjustments (Muller et al., 1983
), if an overall effect was significant but subsequent step-down ANOVAs failed to reach the corrected significance level (but had a p < 0.05), the actual probability values are reported. In all analysis, following significant ANOVAs, post hoc group mean comparisons were performed using a Tukey-Kramer Multiple Comparison Test (
= 0.05) (Kramer, 1956
). These comparisons established which doses of CS2 produced significant changes in FEP peak amplitudes or neurotransmitter levels. All analyses were performed using SAS (PROC GLM) (SAS Institute, Inc., 1989
, 1997
). Group averaged waveforms were calculated from individual animal data, and are reported for illustrative purposes only.
The initial analysis examined all the groups over the two pretreatment days and the pretreatment baseline test on the third day, to assure the similarity of each groups development of peak N166 amplitude prior to treatment with CS2 (Dyer, 1989; Herr et al., 1991
, 1994
). A repeated-measures analysis of variance (ANOVA) was performed for the amplitude of peaks N36 and N166. In this analysis, the animals were coded with their eventual treatment code (representing dose of CS2 and time of testing), even though they were all untreated at this time. Thus, dose of CS2 and time of posttreatment testing were "dummy" between-subject variables, and test day was a repeated within-subject variable.
The data from the post-CS2 treatment times were analyzed in a manner to facilitate comparisons with our previous work (Herr et al., 1992). The previous study employed a repeated-measures design with time as a within-subject factor (e.g., each animal was repeatedly tested at different times following treatment with CS2). Because in this study, the animals were sacrificed at each time point, a within-subjects design was impossible. Therefore, a difference score was calculated for each animal as follows: the third day pretreatment peak value minus the peak value at the time of testing. This difference score was then analyzed using an ANOVA with dose of CS2 and time of testing (sacrifice) as between-subject variables. The NIC groups, which were sacrificed immediately after the pretreatment FEP test on day 3, were dropped from the analysis (no posttreatment data was available). This analysis examined CS2-induced changes in FEP peak amplitudes at different times of posttreatment testing, relative to the animals pretreatment amplitudes as well as non-dose-related changes in FEP peak amplitudes between the test times. Data are reported as mean ± standard error (SE).
The neurochemical data was analyzed using a repeated-measures ANOVA, the dose of CS2, and time of testing as between-subject factors, and the brain region as a within-subject factor. This analysis examined the dose-related changes, differences between times of sacrifice, and regional differences in neurotransmitter levels (as well as the interactions between the three factors). An overall critical = 0.0083 was used in this analysis (0.05/6 compounds). Step-down analysis for each brain region and time of testing were conducted using further Bonferroni-corrected
levels as indicated by the overall ANOVA. This analysis examined changes in neurotransmitter levels produced by treatment with CS2 for each of the brain regions at each of the various times of testing (sacrifice). Data are reported as ng neurotransmitter (or metabolite)/mg protein ± standard error.
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RESULTS |
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Posttreatment FEPs: Peak latencies.
Peak latencies of FEPs were affected by treatment with CS2 (Fig. 1; graphs of mean latencies are not shown). Significant (critical
= 0.0031; 0.5/8 peaks/2 for amplitudes and latencies) dose-related increases in latency relative to the pretreatment values were indicated for peaks N36 and N166 (dose effect: all F values[3,144]
12.55, all p values
0.0001). Treatment with 400 mg/kg CS2 increased these peak latencies relative to the 0, 100, and 200 mg/kg doses. Additionally, treatment with 200 mg/kg CS2 increased peak N36 latency compared to the 0 and 100 mg/kg doses. The time-related increases in latency for peaks N36 (time by dose interaction: F[9,144] = 2.45, p = 0.0127) and N166 (time effect: F[3,144] = 2.87, p = 0.0388) failed to reach corrected significance levels. However, there was evidence of dose-related increases in peak N36 latency at 1, 4, and 8 h (all F values[3,36]
13.05, all p values
0.0001). At each of these times, 400 mg/kg CS2 produced an increase in peak N36 latency relative to pretreatment values, compared to the 0, 100, and 200 mg/kg CS2 doses. Additionally, at 4 h, 200 mg/kg CS2 increased peak N36 latency relative to controls. The latency of peak N166 was increased at 8 h by 400 mg/kg CS2 compared to the 0, 100, and 200 mg/kg doses.
Latencies of other FEP peaks were also affected by treatment with CS2. Significant dose-related increases in latency relative to pretreatment values were observed for peaks P28, P59, N65, P72, N84, and P102 (dose effect: all F values[3,144] 8.63, all p values
0.0001). Treatment with 400 mg/kg CS2 increased the latencies of all these peaks relative to the 0, 100, and 200 mg/kg doses. Additionally, the 200 mg/kg dose of CS2 increased the latency of peak P28 compared to the 0 and 100 mg/kg doses, and increased the latency of peak P102 relative to controls.
Colonic temperature.
Treatment with CS2 decreased colonic temperature in a dose- and time-dependent manner (Fig. 3; time by dose interaction: F[12,180] = 5.89, p
0.0001). Significant dose-related decreases in colonic temperature were observed 1, 4, 8, and 24 h after treatment (dose effect: all F values[3,36]
4.69, all p values
0.0073). No differences in the NIC animals were observed. Treatment with 400 mg/kg CS2 decreased colonic temperature compared to the 0, 100, and 200 mg/kg groups at 1, 4, and 8 h, and compared to the 0 and 100 mg/kg groups at 24 h. Also, the 200 mg/kg dose decreased colonic temperature compared to the 0 and 100 mg/kg groups at 4 h. Thus, the time of maximum depression of colonic temperature was observed 4 h after treatment with CS2.
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Cerebellar NE levels were also decreased in a dose- (F[3,178] = 5.71, p = 0.0009) and time-related (F[4,178] = 6.34, p 0.0001) manner. Significant changes in NE levels resulting from CS2 treatment were observed only at 4 h (F[3,36] = 25.27, p
0.0001). Treatment with 400 mg/kg CS2 decreased cerebellar NE levels compared to the 0, 100, and 200 mg/kg groups.
Levels of NE in the striatum varied over the different times of sacrifice (F[4,178] = 4.49, p = 0.0018). Average levels of NE at 8 h were lower than in the NIC group or at 1 h, and average NE levels at 4 h were less than in the NIC group. However, the decreased levels of striatal NE produced by 400 mg/kg CS2 at 4 (p = 0.0063) and 8 h (p = 0.0069) failed to reach corrected significance levels.
Dopamine and metabolites.
Changes in DA, DOPAC, and HVA produced by treatment with CS2 (Figs. 5, 6
, and 7
) had a differential regional distribution. All three analytes had different levels in the various brain regions (region effect: all F values[3,534]
2,393.87, all p values
0.0001,
s
0.3536), reflecting the much greater levels in the striatum than in the other brain areas. There were no significant changes (critical
= 0.0021; 0.05/6 analytes/4 regions) in the levels of DA or DOPAC observed in the cortex or striatum following CS2 treatment (all F values[12,178]
0.67, all p values
0.7821). Similarly, striatal levels of HVA were not affected by treatment with CS2 (F[12,178] = 1.00, p = 0.4509). Cortical levels of HVA (Fig. 7
) were greater in animals treated with 400 mg/kg CS2 than those treated with 0 or 100 mg/kg (dose effect: F[3,178] = 6.01, p = 0.0006). However, there were no significant treatment-related effects at any of the individual time points (all p values
0.05). In contrast to the relative lack of effects in the cortex and striatum, there were significant CS2-induced increases in DA, DOPAC, and HVA in both the brain stem and cerebellum.
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In the cerebellum, the increases in DA and DOPAC resulting from treatment with CS2 (Figs. 5 and 6
) varied over the different sampling times (all F values[12,178]
3.73, all p values
0.0001). Treatment with 400 mg/kg CS2 increased cerebellar DA levels (Fig. 5
) at 4 h compared to the 0, 100, and 200 mg/kg doses (F[3,36] = 14.72, p
0.0001). Levels of DOPAC (Fig. 6
) in the cerebellum were increased by 400 mg/kg CS2 at 4 h (F[3,36] = 20.76, p
0.0001) compared to the 0, 100, and 200 mg/kg treatments. The increased levels of cerebellar DOPAC at 1 (p = 0.0018) and 8 h (p = 0.0090) failed to reach corrected significance levels. Cerebellar levels of HVA were increased by CS2 (Fig. 7
; dose effect: F[3, 178] = 5.93, p = 0.0007), but the changes over time failed to reach corrected significance levels (p = 0.0083). Treatment with 400 mg/kg CS2 increased levels of cerebellar HVA compared to the 0, 100, and 200 mg/kg doses. However, the only time point which possibly showed an effect of CS2 treatment on cerebellar HVA levels was at 4 h, which failed to meet corrected significance levels (p = 0.0011).
Serotonin and metabolites.
Treatment with CS2 had less effects on serotonin and 5-HIAA levels (Figs. 8 and 9
) than those observed for NE, DA, DOPAC, and HVA. Regional differences in 5-HT levels were observed (region effect: F[3,534] = 933.11, p
0.0001), with the highest levels found in the brain stem, followed by the striatum, cortex, and cerebellum. However, treatment with CS2 did not alter 5-HT levels (Fig. 8
) in any brain region at any time point (all F values [12,178]
0.44, all p values
0.9460).
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Neurochemical correlations with FEP Peaks N36 and N166.
In the cortex (site of generation of FEP peaks N36 and N166), the only neurotransmitter with CS2-induced dose- and time-related changes was NE. Therefore, as a further analysis on an individual animal basis, a linear regression analysis was performed between cortical levels of NE and the amplitudes of peaks N36 and N166 at the times of CS2-induced depressions (4 and 1 h, respectively; Fig. 10). The slope of the regression lines did not differ from zero, indicating that there was no relationship between the amplitude of peak N36 and cortical NE levels at 1 (p = 0.6618) or 4 h (p = 0.0654), or between peak N166 amplitude and cortical NE levels at 1 (p = 0.5979) or 4 h (p = 0.9129) after treatment with CS2.
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DISCUSSION |
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As reported in other studies using rats (Magos and Jarvis, 1970; Magos et al., 1974
; McKenna and DiStefano, 1977
), treatment with CS2 decreased brain levels of NE and altered levels of DA. The mechanism responsible for this effect is believed to be: (1) reaction of CS2 with endogenous amino acids to form dithiocarbamate metabolites, (2) chelation of copper ions by the dithiocarbamate metabolites, and (3) subsequent inhibition of dopamine ß-hydroxylase (DßH; the enzyme responsible for the synthesis of NE from DA), which requires Cu2+ as a necessary cofactor (Bus, 1985
; McKenna and DiStefano, 1977
). The cortex, brain stem, and cerebellum were selected due to their NE terminal innervation or NE cell bodies, and were the brain regions showing the largest changes in monoamine neurotransmitter levels (and metabolite levels). Significantly decreased levels of NE in the cortex, brain stem, and cerebellum were observed 4 h after treatment with CS2 (Fig. 4
). The decreased NE levels were accompanied by increased levels of DA and DOPAC in the brain stem and cerebellum (Figs. 5
and 6
) and HVA in the brain stem (Fig. 7
). These increased levels of DA and its metabolites in the brain stem and cerebellum at 4 h may result from CS2-induced inhibition of NE synthesis, rather than any direct effect on DA neurotransmission. In contrast, the decreases in striatal NE levels failed to reach corrected significance levels (Fig. 4
). At 4 h, there was approximately a 44% decrease in striatal NE compared to about a 68, 58, or 65% decrease in cortical, brain stem, or cerebellar NE, respectively. Thus, the decreased levels of NE produced by treatment with 400 mg/kg CS2 were slightly less in the striatum (which has less NE innervation) than in the other brain regions. In further support of an effect on NE synthesis by CS2, the striatum (which has a large number of DA projections) did not have any changes in the levels of DA or its metabolites. As predicted, 5-HT levels were not altered in any brain region (Fig. 8
). Thus, the data are consistent with a CS2-induced inhibition of DßH, decreased NE synthesis, an accumulation of DA, and metabolism of the excess DA to DOPAC and HVA in NE neurons.
The data presented in this report do not support the hypothesis that CS2-induced decreases in cortical NE are related to the reduced amplitude of FEP peak N166 observed after treatment with CS2. Maximal depression of FEP peak N166 was observed 1 h after treatment (Figs. 1 and 2
). In contrast, CS2-induced decreases in NE levels were not observed until 4 h after treatment (Fig. 4
). At this time, there was approximately a 68% decrease in cortical NE levels compared to controls. This difference in the time-courses of decreases in peak N166 amplitude, followed by later decreases in cortical NE (at a time when peak N166 amplitude was returning to control levels), suggest that changes in NE-induced cortical responsiveness to the flash stimuli were not related to the observed decreases in peak N166 amplitude. This conclusion is also supported by the lack of a correlation between an individual animals peak N166 amplitude and its cortical NE level at 1 or 4 h (Fig. 10
). Similarly, there was no relationship between an individual animals peak N36 amplitude and its cortical NE levels at 1 or 4 h. Because we used tissue homogenates from large portions of the cortex, the levels of NE at the synapse were not quantified and we cannot rule out changes in the ability of NE to modulate FEP peak amplitudes at the synaptic level. However, the data do indicate that the global reductions in NE levels produced by CS2 treatment do not appear to produce the observed changes in the amplitudes of FEP peaks N36 and N166.
Changes in DA function have been postulated to alter FEPs. Previous investigators have shown that treatment with -methyl-para-tryosine (AMT; to deplete NE and DA) increased the latencies of FEP peaks P1 (P28), N1 (N36), and P2 (P59). Importantly, while decreasing the amplitude of peaks N1P2 (N36P59) and P2N2 (P59N84), this treatment failed to alter the amplitude of peak P3N3 (P102N166) (Dyer et al., 1981
). The actions of AMT were attributed to DA depletion in the retina, as increased FEP peak latencies were not observed after electrical stimulation of the optic tract. These same researchers reported that treatment with FLA-63 (bis(4-methyl-1-homopiperazinylthiocarbonly)-disulfide; a DBH inhibitor) failed to alter any FEP peak amplitudes or latencies, suggesting the lack of a role for NE in the effects produced by AMT (Dyer et al., 1981
). In agreement, other researchers have also found that treatment with AMT increases the latencies, and decreases the amplitudes of FEP peaks, and attributed the effects to decreased levels of retinal DA (Onofrj and Bodis-Wollner, 1982
). It should be noted that Onofrj and Bodis-Wollner (1982)
did not quantify NE or DA depletions, and Dyer and coworkers (1981)
only measured DA levels in retinal tissue (>50% depletion), to verify the depletion levels of NE and/or DA.
In contrast to reports of DA depletion increasing FEP peak latencies and decreasing the amplitudes of early peaks, treatment with CS2 increased brain levels of DA and its metabolites at 4 h, when peak N36 amplitude was decreased. However, the changes in DA, DOPAC, and HVA were not observed in the cortex where peak N36 is generated (Bigler, 1977; Branca
k et al., 1990
; Givre et al., 1994
; Kraut et al., 1985
; Mitzdorf, 1986
, 1987
) (Figs. 5
, 6
, and 7
). Additionally, there were no changes in DA or its metabolites observed in the striatum (Figs. 5
, 6
, and 7
), where alterations would presumably reflect direct effects on DA neurons, rather than secondary effects on NE neurons. Therefore, it is unlikely that the decreased amplitude of peak N36 was mediated by changes in DA neurotransmission.
The amplitude of FEP peak N166 is reported to be modified by pharmacologic agents reported to interact with non-monoamine-based neurotransmitter systems. Treatment of rats with physostigmine (a cholinesterase inhibitor) has been shown to decrease the amplitude of peak P3-N3 (P102N166) and/or the PhAD (of which peak N166 is the first portion) (Bigler and Fleming, 1976b; Bigler et al., 1978
). Similarly, the amplitude of peak P3-N3 was reduced by the anticonvulsant trimethadione (Shearer et al., 1976
). Conversely, treatment with pentylenetetrazol (a pro-convulsant) has been shown to increase the amplitude of the PhAD (Bigler, 1977
; Rhodes and Fleming, 1970
). While not conclusively proven, studies such as these suggest a role for the cholinergic and possibly the gamma-aminobutyric acid systems in the expression of peak N166 and/or the PhAD. However, the influence of treatment with CS2 on the function of these neurotransmitter systems remains to be examined.
Treatment with CS2 decreased the animals colonic temperature, similar to the results reported in our previous study (Herr et al., 1992). Although significant hypothermia was observed at all time points, the effect was maximal 4 h after treatment with approximately a 2.4°C decrease in temperature relative to controls (Fig. 3
). Decreased colonic temperature has been shown to increase FEP peak latencies (Dyer and Boyes, 1983
; Fitzgibbon et al., 1984
; Hetzler et al., 1988
). In this study, treatment with CS2 increased average FEP peak latencies relative to their pretreatment values. Similar to the decrease in colonic temperature, the increases in peak latencies were greatest over the 18 h period, but were still detectable at 24 h. The similar time-courses for the decreases in colonic temperature and the increases in peak latencies, coupled with the known effects of decreased body temperature on FEP peak latencies, suggests that the increases in FEP peak latencies may be secondary to CS2-induced hypothermia. However, the decreases in amplitudes of peaks N36 and N166 do not have the same time-course as the CS2-induced reductions in colonic temperature. Decreases in peak N166 amplitude were observed only 1 h, and peak N36 amplitude was decreased only at 4 h after dosing with CS2, in contrast to the reduced colonic temperatures observed at 1, 4, 8, and 24 h after treatment. These difference in time-courses of effects argue that the changes in FEP peak amplitudes were not secondary to depressed colonic temperature. Additionally, it has been shown that hypothermia has minimal effects on FEP peak P1N1 (P28N36) amplitude as long as colonic temperatures remain above 30°C (as were observed in this study, Fig. 3
) (Hetzler et al., 1988
). Other investigators have shown that a 6°C decrease in colonic temperature (to 31°C) can result in an increase in peak P1N1 amplitude (Dyer and Boyes, 1983
). The maximum hypothermia was observed at 4 h after treatment with CS2 (Fig. 3
), at a time when peak N36 amplitude was decreased and the reduction in peak N166 amplitude observed at 1 h was dissipating. Therefore, it would appear that mechanisms other than a reduction in colonic temperature are involved in the decreased amplitudes of FEP peaks N36 and N166 produced by treatment with CS2.
Another possibility is that CS2-induced changes in colonic temperature could influence levels of brain neurotransmitters (and/or metabolites). Most enzymes have an optimum temperature for maximal activity. As such, absolute levels of neurotransmitters would depend on the balance of synthesis and degradation related to the activities of multiple enzymes. In this study, brain levels of NE decreased, levels of DA, DOPAC, HVA, and 5-HIAA increased, while levels of 5-HT were unchanged following treatment with CS2. One might predict similar changes in multiple neurotransmitter systems in response to a generalized decrease in temperature. Additionally, the changes in neurotransmitter levels were statistically significant only at 4 h after treatment with CS2, while significantly decreased colonic temperature was observed 1, 4, 8, and 24 h after dosing with CS2. Coupled with the known inhibition of DBH by dithiocarbamate metabolites of CS2, these observations argue that the observed changes in neurotransmitter levels were the result of toxicological actions of CS2, rather than an indirect result of decreases in colonic temperature.
In summary, we have replicated the differential time-course of CS2-induced depressions of FEP peak N36 and N166 amplitudes. The presence of a large and robust peak N166 at a time when peak N36 is severely depressed argues against the hypothesis that the neurogenerators for these two peaks are arranged in a simple serial relationship. We have also verified that this treatment regime resulted in alterations in brain NE and DA levels. This is the first study to examine the dose- and time-related changes in brain NE levels with respect to visual electrophysiology after CS2 treatment. However, the different time-courses for depression of FEP peak N166 amplitude and decreases in brain NE levels argues that the observed changes in brain monoamine levels were not responsible for the changes in visual system function. Thus, the neurochemical basis for the different time-courses of CS2-induced depression of peaks N36 and N166 remains to be elucidated.
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ACKNOWLEDGMENTS |
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NOTES |
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Portions of this manuscript were presented as a poster at the 27th annual meeting of the Society for Neuroscience (1997, Soc. Neuroscience Abstr. 23(1), 1028).
1 To whom correspondence should be addressed at 109 T.W. Alexander Drive, MD B105-05, NHEERL/NTD/NPTB, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711. Fax: (919) 541-4849. E-mail: herr.david{at}epamail.epa.gov.
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