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Keywords:

  • Methamphetamine;
  • Serotonin;
  • Striatum;
  • Microdialysis;
  • Neurotoxicity

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. METH treatment
  5. HPLC analysis
  6. RESULTS
  7. Basal levels of 5-HT and 5-HIAA
  8. Tissue levels of 5-HT and 5-HIAA
  9. DISCUSSION
  10. Acknowledgements
  11. REFERENCES

Abstract: Repeated administration of methamphetamine to animals can lead to long-lasting decreases in striatal monoamine content. In the present study, the effects of neurotoxic doses of methamphetamine on basal and evoked overflow of striatal serotonin and of its primary metabolite 5-hydroxyindoleacetic acid were examined in awake rats using in vivo microdialysis. Male Fischer-344 rats were administered methamphetamine (5 mg/kg, s.c.) or saline four times in 1 day at 2-h intervals. Microdialysis studies were carried out 1 week, 1 month, and 6 months later. At 1 week posttreatment there were significant decreases in potassium- and amphetamine-evoked overflow of serotonin in the striatum of the methamphetamine-treated animals. Basal extracellular levels of 5-hydroxyindoleacetic acid but not of serotonin were also decreased. Evoked overflow of serotonin recovered by 1 month, and extracellular levels of 5-hydroxyindoleacetic acid had recovered by 6 months. Tissue levels of serotonin and 5-hydroxyindoleacetic acid were decreased at 1 week posttreatment but back to control levels by 1 month after treatment. These results indicate that presynaptic serotonergic functioning is attenuated in the striatum of rats treated 1 week earlier with neurotoxic doses of methamphetamine. However, in the model used, the changes are transient, and recovery can occur within 1-6 months posttreatment.

Repeated administration of methamphetamine (METH) to animals can produce alterations in brain monoamine systems indicative of axonal and nerve terminal degeneration. In the striatum both serotonin [5-hydroxytryptamine (5-HT)] and dopamine (DA) systems are affected. Serotonergic changes include decreases in 5-HT content and in levels of its metabolite 5-hydroxyindoleacetic acid (5-HIAA), reductions in tryptophan hydroxylase activity, and decreases in the uptake of 5-HT (Ricaurte et al., 1980; Bakhit et al., 1981; Seiden and Ricaurte, 1987; Gibb et al., 1994). Similar changes occur regarding striatal DA terminals in animals treated with neurotoxic doses of METH (Ricaurte et al., 1980; Wagner et al., 1980; Seiden and Ricaurte, 1987; Eisch et al., 1992; Gibb et al., 1994). In some cases these changes appear to be long-lasting, and reductions in serotonergic markers in the striatum have been reported for weeks or even months after administration of METH (Ricaurte et al., 1980; Woolverton et al., 1989; Friedman et al., 1998).

Although the neurotoxic effects of METH on 5-HT neurons have been documented in several studies, the functional consequences of METH treatment on 5-HT release have not been extensively examined. Recent reports have indicated that neurotoxic doses of METH can lead to decreases in DA release and uptake in the striatum of intact animals (Cass, 1997; Cass and Manning, 1999). The same functional changes may be true for 5-HT systems as well. Robinson et al. (1990), using in vivo microdialysis, found that basal extracellular levels of 5-HIAA were decreased in rats treated with METH 1 week earlier. Although 5-HT levels were not reported in that study, the reduction in 5-HIAA levels could indicate that extracellular levels of 5-HT are reduced as well. However, compensatory processes may occur in serotonergic terminals after administration of neurotoxins (Stachowiak et al., 1986), similar to those observed in dopaminergic terminals after 6-hydroxydopamine lesions (Zigmond et al., 1990; Robinson et al., 1994). Extracellular levels of 5-HT may therefore be normal in METH-treated animals despite attenuated extracellular levels of 5-HIAA and tissue levels of 5-HT and 5-HIAA. Thus, there is a lack of information available on the consequences of METH treatment on the functioning of 5-HT terminals in the brain.

The purpose of the present study was to evaluate the effects of neurotoxic doses of METH on presynaptic serotonergic terminal functioning in the striatum. In vivo microdialysis in awake rats treated 1 week earlier with METH was used to evaluate basal levels and overflow of 5-HT and 5-HIAA in the striatum following stimulation with systemic administration of amphetamine. To evaluate the time course of any changes observed, basal levels of 5-HT and 5-HIAA and the overflow evoked by local application of potassium and amphetamine were examined with microdialysis in rats treated 1 week, 1 month, and 6 months earlier with neurotoxic doses of METH. Striatal tissue levels of 5-HT and 5-HIAA were determined at the conclusion of each experiment to evaluate further the effects of METH on serotonergic terminals.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. METH treatment
  5. HPLC analysis
  6. RESULTS
  7. Basal levels of 5-HT and 5-HIAA
  8. Tissue levels of 5-HT and 5-HIAA
  9. DISCUSSION
  10. Acknowledgements
  11. REFERENCES

Animals

Adult, male Fischer-344 rats (Harlan Sprague Dawley, Indianapolis, IN, U.S.A.) were used for all experiments. They were housed in groups of two under a 12-h light-dark cycle with food and water freely available. All animal use procedures were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee at the University of Kentucky. Several of the animals used for the dialysis experiments involving local application of stimulants were also used for gathering data on the effects of METH on DA release (Cass and Manning, 1999).

METH treatment

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. METH treatment
  5. HPLC analysis
  6. RESULTS
  7. Basal levels of 5-HT and 5-HIAA
  8. Tissue levels of 5-HT and 5-HIAA
  9. DISCUSSION
  10. Acknowledgements
  11. REFERENCES

Rats weighed 220-290 g on the day of treatment and were housed individually during the injections. They were injected subcutaneously with 5 mg/kg METH-HCl (Sigma Chemical Co., St. Louis, MO, U.S.A.) in saline (1 ml/kg) or with saline alone (1 ml/kg), four times in 1 day at 2-h intervals. Room temperature was 23-24°C, and the initial injection was given between 8:00 and 9:00 a.m.

In vivo microdialysis

For the placement of guide cannulas the rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and positioned in a stereotaxic frame. All surgery was performed using sterile instruments and conditions. The skull was exposed, and a small hole was drilled in the skull over the right striatum. A guide cannula was lowered into the brain so that its tip was positioned 1.2 mm anterior to bregma, 2.5 mm right from midline, and 3.5 mm below the surface of the cortex. The cannula was secured to the skull with anchoring screws and dental acrylic. The incision was closed with sutures and Vetbond tissue adhesive (3M, St. Paul, MN, U.S.A.), and the animals were placed in round microdialysis bowls and allowed to recover. For the rats that were examined 1 week after the METH or saline treatment the cannulas were put in place 2-3 days posttreatment. For the rats that were examined 1 or 6 months posttreatment the guide cannulas were put in place 7 days before the microdialysis experiments. At 1-2 days before the dialysis experiments the rats were tethered to the microdialysis system (BeeKeeper System; BAS, West Lafayette, IN, U.S.A.), without dialysis probes in place, for 4-8 h to help them acclimate to the system.

On the day of the dialysis experiments the animals were connected to the dialysis system, and probes were inserted into the guides (CMA 12 probes, 3-mm-long dialysis membrane; CMA/Microdialysis, Acton, MA, U.S.A.). The probes were perfused with artificial CSF (pH 7.4) containing 145 mM NaCl, 2.7 mM KCl, 1.2 mM CaCl2, 1.0 mM MgCl2, 0.2 mM ascorbic acid, and 2.0 mM NaH2PO4 (Moghaddam and Bunney, 1989). The flow rate was 1.2 μl/min, and fractions of dialysate were collected at 20-min intervals. Following a 3-h equilibration period and the collection of three baseline fractions, the overflow of 5-HT was stimulated either by systemic injection of D-amphetamine sulfate (2.0 mg/kg in 1.0 ml/kg saline, s.c.) or by local application of potassium and D-amphetamine through the dialysis probe. For the experiments with the systemic injection of amphetamine, dialysate samples were collected for 4 h after the injection. For local stimulation the perfusate solution was switched to a 100 mM K+ solution (47.7 mM NaCl, 100 mM KCl, 1.2 mM CaCl2, 1.0 mM MgCl2, 0.2 mM ascorbic acid, and 2.0 mM NaH2PO4, pH 7.4) for a single 20-min fraction and then switched back to the original perfusate for five additional fractions. Amphetamine (100 μM) was then included in the perfusate for one 20-min fraction followed by five final fractions with normal artificial CSF. Dialysate samples were analyzed immediately by HPLC or frozen on dry ice, stored at -80°C, and analyzed within 1 week.

At the end of the experiments the probes were removed, and the animals were anesthetized with CO2 and killed by decapitation. The brains were rapidly removed and chilled in ice-cold saline. A 2-mm-thick coronal slice of brain containing the striatum at the level of the dialysis probe tract was made with the aid of an ice-chilled brain mold (Rodent Brain Matrix; ASI Instruments, Warren, MI, U.S.A.). That half of the section contralateral to the tract was used for measuring tissue content of 5-HT and 5-HIAA. The striatum was dissected out, placed in a preweighed vial, weighed, and frozen on dry ice. Samples were stored at -80°C until assayed for monoamine content by HPLC. The other half of the 2-mm-thick section was used for verifying dialysis probe placement (either by noting blood in the track during dissection or by sectioning with a cryostat if the track was not easily visible).

HPLC analysis

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. METH treatment
  5. HPLC analysis
  6. RESULTS
  7. Basal levels of 5-HT and 5-HIAA
  8. Tissue levels of 5-HT and 5-HIAA
  9. DISCUSSION
  10. Acknowledgements
  11. REFERENCES

Levels of 5-HT and 5-HIAA were determined using HPLC with electrochemical detection as previously described (Cass, 1996, 1997) using an HR-80 reverse-phase C18 column (particle size, 3 μm; 4.6 × 80 mm; ESA, Chelmsford, MA, U.S.A.). The retention times for 5-HIAA and 5-HT were 4.7 and 8.7 min, respectively, and the limit of detection with a signal-to-noise ratio of at least 2:1 was 0.41 pg for 5-HIAA and 0.88 pg for 5-HT (for the dialysis samples this corresponds to ∼0.14 nM for 5-HIAA and 0.33 nM for 5-HT). For dialysate samples 15 μl of dialysate was injected directly onto the HPLC column. For determining tissue monoamine content, the samples were sonicated in 300 μl of cold 0.1 M perchloric acid containing 3,4-dihydroxybenzylamine as an internal standard.

Data analysis

Dialysis data were expressed as concentration of 5-HT or 5-HIAA in the dialysate. Basal levels were defined as the average value in the three fractions preceding stimulation by excess potassium or systemic injection of amphetamine. In a few animals, basal 5-HT levels remained excessively elevated after the 3-h equilibration period. These animals had relatively low levels of evoked overflow of 5-HT compared with basal values and also had noticeable hemorrhaging in the area of the dialysis probe on examination of the brain at the end of the experiments. Because of the possibility that a substantial percentage of the dialysate 5-HT in these animals was nonneuronal in origin, the data from these animals were not used in the final analysis. All probes were calibrated in vitro before use to determine acceptable probes (recovery of 5-HT at least 20%). However, values were not corrected for in vitro recoveries because uncorrected values may be better correlated to true values as it has been shown for striatal DA levels (Glick et al., 1994). Tissue levels of 5-HT and 5-HIAA were expressed as nanograms per gram wet weight of tissue. Unpaired two-tailed t tests and repeated-measures ANOVAs were used for statistical comparison as indicated in Results.

Basal levels of 5-HT and 5-HIAA

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. METH treatment
  5. HPLC analysis
  6. RESULTS
  7. Basal levels of 5-HT and 5-HIAA
  8. Tissue levels of 5-HT and 5-HIAA
  9. DISCUSSION
  10. Acknowledgements
  11. REFERENCES

The mean basal level of 5-HT in the dialysate from the saline- and METH-treated animals ranged from 0.7 to 0.95 nM in all groups (Table 1). There was no significant difference in the data from the animals used for the systemic injection of amphetamine versus the local application of potassium and amphetamine at the 1 week time point, so the basal levels for these groups were pooled together. Basal extracellular levels of 5-HT were not different between the saline and METH groups at 1 week, 1 month, or 6 months posttreatment. In contrast, 5-HIAA levels in the METH-treated animals were significantly lower than those in the saline-treated rats at 1 week posttreatment (Table 1). However, basal levels of 5-HIAA in the METH-treated animals had recovered to control values by 1 month posttreatment.

Table 1. Basal dialysate levels of 5-HT and 5-HIAA from striatum of saline- and METH-treated animals
 Time after treatment
 1 week1 month6 months
  1. Data are mean ± SEM values for 12 animals in the 1-week group and six animals in the 1-month and 6-months groups.

  2. a p < 0.01 versus saline group by two-tailed t test.

5-HT (nM)    
Saline0.95 ± 0.180.94 ± 0.290.86 ± 0.29
METH0.94 ± 0.100.71 ± 0.080.70 ± 0.31
5-HIAA (nM)    
Saline298 ± 19484 ± 50439 ± 26
METH226 ± 16a419 ± 26452 ± 35

Evoked overflow of 5-HT

In the animals administered a subcutaneous injection of amphetamine during the dialysis experiments there was an increase in the extracellular level of 5-HT following the injection in both the saline- and METH-treated animals (Fig. 1). The data were analyzed using a two-way repeated-measures ANOVA with treatment as a between factor and minutes of sample collection as a within factor. There were significant main effects for treatment (p < 0.01) and minutes (p < 0.001) and a significant treatment × minutes interaction (p < 0.001). Amphetamine-evoked overflow of 5-HT was significantly lower in the METH group, compared with the saline group, at 40, 60, and 80 min after the injection (Newman-Keuls post hoc comparisons). Extracellular levels of 5-HIAA also were lower in the METH-treated animals compared with the saline-treated controls (Fig. 1). In contrast to 5-HT levels, 5-HIAA levels were relatively unaffected by the amphetamine injection; however, there was a trend for 5-HIAA levels to increase in the saline-treated animals and decrease in the METH-treated animals. A two-way repeated-measures ANOVA revealed significant main effects for treatment (p < 0.05) and minutes (p < 0.05) and a significant treatment × minutes interaction (p < 0.01). At each individual time point, 5-HIAA levels in the METH group were lower than those of the saline group (Newman-Keuls post hoc comparisons).

image

Figure 1. Dialysate levels of 5-HT (top panel) and 5-HIAA (bottom panel) from striatum of rats treated 1 week earlier with saline or METH. Amphetamine (2.0 mg/kg, s.c.) was injected at time 0 to evoke the overflow of 5-HT (arrows). Data are mean ± SEM (bars) values from six animals in each group. *p < 0.05 versus saline at the same time point (two-way repeated-measures ANOVA followed by Newman-Keuls post hoc comparisons). Saline versus METH is different at each time point for 5-HIAA (Newman-Keuls post hoc comparisons).

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The local application of potassium and amphetamine produced substantial increases in extracellular 5-HT levels in the striatum of both the saline- and METH-treated rats (Fig. 2). For the animals examined 1 week after treatment a two-way repeated-measures ANOVA revealed a significant effect of minutes (p < 0.001) and a significant treatment × minutes interaction (p < 0.01). Potassium-evoked overflow was significantly lower in the METH group, compared with the saline group, at the 20 and 40 min time points, whereas amphetamine-evoked overflow of 5-HT was significantly lower in the METH group at the 160 min time point (Newman-Keuls post hoc comparisons). At both 1 month and 6 months after treatment there was no significant effect of treatment and no significant treatment × minutes interaction.

image

Figure 2. Dialysate levels of 5-HT from striata of animals treated 1 week (top panel), 1 month (middle panel), or 6 months (bottom panel) earlier with saline or METH. Excess potassium (100 mM) was included in the perfusate for 20 min starting at 0 min (bar above K+), and 100 μM amphetamine was included in the perfusate for 20 min starting at 120 min (bar above Amphetamine). Data are mean ± SEM (bars) values from six animals in each group. *p < 0.05 versus saline at the same time point (two-way repeated-measures ANOVA followed by Newman-Keuls post hoc comparisons).

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Figure 3 shows the changes in extracellular levels of 5-HIAA during the experiments where potassium and amphetamine were locally applied to evoke overflow of 5-HT. A significant decrease in 5-HIAA levels occurred in all groups at 40 min following the local application of potassium (one-way repeated-measures ANOVA, p < 0.05), whereas the effects of amphetamine were less dramatic. For the animals examined 1 week after treatment a two-way repeated-measures ANOVA revealed significant main effects of treatment (p < 0.05) and minutes (p < 0.001) and a significant treatment × minutes interaction (p < 0.001). Dialysate levels of 5-HIAA were significantly lower in the METH group, compared with the saline group, at each individual time point (Newman-Keuls post hoc comparisons). By 1 month after treatment 5-HIAA levels had partially recovered. There were significant main effects for treatment (p < 0.05) and minutes (p < 0.001) but not a significant interaction. At 6 months posttreatment there was no difference in 5-HIAA levels between the saline- and METH-treated animals.

image

Figure 3. Dialysate levels of 5-HIAA from the striata of animals treated 1 week (top panel), 1 month (middle panel), or 6 months (bottom panel) earlier with saline or METH. Excess potassium (100 mM) was included in the perfusate for 20 min starting at 0 min (bar above K+), and 100 μM amphetamine was included in the perfusate for 20 min starting at 120 min (bar above Amphetamine). Data are mean ± SEM (bars) values from six animals in each group. *p < 0.05 versus saline group (two-way repeated-measures ANOVA). For the data at 1 week posttreatment, dialysate levels of 5-HIAA were significantly lower in the METH group compared with the saline group at each individual time point (Newman-Keuls post hoc comparisons).

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Tissue levels of 5-HT and 5-HIAA

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. METH treatment
  5. HPLC analysis
  6. RESULTS
  7. Basal levels of 5-HT and 5-HIAA
  8. Tissue levels of 5-HT and 5-HIAA
  9. DISCUSSION
  10. Acknowledgements
  11. REFERENCES

At 1 week after treatment postmortem levels of 5-HT and 5-HIAA in the striatum were decreased by 44 and 40%, respectively, in the METH-treated animals compared with the saline-treated control animals (Fig. 4). However, at both 1 month and 6 months posttreatment there were no significant differences in 5-HT or 5-HIAA levels between the saline- and METH-treated animals.

image

Figure 4. Postmortem levels of 5-HT (top panel) and 5-HIAA (bottom panel) from the striata of rats treated 1 week, 1 month, or 6 months earlier with saline or METH. Data are mean ± SEM (bars) values from 12 animals for the 1-week group and six animals for the 1-month and 6-month groups. *p < 0.001 versus saline group at the same time point (two-tailed t tests).

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. METH treatment
  5. HPLC analysis
  6. RESULTS
  7. Basal levels of 5-HT and 5-HIAA
  8. Tissue levels of 5-HT and 5-HIAA
  9. DISCUSSION
  10. Acknowledgements
  11. REFERENCES

The present results indicate that treatment with neurotoxic doses of METH can lead to significant changes in the functioning of serotonergic terminals in the striatum. Tissue levels and evoked overflow of 5-HT and tissue levels and basal extracellular levels of 5-HIAA were significantly attenuated in the striatum of rats treated 1 week earlier with METH. However, METH-induced changes in tissue levels and evoked overflow of 5-HT were back to control levels by 1 month, and basal and stimulant-evoked changes in extracellular levels of 5-HIAA were back to normal by 6 months after treatment. Thus, with the animal model used, METH-induced changes in presynaptic 5-HT terminal functioning in the striatum are temporary and can completely recover by 6 months after treatment. However, it should be kept in mind that extent of recovery may depend on several factors, including species and duration and severity of METH treatment. For example, in rhesus monkeys treated with a more extensive METH treatment there were still substantial decreases in striatal 5-HT content 4 years after treatment (Woolverton et al., 1989), and Long-Evans rats treated with a higher METH dose than in the present study (12.5 mg/kg; four injections at 2-h intervals) still had a 33% reduction in striatal 5-HT content 237 days after treatment (Friedman et al., 1998). These studies highlight that the extent of the lesion and the recoverability of serotonergic processes will likely depend on the animal model and METH treatment used.

In the present study both potassium- and amphetamine-evoked overflow of 5-HT were attenuated by the METH treatments. This suggests that both calcium-dependent (potassium stimulation) and -independent (amphetamine stimulation) release of 5-HT is altered by the METH. These changes could be due to a decrease in the releasable pools of 5-HT in the striatum, to a loss of 5-HT terminals in the striatum, or to a combination of both processes. Experimental results suggesting that METH may lead to degeneration of 5-HT terminals in the striatum include, in addition to reductions in 5-HT levels, decreases in tryptophan hydroxylase activity and reductions in uptake of 5-HT by striatal synaptosomes (Ricaurte et al., 1980; Bakhit et al., 1981; Gibb et al., 1994). Furthermore, there is evidence of nerve terminal degeneration and accompanying reactive gliosis in the striatum following METH treatment (Ricaurte et al., 1982; Hess et al., 1990; Bowyer et al., 1994; O'Callaghan and Miller, 1994; Pu and Vorhees, 1995). This degeneration and gliosis are probably due, to a large extent, to the loss of DA terminals in the striatum; however, some of the degeneration is likely due to a loss of 5-HT terminals as well. Further evidence for a loss of 5-HT terminals comes from several studies that have demonstrated a loss of 5-HT immunoreactive fibres in many regions of the brain, including the striatum, following treatment with neurotoxic doses of METH (Axt and Molliver, 1991; Axt et al., 1994; Pu and Vorhees, 1995). Taken together with the present results of a decrease in tissue levels and evoked overflow of 5-HT at 1 week after treatment, these findings suggest that METH leads to a functional loss of 5-HT terminals in the striatum.

The results of the present study are similar to those following lesions with the 5-HT neurotoxin 5,7-dihydroxytryptamine. Kirby et al. (1995) found that depletion of striatal 5-HT content by ∼76% with 5,7-dihydroxytryptamine led to a significant decrease in basal extracellular levels of 5-HIAA but had no significant effect on basal 5-HT levels. Fenfluramine-stimulated overflow of 5-HT was also significantly decreased in the 5,7-dihydroxytryptamine-treated animals, similar to amphetamine-evoked overflow in the present study. Kirby et al. (1995) also found that extracellular 5-HIAA levels were minimally or not at all altered following fenfluramine administration even though 5-HT levels increased. This is similar to what has been reported previously in rats treated with saline or neurotoxic doses of METH and given a systemic injection of amphetamine (Robinson et al., 1990) and what is reported in the present study following systemic administration of amphetamine. On the other hand, following the local application of amphetamine there tended to be a reduction in extracellular levels of 5-HIAA (Fig. 3). However, interpretation of the data following local application is difficult as the baseline was slowly decreasing following recovery from the potassium stimulation. Amphetamine at relatively high concentrations can inhibit monoamine oxidase (Robinson, 1985; Cho and Kumagai, 1994), and thus it might be expected to reduce extracellular levels of 5-HIAA. It is possible that following systemic administration of amphetamine the concentration of amphetamine in the brain is insufficient to affect significantly monoamine oxidase activity. The local application of amphetamine would have led to a greater concentration of amphetamine in the vicinity of the dialysis probe that may have reached a level high enough to produce a significant effect on monoamine oxidase activity and lead to a decrease in 5-HIAA levels. The local application of potassium did, however, lead to a significant reduction in extracellular levels of 5-HIAA for a single fraction. This reduction in 5-HIAA levels is more difficult to explain. One possibility is that the prolonged exposure to potassium may have led to reductions in neuronal reuptake or glial uptake of 5-HT due to alterations in the ionic composition of the extracellular milieu or to perturbations of cellular membrane potentials (Mager et al., 1994; Barker et al., 1999). This could lead to a decrease in availability of 5-HT for conversion to 5-HIAA by monoamine oxidase. A similar phenomenon occurs with extracellular levels of the DA metabolite 3,4-dihydroxyphenylacetic acid during local stimulation with potassium (Cass et al., 1998). In any case, extracellular levels of 5-HIAA do not appear to reflect extracellular levels of 5-HT but instead may be more reliable as an indicator of tissue content of 5-HT (Kirby et al., 1995).

The fact that tissue levels and evoked overflow of 5-HT are back to control values by 1 month after METH treatment may indicate that some of the decrease observed at 1 week is due to a transient reduction in the releasable pool of 5-HT that can recover by 1 month. This is supported by immunocytochemical evidence demonstrating that only 30% of rats treated with neurotoxic doses of METH had a persistent loss of 5-HT axons 1-2 weeks after the METH treatment (Axt and Molliver, 1991). An additional factor that could help with recovery is that there may be some sprouting of remaining 5-HT axons that occurs within the first month. Several studies have demonstrated the reappearance of serotonergic axons and terminals over time and the partial to complete recovery of biochemical indices for 5-HT terminals in the brains of rats treated with neurotoxic doses of amphetamine derivatives (Battaglia et al., 1988; Scanzello et al., 1993; Axt et al., 1994; Sabol et al., 1996). Similar processes could be occurring in the striatum of rats treated with neurotoxic doses of METH. Overall, the decrease in 5-HT release and content in the striatum is likely due to a combination of an actual loss of some 5-HT terminals coupled with a decrease in releasable pools of 5-HT in remaining terminals. The relatively rapid recovery of 5-HT levels and release is likely due to compensatory mechanisms occurring in the remaining terminals (Stachowiak et al., 1986; Bendotti et al., 1990) as well as some sprouting or regrowth of axon terminals.

In the animal model used in the present study the severity of the METH-induced lesion to striatal serotonergic terminals appears to be less than the severity of effects on dopaminergic terminals. For example, basal extracellular levels of DA, but not 5-HT, are decreased in the striatum of METH-treated animals 1 week after the treatment (Cass et al., 1998; Cass and Manning, 1999). Additionally, tissue levels and evoked overflow of 5-HT in the present study were back to control levels by 1 month after treatment, whereas recovery of tissue levels and evoked overflow of DA takes from 6 to 12 months (Cass and Manning, 1999). Taken together, the results of these studies indicate that with the METH treatment used, striatal dopaminergic terminals of the Fischer-344 rat are more vulnerable to the effects of neurotoxic doses of METH than are striatal serotonergic terminals. This is similar to what has been reported following repeated doses of METH (10 mg/kg, i.p., four injections at 2-h intervals) in Sprague-Dawley rats (Callahan et al., 1998). The work of Callahan et al. (1998) further suggests that differences in the vulnerability of 5-HT and DA neurons to the toxic effects of METH may be due to differences in the mechanisms that produce toxicity in the two types of neurons. 5-HT terminals appear to be more susceptible to metabolic compromise, whereas DA terminals may be more susceptible to the hyperthermic effects of METH. It should also be pointed out that not all studies have found that DA terminals are more sensitive than 5-HT terminals to the neurotoxic effects of METH. Several groups have reported that indices of 5-HT terminal integrity may be compromised to a greater extent or for a longer interval than those for DA terminals after METH administration (Ricaurte et al., 1980; Gibb et al., 1994; Friedman et al., 1998).

In summary, the results of the present experiments indicate that neurotoxic doses of METH in Fischer-344 rats can lead to a functional loss of 5-HT terminals in the striatum 1 week after treatment. However, recovery of all serotonergic indices examined occurred by 6 months posttreatment. This suggests that compensatory processes occur to normalize serotonergic terminal functioning. These processes may include recovery and up-regulation of 5-HT synthesis and release in existing terminals and sprouting or regrowth of axons from remaining serotonergic fibers. Future studies will be necessary to determine the actual mechanisms for recovery in the present model.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. METH treatment
  5. HPLC analysis
  6. RESULTS
  7. Basal levels of 5-HT and 5-HIAA
  8. Tissue levels of 5-HT and 5-HIAA
  9. DISCUSSION
  10. Acknowledgements
  11. REFERENCES

This work was supported in part by U.S. Public Health Service grant DA10115. I thank Mr. Michael W. Manning for technical assistance with this project and Drs. Alan Frazer and Lynette Daws for their helpful discussions concerning 5-HT neuron functioning.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. METH treatment
  5. HPLC analysis
  6. RESULTS
  7. Basal levels of 5-HT and 5-HIAA
  8. Tissue levels of 5-HT and 5-HIAA
  9. DISCUSSION
  10. Acknowledgements
  11. REFERENCES
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