.
`Ž
`Brain Research 782 1998 83–90
`
`Research report
`Lithium inhibits the reverse tolerance and the c-Fos expression induced by
`methamphetamine in mice
`Misako Namima a,b,), Katsunobu Sugihara b, Koichi Okamoto b
`a Center for Laboratory Animal Science, National Defense Medical College, 3-2, Namiki, Tokorozawa 359, Saitama, Japan
`b Department of Pharmacology, National Defense Medical College, 3-2, Namiki, Tokorozawa 359, Saitama, Japan
`Accepted 14 October 1997
`
`Abstract
`
`To elucidate the mechanism of psychostimulant-induced reverse tolerance, the effects of lithium on ambulatory activity and cerebral
`c-Fos protein expression were investigated in mice injected with methamphetamine 2 mgrkg, s.c., 1–5 times . The ambulatory activity
`Ž
`.
`enhanced by either acute or chronic methamphetamine injection was delayed or diminished by LiCl pretreatment 170 mgrkg, s.c., 1 h
`Ž
`.
`before methamphetamine . The c-Fos expression in the dorsal lateral geniculate nucleus and in the striatum was significantly increased by
`acute but not chronic injection of methamphetamine, and the increases were significantly suppressed by LiCl pretreatment. Although how
`the Li-sensitive c-Fos expressions in the dorsolateral geniculate nucleus and striatum are related to methamphetamine-induced behavioral
`excitation is unclear, these results suggest that lithium at least functionally interferes with the formation of the state of reverse tolerance to
`methamphetamine in the mouse. q 1998 Elsevier Science B.V.
`
`Keywords: Lithium; Methamphetamine; Reverse tolerance; Ambulatory; Activity; c-Fos protein; Striatum; Geniculate nucleus
`
`1. Introduction
`
`2. Materials and methods
`
`Methamphetamine is known to induce hyperkinesia or
`behavioral excitation, and reverse tolerance in animals
`w
`x
`22,27,29,31,48–50 and to increase the expression of c-fos
`w
`x
`mRNA and c-Fos protein in the brain 10,33 . Lithium
`salts are widely used for the treatment of the manic phase
`w
`x
`of manic-depressive psychosis 7,8,53–55 and is known to
`affect psychostimulant-induced locomotor stimulation in
`w
`x
`animals 4–6,9,14,16,18,34,39,40,45,51 . Since the ambu-
`latory activity enhanced by a single administration of
`methamphetamine was reported to be suppressed by pre-
`w
`x
`treatment with lithium in mice 16 , we have surmised that
`studies of the effects of lithium on methamphetamine-in-
`duced behavioral and biochemical changes may provide a
`clue to elucidate the mechanism of not only metham-
`phetamine-induced reverse tolerance but also the antimanic
`effect of lithium. Thus, in the present study, we investi-
`gated, as the first step, the effects of pretreatment with
`LiCl on methamphetamine-stimulated ambulatory activity
`in mice and on the expression of c-Fos protein in mouse
`brain regions.
`
`) Corresponding author. Fax: q81-429-96-5183
`
`0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.
`
`2.1. Drug administration and the ambulatory actiÕity
`
`Ž
`Male mice ddy-strain, 5 weeks old, 10–15 g, Tokyo
`.
`Experimental Animals, Tokyo, Japan were habituated for
`two weeks, with free access to food and water, to the
`animal room conditioned to be 22"28C and regularly
`illuminated from 7:00 to 19:00. For two days before the
`experiment, each mouse was handled appropriately and
`briefly in order to minimize injection-induced increases in
`the basal expression of immediate early genes. For ambula-
`Ž
`.
`tory activity measurement, each mouse was injected s.c.
`Ž
`with a test drug and kept for 15 min in the plastic cage 20
`.
`cm in diameter and 18 cm in depth , and the mice with
`outstandingly low ambulatory activities were eliminated.
`With apparently normally behaving mice, the measurement
`of ambulatory activity was started 15 min after injection.
`The test drugs given were physiological saline 2 mlrkg ,
`Ž
`.
`methamphetamine 2 mgrkg, Dai-Nippon Pharmaceutical,
`Ž
`Osaka, Japan , LiCl 170 mgrkg, Wako Pure Chemicals
`.
`Ž
`Industry, Osaka, Japan , and LiCl 170 mgrkg plus
`.
`Ž
`.
`methamphetamine 2 mgrkg, 1 h after LiCl . For acute
`Ž
`.
`experiments with 20 mice, each test drug was injected
`Ž
`.
`once s.c. , and the ambulatory activity was subsequently
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`M. Namima et al.rBrain Research 782 1998 83–90
`)
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`observed for 180 min. For chronic experiments with 90
`mice,
`the above mentioned injections were repeated 5
`times at intervals of 3 days, and the ambulatory activity
`was observed for 180 min after every injection. The ambu-
`latory activity, which was the number per 10 min of
`horizontal movements of a mouse inside the plastic cage
`
`w x23 , was measured using the ambulometer, AMB-M20
`Ž
`.
`Ohara, Tokyo, Japan , by which the cumulative number of
`the movements was automatically printed out every 10 min
`for a total period of 180 min.
`
`2.2. c-Fos protein-like immunoreactiÕity
`
`The expression of c-Fos protein-like immunoreactivity
`in mouse brain regions was examined following the method
`w
`x
`of Shiosaka and Tohyama 47 . Briefly, the mice which
`had completed the 3 h-measurement of ambulatory activity
`either after a single or repeated drug injection were imme-
`diately anesthetized with pentobarbital and fixed by perfu-
`sion through the left cardiac ventricle with physiological
`Ž
`saline for 20 min,
`then with Zamboni’s solution 2%
`paraformaldehyde and 0.21% picric acid in 0.1 M phos-
`.
`phate-buffered saline, pH 7.4 for 40 min. The resected
`whole brain was soaked in Zamboni’s solution then in
`30% sucrose solution each overnight,
`frozen in dry
`icerisopentane, and 16 mm thick coronal brain sections
`were taken using a cryostat. The sections were washed 3
`times with and then soaked overnight at 48C in the 0.1 M
`phosphate-buffered saline containing 0.3% Triton X-100
`Ž
`.
`pH 7.4 . After 3 times washing with 0.1 M phosphate-
`Ž
`.
`buffered saline pH 7.4 , the sections were treated for 3 h
`Ž
`with the blocking solution 1% non-immunized sheep
`serum, 1% bovine albumin and 0.3% Triton X-100 in 0.1
`.
`M phosphate-buffered saline, pH 7.4 to block nonspecific
`reactions, and then reacted for 48 h at 48C with the first
`Ž
`antibody the rabbit anti-c-Fos polyclonal antibody, Lot.
`.
`No. 40920303, Oncogene Science, Cambridge, MA, USA
`which was diluted 2000-fold with the blocking solution,
`Ž
`then treated for 24 h at 48C with the second antibody the
`biotinylated anti-rabbit IgG antibody, ABC kit, Vector,
`.
`Burlingame, CA, USA which was diluted 250-fold with
`the blocking solution, and finally reacted for 24 h at 48C
`with the avidin–biotin-complex conjugated to horseradish
`Ž
`.
`peroxidase ABC kit which was diluted 250-fold with the
`blocking solution. After 3 times washing with 0.1 M
`phosphate-buffered saline and one washing with 0.05 M
`Ž
`.
`Tris-HCl buffer pH 7.4 , immunopositive cell nuclei in
`brain sections were visualized by the conventional di-
`aminobenzidine–ammonium nickel reaction followed by
`application of hydrogen peroxide. The sections were im-
`mersed in 0.5% gelatin solution then mounted onto slides,
`and the number of c-Fos immunoreactive cell nuclei in an
`area of 0.335 mm s603 mm =555 mm of each brain
`2 Ž
`.
`region was counted from the final brain sections using the
`Ž
`two-dimensional
`image
`analyzer, Luzex-FS Nireco,
`.
`Tokyo, Japan .
`
`2.3. Statistical analysis
`
`Multiple data groups statistically compared first by the
`Ž
`.
`analysis of variance ANOVA using the Statistical Analy-
`Ž
`.
`sis System SAS, SAS Institute, Cary, NC, USA , and, if a
`significant difference was detected among the groups by
`the ANOVA, all paired groups were then examined by the
`post-hoc Fisher’s least significant difference test equipped
`in the SAS–ANOVA. P -0.05 in the Fisher’s test was
`taken as the significant level.
`
`3. Results
`
`3.1. Ambulatory actiÕity
`
`3.1.1. Acute experiments
`Fig. 1 shows the time course changes of the mean
`Ž
`.
`ambulatory activity after a single injection s.c. of saline
`Ž
`.
`Ž
`.
`open squares , LiCl closed squares , methamphetamine
`Ž
`.
`Ž
`open circles , or LiCl plus methamphetamine 1 h after
`.
`LiCl, closed circles
`to four mice for each. A single
`injection of saline 2 mlrkg or LiCl 170 mgrkg did not
`Ž
`.
`Ž
`.
`significantly enhance the ambulatory activity, whereas a
`single injection of methamphetamine 2 mgrkg with or
`Ž
`.
`without LiCl pretreatment 170 mgrkg markedly in-
`Ž
`.
`creased the activity. However, while the ambulatory activ-
`ity after the injection of methamphetamine alone rapidly
`reached a peak in 40 min, the activity after the injection of
`LiCl plus methamphetamine slowly reached a lower peak
`after about 90 min and declined more slowly. The analysis
`of variance using the drug treatment, the observation time
`and the interaction between them as statistical factors
`indicated the presence of a significant difference for the
`drug treatment Fs136.30, d.f.s3, P -0.0001, the d.f.
`Ž
`the residual sum of squaress216 . The post-hoc
`.
`of
`Fisher’s least significant difference test then showed that
`the overall ambulatory activity across the observation pe-
`riod for the methamphetamine group was significantly
`Ž
`.
`different P -0.05 from that for the LiCl plus metham-
`Ž
`.
`phetamine group Fig. 1 . However, Student’s t-test showed
`that the overall mean ambulatory activity of the metham-
`phetamine group 149.47 "120.52 SD , ns72 was not
`Ž
`Ž
`.
`.
`Ž
`.
`significantly different P -0.15 from that for the Li plus
`methamphetamine group 172.54 "98.42, ns72 . Thus,
`Ž
`.
`these statistical results suggest that, although the mouse
`group treated with Li plus methamphetamine showed a
`slightly greater ambulatory activity throughout the obser-
`vation period than that
`treated with methamphetamine
`alone,
`there was a significant difference in their time
`courses, namely, the delayed onset of ambulatory activity
`in the mice pretreated with LiCl and methamphetamine. In
`confirmation of the result in Fig. 1, the chronic experi-
`ments to be described later, which examined again the
`effect of LiCl using a different group of mice, also showed
`a similar delayed development of ambulatory activity in
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`Fig. 1. Changes in the mouse ambulatory activity induced by a single injection of saline, LiCl, methamphetamine, or LiCl plus methamphetamine. I
`saline 2 mlrkg ; B LiCl 170 mgrkg ; ` methamphetamine 2 mgrkg , and v LiCl 170 mgrkg plus methamphetamine 2 mgrkg, 1 h after LiCl .
`Ž
`.
`Ž
`.
`Ž
`.
`Ž
`.
`Ž
`.
`Ordinate, the mean ambulatory activity which was the number of horizontal movements of mice for 10 min counted by the tilting cage method. Abscissa,
`Ž
`.
`Ž
`.
`Ž
`.
`time min after drug injection s.c. . The data shown are the mean "S.E.M. from 4 mice. The overall ambulatory activity across the observation period
`for the methamphetamine group or the LiCl plus methamphetamine group is significantly different from that for the saline group or the LiCl group
`Ž
`.
`P -0.05, post-hoc Fisher’s least significant difference test, 72 observed points from 4 mice for each group . The overall ambulatory activity across the
`Ž
`observation period for the methamphetamine group is significantly different from that for the LiCl plus methamphetamine group P -0.05, post-hoc
`.
`Fisher’s test, 72 observed points from 4 mice for each group . A similar relationship between methamphetamine and LiCl plus methamphetamine is also
`seen in Fig. 2A and B, inverted closed triangles.
`
`the mice treated with LiCl plus methamphetamine as com-
`pared with those injected with methamphetamine alone
`Ž
`.
`Fig. 2A and B, curves for first injections .
`In Fig. 1, the overall ambulatory activity across the
`observation period for the LiCl group or the saline group
`was also significantly different from that for the metham-
`phetamine group or the LiCl plus methamphetamine group
`Ž P -0.05, 72 observed values from 4 mice for each group,
`.
`post-hoc Fisher’s test .
`
`3.1.2. Chronic experiments
`In chronic studies, saline, LiCl, methamphetamine, or
`Ž
`.
`LiCl plus methamphetamine was repeatedly injected s.c.
`5 times at intervals of 3 days to 4—5 mice for each. As
`shown in Fig. 2A and B, when saline alone 2 mlrkg or
`Ž
`.
`LiCl alone 170 mgrkg was repeatedly injected s.c. , the
`Ž
`.
`Ž
`.
`ambulatory activity remained at very low levels and did
`Ž
`not increase as the injection repeated curves overlapping
`the abscissa . When methamphetamine 2 mgrkg was
`.
`Ž
`.
`injected repetitively at intervals of 3 days, the ambulatory
`activity was greatly enhanced and progressively increased
`as the injection repeated, as shown in Fig. 2A. These ten
`curves in Fig. 2A were statistically compared using the
`analysis of variance in SAS and the post-hoc Fisher’s least
`significant difference test in the same manner as that for
`Fig. 1. The post-hoc Fisher’s test showed that the overall
`ambulatory activity across the observation period after the
`Ž
`fifth injection of methamphetamine open circles in Fig.
`.2A was significantly different from that after each of the
`Ž
`.
`Ž
`.
`first
`inverted closed triangles , second closed triangles ,
`
`.
`Ž
`.
`Ž
`third open triangles and fourth closed circles injections
`Ž
`of methamphetamine P -0.05, 72 observed points from 4
`.
`mice for each group . The injection of methamphetamine
`2 mgrkg 1 h after LiCl 170 mgrkg , on the other hand,
`Ž
`.
`Ž
`.
`showed much smaller increases in ambulatory activity than
`the injection of methamphetamine alone. As shown in Fig.
`2B, the ambulatory activity did not show a clear peak and
`was kept nearly flat throughout the observation period of
`180 min. The post-hoc Fisher’s test following the analysis
`of variance of all 20 data groups in Fig. 2A and B showed
`that the overall ambulatory activity across the observation
`period after each of the first to fifth injections of LiCl plus
`methamphetamine was significantly different respectively
`from that after the first
`to fifth injections of metham-
`Ž
`phetamine alone P -0.05, 72 observed points from 4
`.
`mice for each group . Thus, pretreatment with LiCl consis-
`tently suppressed the ambulatory activity enhanced by
`each of the first to fifth subsequent injections of metham-
`phetamine.
`
`3.2. c-fos protein-like immunoreactiÕity
`
`3.2.1. Acute experiments
`Three hours after a single injection of metham-
`2 mgrkg,
`Ž
`.
`the expression of c-fos
`phetamine
`s.c. ,
`protein-like immunoreactivity was found to have increased
`to some extent in the dorsolateral geniculate nucleus, the
`cerebral cortex,
`the caudate putamen,
`the dorsomedial
`hypothalamic nucleus, the habenular nucleus, the amyg-
`dala and the piriform cortex. On the other hand, a single
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`Fig. 2. Changes in the mouse ambulatory activity induced by repetitive chronic injection of saline, LiCl, methamphetamine, or LiCl plus metham-
`phetamine. A, saline alone 2 mlrkg, the group of lower 5 curves , and methamphetamine alone 2 mgrkg, the upper 5 curves . B, LiCl alone 170
`Ž
`.
`Ž
`.
`Ž
`mgrkg, the group of lower 5 curves , and LiCl 170 mgrkg plus methamphetamine 2 mgrkg, 1 h after LiCl, the upper 5 curves . Different symbols
`.
`Ž
`.
`Ž
`.
`show the first to fifth injection at 3 days intervals: %, 1st; ', 2nd; ^, 3rd; v, 4th, and `, 5th injections of methamphetamine in A and of LiCl plus
`methamphetamine in B, and, although not visibly separated, I, 1st; B, 2nd; e, 3rd; l, 4th, and h, 5th injections of saline alone in A and of LiCl alone
`in B. Ordinates: the mean ambulatory activity which was the number of horizontal movements of mice for 10 min counted by the tilting cage method.
`Ž
`.
`Ž
`.
`Ž
`.
`Abscissas: time min after drug injection s.c. . The data shown are the mean "S.E.M. from 4 mice. At every injection, the overall ambulatory activity
`across the observation period for the methamphetamine group in A was significantly different from that for the corresponding LiCl plus methamphetamine
`Ž
`.
`group in B P -0.05, post-hoc Fisher’s least significant difference test, 72 observed points from 4 mice for each group .
`
`injection of saline 2 mlrkg , LiCl 170 mgrkg , or LiCl
`.
`Ž
`.
`Ž
`170 mgrkg plus methamphetamine 2 mgrkg, 1 h after
`Ž
`.
`Ž
`.LiCl
`showed only the sporadic and low expression of
`c-Fos-like immunoreactivity in all these brain regions.
`Counting of immunostained dots inside the 0.335 mm2
`area s555 mm =603 mm of each region in three brain
`Ž
`.
`sections by two-dimensional image analysis showed that
`methamphetamine significantly increased the number of
`
`c-Fos-positive cell nuclei dorsolateral geniculate nucleus
`Ž
`.
`Ž
`.
`Fig. 3A and in the striatum Fig. 3B as each compared
`Ž
`with saline alone or LiCl alone P -0.05 in the post-hoc
`Fisher’s least significant difference test following the anal-
`ysis of variance, ns3 for each . A single injection of
`.
`methamphetamine 1 h after a single injection of LiCl also
`significantly increased c-Fos-positive cell nuclei
`in the
`Ž
`P - 0.05, post-hoc
`dorsolateral geniculate nucleus
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`Fig. 3. The mean number "S.E.M., ns3 of c-Fos protein-like immunoreactive cell nuclei 3 h after a single injection closed columns and 3 h after the
`.
`Ž
`.
`Ž
`last of 5 times repeated injections at 3 days intervals hatched columns of saline 2 mlrkg , LiCl 170 mgrkg , methamphetamine 2 mgrkg , and LiCl
`Ž
`.
`Ž
`.
`Ž
`.
`Ž
`.
`170 mgrkg plus methamphetamine 2 mgrkg, 1 h after LiCl
`Ž
`.
`Ž
`.
`Ž
`.
`Ž .
`in the dorsolateral geniculate nucleus A , the striatium B , the dorsomedial hypothalamic
`nucleus C and the habenular nucleus D . Ordinate: the number of c-Fos immunoreactive nuclei in a unit area of 0.335 mm s555 mm =603 mm ,
`Ž .
`Ž
`.
`2 Ž
`.
`)
`counted using a two-dimensional image analyzer. P -0.05 post-hoc Fisher’s least significant differences test, ns3 for each .
`.
`Ž
`
`Fisher’s test, ns3 for each, Fig. 3A and also in the
`.
`striatum P -0.05, post-hoc Fisher’s test, ns3 for each,
`Ž
`.
`Fig. 3B as compared with saline alone. Moreover, pre-
`treatment with LiCl before methamphetamine reduced
`methamphetamine-induced expression of c-Fos-like im-
`munoreactivity significantly in the dorsolateral geniculate
`nucleus P -0.0001, post-hoc Fisher’s test, ns3 for
`Ž
`each, Fig. 3A, closed columns for methamphetamine and
`Lirmethamphetamine and barely significantly in the stria-
`.
`tum P -0.0572, post-hoc Fisher’s test, ns3 for each,
`Ž
`Fig. 3B, closed columns
`for methamphetamine and
`Lirmethamphetamine . In all other brain regions examined
`.
`Ž
`including the dorsolateral hypothalamic nucleus Fig. 3C,
`.
`Ž
`closed columns and the habenular nucleus Fig. 3D, closed
`.
`columns , acute methamphetamine did not significantly
`induce the expression of c-Fos-like protein, and hence
`Li-pretreatment had no significant effect.
`
`3.2.2. Chronic experiments
`When saline 2 mlrkg , LiCl 170 mgrkg , metham-
`.
`Ž
`Ž
`.
`phetamine 2 mgrkg , or LiCl plus methamphetamine was
`Ž
`.
`repeatedly injected 5 times at
`intervals of 3 days,
`the
`number of c-Fos-positive cell nuclei was expressed most
`Ž
`.
`abundantly in the striatum Fig. 3B, hatched columns ,
`Ž
`followed by the dorsal lateral geniculate nucleus Fig. 3A,
`.
`hatched columns , the dorsomedial hypothalamic nucleus
`Ž
`.
`Ž
`Fig. 3C, hatched columns and the habenular nucleus Fig.
`.
`3D, hatched columns . However, there were no statistically
`significant differences among the four test drugs in the
`number of c-Fos-positive cell nuclei in each of these brain
`regions.
`It was noted, however, that, in the geniculate nucleus
`and the striatum, the expression of c-Fos-like protein was
`significantly small in chronic methamphetamine injection
`Ž
`as compared with its acute injection P -0.05, post-hoc
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`Fisher’s test, ns3 for each, Fig. 3A and B, the closed
`column for methamphetamine and the hatched column for
`Lirmethamphetamine .
`.
`
`4. Discussion
`Ž .
`New findings in the present study are that 1 both the
`rate of increase and the peak level of the mouse ambula-
`tory activity after a single injection of methamphetamine
`Ž
`was apparently suppressed by pretreatment with LiCl cf.
`. Ž .
`Fig. 1 , 2 the expression of c-Fos protein-like immuno-
`reactivity in the mouse dorsolateral geniculate nucleus and
`the striatum was significantly increased after a single
`Ž
`.
`Ž .
`injection of methamphetamine cf. Fig. 3A and B , 3
`these increases in c-Fos were suppressed by pretreatment
`Ž
`.
`Ž .
`with LiCl cf. Fig. 3A and B , and 4 chronic repetitive
`injection of methamphetamine did not significantly in-
`crease the expression of c-Fos protein in all cerebral
`Ž
`.
`regions examined cf. Fig. 3A–D , but the extent of the
`expression was smaller in the chronic injection than in the
`acute injection of methamphetamine in the geniculate nu-
`Ž
`.
`cleus and striatum cf. Fig. 3A and B . As far as we are
`aware, there are no previous studies that have investigated
`the effect of methamphetamine on the regional activation
`of immediate early genes and the effect of lithium on
`methamphetamine-induced regional expression of c-Fos-
`Ž .
`like protein s
`in the mouse brain. In the rat, however,
`acute methamphetamine has been reported to induce the
`expression of c-Fos mRNA and c-Fos protein in the stria-
`w
`x
`tum 33,37 . In addition, it has also been reported that
`w
`x
`w
`x
`w
`x
`amphetamine 17 , cocaine 17 and morphine 32 induce
`the expression of c-Fos in the striatum, and this expression
`is mediated by D1 dopamine receptors and NMDA recep-
`tors. Therefore, there is the possibility that Li antagonized
`the methamphetamine-induced expression of c-Fos through
`the down-regulation of D1 receptors.
`4.1. Effects of LiCl on the increases in ambulatory actiÕity
`and c-Fos-like
`immunoreactiÕity
`induced by acute
`methamphetamine
`.
`Ž
`As described above cf. Figs. 1 and 2 , LiCl pretreat-
`ment seems to attenuate not only the rate of increase but
`also the rate of decline of the ambulatory activity induced
`by a single injection of methamphetamine. A number of
`previous studies have suggested that amphetamine- or
`methamphetamine-induced behavioral excitation in ani-
`mals may involve the activation of catecholaminergic neu-
`w
`x
`rons 13,20,21,26,28,30,36,44 . On the other hand,
`the
`effect of lithium on psychostimulant-induced behavioral
`stimulation has been reported to either involve changes in
`w
`x
`catecholaminergic systems 1,3,15 or may not
`involve
`w
`x
`changes in catecholaminergic systems 2,41,43 . Although
`lithium might interfere either with the methamphetamine-
`induced changes in catecholaminergic neuronal activity or
`with presumable subsequent metabolic pathways leading to
`behavioral excitation, or both, it is not clear whether these
`
`lithium effects are specific or nonspecific for the metham-
`phetamine-induced behavioral excitation, and why the on-
`set and decline of the ambulatory activity were slowed
`Ž
`.
`down by pretreatment with LiCl cf. Fig. 1 . However, the
`prefrontal cortex, especially its dorsolateral
`region is
`thought to play a key role in recognition processes for
`working memories, and these processes are known to be
`w
`x
`mediated by a dopaminergic system 38,56 . In this con-
`the above mentioned
`text,
`there is the possibility that
`Li-induced changes in ambulatory activity may have re-
`sulted from the down-regulation of D1 receptors in the
`dorsolateral geniculate nucleus. Further studies are re-
`quired on these matters.
`The dorsolateral geniculate nucleus and the striatum
`showed statistically significant increases in c-Fos expres-
`Ž
`sion after a single injection of methamphetamine cf. Fig.
`.
`suppressed the
`3A and B . Pretreatment with LiCl
`methamphetamine-induced increases in c-Fos expression
`Ž
`.
`in the dorsolateral geniculate nucleus cf. Fig. 3A and
`Ž
`.
`probably in the striatum cf. Fig. 3B . Since the ambula-
`tory activity was increased by a single injection of
`methamphetamine, and pretreatment with LiCl affected the
`Ž
`onset pattern of this increase as mentioned above cf, Figs.
`.
`1 and 2 , it is tempting to speculate that the expression of
`Ž .
`c-Fos-like protein s in the dorsolateral geniculate nucleus
`andror the striatum may relate to the enhancement of
`ambulatory activity induced by acute methamphetamine.
`However, it is unclear at present by what mechanism acute
`methamphetamine enhances the c-Fos expression in these
`particular brain regions, and how these regional expres-
`sions of c-Fos contribute to methamphetamine-induced
`increase in ambulatory activity. Since each of different
`psychostimulants appears to induce a highly specific pat-
`w
`x
`tern of c-Fos mRNA expression 25 , comparative studies
`with D-amphetamine, cocaine, etc. may provide some clue
`w
`x
`to these questions 17,35 .
`Effects of a single administration of lithium on the
`behavioral excitation induced by psychostimulants in ani-
`mals described in earlier studies are rather confusing prob-
`ably because of differences in animal and drug species
`w
`x
`used 5,13,14,48 . However, we clearly demonstrated in
`the present study that pretreatment with 170 mgrkg LiCl
`modify the enhancing effect of singly injected metham-
`phetamine 2 mgrkg on the mouse ambulatory activity,
`Ž
`.
`although it is not clear whether this is also the case for
`w
`x
`other experimental animals 19,20 , and by what mecha-
`nism LiCl suppresses the methamphetamine-induced ex-
`Ž .
`pression of c-Fos protein s in the dorsolateral geniculate
`nucleus and in the striatum.
`
`4.2. Effect of LiCl on the increases in ambulatory actiÕity
`and c-Fos-like immunoreactiÕity induced by chronic
`methamphetamine
`
`Repetitive injections of methamphetamine significantly
`Ž
`and progressively increased the ambulatory activity cf.
`
`6 of 8
`
`Alkermes, Ex. 1050
`
`

`
`M. Namima et al.rBrain Research 782 1998 83–90
`)
`(
`
`89
`
`.
`Fig. 2A , indicating that behavioral excitation, i.e., a state
`w
`x
`of reverse tolerance 11,29 was progressively produced in
`mice by chronic repetitive injections of methamphetamine
`in confirmation of previous studies with methamphetamine
`w
`x
`21,22,26,49,51 .
`Although repetitively administered lithium salts have
`been reported to suppress the locomotor excitation induced
`by chronic amphetamine or methamphetamine in animals
`w
`x
`in many studies 3,4,6,16,18,42,46,48,52 , the lack of the
`w
`x
`lithium effect has also been reported 9,12 . We demon-
`strated here that pretreatment with LiCl significantly pre-
`vented the progressive enhancement of ambulatory activity
`induced by repetitive injection of methamphetamine in the
`mouse, and this Li effect appeared to be independent of the
`Ž
`.
`times of methamphetamine injection cf. Fig. 2A and B .
`that LiCl can suppress
`the metham-
`This
`suggests
`phetamine-induced behavioral excitation even at an inter-
`mediate stage towards the complete formation of the re-
`verse tolerance.
`In contrast to a single injection, repetitive chronic injec-
`tion of methamphetamine did not significantly change the
`Ž .
`expression of c-Fos-like protein s
`in all brain regions
`examined, but chronic methamphetamine induced a smaller
`amount of c-Fos protein in the geniculate nucleus and
`Ž
`.
`striatum than acute methamphetamine did cf. Fig. 3 . At
`information to explain this
`present, we have no direct
`difference between the acute and chronic metham-
`phetamine treatments in c-Fos expression. However, it is
`conceivable that acute activation by methamphetamine of
`immediate early genes might have shifted to subsequent
`activation of c-Fos-sensitive genes as stimulation with
`methamphetamine repeated, and also that
`the state of
`reverse tolerance might be maintained by the function of
`such delayed genes rather than by the c-Fos gene itself. In
`this connection, the cumulative production of Fos-related
`Ž
`.
`antigens FRAs by chronic administration of cocaine has
`w
`x
`recently been reported by Hope et al. 24 . This possibility
`is to be investigated using antibodies highly specific for
`individual FRAs. However, it may at least be said that
`LiCl suppresses the behavioral excitation even if metham-
`phetamine-induced activation of the c-Fos gene has been
`taken over by the activation of other delayed genes.
`
`Acknowledgements
`
`This work was supported by the grant from the Epilepsy
`Research Foundation of Japan to M. Namima.
`
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