Address correspondence and reprint requests to Kristen A. Keefe, PhD, Department of Pharmacology and Toxicology, University of Utah, 30 South 2000 East, Room 201, Salt Lake City, UT 84112, USA. E-mail: K.Keefe@m.cc.utah.edu
Exposure to repeated high doses of methamphetamine produces long-term toxicity to central monoamine systems and alters striatonigral pathway function 3 weeks after exposure. To determine whether these changes in the striatonigral pathway persist for longer we examined neuropeptide mRNA expression in the striatum and cytochrome oxidase activity in the output nuclei of the basal ganglia after treatment with multiple high doses of methamphetamine. Rats exposed to multiple high doses of methamphetamine had significant depletion in dopamine and serotonin content, decreases in tyrosine hydroxylase immunoreactivity, and decreases in preprotachykinin mRNA expression, 6 and 12 weeks after methamphetamine treatment. Preprotachykinin mRNA expression was significantly reduced by ∼20% in the middle striatum and ∼32% in the caudal striatum, 6 weeks after treatment. Twelve weeks after treatment, preprotachykinin mRNA expression continued to be significantly reduced by ∼20% in the middle striatum and ∼14% in the caudal striatum. Cytochrome oxidase histochemical staining in the entopeduncular nucleus and substantia nigra pars reticulata was not significantly different from that in controls at either timepoint. These data suggest that neurotoxic regimens of methamphetamine induce changes in striatonigral neurons that persist for up to 3 months, although there is some recovery.
Methamphetamine (METH) is an addictive (Johanson et al. 1976) psychomotor stimulant that is widely abused (Johnston et al. 2001). Studies on rats have shown that METH alters the extracellular concentrations of monoamines in the brain, especially in the region of the basal ganglia (Bradberry and Roth 1989; Galloway 1990). Exposure to multiple high doses of METH results in neurotoxic damage to monoamine neurons, leading to long-lasting depletion of dopamine (DA) in the striatum, substantia nigra and neocortex, and serotonin (5-HT) in the frontal cortex, striatum and amygdala (Ricaurte et al. 1980). METH also causes persistent decreases in tyrosine hydroxylase (TH) and tryptophan hydroxylase activity in these brain regions (Kogan et al. 1976; Hotchkiss et al. 1979; Hotchkiss and Gibb 1980). In addition, this neurotoxic regimen of METH has been shown to alter peptide systems (Chapman et al. 2001).
Previous work from our laboratory, as well as that of others, has shown changes in indices of striatonigral efferent neuron function 3 weeks after exposure to a neurotoxic regimen of METH or 6-hydroxydopamine (6-OHDA). Such data illustrate that a partial depletion of DA by 6-OHDA or a partial depletion of both DA and 5-HT by METH decreases preprotachykinin (PPT), but not preproenkephalin, mRNA expression 3 weeks after treatment (Nisenbaum et al. 1996; Chapman et al. 2001). Furthermore, the study by Chapman et al. showed that cytochrome oxidase (CO) histochemical staining in the terminal fields of striatonigral neurons, the entopeduncular nucleus (EPN), and substantia nigra pars reticulata (SNpr), was increased after this METH treatment, suggesting disinhibition and increased activity of these basal ganglia output neurons. Thus, the disruption of monoamine systems induced by METH exposure appears to beassociated with selective alterations in the striatonigral pathway of the basal ganglia 3 weeks later.
The purpose of the present study was to determine whether these post-synaptic consequences of exposure to a neurotoxic regimen of METH persist for a longer period of time, as the deficits in monoamine systems persist for up to 6 months (Bittner et al. 1981). We hypothesized that a persistent partial loss of DA and 5-HT induced by METH would lead to persistent changes in preprotachykinin mRNA expression and cytochrome oxidase staining 6 and 12 weeks after treatment.
Compounds used were obtained from the following sources: (+/–)-METH hydrochloride, National Institutes on Drug Abuse (Rockville, MD, USA); cytochrome c, paraformaldehyde and 3-3′-diaminobenzidine (DAB), Sigma (St Louis, MO, USA); monoclonal mouse anti-TH antibody, DiaSorin Inc. (Stillwater, MN, USA); peroxidase-labeled anti-mouse IgG (H + L) made in horse, Vector Laboratories (Burlingame, CA, USA).
Male Sprague–Dawley rats weighing 290–340 g at the time of the experiment were housed four per cage in hanging wire cages in a temperature-controlled room (22.5–23.2°C) on a 12/12-h light/dark cycle. Rats had free access to food and water. The Institutional Animal Care and Use Committee of the University of Utah approved the animal care and experimental procedures. These procedures were also in agreement with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
The evening before the experiment each rat was weighed. The rats were re-housed in plastic tubs (33 cm length × 28 cm width × 17 cm height) with sawdust bedding (eight rats per tub). On the day of the experiment, rats received a total of four injections of either METH (10 mg/kg, s.c.) or 0.9% saline, with a 2-h interval between each injection. Body temperatures were not recorded, and none of the rats was cooled to prevent METH-induced hyperthermia. Rats remained in the tubs until the following day, at which time they were returned to their home cages (four rats per cage) until they were killed 6 or 12 weeks after treatment with METH. Rats were killed by exposure to CO2 for 1.5 min followed by decapitation. The brains were removed and quickly frozen in isopentane chilled on dry ice and stored at − 20°C.
In situ hybridization histochemistry
Coronal sections (12 µm) were taken from the striatum and substantia nigra of treated and control rats using a cryostat (Cryocut 1800; Cambridge Instruments, Heidelberg, Germany) at − 20°C. The sections were thaw-mounted on to chrome alum–gelatin-coated slides, then stored at − 20°C. Slides were then thawed, post-fixed for 10 min in 4% paraformaldehyde/0.9% NaCl, and placed in a fresh solution of 0.25% acetic anhydride in 0.1 m triethanolamine/0.9% NaCl, pH 8.0, for another 10 min. Sections were dehydrated in a series of alcohols, delipidated in chloroform, then rehydrated in a series of alcohols. Slides were air-dried, then stored at − 20°C.
A ribonucleotide probe was used to determine the expression of PPT mRNA (full length; Krause et al. 1987). The probe was synthesized from the cDNA using [35S]UTP and SP6 RNA polymerase (Roche Molecular Biochemicals, Indianapolis, IN, USA) as described previously (Adams et al. 2000). The labeling reaction was stopped by dilution with Tris (10 mm)–EDTA (1 mm), pH 7.5, and the cRNA probe was then extracted with phenol–chloroform and precipitated with ethanol. The ethanol-precipitated cRNA was pelleted by centrifugation (16 000 g for 15 min) then resuspended in Tris–EDTA, pH 8.0, with 100 mm dithiothreitol. The radiolabeled ribonucleotide probe was mixed with nuclease-free water and RNA mix (final concentrations: 100 µg/mL salmon sperm DNA, 250 µg/mL yeast total RNA and 250 µg/mL yeast tRNA), heated at 65°C for 5 min, and then cooled for 1 min on wet ice. Tothis mixture was added (final concentrations): dithiothreitol (100 mm), sodium dodecyl sulfate (0.2% w/v), sodium thiosulfate (0.1% w/v) and hybridization buffer. The hybridization buffer consisted of the following (final concentrations): Tris buffer (23.8 mm, pH 7.4), EDTA (1.2 mm, pH 8.0), NaCl (357 mm), dextran sulfate (11.9%, w/v), Denhardt's solution (1.2 ×) and formamide (59.5%, v/v). Ninety μl of the probe/hybridization buffer was applied to each slide containing four different striatal sections and covered with a glass coverslip. Slides were hybridized in humid chambers at 55°C overnight for 12–18 h. Slides were then washed four times in 1 × SSC (0.15 m NaCl/0.015 m sodium citrate). Next, the slides were incubated in RNase A (5 µg/mL; Roche Molecular Biochemicals) in buffer containing 0.5 m NaCl, 10 mm Tris (pH 7.5) and 0.25 m EDTA (pH 8.0) for 15 min at room temperature (∼25°C). After incubation, slides were washed four times for 20 min each in 0.2 × SSC at 60°C. Slides were quickly rinsed in deionized water, then air-dried. Radiolabeled slides were apposed to radiographic film (Kodak Biomax MR; Eastman Kodak, NY, USA) for 7 days.
An oligonucleotide probe was used for the detection of TH mRNA (bases 1441–1488; Grima et al. 1985). The oligonucleotide probe was synthesized by the DNA/peptide facility at the University of Utah. The probe was end-labeled with 35S-dATP and terminal deoxynucleotidyl transferase (Roche Molecular Biochemicals) as described previously (Adams et al. 2000). For hybridization, 200 µL of the radiolabeled probe was mixed with 5 mL of hybridization buffer and 100 µL of 5 m dithiothreitol. The hybridization buffer consisted of the following (final concentrations): Tris buffer (80 mm, pH 7.5), NaCl (0.6 m), EDTA (4 mm), sodium pyrophosphate (0.1% w/v), 50% dextran sulfate (10% w/v), 10% sodium dodecyl sulfate (0.2% w/v), heparin sulfate (0.2% w/v) and formamide (50% v/v). A 90-μL aliquot of the probe/hybridization buffer was applied to each slide containing four different nigral sections, and the sections were covered with a glass coverslip. Slides were hybridized at 37°C overnight for 12–18 h. Slides were then washed four times in 1 × SSC at room temperature, three times for 20 min each in 2 × SSC + 50% formamide at 38°C, and then twice for 30 min in 1 × SSC at room temperature. Slides were quickly rinsed in deionized water, then air-dried. Radiolabeled slides were apposed to radiographic film (Kodak Biomax MR) for 3–4 days.
Frozen brains from control and treated rats were sectioned coronally at 20 µm from the striatum through the substantia nigra in a cryostat (Cryocut). Sections were thaw-mounted on gelatin–chrome alum-coated slides, then stored at − 20°C. The CO histochemistry technique was modified from Wong-Riley (1979) and performed on slides containing sections from the caudal striatum (− 0.92 mm posterior from bregma), globus pallidus (GP; – 0.92 mm posterior), EPN (− 2.30 mm posterior), subthalamic nucleus (STN; − 3.60 and −3.80 mm posterior) and the SNpr (− 4.80, − 5.20, − 5.30 and − 5.80 mm posterior to bregma). Slides were thawed, then incubated in 300 mL of 0.1 m phosphate-buffered saline (PBS), pH 7.4, containing 180 mg DAB and 45 mg cytochrome c (from horse) for 2 h at 37°C. Slides were washed three times for 10 min in 0.1 m PBS, pH 7.4, briefly rinsed in deionized water, and covered using glass coverslips and Permount (Fisher Scientific, NJ, USA).
This procedure was performed using a modification of the peroxidase anti-peroxidase method Sternberger (1979) on non-perfused, 12-µm thick coronal sections of striatal tissue mounted on slides. The slides were thawed, washed twice for 5 min in 0.1 m PBS, pH 7.4, and fixed in 4% paraformaldehyde/0.9% NaCl for 10 min. The sections were rinsed three times for 5 min in PBS, dried, then circled using a PAP-pen (ImmunoStar, Hudson, WI, USA). Non-specific binding was blocked with 10% normal horse serum (Sigma) in PBS/0.3% Triton for 2 h at room temperature. The horse serum then was removed from the slides, and the slides incubated in humid chambers overnight at 4°C with monoclonal mouse anti-TH antibody (1 : 300 dilution) in PBS/0.3% Triton. The following day, slides were washed three times for 5 min in PBS, then incubated with anti-mouse IgG (H + L) peroxidase made in horse (1 : 150 dilution) in PBS/0.3% Triton for 1.5 h at room temperature. The slides were rinsed three times for 5 min in PBS then incubated in 0.1% DAB/0.005% hydrogen peroxide/PBS solution for 4–8 min. Slides were dehydrated in ethanol (70, 95 and 100%), incubated in xylene for 1 min, then covered using glass coverslips and Permount.
DA and 5-HT tissue content
A blunt-tip, 16-G needle was used to collect 1-mm3 tissue punches from the frontal cortex and the caudate putamen during sectioning of the brains for in situ hybridization, TH immunohistochemistry and CO histochemistry. Two punches were taken from the region of the frontal cortex, and one punch was taken from both the dorsomedial and dorsolateral striatum. Sections were cut coronally until the landmark of the corpus callosum became apparent (3.7 mm anterior to bregma) at which point tissue punches were collected from the frontal cortex. In addition, sections were cut coronally until the corpus callosum from the left and right hemispheres of the brain merged and the lateral ventricles were extended to the anterior commissure (0.7 mm anterior to bregma). At that point, tissue punches were collected from the striatum. Tissue punches were sonicated in tissue buffer (0.05 m sodium phosphate/0.03 m citric acid with 25% methanol (v/v), pH 2.48), then centrifuged to separate the supernatant from the protein. Twenty microliters of the supernatant was injected on to a HPLC system (Dynamax AI-200 autosampler and SD-200 pump; Varian, Walnut, CA, USA) coupled to an electrochemical detector (potential for oxidation at working electrode surface (Eox) = + 0.65 V; Decade, Antec-Leyden, Zoeterwoude, The Netherlands) to quantitate the DA and 5-HT levels. A Whatman (Clifton, NJ, USA) PartiSphere C-18 column (250 × 4.6 mm, 5-µm) was used to separate the monoamines. The mobile phase consisted of MeOH (23% v/v), sodium octyl sulfate (0.03% w/v), EDTA (0.1 mm), sodium phosphate dibasic (0.05 m) and citric acid (0.03 m). The pH of the mobile phase was 2.87, and the flow rate was 0.5 mL/min. Protein content was determined using the Lowry assay (Lowry et al. 1951).
Autoradiograms from in situ hybridization histochemistry and slides from TH and CO histochemistry were analyzed using the Macintosh-based image analysis program, Image (NIH; http://rsb.info.nih.gov/nih-image/). The images were captured with a video camera (CCD-72SX; DAGE MTI, Michigan City, IN, USA) and stored on computer. The linear range for the video camera and video capture card was established by measuring the mean gray values for a range of known optical density values on a calibrated photographic step tablet (Eastman Kodak Co., NY, USA). Light intensity was then adjusted so that the optical density measurements from the film autoradiograms and slides from TH and CO histochemistry were within the linear parameters of the system. Lighting and camera conditions remained constant during the process of capturing images and collecting density measurements. Mean gray values were analyzed in the medial and lateral halves of the rostral and middle striatum, the dorsal and ventral halves of the caudal striatum, or the whole striatum for each region. Striatal regions were designated as the area below the corpus callosum and above the anterior commissure. Densitometric analysis was performed on sections (relative to bregma) from the rostral (1.7 mm anterior), middle (0.7 mm anterior) and caudal (− 0.92 mm posterior) regions of the dorsal striatum, ventral tegmental area (VTA), substantia nigra pars compacta (SNpc), EPN, STN and the SNpr. To correct for background labeling, the average gray value of the white matter was subtracted from that of the region of interest. For CO histochemical analysis, regions of interest were outlined and measured without the subtraction of white matter. Data from the METH experiments were analyzed using unpaired t-tests for the areas of interest. Statistical significance was set at p ≤ 0.05.
DA and 5-HT depletion
Multiple high doses of METH decreased DA and 5-HT tissue content, TH mRNA expression in the SNpc, and TH immunoreactivity (IR) in the striatum and SNpc at both 6 and 12 weeks after treatment. DA was decreased in the frontal cortex, the dorsomedial striatum and the dorsolateral striatum at both timepoints (Fig. 1a). 5-HT was decreased in the dorsomedial and dorsolateral striatum at both timepoints (Fig. 1b). There were no significant changes in 5-HT content in the frontal cortex. In situ hybridization histochemistry revealed a significant decrease in TH mRNA expression in the SNpc; however, the decrease in the VTA was not statistically significant (Table 1). In addition, immunohistochemical analysis revealed decreases in TH-IR throughout the striatum and in the SNpc after both survival times. As was the case with TH mRNA expression, there were no significant changes in TH-IR in the VTA (Table 1).
Table 1. Effects of multiple high doses of METH on TH mRNA and TH immunoreactivity 6 and 12 weeks post-treatment
Values are mean gray values (arbitrary units ± SEM) obtained from densitometric analysis of film autoradiograms and immunohistochemically stained slides. Numbers in parentheses indicate the number of rats per group. *Significantly different from control, p≤0.05.
Six weeks after METH exposure, the signal corresponding to PPT mRNA expression was decreased 20% in the medial and 18% in the lateral regions of the middle striatum (Figs 2a and 3a). In the caudal striatum, the PPT mRNA signal was decreased 33% and 31% in the dorsal and ventral regions, respectively (Figs 2a and 4a). Twelve weeks after treatment, the density of PPT mRNA labeling was decreased 18% in the medial and 22% in the lateral regions of the middle striatum (Figs 2b and 3b). In the caudal striatum, the signal was decreased 14% in the dorsal region and 13% in the ventral region (Figs 2b and 4b). There were no changes in PPT mRNA labeling in the rostral striatum at either 6 or 12 weeks (Figs 2a and 2b).
There were no changes in CO activity in the GP, STN, EPN and SNpr after exposure to multiple doses of METH at either the 6- or 12-week timepoints (Table 2).
Table 2. Effects of multiple high doses of METH on cytochrome oxidase staining in striatum and striatal output nuclei
Values are mean gray values (arbitrary units ± SEM) obtained from densitometric analysis of histochemically stained slides. Numbers in parentheses indicate the number of rats per group.
41.0 ± 0.7
42.3 ± 0.9
40.7 ± 1.3
40.0 ± 0.7
22.0 ± 0.8
22.7 ± 0.6
23.4 ± 1.1
22.2 ± 0.5
105.1 ± 2.6
111.4 ± 3.7
107.7 ± 2.9
104.1 ± 5.6
103.1 ± 2.1
104.3 ± 1.4
105.5 ± 2.8
100.6 ± 2.0
Substantia nigra pars reticulata
78.6 ± 1.9
78.0 ± 1.7
70.2 ± 1.9
66.7 ± 1.5
The purpose of the present study was to determine whether post-synaptic consequences of METH-induced toxicity to central monoamine systems persist for several months, as the loss of monoamines in rodents has been shown to last for 6 months or more (Bittner et al. 1981; Friedman et al. 1998). The data from the current study illustrate that there are persistent decreases in PPT mRNA expression in the middle and caudal regions of the striatum 6 and 12 weeks after exposure to a neurotoxic regimen of METH. However, PPT mRNA expression in the rostral region of striatum was not different from control. Thus, some residual deficits in striatonigral neurons are still evident after 3 months.
The decrease in PPT mRNA expression observed after exposure to multiple high doses of METH may arise as a consequence of the reduction in monoamine neurotransmission associated with such treatment, since this dosing paradigm of METH results in a loss of monoamines, as shown in the present work, as well as that of others (Hotchkiss and Gibb 1980; Ricaurte et al. 1980). Previous studies have shown that a partial loss of the DA innervation of the striatum induced by the neurotoxin 6-OHDA also decreases PPT mRNA expression in striatonigral efferent neurons when examined 3 weeks later (Nisenbaum et al. 1996). In addition, decreases in 5-HT synthesis or lesion of 5-HT systems with the neurotoxin 5,7-dihydroxytryptamine also reduce PPT mRNA expression (Walker et al. 1991). Furthermore, stimulation of 5-HT receptors can alter the decrease in PPT mRNA expression induced by DA-depleting brain lesions (Gresch and Walker 1999). These data suggest that the decrease in PPT mRNA expression observed in the present study probably arose as a consequence of the loss of monoamine neurotransmission induced by the METH regimen used. The extent to which restoration of DA or 5-HT tone can reverse the observed decreases in PPT mRNA expression remains to be determined.
Although a significant decrease in PPT mRNA expression was apparent in the middle and caudal striatum throughout this experiment, there was no significant decrease in PPT mRNA expression in the rostral striatum. This is in contrast to our previous findings that showed a significant decrease in PPT mRNA expression in rostral, as well as middle and caudal, striatum 3 weeks after exposure to a neurotoxic regimen of METH (Chapman et al. 2001). This apparent recovery of PPT mRNA expression in rostral striatum by 6 weeks after exposure to METH may be a consequence of the differential sensitivity of rostral and caudal striatal regions to monoamine loss, and the recovery of monoamine innervation of the striatum over time. These data parallel the pattern of sensitivity to monoamine loss reported previously for 6-OHDA-induced loss of striatal DA (Marshall 1979; Marshall et al. 1980). These data support a rostral–caudal gradient in the sensitivity of the striatum to DA or monoamine loss, consistent with our present and previous (Chapman et al. 2001) observations that PPT mRNA expression in rostral striatum is less sensitive to monoamine loss and recovers more quickly than does PPT mRNA expression in caudal striatum after exposure to neurotoxic doses of METH.
The significance of the persistent decrease in PPT mRNA levels observed in this study, as well as in our earlier work (Chapman et al. 2001), is currently not known. At 3 weeks after exposure to a neurotoxic regimen of METH, we observed an increase in CO histochemical staining in the SNpr and EPN, suggesting a generalized decrease in striatonigral neuron activity. In the present study, however, CO staining in these nuclei was not different in METH-treated rats from that in controls. At these later timepoints, the decrease in PPT mRNA expression may therefore reflect only a decrease in signaling via substance P or neurokinin A, the peptides encoded by PPT-A mRNA in striatum, rather than a generalized alteration in the firing of striatonigral efferent neurons. Loss of neuromodulatory neuropeptide signaling alone, in the absence of altered GABA signaling, would be less likely to sustain the chronic changes in firing rates of nigral and entopeduncular neurons necessary for altered CO staining. Such normalization of CO activity and presumably tonic neuronal firing, however, does not preclude an important role for altered neuropeptide signaling in the long-term consequences of METH exposure, as alterations in substance P signaling affect behavioral recovery from 6-OHDA-induced DA loss (Mattioli et al. 1992). Clearly, additional studies are required to determine whether substance P or neurokinin A signaling is altered in the SNpr and EPN after exposure to a neurotoxic regimen of METH.
The functional implications of the METH-induced changes in monoamines and PPT mRNA expression are presently unknown. However, the rats reported in this study that were killed 6 weeks after exposure to a neurotoxic regimen of METH were impaired on a sequential motor learning task when tested 3–5 weeks after exposure (Chapman et al. 2001). These data therefore suggest that the monoamine loss or a loss of neurokinin signaling per se may contribute to the long-term behavioral consequences observed in rats weeks to months after exposure to METH (Walsh and Wagner 1992; Friedman et al. 1998; Chapman et al. 2001). As noted above, previous work has demonstrated that substance P, given after administration of the DA neurotoxin 6-OHDA, promotes functional recovery (Pelleymounter et al. 1988; Mattioli et al. 1992; Nikolaus et al. 1997). Therefore, a persistent loss of neurokinin signaling as a consequence of exposure to a neurotoxic regimen of METH may contribute to the behavioral deficits associated with such exposure in both rodents and humans (Walsh and Wagner 1992; Friedman et al. 1998; Rogers et al. 1999; Chapman et al. 2001; Volkow et al. 2001a,b). Further studies are needed to determine the extent to which the loss of monoamines generally, or a decrease in neurokinin signaling specifically, contributes to the long-term functional deficits that have been reported to be associated with psychostimulant toxicity.
In summary, multiple high doses of METH not only result in neurotoxic consequences to striatal monoamines but also reduce PPT mRNA expression in striatonigral efferent neurons 6 and 12 weeks after treatment. The present data also suggest that there is some regional recovery of PPT mRNA expression over time, most notably in the rostral striatum. These data support our previous findings that the METH-induced toxicity to central monoamine systems is associated with alterations in striatonigral efferent neurons and further extend those findings by demonstrating that post-synaptic consequences persist for up to 3 months after exposure. The role of these prolonged alterations in the long-term behavioral consequences of METH-induced neurotoxicity remains to be determined.
We thank David E. Chapman for treating the rats in the 6-week study. This work was supported by NIH grants 5 T32 G07559-19, DA 00378 (GRH), DA 09407 (KAK) and an American Psychological Association Minority Fellowship in Neuroscience (KLJ-D).