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Seizure Activity and Hyperthermia Potentiate the Increases in Dopamine and Serotonin Extracellular Levels in the Amygdala during Exposure to d-Amphetamine

Seizure Activity and Hyperthermia Potentiate the Increases in Dopamine and Serotonin... Abstract Behavioral stereotypy, hyperthermia, and convulsive activity produced by exposure to multiple doses of d-amphetamine (AMPH) were related to changes in the extracellular levels of dopamine and serotonin (5-HT) in the amygdala, using the technique of microdialysis in awake and freely moving rats. Hyperactivity and stereotypy, as well as increases in microdialysis dopamine levels ranging from 100–300% of pre-AMPH basal microdialysate levels (BL), occurred during exposure to 3 doses of 2.5 mg/kg (3 × 2.5 mg/kg) AMPH. Three doses of 5 mg/kg produced a more intense stereotypic behavior as well as hyperthermia, and resulted in large increases in the peak dopamine levels (700% BL) while 5-HT levels were increased to a lesser extent (300% BL). The highest doses tested of 3 × 15 mg/kg produced convulsive activity, seizures, intense stereotypy and hyperthermia with peak microdialysate dopamine (1300% BL) and 5-HT levels (1800% BL) that were 2-fold and 6-fold greater, respectively, than those at the 3 × 5-mg/kg doses. Microdialysate glutamate levels were not changed by AMPH exposure. Rats that did not become hyperthermic when dosed with 15 mg/kg AMPH in a cold environment (10°C) exhibited some hyperactivity and stereotypic behavior, but not overt convulsive behavior. Dopamine and 5-HT levels in these rats were significantly reduced by about 75% and 60%, respectively, compared to the room-temperature group. Inclusion of 2 μM tetrodotoxin (TTX) in the microdialysis buffer significantly reduced the 15-mg/kg AMPH-induced increases in dopamine by 30% and the increase in 5-HT levels by 70% at room temperature. These results indicate that a smaller portion of the dopamine release evoked by doses of AMPH that induce seizure activity is neuronal impulse-dependent while the majority of 5-HT released is impulse-dependent. Irrespective of impulse activity, the hyperthermia alone markedly potentiated dopamine release but had a lesser effect on 5-HT release. Thus, there are differences in the regulation of dopamine and serotonin release in the amygdala from high doses of AMPH, which are known to produce neurotoxicity. Further studies are necessary to determine the impact of these differences in release on AMPH neurotoxicity. seizures, neurotoxicity, amphetamine, amygdala, dopamine, serotonin Exposure to amphetamine (AMPH) and methamphetamine (METH), at moderate to high doses, can produce intense stereotypic behavior (Koob, 1998; Nakatani and Hara, 1998; Seiden et al., 1993), fear and aggression (Cherek et al., 1989; Seiden et al., 1993; Siegel et al., 1997) and seizures (Callaway and Clark, 1994; Derlet et al., 1992; Kalant and Kalant, 1975) in humans as well as laboratory animals. A state produced in humans from METH intoxication, termed a “delirium and twilight state,” in which consciousness is compromised, may indicate that neuronal activity very similar to limbic seizures can occur without overt generalized motor seizures (Nakatani and Hara, 1998). Also, exposure to multiple doses of either AMPH or METH that result in seizure activity produce neuronal degeneration in the limbic system and thalamus of rodent (Bowyer et al., 1998; Schmued and Bowyer, 1997). In particular, the amygdala has been implicated as playing an important role in the previously described behaviors as well as in animal models of limbic seizures (Kelly and McIntyre, 1996; Koob, 1998; McNamara, 1986; Siegel et al., 1997). Normally, minimal somatic neuronal degeneration is seen in amygdala of rodents exposed to seizure-genic doses of AMPH and METH, although there may be some terminal damage in this area (Bowyer et al., 1998; Schmued and Bowyer, 1997). However, there are extensive connections between the amygdala and the limbic areas sensitive to neurodegeneration produced by AMPH-induced seizures (Amaral and Witter, 1995; Jakab and Leranth, 1995; Shipley et al., 1995). Reciprocal innervation between the amygdala and ventral tegmental area (VTA) and substantia nigra compacta (SNC) also exist (Ben-Ari et al., 1975; Fallon and Loughlin, 1995; Fuxe et al., 1974). Direct and multisynaptic pathways are present between the amygdala and the dorsal raphe nucleus (DRN), involving 5-hydroxytryptamine (serotonin) (5-HT) receptors (Bufton et al., 1993; Cheng et al., 1998; Morales et al., 1996; Rainnie, 1999; Tecott et al., 1993). Therefore, it is likely that doses of AMPH, which produce intense stereotypic activity and seizures, directly or indirectly increase neuronal impulses in the VTA, SNC, and DRN, as well as increase dopamine and serotonin release in the amygdala. In support of this hypothesis, seizures induced by electroconvulsive shock produce short-term increases in tyrosine hydroxylase mRNA and enzyme activity in the SNC, VTA, and amygdala (Leviel et al., 1990), which is indicative of increased neuronal activity due to seizures in these brain regions. Although there are a few studies on 5-HT release in the amygdala during seizure activity, kainate microinjection into the DRN nucleus results in significant 5-HT release in the amygdala (Viana et al., 1997). The kindling of seizures through electrical stimulation of the amygdala also has been proposed to result in a marked increase in gamma-aminobutyric acid (GABA) stimulation to the DRN nucleus, which initially acts as a type of feedback inhibition (During et al., 1992). The present investigation evaluated the effects of doses of AMPH that produce intense stereotypic behavior and convulsive behavior upon the release of dopamine and 5-HT in the basolateral amygdaloid complex. This is a necessary initial step towards elucidating the role of dopamine and 5-HT release in the amygdala in either the generation of amphetamine-induced seizures or the behaviors induced as a result of the seizure activity. The magnitude of neurodegeneration in the limbic system from AMPH or METH is dependent on the severity of seizure activity during exposure (Bowyer et al., 1998; Schmued and Bowyer, 1997). Thus, elucidating the role of dopamine and serotonin in the generation of seizure activity during exposure to amphetamines is important in understanding their neurotoxicity. In vivo microdialysis in the amygdala of awake and freely moving rats was used to determine whether a portion of the dopamine, released in the amygdala during exposure to multiple doses of AMPH and known to cause seizures and neurotoxicity, is tetrodotoxin (TTX) and is impulse-dependent. In other experiments, some animals given high doses of AMPH were placed in a cold room (10°C) to block the hyperthermia produced by a high dose of AMPH, to determine the temperature dependency of the dopamine release in the amygdala during AMPH exposure. The role of ambient temperature was assessed because the magnitude of increased extracellular levels of dopamine in the caudate/putamen (CPu) during AMPH exposure is temperature-dependent and hyperthermia almost invariably precedes convulsive behavior (Bowyer et al., 1993, 1998, 2000). The doses chosen in the present studies ranged from that necessary to induce hyperactivity and some stereotypy (2.5 mg/kg) to those that produce seizures and neurodegeneration (15 mg/kg). MATERIALS AND METHODS Animals, housing conditions, and dosing. Male Sprague-Dawley rats (Crl:COBS CD [SD] BR), 4–5 months old, were obtained from the breeding colony of the National Center for Toxicological Research (NCTR). The Institutional Animal Care and Use Committee of the NCTR approved all the procedures involving animals. Studies were carried out in accordance with the declaration of Helsinki and the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. Rats used for microdialysis were individually housed starting on the day of guide-cannula implantation until sacrifice at several hours to 1 day post microdialysis. Each rat was transferred into a microdialysis bowl 0.5 h before initiation of microdialysis. For these experiments, the d-amphetamine sulfate and TTX were purchased from Sigma Chemical Company (St. Louis, MO). The d-amphetamine was dissolved in normal saline and injected ip. The TTX was initially dissolved in 1 mM citric acid at a concentration of 1 mM, to form a stock solution. This stock solution was used to make a 2-μM solution of TTX and citrate in the microdialysis buffer. A 2-μM citrate in the microdialysis buffer was used as an appropriate control for the 2 μM TTX animals. In the first experiment, the results of which are shown in Figures 1 through 3 and at the top of Table 1, rats were given the same dose of either AMPH or saline every 2 h for a total of 3 doses. The doses tested were 3 × 2.5 mg/kg, 3 × 5 mg/kg, 3 × 15 mg/kg AMPH, or 3 × saline. In the second experiment, the results of which are shown in Figure 4 and at the bottom of Table 1, rats were given a single dose of 15 mg/kg AMPH at either room temperature (23°C) or in a cold environment (10°C). Tetrodotoxin was included in the microdialysis buffer of some of the animals dosed at room temperature. Brain microdialysis. Implantation of microdialysis guide cannulae and amygdaloid microdialysis were carried out in the manner previously described (Clausing et al., 1995). CMA microdialysis equipment (Carnegie Medicine, Stockholm, Sweden) was used and CMA/12 guide cannulae were implanted at an angle of 8° lateral to horizontal into the basolateral amygdaloid complex. The desired coordinates of the tip of the guide cannulae were AP–3.0 mm, LAT 4.5 mm, DV 7.5 mm relative to bregma (Paxinos and Watson, 1995). The microdialysis buffer was composed of 145 mM NaCl, 1.5 mM KCl, 1.5 mM MgCl2*6 H2O, 1.25 mM CaCl2*2 H2O, 1 mM glucose, 1.5 mM K2HPO4, adjusted to pH 7.0 with HCl. After surgery to implant the microdialysis guide cannula, each rat was allowed a recovery period of 5 to 7 days. On the morning of the experiment, each rat was hand-held as the CMA/12 dialysis probe (2 mm probe tip) was carefully inserted through the guide cannula into the right amygdala. Microdialysate flow rate was held at 1.0 μl/min throughout, and fractions were collected every 20 min. In order that the aromatic monoamine levels in the microdialysate reached a relatively stable baseline, dosing did not begin until 2 h or more after probe insertion. Tubes that collected the fractions each contained 2 μl of 0.25 M phosphoric acid to acidify and stabilize the aromatic monoamines in the microdialysate as the fraction was collected. Each 20-min aliquot was immediately frozen on dry ice. The frozen aliquots were then transferred to a –150°C freezer until analysis. Experiments were carried out either at room temperatures of 22–23°C or in a cold room held at 10 to 11°C. Rectal temperatures (core body temperatures) were determined as previously described by Bowyer et al. (2000). However, these rectal temperatures were only taken when rats became sluggish or collapsed from hyperthermia or at the end of the experiment, in order to avoid damaging the microdialysis equipment and implanted probe. To prevent the lethal effects of severe hyperthermia, when the temperature of a rat exceeded 41.3°C, crushed ice was placed in the microdialysis bowl for 15 to 25 min, removed, and the inside of the bowl dried. After microdialysis, each rat was sacrificed, and the brain removed and fixed in 4% formalin for later verification of microdialysis probe location as previously described (Clausing et al., 1995). We used only those animals in which the microdialysis probe tip was within 0.7 mm of the ideal AP and LAT coordinates and 0.4 mm of the ideal DV coordinates. In vitro probe recovery was performed after the probe was used in vivo to assess its functionality. The results of the recovery tests were used to exclude data from probes with poor efficiency. The in vitro probe recoveries for dopamine, DOPAC, homovanillic acid (HVA), 5-HT, and 5-hydroxy-indole acetic acid (5-HIAA) for the probes ranged from 14 to 20% [concentration in collected sample × 100)/concentration in standard solution] at 23°C and 1-μl/min flow rate. HPLC-quantitation of neurotransmitter, metabolites and AMPH levels in amygdaloid microdialysate. The frozen aliquots were rapidly thawed (for less than 30 s) and 11 μl (1/2 the total) of the microdialysate aliquot was added to 15 μl HPLC mobile phase. The sample was then immediately injected onto the HPLC for analysis. Microdialysate levels of dopamine, DOPAC, HVA, 3-methoxytryamine, 5-HT, and 5-HIAA were determined using reverse-phase HPLC, by methods similar to Stephans et al. (1998). The mobile phase consisted of 40 mM sodium acetate, 25 mM citric acid, 1.5 mM octane sulfonic acid, 1.5 mM EDTA and 6% methanol at pH of 3.8, adjusted using acetic acid. A Supelcosil LC18 3 μm analytical column (75 × 2.1 mm (Supelco, Bellefonte, PA) was used for separation, and a BAS-LC4B amperometric detector with a BAS-LC-17 oxidative flow cell was used for detection, with the potential working electrode potential set at 0.65V. Dopamine, DOPAC (dihydroxyphenylacetic acid), and 5-HIAA could be quantitated at 0.33 pg/10 μl microdialysate, while 5-HT and HVA were measurable at the 0.75 pg/10 μl levels and 3-methoxytyramine at the 2 pg/10 μl level. The HPLC retention times were 3.25 min (DOPAC), 4.7 min (dopamine), 5.95 min (5-HIAA), 10.4 min (HVA), 13.1 min (5-HT), and 15.4 min (3-methoxytyramine. The mobile phase was adjusted to insure that the unknown contaminants that frequently appear at the high sensitivity settings needed to measure biological amines in the amygdala were sufficiently separated from dopamine and 5-HT. In some animals, glutamate, as well as AMPH, levels were determined in amygdaloid microdialysate using the remaining 11 μl of the microdialysate fraction aliquot not used for aromatic monoamine analysis (Fig. 3). Methods adapted by Bowyer et al. (2000), which were originally developed as methods for detecting amino acids (Godel et al., 1984), were used for detecting both glutamate and AMPH. The only changes necessary to detect glutamate, as well as AMPH, was an alteration of the elution gradient and a shift to a more basic pH. In brief, AMPH levels were determined by fluorescent detection after o-phthaldialdehyde/3-mercaptopropionic acid-derivatization and separation on a Supelcosil® LC-18 5 μm analytical column 150 mm × 4.6 mm (Supelco, Bellefonte, PA) running a gradient with 95% KH2PO4 (0.05 M, pH 7.4) + 5% methanol (mobile phase A) versus 35% KH2PO4 (0.05 M, pH 7.4) + 65% methanol (mobile phase B). Flow rate was 1.5 ml/min, and the fluorescent detector was set at γex = 340 nm and γem = 440 nm. Microdialysis samples were derivatized and injected directly using a CMA/200 autosampler. Statistics. The microdialysate values for the neurotransmitters and metabolites shown in Figures 1 through 3 were calculated in the following manner. The pre-stimulus basal microdialysate levels (BL) for each animal were determined by averaging the levels, for any particular neurotransmitter or metabolite, in the last 2 fractions (normally the 5th and 6th) prior to AMPH exposure. The values for the individual fractions, after AMPH exposure, were then calculated as a percentage of the BL. The arithmetic mean ± standard error of the mean (SEM) for each fraction was then determined from the individual values (% BL) in each group. Absolute (pgrams/10 μl microdialysate) dopamine and 5-HT levels are shown in Figure 4, because the cold environment and the tetrodotoxin reduced pre-AMPH levels to below the threshold of quantitation for over one-half the animals in these groups. Multiple groups were analyzed by a repeated-measures 2way analysis of variance (ANOVA). A post hoc Tukey's least-significant difference test was applied if significant main effects were observed. Significance was set at p ≤ 0.05. RESULTS Behavioral Effects of AMPH The behavioral observations made on the rats dosed with either 3 doses (first experiment) or 1 dose (second experiment) of AMPH are noted in Table 1. Rats administered doses of saline (3× saline) did not show behaviors similar to the AMPH groups but did show an increase in motor activity and grooming from 2 to 10 min after saline injections. Hyperactivity was noted in all the rats receiving AMPH but was not included in the table. Rats that received multiple injections were not monitored for body temperatures until they became very sluggish or inactive (see Materials and Methods for explanations) from the AMPH exposure. Therefore, the degree of severe hyperthermia that is noted in Table 1 is, in some cases, an underestimation of its occurrence. Only rats given 15 mg/kg AMPH exhibited overt behavioral seizure activity. The Effects of Multiple Doses of AMPH on Dopamine, 5-HT, and Metabolites The effects of the multiple injections of saline or the various doses of AMPH on dopamine levels in the microdialysate can be seen in Figure 1. Although not shown, the pre-AMPH-basal microdialysate dopamine levels (-0.33- and 0.0-h time points) ranged from 0.6 ± 0.4 to 1.2 ± 0.5 pg/10 μl among the 4 groups, and these basal levels did not vary significantly. However, after AMPH exposure, the levels for the 3 × 5-mg/kg and 3 × 15-mg/kg groups were significantly greater than 3 × saline (see Fig. 1). The increases due to 3 × 2.5-mg/kg AMPH were not quite statistically significant over all 3 doses. Basal microdialysate DOPAC levels ranged from 32.6 ± 9.0 to 45.9 ± 11.0 pg/10 μl for the groups, and did not vary significantly among the groups prior to AMPH exposure. Although 3 × 15 mg/kg AMPH did not significantly reduce DOPAC levels over the entire AMPH exposure interval, these decreases were significant if they were assessed after the third dose (Fig. 1). Basal microdialysate HVA levels ranged from 23.3 ± 7.3 to 35.9 ± 8.7 pg/10 μl for the groups, and did not vary significantly among the groups prior to AMPH exposure. Although the increases in the microdialysate HVA levels after multiple injections of either 3 × 5 or 3 × 15 mg/kg AMPH appeared greater than control, only the increases produced by the 3 × 15 mg/kg AMPH were significant over all 3 injections (Fig. 1). The 5-HT levels were significantly increased by AMPH in the 15-mg/kg group compared to controls and other AMPH groups, but the 2.5- and 5.0-mg/kg groups were not significantly greater than the controls over all 3 injections of AMPH (see Fig. 2). However, the increases of 5-HT in the second and third microdialysate fractions after the last 2 doses of 5.0 mg/kg AMPH were significantly (p < 0.05) greater than control. Although basal microdialysate 5-HT levels (from 0.9 ± 0.4 to 2.3 ± 1.3 pg/10 μl) for all groups prior to AMPH exposure was more variable than dopamine, this was not significant. The 5-HIAA levels were not significantly affected by AMPH exposure (see Fig. 2). Basal microdialysate 5-HIAA levels prior to AMPH exposure ranged from 277.1 ± 61.3 to 403.1 ± 60.7 pg/10 μl for the groups, and did not vary significantly among the groups. The levels of glutamate in the microdialysate of the 3 × 5-mg/kg or 3 × 15-mg/kg AMPH groups were not significantly elevated over the duration of dosing when compared to the 3× saline (Fig. 3, top graph). Basal microdialysate glutamate concentrations ranged from 2.2 ± 0.9 to 3.3 ± 1.0 μM for the groups, and did not vary significantly among the groups prior to AMPH exposure. The levels of AMPH in the amygdaloid microdialysis after 3 × 5 mg/kg or 3 × 15 mg/kg AMPH are shown in the bottom graph of Figure 3. TTX and Environmental Temperature Effects on Dopamine and 5-HT Levels during AMPH Exposure A final experiment was conducted to determine how much of the increase in dopamine and 5-HT in the microdialysate after doses of 15 mg/kg AMPH was impulse-dependent and related to body temperature. TTX (2 μM) was included in the microdialysis buffer to block the impulse-mediated release of dopamine and 5-HT release from AMPH exposure at room temperature (23°C). Another group was dosed in a cold room held at a temperature of between 10 and 11°C to prevent hyperthermia during AMPH exposure. TTX was introduced at 1.33 h, and the rats were subsequently dosed with 15 mg/kg AMPH at the 3-h time point. Control rats were switched to a microdialysis buffer with 2 μM citrate at 1.33 h, and the rats were subsequently dosed with 15 mg/kg AMPH at the 3-h time point. Cold-room rats were placed in the cold room 0.5 h prior to the start of microdialysis, and subsequently dosed with 15 mg/kg AMPH at the 3-h time point. All the rats injected with one dose of 15 mg/kg AMPH at room temperature exhibited the stereotypic behaviors, hyperthermia, and some seizure-like activity. The inclusion of TTX in the microdialysis buffer did not notably alter the behavioral response to 15 mg/kg AMPH. However, the cold environment significantly altered the behavioral response to one dose of 15 mg/kg AMPH (Table 1). Five of the 6 rats dosed at 10°C exhibited stereotypic behavior such as repetitive licking, grooming, and slow circling but did not exhibit retrograde propulsion or convulsive behavior. The temperatures of these 5 rats ranged from 34.8°C to 37.5°C at 2 h after dosing. Microdialysate DOPAC levels prior to AMPH exposure were not affected by either inclusion of TTX in the microdialysis buffer (48.2 ± 11.5 pg/10 μl, n = 12) or a cold environmental temperature (39.5 ± 12.2 pg/10 μl, n = 5) compared to control (35.2 ± 6.5 pg/10 μl, n = 8). Although pre-AMPH HVA levels were higher in the tetrodotoxin group (58.5 ± 9.7 pg/10 μl), this was not statistically significant relative to the cold room (30.8 ± 8.7 pg/10 μl) and 23°C (31.4 ± 7.0 pg/10 μl). Also, 5-HIAA levels were not affected by TTX (303.1 ± 39.2 pg/10 μl) or a cold environment (294.3 ± 28.1 pg/10 μl) compared to control (338.5 ± 42.4 pg/10 μl). TTX or a cold environment significantly reduced both the basal and AMPH-induced increases in dopamine levels in the microdialysate (Fig. 4, top graph). The basal dopamine levels over the 3 fractions for the 3 groups is not readily apparent, but the levels for both the TTX (0.23 ± 0.05 pg/10 μl, mean ± SEM, n = 8) and cold-room (0.20 ± 0.09 pg/10 μl, mean ± SEM, n = 12) groups were significantly less than the control (0.50 ± 0.07 pg/10 μl, mean ± SEM, n = 5). After AMPH exposure, not only were the microdialysate dopamine levels for both the TTX and cold-room groups significantly less than control, but the dopamine levels in the cold-room group were also significantly less than the TTX group. The cold environment or tetrodotoxin did not affect the changes in DOPAC levels after AMPH exposure. However, a 2-way RM ANOVA indicated that the increases in HVA levels, after AMPH exposure in the cold (110 ± 6% or less over all 6 fractions), were significantly less in the cold-room group (p < 0.01) compared to the 23°C group (averaged 175 ± 9% over all 6 fractions). Baseline 5-HT release was decreased significantly by TTX (0.5 ± 0.1 pg/10 μl, mean ± SEM) compared to control (1.1 ± 0.2 pg/10 μl, mean ± SEM) but was not significantly affected by a cold environment (0.8 ± 0.2 pg/10 μl, mean SEM). TTX inhibited AMPH-induced increases in the 5-HT levels in microdialysate (Fig. 4, bottom graph) to a greater extent than that observed on dopamine release. Cold environmental temperatures also significantly inhibited 5-HT levels with more inhibition evident at the later time points after AMPH exposure. Although not shown in Figure 4, the AMPH levels of 3.8 ± 0.8 μM (mean ± SEM, n = 5) 40 min after dosing the amygdaloid microdialysate for the 10°C group were comparable to the 4.2 ± 0.6 μM (mean ± SEM, n = 8) for the controls. Neither the cold environment nor tetrodotoxin affected the 5-HIAA levels after AMPH exposure. DISCUSSION The results of the present study indicate that much of the dopamine and 5-HT released in the amygdala as a result of exposure to moderate to high doses of AMPH is neuronal impulse- and temperature-dependent. Our results are in general agreement with previous in vivo microdialysis studies that examined dopamine release during exposure to low doses of AMPH (Harmer et al., 1997; Young and Rees, 1998)—that the increases in dopamine levels in microdialysate are produced by doses of 2.5 mg/kg of AMPH or less are modest to minimal. However, this may be dependent on the region of amygdala examined (Young and Rees, 1998). The lesser increases in microdialysate dopamine levels in the amygdala relative to the caudate-putamen (CPu) induced by AMPH might be expected from the lower density of dopaminergic terminals (Ben-Ari et al., 1975; Bjorklund et al., 1979; Fuxe et al., 1974; Hokfelt et al., 1977). Alternatively, it may that a lesser density of plasma-membrane dopamine transporters on the terminals in the amygdala may be responsible for differences in release (Jones et al., 1995). It is unlikely that the differences between CPu and amygdala are due to extracellular levels of AMPH. The peak levels of 0.9 to 1.3 μM AMPH that were observed in amygdaloid microdialysate in the present study after 5 mg/kg doses of AMPH were very comparable to the 0.75 to 1.4 μM AMPH levels reported for CPu microdialysate (Clausing et al., 1995). The 5-HT release in amygdaloid microdialysate after 3 × 15 mg/kg AMPH was 5- to 10-fold greater than that produced by 3 × 5 mg/kg while the 3 × 15 mg/kg dose produced only a 2-fold greater dopamine release than the 3 × 5 mg/kg dose. The greater increase in the 5-HT release compared to the dopamine release produced by 3 × 15 mg/kg AMPH may be due in part to the levels of AMPH being above the km for the plasma membrane dopamine transporter, but at or below the km for the plasma membrane serotonin transporters (Azzaro and Rutledge, 1973; Shaskan and Snyder, 1970). However, the greater increase in 5-HT levels relative to dopamine levels in amygdaloid microdialysis produced by 3 × 15 mg/kg AMPH may also be due to the 5-HT release being more impulse-dependent. Behavioral signs of seizure activity occurred frequently with the 3 × 15 mg/kg dose but seldom occurred with the 3 × 5 mg/kg AMPH (Table 1; Bowyer et al., 1998). Increased neuronal activity within the amygdala due to seizure activity could be either directly or indirectly responsible for increased dopamine and 5-HT release at the higher doses. The results of the present study indicate that about 70% of the 5-HT and 30% of the dopamine release induced by 3 × 15 mg/kg AMPH are impulse-dependent. However, it is possible that 3 × 5 mg/kg AMPH may also produce a substantial impulse-dependent release in the amygdala. Understanding the mechanisms by which AMPH-induced seizure activity would increase impulse-dependent dopamine and 5-HT release in the amygdala are important, since the amygdala is likely to be involved in the generation of AMPH-induced seizures and the associated behaviors. There is indirect evidence implicating the involvement of the basolateral amygdala in AMPH- and METH-induced seizures. The amygdala is a site where chemical and electrical stimulation can readily generate or “kindle” limbic type seizures in animals (Goddard et al., 1969; Kelly and McIntyre, 1996). Furthermore, the behavioral expression of seizure activity evoked by stimulation of the amygdala is very similar to that of AMPH-induced seizures. In addition, chronic electrical stimulation of the amygdala leads to spontaneous limbic behaviors and seizure activity (Michalakis et al., 1998) that are very similar to AMPH-induced seizures (Bowyer et al., 1998; Schmued and Bowyer, 1997). Brain regions necessary for the generation and development of amygdaloid-kindled seizures, such as the anterior piriform cortex and insular cortex (Loscher and Ebert, 1996; Mohapel and Corcoran, 1996; Piredda and Gale, 1985; Tortorella et al., 1997) are areas sensitive to neurodegeneration produced by AMPH-induced seizures (Bowyer et al., 1998). This indicates that these limbic areas are excessively stimulated and may be involved in generating AMPH-induced seizures. Some of the amygdala interconnections with other areas of the limbic system include basolateral amygdaloid efferents to the CA1 of hippocampus and afferents from the CA1 and subiculum (Amaral and Witter, 1995). The amygdala also has afferent and efferent connections with the lateral and medial septum (Jakab and Leranth, 1995). The medial and posterior cortical amygdaloid nuclei are also reciprocally innervated with the accessory olfactory bulbs (Shipley et al., 1995). Particularly relevant to the present study are the serotonergic projections from the DRN and the dopaminergic afferents from the ventral tegmentum (VTA) and substantia nigra compacta (SNC). There is a significant 5-HT innervation of the amygdala by the DRN (D'Amato et al., 1987; Savaki et al., 1985; Sette et al., 1981), and a reciprocal innervation between the amygdala and the SNC (Ben-Ari et al., 1975; Bjorklund et al., 1979; Fallon and Loughlin, 1995; Fuxe et al., 1974; Hokfelt et al., 1977). The exact mechanisms by which AMPH-induced seizure activity increase the rates of neuronal depolarization of dopaminergic neurons in the VTA/SNC and serotonergic neurons in the DRN are not known. However, repetitive electroconvulsive shock appears to increase dopamine release in the VTA and SN (Leviel et al., 1990), and kainate injection into the DRN evokes 5-HT release in the amygdala (Viana et al., 1997). Also, recent data indicate that 5-HT receptors in the amygdala can modulate 5-HT release through interneurons and GABAergic projections to the DRN (Bufton et al., 1993; Cheng et al., 1998; Morales et al., 1996; Rainnie, 1999, Tecott et al., 1993). Based on the numerous neuronal interactions between 5-HT and dopamine-containing brain regions and their interconnections with the amygdala, it is not surprising that much of the 5-HT and some of the dopamine release during AMPH-induced seizures are TTX-dependent. This impulse-dependent dopamine and 5-HT release would be expected to be additive with the more traditional non-impulse-dependent release produced by AMPH (Seiden et al., 1993). Since amphetamines can release 5-HT from blood platelets (Bak et al., 1967; Lemmer, 1973; Paasonen, 1965), it is possible that a portion of the 5-HT in the amygdaloid microdialysis could be platelet-derived rather than from nerve-terminal release. However, the majority of 5-HT release we see in the amygdala is likely neuronal and not platelet-derived because the release of 5-HT from platelets should not be sensitive to inhibition by TTX. Much of the dopamine and 5-HT release in the amygdala caused by high to moderate doses of AMPH are also sensitive to body temperature (Fig. 4). This is expected because AMPH induction of seizures in the rat is temperature-dependent, and AMPH-induced dopamine release in the CPu is also influenced by body temperature (Bowyer et al., 1993, 2000). In the present studies, the low environmental temperatures that blocked AMPH-induced hyperthermia also reduced the AMPH-induced release of dopamine by 75% and 5-HT by 50%. Furthermore, the stereotypic behaviors and seizure activity induced by 15 mg/kg d-AMPH were greatly diminished in the cool environment. These results were not due to the reduction of AMPH levels by the cool environment since no significant difference between the peak levels of AMPH in the microdialysate were observed at the two environmental temperatures. The increase in the 5-HT levels in the amygdala might actually produce a “negative-feedback” effect on seizure activity. In general, elevated 5-HT receptor levels and receptor stimulation have been reported to inhibit seizure activity (Dailey et al., 1996; Heisler et al., 1998). The lack of an increase in extracellular glutamate was, at first glance, somewhat surprising given that previous reports showed that high doses of AMPH or METH produce large increases in extracellular striatal glutamate (Abekawa et al., 1994; Nash and Yamamoto, 1992, 1993). Nevertheless, the present findings are consistent with minimal neurotoxicity observed in this region and parallel a similar relationship between the lack of change in glutamate and the absence of dopamine depletions observed in the nucleus accumbens (Abekawa et al., 1994). In summary, these results indicate that some of the dopamine and the majority of 5-HT released during exposure to convulsive doses of AMPH are neuronal impulse-dependent and related to seizure activity. In addition, the hyperthermia that occurs concurrently with seizure activity may, by itself, markedly potentiate both dopamine and 5-HT release. Further studies will be necessary to determine the roles of the dopamine and 5-HT release in amygdala in the generation and behavioral expression of seizures, and the neurodegeneration within the limbic system that is seizure-dependent. TABLE 1 The Effects of Various Doses of d-Amphetamine on Behavior     Stereotypic behaviors  Convulsive-like behaviors   Dose  n  Continuousa  Retrograde propulsions  Running fits, WDS, MCb  Myoclonic seizures  Severe hyperthermiac  aContinuous behavior includes nodding, grooming, and chewing; Retrograde Propulsions include head weaving and forepaw treading.  bWDS denotes wet-dog shakes.; MC denotes incidences of single, brief instances of myoclonus.  cSevere hyperthermia indicates animal's temperature was recorded to be above 41.0°C.  3 × 2.5 mg/kg  6  100%  0%  0%  0%  0%   3 × 5 mg/kg  8  100%  87%  13%  0%  67%   3 × 15 mg/kg  7  100%  100%  100%  57%  71%   1 × 15 mg/kg  8  100%  100%  88%  38%  50%   1 × 15 mg/kg + TTX  12  100%  100%  92%  42%  58%   1 × 15 mg/kg (10°C)  5  100%  40%  20%  0%  0%      Stereotypic behaviors  Convulsive-like behaviors   Dose  n  Continuousa  Retrograde propulsions  Running fits, WDS, MCb  Myoclonic seizures  Severe hyperthermiac  aContinuous behavior includes nodding, grooming, and chewing; Retrograde Propulsions include head weaving and forepaw treading.  bWDS denotes wet-dog shakes.; MC denotes incidences of single, brief instances of myoclonus.  cSevere hyperthermia indicates animal's temperature was recorded to be above 41.0°C.  3 × 2.5 mg/kg  6  100%  0%  0%  0%  0%   3 × 5 mg/kg  8  100%  87%  13%  0%  67%   3 × 15 mg/kg  7  100%  100%  100%  57%  71%   1 × 15 mg/kg  8  100%  100%  88%  38%  50%   1 × 15 mg/kg + TTX  12  100%  100%  92%  42%  58%   1 × 15 mg/kg (10°C)  5  100%  40%  20%  0%  0%  View Large FIG. 1. View largeDownload slide The effects of multiple doses of d-amphetamine on dopamine and metabolite levels in amygdaloid microdialysate. The changes in the microdialysate levels of dopamine, DOPAC, and HVA after 3 doses of saline or 3 doses of 2.5 mg/kg, 5 mg/kg, or 15 mg/kg of AMPH administered at a room temperature of 23°C are shown from the first experiment. Values represent arithmetic means ± SEM. There were 8 rats in the saline group, 6 in the 2.5 mg/kg, 8 in the 5-mg/kg, and 7 in the 15-mg/kg group. Repeated-measures 2-way ANOVA indicated that none of the baseline dopamine, DOPAC, or HVA levels for the AMPH groups varied significantly from the 3× saline group. However, ANOVA did show that the differences in the mean values of dopamine between treatment groups was greater than expected by chance (p = 0.003), during AMPH exposure. Tukey's post hoc test showed that the levels for the 3 × 5-mg/kg and 3 × 15-mg/kg groups differed significantly (p = 0.046 and p = 0.003, respectively) from 3× saline during AMPH dosing (time points 0.33 h to 5.67 h). The dopamine levels for the 3 × 15-mg/kg group were also significantly greater (p = 0.028) than the 2.5-mg/kg group. Only the DOPAC levels in the 15-mg/kg group were reduced over time, due to AMPH exposure, compared to control, but this was significant only after the third dose (see text). Repeated-measures 1-way ANOVA indicated that the difference in the means for the HVA levels of the treatment groups was significantly greater (p = 0.034) than expected by chance. Post hoc tests indicated that the mean HVA levels for only the 3 × 15-mg/kg group were significantly greater (p = 0.04) than 3× saline over all 3 doses. FIG. 1. View largeDownload slide The effects of multiple doses of d-amphetamine on dopamine and metabolite levels in amygdaloid microdialysate. The changes in the microdialysate levels of dopamine, DOPAC, and HVA after 3 doses of saline or 3 doses of 2.5 mg/kg, 5 mg/kg, or 15 mg/kg of AMPH administered at a room temperature of 23°C are shown from the first experiment. Values represent arithmetic means ± SEM. There were 8 rats in the saline group, 6 in the 2.5 mg/kg, 8 in the 5-mg/kg, and 7 in the 15-mg/kg group. Repeated-measures 2-way ANOVA indicated that none of the baseline dopamine, DOPAC, or HVA levels for the AMPH groups varied significantly from the 3× saline group. However, ANOVA did show that the differences in the mean values of dopamine between treatment groups was greater than expected by chance (p = 0.003), during AMPH exposure. Tukey's post hoc test showed that the levels for the 3 × 5-mg/kg and 3 × 15-mg/kg groups differed significantly (p = 0.046 and p = 0.003, respectively) from 3× saline during AMPH dosing (time points 0.33 h to 5.67 h). The dopamine levels for the 3 × 15-mg/kg group were also significantly greater (p = 0.028) than the 2.5-mg/kg group. Only the DOPAC levels in the 15-mg/kg group were reduced over time, due to AMPH exposure, compared to control, but this was significant only after the third dose (see text). Repeated-measures 1-way ANOVA indicated that the difference in the means for the HVA levels of the treatment groups was significantly greater (p = 0.034) than expected by chance. Post hoc tests indicated that the mean HVA levels for only the 3 × 15-mg/kg group were significantly greater (p = 0.04) than 3× saline over all 3 doses. FIG. 2. View largeDownload slide The effects of multiple doses of d-amphetamine on 5-HT and 5-HIAA levels in amygdaloid microdialysate. The changes in the microdialysate levels of 5-HT and 5-HIAA after 3 doses of saline or 3 doses of 2.5 mg/kg, 5 mg/kg or 15 mg/kg of AMPH administered at room temperature 23°C are shown from the first experiment. Values shown represent arithmetic means ± SEM. Repeated measures 2-way ANOVA indicated that the difference in the means for the 5-HT levels of the treatment groups was significant (p < 0.001). A post hoc test indicated that the mean 5-HT levels for the 3 × 15-mg/kg group was significantly greater than the 3× saline (p < 0.001) and the 3 × 2.5-mg/kg group (p = 0.004 and p = 0.005, respectively). However, over all 3 injections, neither the 3 × 2.5 nor the 3 × 5 mg/kg increased 5-HT levels significantly (see text). Two-way ANOVA indicated that the difference in the mean values for 5-HIAA levels in the treatment groups was not significant (p = 0.51). FIG. 2. View largeDownload slide The effects of multiple doses of d-amphetamine on 5-HT and 5-HIAA levels in amygdaloid microdialysate. The changes in the microdialysate levels of 5-HT and 5-HIAA after 3 doses of saline or 3 doses of 2.5 mg/kg, 5 mg/kg or 15 mg/kg of AMPH administered at room temperature 23°C are shown from the first experiment. Values shown represent arithmetic means ± SEM. Repeated measures 2-way ANOVA indicated that the difference in the means for the 5-HT levels of the treatment groups was significant (p < 0.001). A post hoc test indicated that the mean 5-HT levels for the 3 × 15-mg/kg group was significantly greater than the 3× saline (p < 0.001) and the 3 × 2.5-mg/kg group (p = 0.004 and p = 0.005, respectively). However, over all 3 injections, neither the 3 × 2.5 nor the 3 × 5 mg/kg increased 5-HT levels significantly (see text). Two-way ANOVA indicated that the difference in the mean values for 5-HIAA levels in the treatment groups was not significant (p = 0.51). FIG. 3. View largeDownload slide Glutamate and amphetamine levels in microdialysate after either 3 × 5 mg/kg or 3 × 15 mg/kg d-amphetamine. The levels of glutamate (upper) and AMPH (lower) in amygdaloid microdialysate after 3 doses of saline or 3 doses of either 5 mg/kg or 15 mg/kg AMPH are plotted. Values shown represent arithmetic means ± SEM. The levels of glutamate and AMPH were determined from the microdialysate of the same rats used in the first experiment to generate Figures 1 and 2. The levels of glutamate in the microdialysis were not significantly affected by either dose of AMPH. The AMPH levels were significantly higher in the 15-mg/kg group than the 5-mg/kg group (p < 0.001). FIG. 3. View largeDownload slide Glutamate and amphetamine levels in microdialysate after either 3 × 5 mg/kg or 3 × 15 mg/kg d-amphetamine. The levels of glutamate (upper) and AMPH (lower) in amygdaloid microdialysate after 3 doses of saline or 3 doses of either 5 mg/kg or 15 mg/kg AMPH are plotted. Values shown represent arithmetic means ± SEM. The levels of glutamate and AMPH were determined from the microdialysate of the same rats used in the first experiment to generate Figures 1 and 2. The levels of glutamate in the microdialysis were not significantly affected by either dose of AMPH. The AMPH levels were significantly higher in the 15-mg/kg group than the 5-mg/kg group (p < 0.001). FIG. 4. View largeDownload slide The effect of either a cold environment or TTX on dopamine and 5-HT levels in microdialysate after 15 mg/kg d-amphetamine. In the second experiment, the levels of dopamine (upper) and 5-HT (lower) after one 15-mg/kg dose of AMPH were determined at room temperature, in a cold environment, and after including 2 μM TTX in the microdialysis buffer. There were 8 rats in the room-temperature group, 12 rats in the TTX group, and 5 rats in the cold-room group. Values shown represent arithmetic means ± SEM. (Upper) Repeated-measures 2-way ANOVA with time of microdialysate collection and treatment (TTX, 23°C or a cold environment) for the pre-AMPH fractions collected between the –1.0 and 0.0 h of microdialysis showed that the mean dopamine levels among the treatment groups significantly (p = 0.01) differed. The basal dopamine levels over the 3 fractions for both the TTX and cold-room groups were significantly less (p = 0.01 and p = 0.04, respectively) than the 23°C group. Repeated-measures 2-way ANOVA after AMPH exposure showed that the differences in the mean levels among the treatment groups were significantly greater (p < 0.001) than expected by chance. Also the mean values among the different levels of fractions were significantly greater than expected (p < 0.001). The microdialysate dopamine levels for both the TTX and cold room groups were significantly less than the 23°C group (p = 0.026 and p < 0.001, respectively). The cold-room group was also significantly less (p = 0.031) than the TTX group. (Lower) Repeated-measures 2-way ANOVA with time of microdialysate collection and treatment (TTX, 23°C or a cold environment) for the pre-AMPH fractions collected between the –1.0 and 0.0 h of microdialysis showed that differences in the mean 5-HT levels among the treatment groups were significantly greater (p = 0.03) than expected by chance. Baseline 5-HT release was decreased significantly (p = 0.002) by TTX but the cold environment did not significantly reduce the basal 5-HT concentrations. Repeated-measures 2-way ANOVA after AMPH exposure showed that differences in the mean 5-HT levels among the treatment groups were significantly greater (p < 0.001) than expected by chance. Both TTX (p < 0.001) and the cold environment (p = 0.006) inhibited AMPH-induced increases in the 5-HT levels in microdialysate compared to the 23°C group. FIG. 4. View largeDownload slide The effect of either a cold environment or TTX on dopamine and 5-HT levels in microdialysate after 15 mg/kg d-amphetamine. In the second experiment, the levels of dopamine (upper) and 5-HT (lower) after one 15-mg/kg dose of AMPH were determined at room temperature, in a cold environment, and after including 2 μM TTX in the microdialysis buffer. There were 8 rats in the room-temperature group, 12 rats in the TTX group, and 5 rats in the cold-room group. Values shown represent arithmetic means ± SEM. (Upper) Repeated-measures 2-way ANOVA with time of microdialysate collection and treatment (TTX, 23°C or a cold environment) for the pre-AMPH fractions collected between the –1.0 and 0.0 h of microdialysis showed that the mean dopamine levels among the treatment groups significantly (p = 0.01) differed. The basal dopamine levels over the 3 fractions for both the TTX and cold-room groups were significantly less (p = 0.01 and p = 0.04, respectively) than the 23°C group. Repeated-measures 2-way ANOVA after AMPH exposure showed that the differences in the mean levels among the treatment groups were significantly greater (p < 0.001) than expected by chance. Also the mean values among the different levels of fractions were significantly greater than expected (p < 0.001). The microdialysate dopamine levels for both the TTX and cold room groups were significantly less than the 23°C group (p = 0.026 and p < 0.001, respectively). The cold-room group was also significantly less (p = 0.031) than the TTX group. (Lower) Repeated-measures 2-way ANOVA with time of microdialysate collection and treatment (TTX, 23°C or a cold environment) for the pre-AMPH fractions collected between the –1.0 and 0.0 h of microdialysis showed that differences in the mean 5-HT levels among the treatment groups were significantly greater (p = 0.03) than expected by chance. Baseline 5-HT release was decreased significantly (p = 0.002) by TTX but the cold environment did not significantly reduce the basal 5-HT concentrations. Repeated-measures 2-way ANOVA after AMPH exposure showed that differences in the mean 5-HT levels among the treatment groups were significantly greater (p < 0.001) than expected by chance. Both TTX (p < 0.001) and the cold environment (p = 0.006) inhibited AMPH-induced increases in the 5-HT levels in microdialysate compared to the 23°C group. 1 To whom correspondence should be addressed at NCTR, HFT-132, Jefferson, AR 72079–9502. Fax: (870) 543-7745. E-mail: jbowyer@nctr.fda.gov. 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Seizure Activity and Hyperthermia Potentiate the Increases in Dopamine and Serotonin Extracellular Levels in the Amygdala during Exposure to d-Amphetamine

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Oxford University Press
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© 2001 Society of Toxicology
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1096-6080
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1096-0929
DOI
10.1093/toxsci/60.1.103
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Abstract

Abstract Behavioral stereotypy, hyperthermia, and convulsive activity produced by exposure to multiple doses of d-amphetamine (AMPH) were related to changes in the extracellular levels of dopamine and serotonin (5-HT) in the amygdala, using the technique of microdialysis in awake and freely moving rats. Hyperactivity and stereotypy, as well as increases in microdialysis dopamine levels ranging from 100–300% of pre-AMPH basal microdialysate levels (BL), occurred during exposure to 3 doses of 2.5 mg/kg (3 × 2.5 mg/kg) AMPH. Three doses of 5 mg/kg produced a more intense stereotypic behavior as well as hyperthermia, and resulted in large increases in the peak dopamine levels (700% BL) while 5-HT levels were increased to a lesser extent (300% BL). The highest doses tested of 3 × 15 mg/kg produced convulsive activity, seizures, intense stereotypy and hyperthermia with peak microdialysate dopamine (1300% BL) and 5-HT levels (1800% BL) that were 2-fold and 6-fold greater, respectively, than those at the 3 × 5-mg/kg doses. Microdialysate glutamate levels were not changed by AMPH exposure. Rats that did not become hyperthermic when dosed with 15 mg/kg AMPH in a cold environment (10°C) exhibited some hyperactivity and stereotypic behavior, but not overt convulsive behavior. Dopamine and 5-HT levels in these rats were significantly reduced by about 75% and 60%, respectively, compared to the room-temperature group. Inclusion of 2 μM tetrodotoxin (TTX) in the microdialysis buffer significantly reduced the 15-mg/kg AMPH-induced increases in dopamine by 30% and the increase in 5-HT levels by 70% at room temperature. These results indicate that a smaller portion of the dopamine release evoked by doses of AMPH that induce seizure activity is neuronal impulse-dependent while the majority of 5-HT released is impulse-dependent. Irrespective of impulse activity, the hyperthermia alone markedly potentiated dopamine release but had a lesser effect on 5-HT release. Thus, there are differences in the regulation of dopamine and serotonin release in the amygdala from high doses of AMPH, which are known to produce neurotoxicity. Further studies are necessary to determine the impact of these differences in release on AMPH neurotoxicity. seizures, neurotoxicity, amphetamine, amygdala, dopamine, serotonin Exposure to amphetamine (AMPH) and methamphetamine (METH), at moderate to high doses, can produce intense stereotypic behavior (Koob, 1998; Nakatani and Hara, 1998; Seiden et al., 1993), fear and aggression (Cherek et al., 1989; Seiden et al., 1993; Siegel et al., 1997) and seizures (Callaway and Clark, 1994; Derlet et al., 1992; Kalant and Kalant, 1975) in humans as well as laboratory animals. A state produced in humans from METH intoxication, termed a “delirium and twilight state,” in which consciousness is compromised, may indicate that neuronal activity very similar to limbic seizures can occur without overt generalized motor seizures (Nakatani and Hara, 1998). Also, exposure to multiple doses of either AMPH or METH that result in seizure activity produce neuronal degeneration in the limbic system and thalamus of rodent (Bowyer et al., 1998; Schmued and Bowyer, 1997). In particular, the amygdala has been implicated as playing an important role in the previously described behaviors as well as in animal models of limbic seizures (Kelly and McIntyre, 1996; Koob, 1998; McNamara, 1986; Siegel et al., 1997). Normally, minimal somatic neuronal degeneration is seen in amygdala of rodents exposed to seizure-genic doses of AMPH and METH, although there may be some terminal damage in this area (Bowyer et al., 1998; Schmued and Bowyer, 1997). However, there are extensive connections between the amygdala and the limbic areas sensitive to neurodegeneration produced by AMPH-induced seizures (Amaral and Witter, 1995; Jakab and Leranth, 1995; Shipley et al., 1995). Reciprocal innervation between the amygdala and ventral tegmental area (VTA) and substantia nigra compacta (SNC) also exist (Ben-Ari et al., 1975; Fallon and Loughlin, 1995; Fuxe et al., 1974). Direct and multisynaptic pathways are present between the amygdala and the dorsal raphe nucleus (DRN), involving 5-hydroxytryptamine (serotonin) (5-HT) receptors (Bufton et al., 1993; Cheng et al., 1998; Morales et al., 1996; Rainnie, 1999; Tecott et al., 1993). Therefore, it is likely that doses of AMPH, which produce intense stereotypic activity and seizures, directly or indirectly increase neuronal impulses in the VTA, SNC, and DRN, as well as increase dopamine and serotonin release in the amygdala. In support of this hypothesis, seizures induced by electroconvulsive shock produce short-term increases in tyrosine hydroxylase mRNA and enzyme activity in the SNC, VTA, and amygdala (Leviel et al., 1990), which is indicative of increased neuronal activity due to seizures in these brain regions. Although there are a few studies on 5-HT release in the amygdala during seizure activity, kainate microinjection into the DRN nucleus results in significant 5-HT release in the amygdala (Viana et al., 1997). The kindling of seizures through electrical stimulation of the amygdala also has been proposed to result in a marked increase in gamma-aminobutyric acid (GABA) stimulation to the DRN nucleus, which initially acts as a type of feedback inhibition (During et al., 1992). The present investigation evaluated the effects of doses of AMPH that produce intense stereotypic behavior and convulsive behavior upon the release of dopamine and 5-HT in the basolateral amygdaloid complex. This is a necessary initial step towards elucidating the role of dopamine and 5-HT release in the amygdala in either the generation of amphetamine-induced seizures or the behaviors induced as a result of the seizure activity. The magnitude of neurodegeneration in the limbic system from AMPH or METH is dependent on the severity of seizure activity during exposure (Bowyer et al., 1998; Schmued and Bowyer, 1997). Thus, elucidating the role of dopamine and serotonin in the generation of seizure activity during exposure to amphetamines is important in understanding their neurotoxicity. In vivo microdialysis in the amygdala of awake and freely moving rats was used to determine whether a portion of the dopamine, released in the amygdala during exposure to multiple doses of AMPH and known to cause seizures and neurotoxicity, is tetrodotoxin (TTX) and is impulse-dependent. In other experiments, some animals given high doses of AMPH were placed in a cold room (10°C) to block the hyperthermia produced by a high dose of AMPH, to determine the temperature dependency of the dopamine release in the amygdala during AMPH exposure. The role of ambient temperature was assessed because the magnitude of increased extracellular levels of dopamine in the caudate/putamen (CPu) during AMPH exposure is temperature-dependent and hyperthermia almost invariably precedes convulsive behavior (Bowyer et al., 1993, 1998, 2000). The doses chosen in the present studies ranged from that necessary to induce hyperactivity and some stereotypy (2.5 mg/kg) to those that produce seizures and neurodegeneration (15 mg/kg). MATERIALS AND METHODS Animals, housing conditions, and dosing. Male Sprague-Dawley rats (Crl:COBS CD [SD] BR), 4–5 months old, were obtained from the breeding colony of the National Center for Toxicological Research (NCTR). The Institutional Animal Care and Use Committee of the NCTR approved all the procedures involving animals. Studies were carried out in accordance with the declaration of Helsinki and the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. Rats used for microdialysis were individually housed starting on the day of guide-cannula implantation until sacrifice at several hours to 1 day post microdialysis. Each rat was transferred into a microdialysis bowl 0.5 h before initiation of microdialysis. For these experiments, the d-amphetamine sulfate and TTX were purchased from Sigma Chemical Company (St. Louis, MO). The d-amphetamine was dissolved in normal saline and injected ip. The TTX was initially dissolved in 1 mM citric acid at a concentration of 1 mM, to form a stock solution. This stock solution was used to make a 2-μM solution of TTX and citrate in the microdialysis buffer. A 2-μM citrate in the microdialysis buffer was used as an appropriate control for the 2 μM TTX animals. In the first experiment, the results of which are shown in Figures 1 through 3 and at the top of Table 1, rats were given the same dose of either AMPH or saline every 2 h for a total of 3 doses. The doses tested were 3 × 2.5 mg/kg, 3 × 5 mg/kg, 3 × 15 mg/kg AMPH, or 3 × saline. In the second experiment, the results of which are shown in Figure 4 and at the bottom of Table 1, rats were given a single dose of 15 mg/kg AMPH at either room temperature (23°C) or in a cold environment (10°C). Tetrodotoxin was included in the microdialysis buffer of some of the animals dosed at room temperature. Brain microdialysis. Implantation of microdialysis guide cannulae and amygdaloid microdialysis were carried out in the manner previously described (Clausing et al., 1995). CMA microdialysis equipment (Carnegie Medicine, Stockholm, Sweden) was used and CMA/12 guide cannulae were implanted at an angle of 8° lateral to horizontal into the basolateral amygdaloid complex. The desired coordinates of the tip of the guide cannulae were AP–3.0 mm, LAT 4.5 mm, DV 7.5 mm relative to bregma (Paxinos and Watson, 1995). The microdialysis buffer was composed of 145 mM NaCl, 1.5 mM KCl, 1.5 mM MgCl2*6 H2O, 1.25 mM CaCl2*2 H2O, 1 mM glucose, 1.5 mM K2HPO4, adjusted to pH 7.0 with HCl. After surgery to implant the microdialysis guide cannula, each rat was allowed a recovery period of 5 to 7 days. On the morning of the experiment, each rat was hand-held as the CMA/12 dialysis probe (2 mm probe tip) was carefully inserted through the guide cannula into the right amygdala. Microdialysate flow rate was held at 1.0 μl/min throughout, and fractions were collected every 20 min. In order that the aromatic monoamine levels in the microdialysate reached a relatively stable baseline, dosing did not begin until 2 h or more after probe insertion. Tubes that collected the fractions each contained 2 μl of 0.25 M phosphoric acid to acidify and stabilize the aromatic monoamines in the microdialysate as the fraction was collected. Each 20-min aliquot was immediately frozen on dry ice. The frozen aliquots were then transferred to a –150°C freezer until analysis. Experiments were carried out either at room temperatures of 22–23°C or in a cold room held at 10 to 11°C. Rectal temperatures (core body temperatures) were determined as previously described by Bowyer et al. (2000). However, these rectal temperatures were only taken when rats became sluggish or collapsed from hyperthermia or at the end of the experiment, in order to avoid damaging the microdialysis equipment and implanted probe. To prevent the lethal effects of severe hyperthermia, when the temperature of a rat exceeded 41.3°C, crushed ice was placed in the microdialysis bowl for 15 to 25 min, removed, and the inside of the bowl dried. After microdialysis, each rat was sacrificed, and the brain removed and fixed in 4% formalin for later verification of microdialysis probe location as previously described (Clausing et al., 1995). We used only those animals in which the microdialysis probe tip was within 0.7 mm of the ideal AP and LAT coordinates and 0.4 mm of the ideal DV coordinates. In vitro probe recovery was performed after the probe was used in vivo to assess its functionality. The results of the recovery tests were used to exclude data from probes with poor efficiency. The in vitro probe recoveries for dopamine, DOPAC, homovanillic acid (HVA), 5-HT, and 5-hydroxy-indole acetic acid (5-HIAA) for the probes ranged from 14 to 20% [concentration in collected sample × 100)/concentration in standard solution] at 23°C and 1-μl/min flow rate. HPLC-quantitation of neurotransmitter, metabolites and AMPH levels in amygdaloid microdialysate. The frozen aliquots were rapidly thawed (for less than 30 s) and 11 μl (1/2 the total) of the microdialysate aliquot was added to 15 μl HPLC mobile phase. The sample was then immediately injected onto the HPLC for analysis. Microdialysate levels of dopamine, DOPAC, HVA, 3-methoxytryamine, 5-HT, and 5-HIAA were determined using reverse-phase HPLC, by methods similar to Stephans et al. (1998). The mobile phase consisted of 40 mM sodium acetate, 25 mM citric acid, 1.5 mM octane sulfonic acid, 1.5 mM EDTA and 6% methanol at pH of 3.8, adjusted using acetic acid. A Supelcosil LC18 3 μm analytical column (75 × 2.1 mm (Supelco, Bellefonte, PA) was used for separation, and a BAS-LC4B amperometric detector with a BAS-LC-17 oxidative flow cell was used for detection, with the potential working electrode potential set at 0.65V. Dopamine, DOPAC (dihydroxyphenylacetic acid), and 5-HIAA could be quantitated at 0.33 pg/10 μl microdialysate, while 5-HT and HVA were measurable at the 0.75 pg/10 μl levels and 3-methoxytyramine at the 2 pg/10 μl level. The HPLC retention times were 3.25 min (DOPAC), 4.7 min (dopamine), 5.95 min (5-HIAA), 10.4 min (HVA), 13.1 min (5-HT), and 15.4 min (3-methoxytyramine. The mobile phase was adjusted to insure that the unknown contaminants that frequently appear at the high sensitivity settings needed to measure biological amines in the amygdala were sufficiently separated from dopamine and 5-HT. In some animals, glutamate, as well as AMPH, levels were determined in amygdaloid microdialysate using the remaining 11 μl of the microdialysate fraction aliquot not used for aromatic monoamine analysis (Fig. 3). Methods adapted by Bowyer et al. (2000), which were originally developed as methods for detecting amino acids (Godel et al., 1984), were used for detecting both glutamate and AMPH. The only changes necessary to detect glutamate, as well as AMPH, was an alteration of the elution gradient and a shift to a more basic pH. In brief, AMPH levels were determined by fluorescent detection after o-phthaldialdehyde/3-mercaptopropionic acid-derivatization and separation on a Supelcosil® LC-18 5 μm analytical column 150 mm × 4.6 mm (Supelco, Bellefonte, PA) running a gradient with 95% KH2PO4 (0.05 M, pH 7.4) + 5% methanol (mobile phase A) versus 35% KH2PO4 (0.05 M, pH 7.4) + 65% methanol (mobile phase B). Flow rate was 1.5 ml/min, and the fluorescent detector was set at γex = 340 nm and γem = 440 nm. Microdialysis samples were derivatized and injected directly using a CMA/200 autosampler. Statistics. The microdialysate values for the neurotransmitters and metabolites shown in Figures 1 through 3 were calculated in the following manner. The pre-stimulus basal microdialysate levels (BL) for each animal were determined by averaging the levels, for any particular neurotransmitter or metabolite, in the last 2 fractions (normally the 5th and 6th) prior to AMPH exposure. The values for the individual fractions, after AMPH exposure, were then calculated as a percentage of the BL. The arithmetic mean ± standard error of the mean (SEM) for each fraction was then determined from the individual values (% BL) in each group. Absolute (pgrams/10 μl microdialysate) dopamine and 5-HT levels are shown in Figure 4, because the cold environment and the tetrodotoxin reduced pre-AMPH levels to below the threshold of quantitation for over one-half the animals in these groups. Multiple groups were analyzed by a repeated-measures 2way analysis of variance (ANOVA). A post hoc Tukey's least-significant difference test was applied if significant main effects were observed. Significance was set at p ≤ 0.05. RESULTS Behavioral Effects of AMPH The behavioral observations made on the rats dosed with either 3 doses (first experiment) or 1 dose (second experiment) of AMPH are noted in Table 1. Rats administered doses of saline (3× saline) did not show behaviors similar to the AMPH groups but did show an increase in motor activity and grooming from 2 to 10 min after saline injections. Hyperactivity was noted in all the rats receiving AMPH but was not included in the table. Rats that received multiple injections were not monitored for body temperatures until they became very sluggish or inactive (see Materials and Methods for explanations) from the AMPH exposure. Therefore, the degree of severe hyperthermia that is noted in Table 1 is, in some cases, an underestimation of its occurrence. Only rats given 15 mg/kg AMPH exhibited overt behavioral seizure activity. The Effects of Multiple Doses of AMPH on Dopamine, 5-HT, and Metabolites The effects of the multiple injections of saline or the various doses of AMPH on dopamine levels in the microdialysate can be seen in Figure 1. Although not shown, the pre-AMPH-basal microdialysate dopamine levels (-0.33- and 0.0-h time points) ranged from 0.6 ± 0.4 to 1.2 ± 0.5 pg/10 μl among the 4 groups, and these basal levels did not vary significantly. However, after AMPH exposure, the levels for the 3 × 5-mg/kg and 3 × 15-mg/kg groups were significantly greater than 3 × saline (see Fig. 1). The increases due to 3 × 2.5-mg/kg AMPH were not quite statistically significant over all 3 doses. Basal microdialysate DOPAC levels ranged from 32.6 ± 9.0 to 45.9 ± 11.0 pg/10 μl for the groups, and did not vary significantly among the groups prior to AMPH exposure. Although 3 × 15 mg/kg AMPH did not significantly reduce DOPAC levels over the entire AMPH exposure interval, these decreases were significant if they were assessed after the third dose (Fig. 1). Basal microdialysate HVA levels ranged from 23.3 ± 7.3 to 35.9 ± 8.7 pg/10 μl for the groups, and did not vary significantly among the groups prior to AMPH exposure. Although the increases in the microdialysate HVA levels after multiple injections of either 3 × 5 or 3 × 15 mg/kg AMPH appeared greater than control, only the increases produced by the 3 × 15 mg/kg AMPH were significant over all 3 injections (Fig. 1). The 5-HT levels were significantly increased by AMPH in the 15-mg/kg group compared to controls and other AMPH groups, but the 2.5- and 5.0-mg/kg groups were not significantly greater than the controls over all 3 injections of AMPH (see Fig. 2). However, the increases of 5-HT in the second and third microdialysate fractions after the last 2 doses of 5.0 mg/kg AMPH were significantly (p < 0.05) greater than control. Although basal microdialysate 5-HT levels (from 0.9 ± 0.4 to 2.3 ± 1.3 pg/10 μl) for all groups prior to AMPH exposure was more variable than dopamine, this was not significant. The 5-HIAA levels were not significantly affected by AMPH exposure (see Fig. 2). Basal microdialysate 5-HIAA levels prior to AMPH exposure ranged from 277.1 ± 61.3 to 403.1 ± 60.7 pg/10 μl for the groups, and did not vary significantly among the groups. The levels of glutamate in the microdialysate of the 3 × 5-mg/kg or 3 × 15-mg/kg AMPH groups were not significantly elevated over the duration of dosing when compared to the 3× saline (Fig. 3, top graph). Basal microdialysate glutamate concentrations ranged from 2.2 ± 0.9 to 3.3 ± 1.0 μM for the groups, and did not vary significantly among the groups prior to AMPH exposure. The levels of AMPH in the amygdaloid microdialysis after 3 × 5 mg/kg or 3 × 15 mg/kg AMPH are shown in the bottom graph of Figure 3. TTX and Environmental Temperature Effects on Dopamine and 5-HT Levels during AMPH Exposure A final experiment was conducted to determine how much of the increase in dopamine and 5-HT in the microdialysate after doses of 15 mg/kg AMPH was impulse-dependent and related to body temperature. TTX (2 μM) was included in the microdialysis buffer to block the impulse-mediated release of dopamine and 5-HT release from AMPH exposure at room temperature (23°C). Another group was dosed in a cold room held at a temperature of between 10 and 11°C to prevent hyperthermia during AMPH exposure. TTX was introduced at 1.33 h, and the rats were subsequently dosed with 15 mg/kg AMPH at the 3-h time point. Control rats were switched to a microdialysis buffer with 2 μM citrate at 1.33 h, and the rats were subsequently dosed with 15 mg/kg AMPH at the 3-h time point. Cold-room rats were placed in the cold room 0.5 h prior to the start of microdialysis, and subsequently dosed with 15 mg/kg AMPH at the 3-h time point. All the rats injected with one dose of 15 mg/kg AMPH at room temperature exhibited the stereotypic behaviors, hyperthermia, and some seizure-like activity. The inclusion of TTX in the microdialysis buffer did not notably alter the behavioral response to 15 mg/kg AMPH. However, the cold environment significantly altered the behavioral response to one dose of 15 mg/kg AMPH (Table 1). Five of the 6 rats dosed at 10°C exhibited stereotypic behavior such as repetitive licking, grooming, and slow circling but did not exhibit retrograde propulsion or convulsive behavior. The temperatures of these 5 rats ranged from 34.8°C to 37.5°C at 2 h after dosing. Microdialysate DOPAC levels prior to AMPH exposure were not affected by either inclusion of TTX in the microdialysis buffer (48.2 ± 11.5 pg/10 μl, n = 12) or a cold environmental temperature (39.5 ± 12.2 pg/10 μl, n = 5) compared to control (35.2 ± 6.5 pg/10 μl, n = 8). Although pre-AMPH HVA levels were higher in the tetrodotoxin group (58.5 ± 9.7 pg/10 μl), this was not statistically significant relative to the cold room (30.8 ± 8.7 pg/10 μl) and 23°C (31.4 ± 7.0 pg/10 μl). Also, 5-HIAA levels were not affected by TTX (303.1 ± 39.2 pg/10 μl) or a cold environment (294.3 ± 28.1 pg/10 μl) compared to control (338.5 ± 42.4 pg/10 μl). TTX or a cold environment significantly reduced both the basal and AMPH-induced increases in dopamine levels in the microdialysate (Fig. 4, top graph). The basal dopamine levels over the 3 fractions for the 3 groups is not readily apparent, but the levels for both the TTX (0.23 ± 0.05 pg/10 μl, mean ± SEM, n = 8) and cold-room (0.20 ± 0.09 pg/10 μl, mean ± SEM, n = 12) groups were significantly less than the control (0.50 ± 0.07 pg/10 μl, mean ± SEM, n = 5). After AMPH exposure, not only were the microdialysate dopamine levels for both the TTX and cold-room groups significantly less than control, but the dopamine levels in the cold-room group were also significantly less than the TTX group. The cold environment or tetrodotoxin did not affect the changes in DOPAC levels after AMPH exposure. However, a 2-way RM ANOVA indicated that the increases in HVA levels, after AMPH exposure in the cold (110 ± 6% or less over all 6 fractions), were significantly less in the cold-room group (p < 0.01) compared to the 23°C group (averaged 175 ± 9% over all 6 fractions). Baseline 5-HT release was decreased significantly by TTX (0.5 ± 0.1 pg/10 μl, mean ± SEM) compared to control (1.1 ± 0.2 pg/10 μl, mean ± SEM) but was not significantly affected by a cold environment (0.8 ± 0.2 pg/10 μl, mean SEM). TTX inhibited AMPH-induced increases in the 5-HT levels in microdialysate (Fig. 4, bottom graph) to a greater extent than that observed on dopamine release. Cold environmental temperatures also significantly inhibited 5-HT levels with more inhibition evident at the later time points after AMPH exposure. Although not shown in Figure 4, the AMPH levels of 3.8 ± 0.8 μM (mean ± SEM, n = 5) 40 min after dosing the amygdaloid microdialysate for the 10°C group were comparable to the 4.2 ± 0.6 μM (mean ± SEM, n = 8) for the controls. Neither the cold environment nor tetrodotoxin affected the 5-HIAA levels after AMPH exposure. DISCUSSION The results of the present study indicate that much of the dopamine and 5-HT released in the amygdala as a result of exposure to moderate to high doses of AMPH is neuronal impulse- and temperature-dependent. Our results are in general agreement with previous in vivo microdialysis studies that examined dopamine release during exposure to low doses of AMPH (Harmer et al., 1997; Young and Rees, 1998)—that the increases in dopamine levels in microdialysate are produced by doses of 2.5 mg/kg of AMPH or less are modest to minimal. However, this may be dependent on the region of amygdala examined (Young and Rees, 1998). The lesser increases in microdialysate dopamine levels in the amygdala relative to the caudate-putamen (CPu) induced by AMPH might be expected from the lower density of dopaminergic terminals (Ben-Ari et al., 1975; Bjorklund et al., 1979; Fuxe et al., 1974; Hokfelt et al., 1977). Alternatively, it may that a lesser density of plasma-membrane dopamine transporters on the terminals in the amygdala may be responsible for differences in release (Jones et al., 1995). It is unlikely that the differences between CPu and amygdala are due to extracellular levels of AMPH. The peak levels of 0.9 to 1.3 μM AMPH that were observed in amygdaloid microdialysate in the present study after 5 mg/kg doses of AMPH were very comparable to the 0.75 to 1.4 μM AMPH levels reported for CPu microdialysate (Clausing et al., 1995). The 5-HT release in amygdaloid microdialysate after 3 × 15 mg/kg AMPH was 5- to 10-fold greater than that produced by 3 × 5 mg/kg while the 3 × 15 mg/kg dose produced only a 2-fold greater dopamine release than the 3 × 5 mg/kg dose. The greater increase in the 5-HT release compared to the dopamine release produced by 3 × 15 mg/kg AMPH may be due in part to the levels of AMPH being above the km for the plasma membrane dopamine transporter, but at or below the km for the plasma membrane serotonin transporters (Azzaro and Rutledge, 1973; Shaskan and Snyder, 1970). However, the greater increase in 5-HT levels relative to dopamine levels in amygdaloid microdialysis produced by 3 × 15 mg/kg AMPH may also be due to the 5-HT release being more impulse-dependent. Behavioral signs of seizure activity occurred frequently with the 3 × 15 mg/kg dose but seldom occurred with the 3 × 5 mg/kg AMPH (Table 1; Bowyer et al., 1998). Increased neuronal activity within the amygdala due to seizure activity could be either directly or indirectly responsible for increased dopamine and 5-HT release at the higher doses. The results of the present study indicate that about 70% of the 5-HT and 30% of the dopamine release induced by 3 × 15 mg/kg AMPH are impulse-dependent. However, it is possible that 3 × 5 mg/kg AMPH may also produce a substantial impulse-dependent release in the amygdala. Understanding the mechanisms by which AMPH-induced seizure activity would increase impulse-dependent dopamine and 5-HT release in the amygdala are important, since the amygdala is likely to be involved in the generation of AMPH-induced seizures and the associated behaviors. There is indirect evidence implicating the involvement of the basolateral amygdala in AMPH- and METH-induced seizures. The amygdala is a site where chemical and electrical stimulation can readily generate or “kindle” limbic type seizures in animals (Goddard et al., 1969; Kelly and McIntyre, 1996). Furthermore, the behavioral expression of seizure activity evoked by stimulation of the amygdala is very similar to that of AMPH-induced seizures. In addition, chronic electrical stimulation of the amygdala leads to spontaneous limbic behaviors and seizure activity (Michalakis et al., 1998) that are very similar to AMPH-induced seizures (Bowyer et al., 1998; Schmued and Bowyer, 1997). Brain regions necessary for the generation and development of amygdaloid-kindled seizures, such as the anterior piriform cortex and insular cortex (Loscher and Ebert, 1996; Mohapel and Corcoran, 1996; Piredda and Gale, 1985; Tortorella et al., 1997) are areas sensitive to neurodegeneration produced by AMPH-induced seizures (Bowyer et al., 1998). This indicates that these limbic areas are excessively stimulated and may be involved in generating AMPH-induced seizures. Some of the amygdala interconnections with other areas of the limbic system include basolateral amygdaloid efferents to the CA1 of hippocampus and afferents from the CA1 and subiculum (Amaral and Witter, 1995). The amygdala also has afferent and efferent connections with the lateral and medial septum (Jakab and Leranth, 1995). The medial and posterior cortical amygdaloid nuclei are also reciprocally innervated with the accessory olfactory bulbs (Shipley et al., 1995). Particularly relevant to the present study are the serotonergic projections from the DRN and the dopaminergic afferents from the ventral tegmentum (VTA) and substantia nigra compacta (SNC). There is a significant 5-HT innervation of the amygdala by the DRN (D'Amato et al., 1987; Savaki et al., 1985; Sette et al., 1981), and a reciprocal innervation between the amygdala and the SNC (Ben-Ari et al., 1975; Bjorklund et al., 1979; Fallon and Loughlin, 1995; Fuxe et al., 1974; Hokfelt et al., 1977). The exact mechanisms by which AMPH-induced seizure activity increase the rates of neuronal depolarization of dopaminergic neurons in the VTA/SNC and serotonergic neurons in the DRN are not known. However, repetitive electroconvulsive shock appears to increase dopamine release in the VTA and SN (Leviel et al., 1990), and kainate injection into the DRN evokes 5-HT release in the amygdala (Viana et al., 1997). Also, recent data indicate that 5-HT receptors in the amygdala can modulate 5-HT release through interneurons and GABAergic projections to the DRN (Bufton et al., 1993; Cheng et al., 1998; Morales et al., 1996; Rainnie, 1999, Tecott et al., 1993). Based on the numerous neuronal interactions between 5-HT and dopamine-containing brain regions and their interconnections with the amygdala, it is not surprising that much of the 5-HT and some of the dopamine release during AMPH-induced seizures are TTX-dependent. This impulse-dependent dopamine and 5-HT release would be expected to be additive with the more traditional non-impulse-dependent release produced by AMPH (Seiden et al., 1993). Since amphetamines can release 5-HT from blood platelets (Bak et al., 1967; Lemmer, 1973; Paasonen, 1965), it is possible that a portion of the 5-HT in the amygdaloid microdialysis could be platelet-derived rather than from nerve-terminal release. However, the majority of 5-HT release we see in the amygdala is likely neuronal and not platelet-derived because the release of 5-HT from platelets should not be sensitive to inhibition by TTX. Much of the dopamine and 5-HT release in the amygdala caused by high to moderate doses of AMPH are also sensitive to body temperature (Fig. 4). This is expected because AMPH induction of seizures in the rat is temperature-dependent, and AMPH-induced dopamine release in the CPu is also influenced by body temperature (Bowyer et al., 1993, 2000). In the present studies, the low environmental temperatures that blocked AMPH-induced hyperthermia also reduced the AMPH-induced release of dopamine by 75% and 5-HT by 50%. Furthermore, the stereotypic behaviors and seizure activity induced by 15 mg/kg d-AMPH were greatly diminished in the cool environment. These results were not due to the reduction of AMPH levels by the cool environment since no significant difference between the peak levels of AMPH in the microdialysate were observed at the two environmental temperatures. The increase in the 5-HT levels in the amygdala might actually produce a “negative-feedback” effect on seizure activity. In general, elevated 5-HT receptor levels and receptor stimulation have been reported to inhibit seizure activity (Dailey et al., 1996; Heisler et al., 1998). The lack of an increase in extracellular glutamate was, at first glance, somewhat surprising given that previous reports showed that high doses of AMPH or METH produce large increases in extracellular striatal glutamate (Abekawa et al., 1994; Nash and Yamamoto, 1992, 1993). Nevertheless, the present findings are consistent with minimal neurotoxicity observed in this region and parallel a similar relationship between the lack of change in glutamate and the absence of dopamine depletions observed in the nucleus accumbens (Abekawa et al., 1994). In summary, these results indicate that some of the dopamine and the majority of 5-HT released during exposure to convulsive doses of AMPH are neuronal impulse-dependent and related to seizure activity. In addition, the hyperthermia that occurs concurrently with seizure activity may, by itself, markedly potentiate both dopamine and 5-HT release. Further studies will be necessary to determine the roles of the dopamine and 5-HT release in amygdala in the generation and behavioral expression of seizures, and the neurodegeneration within the limbic system that is seizure-dependent. TABLE 1 The Effects of Various Doses of d-Amphetamine on Behavior     Stereotypic behaviors  Convulsive-like behaviors   Dose  n  Continuousa  Retrograde propulsions  Running fits, WDS, MCb  Myoclonic seizures  Severe hyperthermiac  aContinuous behavior includes nodding, grooming, and chewing; Retrograde Propulsions include head weaving and forepaw treading.  bWDS denotes wet-dog shakes.; MC denotes incidences of single, brief instances of myoclonus.  cSevere hyperthermia indicates animal's temperature was recorded to be above 41.0°C.  3 × 2.5 mg/kg  6  100%  0%  0%  0%  0%   3 × 5 mg/kg  8  100%  87%  13%  0%  67%   3 × 15 mg/kg  7  100%  100%  100%  57%  71%   1 × 15 mg/kg  8  100%  100%  88%  38%  50%   1 × 15 mg/kg + TTX  12  100%  100%  92%  42%  58%   1 × 15 mg/kg (10°C)  5  100%  40%  20%  0%  0%      Stereotypic behaviors  Convulsive-like behaviors   Dose  n  Continuousa  Retrograde propulsions  Running fits, WDS, MCb  Myoclonic seizures  Severe hyperthermiac  aContinuous behavior includes nodding, grooming, and chewing; Retrograde Propulsions include head weaving and forepaw treading.  bWDS denotes wet-dog shakes.; MC denotes incidences of single, brief instances of myoclonus.  cSevere hyperthermia indicates animal's temperature was recorded to be above 41.0°C.  3 × 2.5 mg/kg  6  100%  0%  0%  0%  0%   3 × 5 mg/kg  8  100%  87%  13%  0%  67%   3 × 15 mg/kg  7  100%  100%  100%  57%  71%   1 × 15 mg/kg  8  100%  100%  88%  38%  50%   1 × 15 mg/kg + TTX  12  100%  100%  92%  42%  58%   1 × 15 mg/kg (10°C)  5  100%  40%  20%  0%  0%  View Large FIG. 1. View largeDownload slide The effects of multiple doses of d-amphetamine on dopamine and metabolite levels in amygdaloid microdialysate. The changes in the microdialysate levels of dopamine, DOPAC, and HVA after 3 doses of saline or 3 doses of 2.5 mg/kg, 5 mg/kg, or 15 mg/kg of AMPH administered at a room temperature of 23°C are shown from the first experiment. Values represent arithmetic means ± SEM. There were 8 rats in the saline group, 6 in the 2.5 mg/kg, 8 in the 5-mg/kg, and 7 in the 15-mg/kg group. Repeated-measures 2-way ANOVA indicated that none of the baseline dopamine, DOPAC, or HVA levels for the AMPH groups varied significantly from the 3× saline group. However, ANOVA did show that the differences in the mean values of dopamine between treatment groups was greater than expected by chance (p = 0.003), during AMPH exposure. Tukey's post hoc test showed that the levels for the 3 × 5-mg/kg and 3 × 15-mg/kg groups differed significantly (p = 0.046 and p = 0.003, respectively) from 3× saline during AMPH dosing (time points 0.33 h to 5.67 h). The dopamine levels for the 3 × 15-mg/kg group were also significantly greater (p = 0.028) than the 2.5-mg/kg group. Only the DOPAC levels in the 15-mg/kg group were reduced over time, due to AMPH exposure, compared to control, but this was significant only after the third dose (see text). Repeated-measures 1-way ANOVA indicated that the difference in the means for the HVA levels of the treatment groups was significantly greater (p = 0.034) than expected by chance. Post hoc tests indicated that the mean HVA levels for only the 3 × 15-mg/kg group were significantly greater (p = 0.04) than 3× saline over all 3 doses. FIG. 1. View largeDownload slide The effects of multiple doses of d-amphetamine on dopamine and metabolite levels in amygdaloid microdialysate. The changes in the microdialysate levels of dopamine, DOPAC, and HVA after 3 doses of saline or 3 doses of 2.5 mg/kg, 5 mg/kg, or 15 mg/kg of AMPH administered at a room temperature of 23°C are shown from the first experiment. Values represent arithmetic means ± SEM. There were 8 rats in the saline group, 6 in the 2.5 mg/kg, 8 in the 5-mg/kg, and 7 in the 15-mg/kg group. Repeated-measures 2-way ANOVA indicated that none of the baseline dopamine, DOPAC, or HVA levels for the AMPH groups varied significantly from the 3× saline group. However, ANOVA did show that the differences in the mean values of dopamine between treatment groups was greater than expected by chance (p = 0.003), during AMPH exposure. Tukey's post hoc test showed that the levels for the 3 × 5-mg/kg and 3 × 15-mg/kg groups differed significantly (p = 0.046 and p = 0.003, respectively) from 3× saline during AMPH dosing (time points 0.33 h to 5.67 h). The dopamine levels for the 3 × 15-mg/kg group were also significantly greater (p = 0.028) than the 2.5-mg/kg group. Only the DOPAC levels in the 15-mg/kg group were reduced over time, due to AMPH exposure, compared to control, but this was significant only after the third dose (see text). Repeated-measures 1-way ANOVA indicated that the difference in the means for the HVA levels of the treatment groups was significantly greater (p = 0.034) than expected by chance. Post hoc tests indicated that the mean HVA levels for only the 3 × 15-mg/kg group were significantly greater (p = 0.04) than 3× saline over all 3 doses. FIG. 2. View largeDownload slide The effects of multiple doses of d-amphetamine on 5-HT and 5-HIAA levels in amygdaloid microdialysate. The changes in the microdialysate levels of 5-HT and 5-HIAA after 3 doses of saline or 3 doses of 2.5 mg/kg, 5 mg/kg or 15 mg/kg of AMPH administered at room temperature 23°C are shown from the first experiment. Values shown represent arithmetic means ± SEM. Repeated measures 2-way ANOVA indicated that the difference in the means for the 5-HT levels of the treatment groups was significant (p < 0.001). A post hoc test indicated that the mean 5-HT levels for the 3 × 15-mg/kg group was significantly greater than the 3× saline (p < 0.001) and the 3 × 2.5-mg/kg group (p = 0.004 and p = 0.005, respectively). However, over all 3 injections, neither the 3 × 2.5 nor the 3 × 5 mg/kg increased 5-HT levels significantly (see text). Two-way ANOVA indicated that the difference in the mean values for 5-HIAA levels in the treatment groups was not significant (p = 0.51). FIG. 2. View largeDownload slide The effects of multiple doses of d-amphetamine on 5-HT and 5-HIAA levels in amygdaloid microdialysate. The changes in the microdialysate levels of 5-HT and 5-HIAA after 3 doses of saline or 3 doses of 2.5 mg/kg, 5 mg/kg or 15 mg/kg of AMPH administered at room temperature 23°C are shown from the first experiment. Values shown represent arithmetic means ± SEM. Repeated measures 2-way ANOVA indicated that the difference in the means for the 5-HT levels of the treatment groups was significant (p < 0.001). A post hoc test indicated that the mean 5-HT levels for the 3 × 15-mg/kg group was significantly greater than the 3× saline (p < 0.001) and the 3 × 2.5-mg/kg group (p = 0.004 and p = 0.005, respectively). However, over all 3 injections, neither the 3 × 2.5 nor the 3 × 5 mg/kg increased 5-HT levels significantly (see text). Two-way ANOVA indicated that the difference in the mean values for 5-HIAA levels in the treatment groups was not significant (p = 0.51). FIG. 3. View largeDownload slide Glutamate and amphetamine levels in microdialysate after either 3 × 5 mg/kg or 3 × 15 mg/kg d-amphetamine. The levels of glutamate (upper) and AMPH (lower) in amygdaloid microdialysate after 3 doses of saline or 3 doses of either 5 mg/kg or 15 mg/kg AMPH are plotted. Values shown represent arithmetic means ± SEM. The levels of glutamate and AMPH were determined from the microdialysate of the same rats used in the first experiment to generate Figures 1 and 2. The levels of glutamate in the microdialysis were not significantly affected by either dose of AMPH. The AMPH levels were significantly higher in the 15-mg/kg group than the 5-mg/kg group (p < 0.001). FIG. 3. View largeDownload slide Glutamate and amphetamine levels in microdialysate after either 3 × 5 mg/kg or 3 × 15 mg/kg d-amphetamine. The levels of glutamate (upper) and AMPH (lower) in amygdaloid microdialysate after 3 doses of saline or 3 doses of either 5 mg/kg or 15 mg/kg AMPH are plotted. Values shown represent arithmetic means ± SEM. The levels of glutamate and AMPH were determined from the microdialysate of the same rats used in the first experiment to generate Figures 1 and 2. The levels of glutamate in the microdialysis were not significantly affected by either dose of AMPH. The AMPH levels were significantly higher in the 15-mg/kg group than the 5-mg/kg group (p < 0.001). FIG. 4. View largeDownload slide The effect of either a cold environment or TTX on dopamine and 5-HT levels in microdialysate after 15 mg/kg d-amphetamine. In the second experiment, the levels of dopamine (upper) and 5-HT (lower) after one 15-mg/kg dose of AMPH were determined at room temperature, in a cold environment, and after including 2 μM TTX in the microdialysis buffer. There were 8 rats in the room-temperature group, 12 rats in the TTX group, and 5 rats in the cold-room group. Values shown represent arithmetic means ± SEM. (Upper) Repeated-measures 2-way ANOVA with time of microdialysate collection and treatment (TTX, 23°C or a cold environment) for the pre-AMPH fractions collected between the –1.0 and 0.0 h of microdialysis showed that the mean dopamine levels among the treatment groups significantly (p = 0.01) differed. The basal dopamine levels over the 3 fractions for both the TTX and cold-room groups were significantly less (p = 0.01 and p = 0.04, respectively) than the 23°C group. Repeated-measures 2-way ANOVA after AMPH exposure showed that the differences in the mean levels among the treatment groups were significantly greater (p < 0.001) than expected by chance. Also the mean values among the different levels of fractions were significantly greater than expected (p < 0.001). The microdialysate dopamine levels for both the TTX and cold room groups were significantly less than the 23°C group (p = 0.026 and p < 0.001, respectively). The cold-room group was also significantly less (p = 0.031) than the TTX group. (Lower) Repeated-measures 2-way ANOVA with time of microdialysate collection and treatment (TTX, 23°C or a cold environment) for the pre-AMPH fractions collected between the –1.0 and 0.0 h of microdialysis showed that differences in the mean 5-HT levels among the treatment groups were significantly greater (p = 0.03) than expected by chance. Baseline 5-HT release was decreased significantly (p = 0.002) by TTX but the cold environment did not significantly reduce the basal 5-HT concentrations. Repeated-measures 2-way ANOVA after AMPH exposure showed that differences in the mean 5-HT levels among the treatment groups were significantly greater (p < 0.001) than expected by chance. Both TTX (p < 0.001) and the cold environment (p = 0.006) inhibited AMPH-induced increases in the 5-HT levels in microdialysate compared to the 23°C group. FIG. 4. View largeDownload slide The effect of either a cold environment or TTX on dopamine and 5-HT levels in microdialysate after 15 mg/kg d-amphetamine. In the second experiment, the levels of dopamine (upper) and 5-HT (lower) after one 15-mg/kg dose of AMPH were determined at room temperature, in a cold environment, and after including 2 μM TTX in the microdialysis buffer. There were 8 rats in the room-temperature group, 12 rats in the TTX group, and 5 rats in the cold-room group. Values shown represent arithmetic means ± SEM. (Upper) Repeated-measures 2-way ANOVA with time of microdialysate collection and treatment (TTX, 23°C or a cold environment) for the pre-AMPH fractions collected between the –1.0 and 0.0 h of microdialysis showed that the mean dopamine levels among the treatment groups significantly (p = 0.01) differed. The basal dopamine levels over the 3 fractions for both the TTX and cold-room groups were significantly less (p = 0.01 and p = 0.04, respectively) than the 23°C group. Repeated-measures 2-way ANOVA after AMPH exposure showed that the differences in the mean levels among the treatment groups were significantly greater (p < 0.001) than expected by chance. Also the mean values among the different levels of fractions were significantly greater than expected (p < 0.001). The microdialysate dopamine levels for both the TTX and cold room groups were significantly less than the 23°C group (p = 0.026 and p < 0.001, respectively). The cold-room group was also significantly less (p = 0.031) than the TTX group. (Lower) Repeated-measures 2-way ANOVA with time of microdialysate collection and treatment (TTX, 23°C or a cold environment) for the pre-AMPH fractions collected between the –1.0 and 0.0 h of microdialysis showed that differences in the mean 5-HT levels among the treatment groups were significantly greater (p = 0.03) than expected by chance. Baseline 5-HT release was decreased significantly (p = 0.002) by TTX but the cold environment did not significantly reduce the basal 5-HT concentrations. Repeated-measures 2-way ANOVA after AMPH exposure showed that differences in the mean 5-HT levels among the treatment groups were significantly greater (p < 0.001) than expected by chance. Both TTX (p < 0.001) and the cold environment (p = 0.006) inhibited AMPH-induced increases in the 5-HT levels in microdialysate compared to the 23°C group. 1 To whom correspondence should be addressed at NCTR, HFT-132, Jefferson, AR 72079–9502. Fax: (870) 543-7745. E-mail: jbowyer@nctr.fda.gov. 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