OtherNEUROPHARMACOLOGY

Resistance of Neuronal Nitric Oxide Synthase-Deficient Mice to Methamphetamine-Induced Dopaminergic Neurotoxicity

Yossef Itzhak, Carlos Gandia, Paul L. Huang and Syed F. Ali
Journal of Pharmacology and Experimental Therapeutics March 1998, 284 (3) 1040-1047;

Abstract

Methamphetamine (METH) is a powerful psychostimulant that produces dopaminergic neurotoxicity manifested by a decrease in the levels of dopamine, tyrosine hydroxylase activity and dopamine transporter (DAT) binding sites in the nigrostriatal system. We have recently reported that blockade of the neuronal nitric oxide synthase (nNOS) isoform by 7-nitroindazole provides protection against METH-induced neurotoxicity in Swiss Webster mice. The present study was undertaken to investigate the effect of a neurotoxic dose of METH on mutant mice lacking the nNOS gene [nNOS(−/−)] and wild-type controls. In addition, we sought to investigate the behavioral outcome of exposure to a neurotoxic dose of METH. Homozygote nNOS(−/−), heterozygote nNOS(+/−) and wild-type animals were administered either saline or METH (5 mg/kg × 3). Dopamine, DOPAC and HVA levels, as well as DAT binding site levels, were determined in striatal tissue derived 72 h after the last METH injection. This regimen of METH given to nNOS(−/−) mice affected neither the tissue content of dopamine and its metabolites nor the number of DAT binding sites. Although a moderate reduction in the levels of dopamine (35%) and DAT binding sites (32%) occurred in striatum of heterozygote nNOS(+/−) mice, a more profound depletion of the dopaminergic markers (up to 68%) was observed in the wild-type animals. METH-induced hyperthermia was observed in all animal strains examined except the nNOS(−/−) mice. Investigation of the animals’ spontaneous locomotor activity before and after administration of the neurotoxic dose of METH (5 mg/kg × 3) revealed no differences. A low dose of METH (1.0 mg/kg) administered to naive animals (nNOS(−/−) and wild-type) resulted in a similar intensity of locomotor stimulation. However, 68 to 72 h after exposure to the high-dose METH regimen, a marked sensitized response to a challenge METH injection was observed in the wild-type mice but not in the nNOS(−/−) mice. Taken together, these results indicate that nNOS(−/−) mice are protected against METH-induced dopaminergic neurotoxicity and locomotor sensitization. It also appears that a partial deficit of dopaminergic transmission in wild-type animals does not prevent the development of sensitization to METH, whereas a deficit in nNOS may attenuate this process.

METH is a synthetic drug that is closely related chemically to amphetamine but has a higher potential for abuse as a psychostimulant. Amphetamines cause a massive release of newly synthesized dopamine from presynaptic vesicles and an increase in extracellular dopamine concentration in the nigrostriatal system. High doses of amphetamines cause long-lasting neurotoxicity associated with a marked decrease in dopamine level (Kogan et al., 1976; Fuller and Hemrick-Luecke, 1980; Gibbet al., 1990; Robinson et al., 1990), dopamine transporter binding sites (Wagner et al., 1980) and tyrosine hydroxylase activity (Kogan et al., 1976; Gibb et al., 1990) in the striatum. Recent studies demonstrated a similar depletion in striatal dopamine nerve terminal markers in post-mortem, chronic METH users (Wilson et al., 1996). The damage to nigrostriatal dopaminergic neurons caused by METH and that caused by the neurotoxin MPTP represent experimental animal models of Parkinson’s disease. The involvement of EAA transmission in METH-induced neurotoxicity is suggested by two major findings. First, blockade of the NMDA type of glutamate receptors by dizocilpine (MK-801) attenuates METH-induced neurotoxicity (Sonsalla et al., 1989; Farfel et al., 1992; Weihmuller et al., 1992; Ali et al., 1994). Second, repeated administration of METH causes an increase in glutamate release in the striatum (Nash and Yamamoto, 1992). The fact that EAA receptor antagonists modulate the development of amphetamine-induced behavioral sensitization further supports the role of glutamatergic neurotransmission in the effects of these psychostimulants (Karleret al., 1990).

NMDA receptor activation is thought to be associated with stimulation of the nNOS isoform. Interaction of glutamate, for instance, with the NMDA receptor complex opens channels that admit calcium into the cell; binding of calcium to calmodulin activates nNOS, which produces NO, and subsequently elicits the accumulation of cGMP (Garthwaite, 1991;Snyder, 1992). NO-mediated neurotoxicity may arise from the formation of free radicals (peroxynitrite and breakdown products) (Beckmanet al., 1990) and activation of the nuclear enzyme poly (ADP-ribose) synthase that leads to energy depletion and cell death (Zhang et al., 1994). The relationship between NMDA receptor activation and the stimulation of NO production prompted us to investigate the role of nNOS first in METH-induced dopaminergic neurotoxicity and second in the behavioral sensitization caused by repeated administration of relatively low doses of METH. We reported that blockade of brain NOS by 7-NI prevented METH-induced dopaminergic neurotoxicity (Itzhak and Ali, 1996) and the development of locomotor sensitization (Itzhak, 1997) in Swiss Webster mice. The purpose of the present study was two-fold: to investigate the susceptibility to METH-induced dopaminergic neurotoxicity of mutant mice that lack the nNOS gene and to determine what the behavioral consequence of exposure to a potentially neurotoxic dose of METH is. We report that mice lacking the nNOS gene are protected against METH-induced dopaminergic neurotoxicity. In addition, despite the fact that wild-type animals displayed obvious dopaminergic deficit after exposure to a high dose of METH, they still developed a marked sensitized response to a challenge METH injection; however, nNOS knockout mice did not show such sensitized response to METH.

Materials and Methods

Materials.

(d)Methamphetamine-HCl, desipramine-HCl and benztropine mesylate were purchased from Sigma (St. Louis, MO). [3H]Mazindol (24.0 Ci/mmole) was purchased from New England Nuclear (Wilmington, DE). METH solution was prepared in saline (0.9% NaCl).

Animals.

Homozygote neuronal NOS knockout mice [nNOS(−/−)] were obtained from a breeding colony established at the University of Miami School of Medicine using homozygote animals that were previously generated by homologous recombination (Huang et al., 1993) at Massachusetts General Hospital, Charlestown, MA. Because the nNOS gene mutation was made on a SV129 agouti mouse and C57BL/6 mouse background, three different animal strains (purchased from Jackson Laboratories) were used as wild-type controls: C57BL/6, SV129 and B6J/SV129 F2 strains; the latter represents F2 progeny of cross-breeding between C57BL/6 and SV129 strains. F1 progeny of heterozygote nNOS knockout mice [nNOS(+/−)] were generated from the cross-breeding of wild-type C57BL/6 males and nNOS(−/−) females. For the present study, only male mice 10 to 12 weeks of age (23–28 g) were used. All animals were maintained on a 12-h light/dark lighting schedule, kept at a room temperature of 21°C and housed in groups of 4 to 5 with free access to food and water. The principles of laboratory animal care (NIH publication No. 85-23, revised 1985) were followed.

Administration of METH for the determination of dopaminergic neurotoxicity.

Each of the five groups of mice, nNOS(−/−), nNOS(+/−), B6/SV129, C57BL/6 and SV129, were divided into two subgroups each containing 10 to 15 mice. One group received METH (5 mg/kg) i.p. three times, every 3 h, and the second group received three saline injections. We have previously found that this schedule of METH administration to Swiss Webster mice produced 68, 44 and 55% decreases in the concentration of striatal dopamine, DOPAC and HVA, respectively, and 48% decrease in the number of DAT binding sites (Itzhak and Ali, 1996). Animals were sacrificed 72 h after the last METH injection. The brain was rapidly removed, and the caudate putamen was dissected and stored at −80°C. Striatal tissue from one hemisphere was used for monoamine assays, and the tissue from the other hemisphere was used for [3H]mazindol binding assays.

Determination of neurotransmitter concentrations.

Concentrations of dopamine and its metabolites DOPAC and HVA were quantitated by a modified HPLC method combined with electrochemical detection as described by Ali et al. (1994). Each striatum was weighed in a measured volume (20% w/v) of 0.2 N perchloric acid containing 100 ng/ml of the internal standard 3,4-dihydroxybenzylamine. The tissue was next disrupted by ultrasonication and centrifuged at 4°C (15,000 × g; 7 min). Then 150 μl of the supernatant was removed and filtered through a 0.2-μm Nylon-66 microfilter (MF-1 centrifugal filter, Bioanalytical System, W. Lafayette, IN). Aliquots of 25 μl representing 2.5 mg of brain tissue were injected directly onto the HPLC/EC system for separation of the analytes. The concentrations of dopamine, DOPAC and HVA were calculated using standard curves that were generated by determining, in triplicate, the ratio between three different known amounts of the amine or its metabolites and a constant amount of internal standard.

Binding of [3H]mazindol to the dopamine transporter.

Striatal tissue from one hemisphere of 3 to 4 mice was pooled together, homogenized in 20 volumes of Tris-HCl buffer (50 mM; pH 7.7) containing 300 mM NaCl and 5 mM KCl and centrifuged (40,000 × g; 15 min; 4°C). The pellet was resuspended in 6 volumes of sucrose (0.32 M), and aliquots were stored at −80°C. For binding assays, the tissue was resuspended in 15 volumes of the buffer to yield a protein concentration of 0.3 to 0.4 mg/ml. Saturation binding experiments were carried out in a final volume of 0.5 ml containing various concentrations of [3H]mazindol (1.0, 2.0, 4.0, 6.0, 8.0 and 10.0 nM) and desipramine (300 nM; to occlude binding to the norepinephrine transporter). Nonspecific binding was determined in the presence of 20 μM benztropine. The reaction mixture was incubated for 60 min at 4°C, and stopped by a rapid vacuum filtration (Brandel M-12) through Whatman GF/B filters presoaked for 15 min in buffer containing polyethylenimine (0.05%). The filters were washed twice with 4 ml ice-cold buffer, and radioactivity was determined by scintillation counting. Protein concentration was determined by the method of Lowryet al. (1951). The binding parameters of [3H]mazindol, e.g., the maximal number of binding sites (Bmax) and the dissociation constant (Kd ), were determined by the LIGAND program, version 2.3.10.

Measurement of body temperature.

The animals’ body temperature was measured before the first injection of METH (5 mg/kg) and every 30 min thereafter, for a total period of 120 min. The temperature measurement was taken by Thermoscan Instant Thermometer (Thermoscan, San Diego, CA), which was positioned in the animal’s ear for only 1 s. The variation between the readings from the two ears usually did not exceed 0.3°C, and the average temperature determined from both ears was considered as n = 1.

Measurement of locomotor activity.

To investigate the consequence of administration of the high dose of METH (5 mg/kg × 3) on the animals’ locomotor activity, we used three groups of animals for this set of experiments: nNOS(−/−), B6/SV129 and C57BL/6 (n = 20 for each strain). Experiments were conducted in a manner similar to the procedure previously described (Itzhak, 1997). On day 1, before any drug treatment was initiated, each mouse was placed in the test cage; this was a standard transparent rectangular rodent cage 42 × 42 × 20 cm high. The animals’ spontaneous activity was measured for a 60-min period. Then one group (n = 10) received a saline injection, and a second group (n = 10) received a relatively low dose of METH (1 mg/kg); the animals’ locomotor activity was then measured for a 60-min period. One hour later, the animals that received 1 mg/kg METH received three additional injections of METH (5 mg/kg each) in 3-h intervals, as described above for the neurochemical experiments. On day 4 (68–72 h after the last METH injection), the animals’ spontaneous activity was measured for a 60-min period. Then the animals were challenged with METH (1 mg/kg), and their locomotor activity was recorded for a 60-min period.

The animals’ activity was monitored by activity meter, Opto-Varimex Mini (Columbus Instruments, Columbus, OH), which consists of an array of 15 infrared emitter/detector pairs, spaced at 2.65-cm intervals, measuring activity along a single axis of motion. Each emitter and detector were mounted alongside the length of the cage (42 cm long). Both total counts and ambulatory counts were recorded and transferred by a computer interface to an IBM computer. Ambulatory counts correspond to horizontal activity, whereas the difference between the total and ambulatory counts corresponds to vertical activity. On the basis of our previously observations (Itzhak, 1997) and the current results, it appears that the fraction of nonambulatory counts (25–30% of the total counts) increases or decreases in parallel to corresponding changes in ambulation counts. Because of this relationship, only ambulatory counts are reported. Counts were registered every 10 min for a period of 60 to 120 min.

Statistical analysis.

Analysis of the differences in outcome between saline and METH treatments within each mouse strain was performed by Student’s t test. The results of locomotor activity were analyzed by a two-way ANOVA (drug treatment × time or strain × time) with time as the repeated measure (e.g., comparison between locomotor activity on day 1 and day 4). Bonferroni multiple comparison adjustment was performed to determine differences between specific groups.

Results

Content of striatal dopamine and its metabolites.

The concentrations of dopamine and its metabolites DOPAC and HVA in striatal tissue of mice, treated with either saline or METH (5 mg/kg × 3), are summarized in figure1. The administration of METH to nNOS(−/−) mice produce no significant change in tissue content of dopamine, DOPAC or HVA compared with the levels obtained after saline administration to nNOS(−/−) mice. However, the same treatment delivered to heterozygote nNOS(+/−) mice revealed a 35% decrease in dopamine concentration (P < .05) compared with control. Administration of METH to wild-type B6/SV129 mice resulted in 45, 37 and 35% decreases in the concentration of dopamine, DOPAC and HVA, respectively, compared with saline treatment (P < .01; fig. 1). The same treatment given to C57BL/6 mice caused 68, 61, and 45% losses in dopamine, DOPAC and HVA concentrations, respectively, compared with saline treatment (P < .01; fig. 1). Finally, administration of METH to SV129 mice produced 37, 35 and 39% decreases in the levels of dopamine, DOPAC and HVA, respectively, compared with the saline treatment (P < .01; fig. 1). These findings suggest that nNOS(−/−) mice are markedly protected against METH-induced depletion of dopamine and its metabolites.

Figure 1
Figure 1

Concentration of dopamine (panel A), DOPAC (panel B) and HVA (panel C) in mouse striatum. Homozygote nNOS(−/−), heterozygote nNOS(+/−) and wild-type B6/SV129, C57BL/6 and SV129 mice were administered either saline (control; n = 10–12 for each mouse strain) or METH (5 mg/kg × 3; n = 10–15 for each mouse strain). The concentrations of dopamine, DOPAC and HVA were determined 72 h after the last METH injection. Results are presented as mean ± S.E.M. * P < .05; ** P < .01 Student’s t test for the comparison between control and METH for each mouse strain.

Dopamine transporter binding sites.

Saturation binding experiments of [3H]mazindol to the DAT were carried out to assess further the dopaminergic neurotoxicity caused by administration of high doses of METH. The binding parameters of [3H]mazindol (in the presence of 300 nM desipramine) to striatal tissue are summarized in table1. Administration of METH to B6/SV129, C57BL/6 and SV129 mice resulted in 60, 66 and 49% decreases, respectively, in the number of [3H]mazindol binding sites, compared with saline administration. However, administration of METH to heterozygote nNOS(+/−) and homozygote nNOS(−/−) mice resulted in only 32 and 12% decreases, respectively, in [3H]mazindol binding sites, compared with saline treatment (table 1). These findings suggest, again, that nNOS(−/−) mice are relatively protected against METH-induced loss of DAT binding sites.

View this table:
Table 1

Binding parameters of [3H]mazindol to striatal tissue of different mouse strains

Body temperature.

We observed, 30 and 60 min after the administration of the first dose of METH (5 mg/kg) to heterozygote nNOS(+/−), B6/SV129, C57BL/6 and SV129 mice, a marked increase in body temperature, compared with the animals’ temperature measured before METH administration (36.11 ± 0.15°C). As indicated in figure2, the maximal hyperthermic effect was observed 60 min after METH administration (38.23 ± 0.2 to 38.74 ± 0.15°C). However, METH administration had no significant effect on the body temperature of homozygote nNOS(−/−) mice (fig. 2). In some experiments, the animals’ temperatures were also measured after the third injection of METH. Results indicated no significant differences between the effect of the first and the third METH injection on the animals’ temperature (data not shown).

Figure 2
Figure 2

Effect of METH on animals’ body temperature. The mice’s body temperatures (n = 10–15 for each strain) were taken 0 to 10 min before the administration of METH (5 mg/kg) and every 30 min thereafter for a total period of 120 min. Results are presented as mean ± S.E.M. A significant (** P < .01 paired Student’s t test) increase in body temperature was observed 30 and 60 min after METH administration in all mouse strains examined except the homozygote nNOS(−/−).

Locomotor activity.

Because we observed that the administration of high doses of METH (5 mg/kg × 3) had differential effects on homozygote nNOS(−/−) and wild-type animals, we sought to determine how the absence and presence of nNOS and dopaminergic neurotoxicity would correlate with the animals’ spontaneous locomotor activity, as well as with METH-induced locomotor stimulation. To this end, we chose to investigate 3 out of the 5 animal strains we had. Homozygote nNOS(−/−), B6/SV129, and C57BL/6 were chosen because the nNOS(−/−) mice displayed practically no dopaminergic neurotoxicity, whereas the B6/SV129 and C57BL/6 mice developed the greatest dopaminergic neurotoxicity (fig. 1; table 1). Moreover, except for the deletion of the nNOS gene, the genetic backgrounds of the B6/SV129 and nNOS(−/−) mice are the most similar. The spontaneous locomotor activity of the three groups of mice before the administration of any drug had a typical pattern. Initially, the animals’ locomotor activity was relatively high, and then, as the animals became familiar with the new environment (the test cage), locomotor activity declined over time (fig.3). A two-way ANOVA (strain × time) revealed nonsignificant strain effect (P = .09) [time effect (P = .0098) and interaction (P = .142)]. Spontaneous locomotor activity was determined again on day 4, after the administration of METH (1 mg/kg followed by 5 mg/kg × 3) on day 1. Results presented in figure 3 (bottom panel) indicate that spontaneous locomotor activity after the administration of METH on day 1 did not change compared with that recorded before the administration of METH. The following cumulative ambulation counts were registered for the 60-min period for the three animal strains on day 1 and day 4. nNOS(−/−): day 1, 4893 ± 381; day 4, 5125 ± 423. B6/SV129: day 1, 4186 ± 526; day 4, 4462 ± 582. C57BL/6: day 1, 4738 ± 320; day 4, 4269 ± 501. Thus ambulation counts on days 1 and 4 were not significantly different from each other for any of the groups tested.

Figure 3
Figure 3

Spontaneous locomotor activity of homozygote nNOS(−/−) and wild-type B6/SV129 and C57BL/6 mice (n= 20 for each strain) before (day 1) and after (day 4) the administration of a neurotoxic dose of METH. On day 1, each mouse was placed in a test cage, and ambulatory counts were registered every 10 min for a 60-min period. On day 1, a two-way ANOVA (strain × time) revealed a nonsignificant strain effect (P = .09), a significant time effect (comparison between the first and last 10-min intervals P = .0098) and nonsignificant interaction (P = .142). Spontaneous locomotor activity was measured again on day 4 (after the administration of 1.0 mg/kg and 5 mg/kg × 3 METH on day 1). A two-way ANOVA (strain × time) for comparison between locomotor activity on day 1 and that on 4 revealed nonsignificant strain (P = .12) and time (P = .18) effects.

METH-induced locomotor activity was also investigated before and after the administration of the high dose of METH (5 mg/kg × 3). On day 1, the administration of 1 mg/kg METH caused a marked increase in locomotor activity in all three groups tested. Total ambulation counts registered for a 60-min period after saline or METH (1 mg/kg) administration to nNOS(−/−), B6/SV129 and C57BL/6 mice are summarized in figure 4. The increases in locomotor activity after METH administration to the three groups on day 1 were very similar in magnitude (fig. 4). METH-induced locomotor activity was recorded again on day 4, after the administration of METH (5 mg/kg × 3) on day 1. Results presented in figure 4 indicate that the challenge METH (1 mg/kg) administered on day 4 had a differential effect on the nNOS(−/−) mice compared with the two wild-type groups, B6/SV129 and C57BL/6. METH challenge given to nNOS(−/−) on day 4 resulted in locomotor activity similar in intensity to that on day 1 (fig. 4). However, METH challenge given to B6/SV129 and C57BL/6 mice resulted in a significant sensitized response. METH-induced ambulation counts on day 4 were almost double the counts recorded on day 1 (B6/SV129: day 1, 4589 ± 645; day 4, 9176 ± 1020; P = .001. C57BL/6: day 1, 5971 ± 831; day 4, 10,268 ± 750 P = .001). These findings indicate that mice deficient in nNOS did not develop sensitization to subsequent METH administration, whereas the wild-type animals developed robust sensitization to METH despite the fact that the prior METH exposure produced a 60 to 66% decrease in dopamine transporter binding sites (table 1).

Figure 4
Figure 4

Effect of METH on locomotor activity of the mice. Homozygote nNOS(−/−) and wild-type B6/SV129 and C57BL/6 mice were divided into two groups (n = 10 each). On day 1, animals were placed in the test cage for 60 min and then received either saline or METH (1.0 mg/kg), and ambulation counts were recorded for a 60-min period. On day 4 (after exposure to the neurotoxic dose of METH on day 1), animals were placed in the test cage for 60 min and then received METH (1.0 mg/kg), and ambulation counts were recorded for a 60-min period. A two-way ANOVA (drug treatment × time) with time as the repeated measure yielded a significant interaction (P = .0001). Bonferroni multiple comparison adjustment was performed to determine specific group differences. On day 1, METH vs.saline: *a P = .0001. Comparison between day 1 and day 4 for the METH group: *b P = .0001.

Discussion

The present study suggests that the nNOS isoform plays a major role in the dopaminergic neurotoxicity produced by the administration of a high dose of METH. Mice lacking the nNOS gene were protected against METH-induced depletion of dopamine, DOPAC and HVA and of dopamine transporter binding sites. nNOS(−/−) mice did not display any overt behavioral deficits compared with wild-type animals, and they were responsive to METH-induced locomotor stimulation. However, unlike wild-type animals, nNOS(−/−) mice did not develop behavioral sensitization as a consequence of the prior exposure to METH. Thus our data underscore the role of nNOS in METH-induced dopaminergic neurotoxicity and behavioral sensitization.

nNOS and dopaminergic neurotoxicity.

Two major findings suggest that mice lacking the nNOS gene are protected against METH-induced dopaminergic neurotoxicity. First, the administration of a high dose of METH (5 mg/kg × 3) to nNOS(−/−) mice did not produce depletion in striatal content of dopamine, DOPAC and HVA, where administering the same treatment to heterozygote nNOS(+/−) mice produced a 35% depletion in dopamine. A greater decrease in dopamine concentration and in that of its metabolites was observed after the administration of METH to wild-type animals (fig. 1). Second, METH administration to homozygote nNOS(−/−), heterozygote nNOS(+/−) and wild-type animals resulted in 12, 35 and 49 to 66% depletion in DAT binding sites, respectively (table 1).

In the present study, we investigated three different animal strains as the wild-type controls—B6/SV129, C57BL/6 and SV129—because the genetic background of our nNOS(−/−) mice includes contributions from both SV129 and C57BL/6 strains. Thus the B6/SV129 strain may be considered congenic to the nNOS(−/−) mice. We also studied the C57BL/6 and SV129 strains because other investigators often use only these two animal strains as “wild-type” controls (e.g.,Huang et al., 1994). Clearly, the genetic background of the mouse may have profound implications for both METH-induced neurotoxicity and locomotor stimulation. In fact, it appears that the SV129 strain was somewhat less susceptible to METH-induced dopaminergic neurotoxicity than were C57BL/6 and B6/SV129 mice (fig. 1; table 1), and it has been reported that SV129 mice are insensitive to MPTP-induced neurotoxicity (Przedborski et al., 1996). Results in figure 1 also indicate that control tissue contents of dopamine and its metabolites in homozygote nNOS(−/−) and heterozygote nNOS(+/−) mice were lower than in wild-type animals. Further studies are required to determine whether deletion of the nNOS gene is related to the reduced level of striatal dopamine and whether diminished basal dopamine level is correlated with protection against the development of dopaminergic neurotoxicity. On the other hand, the finding that the spontaneous and METH-induced (day 1) locomotor activities of the mutant and wild-type animals were the same (e.g., figs. 3 and 4) suggests that the strain difference in tissue content of dopamine has no effect on the expression of locomotor activity.

Increasing evidence now supports the role of brain NOS in dopaminergic neurotoxicity. First, we (Itzhak and Ali, 1996) and others (Di Monteet al., 1996) have recently reported that pharmacological manipulation of brain NOS by 7-NI, a relatively selective inhibitor of nNOS, provided complete protection against METH-induced neurotoxicity in Swiss Webster mice. Also, L-NG-nitro-arginine methyl ester was reported to protect against METH-induced dopaminergic neurotoxicity in rats (Abekawa et al., 1996). Second, it has been reported that blockade of NOS by L-NG-nitro-arginine and monomethyl-L-arginine in primary cultures of mesencephalic cells protected against METH-induced neurotoxicity in vitro (Shenget al., 1996). Third, 7-NI blocked MPTP-induced neurotoxicity in vivo (Schulz et al., 1995;Hantraye et al., 1996; Przedborski et al., 1996), and a significant attenuation of MPTP-induced neurotoxicity was observed in mice lacking the nNOS gene (Przedborski et al., 1996). Fourth, striatal malonate lesions were significantly attenuated in nNOS(−/−) mice (Schulz et al., 1996).

It is hypothesized that NO, superoxide radicals, and peroxynitrite may be involved in both METH- (Sheng et al., 1996) and MPTP- (Schulz et al., 1995; Przedborski et al., 1996) induced neurotoxicity. The results of our previous study (Itzhak and Ali, 1996) and of the current investigation are in accord with these reports and suggest that the deficiency in neuronal NO in nNOS(−/−) mice may be associated with the reduced formation of free radicals thought to contribute to METH toxicity (De Vito and Wagner, 1989; Cadetet al., 1995).

The role of temperature in METH-induced neurotoxicity was extensively investigated (e.g., Bowyer et al., 1992; Aliet al., 1994; Miller and O’Callagahan, 1994; Kupermanet al., 1997). It has been reported that both lowering of the ambient temperature and administration of agents that cause hypothermia significantly reduce METH-induced neurotoxicity (Bowyeret al., 1992; Ali et al., 1994; Miller and O’Callagahan, 1994). In the present study, the administration of METH to the different animal strains examined caused a significant hyperthermia (increase of 2.8 ± 0.12°C) in all animal strains except the nNOS(−/−) (fig. 2). The finding that nNOS(−/−) mice were protected against METH-induced neurotoxicity and hyperthermia supports the hypothesis that these may be related occurrences. However, it appears that 7-NI, which blocks brain NOS and affords protection against METH-induced neurotoxicity, did not alter METH-induced hyperthermia in Swiss Webster mice (Itzhak and Ali, 1996; Di Monteet al., 1996). Thus, although some evidence supports the association between METH-induced hyperthermia and neurotoxicity, other findings do not always support this relationship.

Behavioral consequences of the administration of a high dose of METH.

The development of behavioral sensitization to amphetamines is usually investigated via the administration of intermittent relatively low, non-neurotoxic doses of amphetamines. The induction of behavioral sensitization to amphetamines has been linked to amphetamine-induced psychosis in humans (Robinson and Becker, 1986;Segal and Kuczenski, 1997) and to the development of drug craving (Robinson and Berridge, 1993).

In the present study, we sought to investigate the behavioral outcome (e.g., locomotor activity) of exposure to a neurotoxic dose of METH. We determined the spontaneous and the METH-induced locomotor activity of nNOS(−/−), B6/SV129 and C57BL/6 mice before and after exposure to the high dose of METH. We had chosen the nNOS(−/−) mice because they were protected against METH-induced neurotoxicity, whereas C57BL/6 and B6/SV129 mice endured the greatest dopaminergic neurotoxicity. Results presented in figure 3 demonstrate that there was no significant strain difference in the animals’ spontaneous locomotor activity. Also, the neurotoxic regimen of METH did not produce behavioral deficits in any of the mouse strains tested (fig. 3, comparison between spontaneous locomotor activity on day 1 and day 4). Thus mice lacking the nNOS gene, or mice that endure more than 50% decrease in striatal dopamine and dopamine transporter (e.g., B6/SV129 and C57BL/6), did not express any overt abnormalities in spontaneous locomotor activity. The finding that the nNOS(−/−) and wild-type mice were similar in their initial response to METH (1 mg/kg) (fig. 4; day 1) suggests that deletion of nNOS had no effect on the locomotor activity generated after a single administration of METH.

The only distinction between nNOS(−/−) mice and the wild-type controls was their response to the challenge METH injection given on day 4. Whereas B6/SV129 and C57BL/6 mice developed marked locomotor sensitization to the challenge METH, nNOS(−/−) mice did not develop such sensitization. These findings suggest two things: First, nNOS(−/−) mice are protected against METH-induced dopaminergic neurotoxicity and locomotor sensitization. Second, despite the fact that the prior exposure of wild-type animals to METH resulted in a marked deficiency in striatal dopamine content and DAT binding sites, the animals became sensitized to the subsequent METH injection. This finding supports previous studies demonstrating that rats may develop sensitization to amphetamine even when striatal dopamine is depleted by over 80% (Robinson et al., 1990). Because nucleus accumbens dopamine levels were less severely depleted by prior METH exposure, the authors suggested that the behavioral sensitization they observed may be associated with enhanced dopamine release in the nucleus accumbens (Robinson et al., 1990). In the present study, we measured the level of dopamine and DAT binding sites in the mouse striatum, which includes the caudate putamen and the nucleus accumbens. Thus it is unclear whether the lack of effect of depletion of dopamine and DAT on the psychomotor stimulating effect of METH in wild-type animals is due to the resistance of the nucleus accumbens to METH-induced neurotoxicity. Although it is thought that psychostimulant-induced increase in extracellular dopamine level within the nucleus accumbens is a major mechanism in sensitization (Robinson and Becker, 1986;Kalivas and Stewart, 1991; White and Wolf 1991), other studies have shown persistent behavioral sensitization that coincides with a diminished dopamine response in the nucleus accumbens (Segal and Kuczenski 1992; Kalivas and Duffy 1993).

It also appears that a partial depletion of DAT binding sites may not modify the response to psychostimulants. For instance, whereas homozygote mice (−/−) lacking the DAT binding site were insensitive to the effects of cocaine and amphetamine,heterozygote mice (+/−) responded to these psychostimulants, as did the wild-type controls (Giros et al., 1996). Regardless of the role of DAT in the development of behavioral sensitization to METH, the present study suggests that the deficit in nNOS in the mutant mice attenuated this process.

Our suggestion that brain NOS plays a role in the psychomotor stimulating effect of METH stems also from our recent studies showing that 7-NI attenuated the induction of locomotor sensitization produced by repeated administration of a low dose of METH (1 mg/kg) for 5 days (Itzhak, 1997). Moreover, 7-NI pretreatment also blocked the development of cross-sensitization between cocaine and METH (Itzhak, 1997). Thus both the pharmacological manipulation of brain NOS and the deletion of the gene that encodes for nNOS appear to protect against the development of locomotor sensitization to METH.

Dopamine/glutamate/NO interactions.

The mechanism underlying the protection against METH-induced dopaminergic neurotoxicity afforded by deletion of nNOS remains to be investigated. Because evidence suggests that METH-induced neurotoxicity is associated with an increase in glutamate release in the striatum (Nash and Yamamoto, 1992), and because NOS inhibitors attenuate NMDA receptor-mediated neurotoxicity (Garthwaite, 1991; Snyder, 1992; Dawson et al., 1993), it is possible that deletion of nNOS provides protection against the glutamatergic-mediated neurotoxicity that arises from the administration of a high dose of METH. Although dopaminergic neurotoxicity may correlate with increased glutamatergic activity, amphetamine-induced behavioral sensitization (by the administration of relatively low doses) may not necessarily overlap with an increase in extracellular glutamate level in rat ventral tegmental area and nucleus accumbens (Xue et al., 1996). Nevertheless, the interactions between nigrostriatal dopamine and corticostriatal glutamate transmission, and particularly the hypothesis that dopamine depletion is associated with a subsequent increase in glutamatergic transmission (Carlsson and Carlsson, 1990), may support the following theory: METH-induced dopamine depletion is associated with increased glutamatergic activity, which subsequently leads to stimulation of nNOS. The increase in NO levels may further modulate the release of dopamine and glutamate (Lonart et al., 1993; Montagueet al., 1994), which contribute to both neurotoxicity and behavioral sensitization.

Another possibility is that direct dopamine/NO interactions may modulate the outcome of psychostimulant action. For instance, it has been proposed that diminishing brain NO levels attenuate psychostimulant-induced dopamine release (Bowyer et al., 1995). Several studies have indicated that NO causes the release of striatal dopamine and that blockade of NOS, in vitro andin vivo, diminishes dopamine release (Strasser et al., 1994; Lonart et al., 1993; Zhu and Luo, 1992). However, this subject remains controversial, because other studies suggest that NOS inhibitors cause an increase in dopamine release (Silva et al., 1995; Shibata et al., 1996), and our previous studies indicated that 7-NI had no significant effect on the content of striatal dopamine and its metabolites (Itzhak and Ali, 1996). Additional studies are required to determine whether, and how, dopamine/NO or dopamine/glutamate/NO interactions underlie the mechanism by which the absence of nNOS affords protection against METH effects.

In summary, the present study provides evidence that the nNOS isoform plays a role in the dopaminergic neurotoxicity and the psychomotor stimulation produced by methamphetamine. These findings may have implications for the development of new therapeutics for management of the effects of psychostimulants, as well as better medications for the treatment of neurodegenerative disorders such as Parkinson’s disease.

Footnotes

  • Send reprint requests to: Yossef Itzhak, Ph.D., Department of Biochemistry and Molecular Biology (R-629), University of Miami School of Medicine, P.O. Box 016129, Miami, FL 33101.

  • ↵1 This study was supported by the National Institute on Drug Abuse (DA08584, to Y.I.) and by the National Institute of Neurological Disease and Stroke (NS33335, to P.L.H.).

  • Abbreviations:
    METH
    methamphetamine
    DAT
    dopamine transporter
    nNOS
    neuronal nitric oxide synthase
    NO
    nitric oxide
    DOPAC
    3,4-dihydroxyphenylacetic acid
    HVA
    homovanillic acid
    7-NI
    7-nitroindazole
    NMDA
    N-methyl-D-aspartate
    ANOVA
    analysis of variance
    MPTP
    1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
    EAA
    exitatory amino acid
    • Received August 6, 1997.
    • Accepted November 17, 1997.

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OtherNEUROPHARMACOLOGY

Resistance of Neuronal Nitric Oxide Synthase-Deficient Mice to Methamphetamine-Induced Dopaminergic Neurotoxicity

Yossef Itzhak, Carlos Gandia, Paul L. Huang and Syed F. Ali
Journal of Pharmacology and Experimental Therapeutics March 1, 1998, 284 (3) 1040-1047;
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