Effects of mechanical stress and deficiency of dihydrotestosterone or 17ß-estradiol on Temporomandibular Joint Osteoarthritis in mice.

OBJECTIVE: To observe and analyze the interaction between excessive mechanical stress (MS) and decreased sex hormones on Temporomandibular Joint Osteoarthritis (TMJ-OA), and to discover TMJ-OA disease susceptibility genes by molecular biological analysis to elucidate part of the mechanism of TMJ-OA onset. DESIGN: For experimental groups, orchiectomy (ORX) or ovariectomy (OVX) was performed on sexually mature 8-week-old mice. A metal plate was attached to the posterior surface of the maxillary incisors to apply excessive MS on mandibular condyles. Male mice were divided into control, ORX, MS, and ORX + MS groups, while female mice were divided into control, OVX, MS, and OVX + MS groups. Mandibular condyles were evaluated by histology and molecular biology. RESULTS: Histomorphometric analysis of the TMJ in ORX + MS and OVX + MS groups revealed the thinnest chondrocyte layers, highest modified Mankin scores, and significant increases in the number of osteoclasts. Gene expression analysis indicated upregulation of Angptl7 and Car1 genes in the mandibular condyles of mice subjected to the combined effects of excessive MS and reduced sex hormones. In vitro analysis suggested that cartilage-like cells overexpressing Angptl7 enhanced calcification, and osteoblast-like cells overexpression Car1 suppressed cell proliferation and calcification. CONCLUSIONS: A severe TMJ-OA mouse model was successfully developed by applying excessive MS on the mandibular condyle of male and female mice with reduced sex hormones. Disease-susceptibility genes Angptl7 and Car1 were newly discovered in the experimental groups, suggesting their involvement in the onset mechanism of TMJ-OA.

DNA methylation of the BMAL1 promoter.

We previously analyzed transcriptional regulation of the BMAL1 gene, a critical component of the mammalian clock system and found that the BMAL1 gene is expressed with circadian oscillation and that its regulatory region is located in hypomethylated CpG islands with an open chromatin structure. Here, we found that the BMAL1 gene is not expressed with circadian oscillation in CPT-K cells because the CpG islands located in the BMAL1 promoter are hypermethylated and that 5-aza-2'-deoxycytidine (aza-dC) recovered BMAL1 expression. In contrast, CpG islands in the PER2 promoter were hypomethylated, the PER2 gene was expressed and aza-dC enhanced PER2 gene expression in CPT-K cells. Reporter gene assays showed that intracellular transcriptional machinery for the BMAL1 gene is active, suggesting that BMAL1 inactivation is caused by DNA methylation and not by malfunctional promoter activity. Incubating CPT-K cells with aza-dC also increased CRY1 expression, whereas CLOCK expression was not altered and the CRY1 promoter was unmethylated. These results suggest that aza-dC induces BMAL1 expression via DNA demethylation in the BMAL1 promoter and enhances PER2 and CRY1 transcription. Finally, aza-dC recovered the circadian oscillation of BMAL1 transcription. These results suggest that DNA methylation of the BMAL1 gene is critical for interfering with circadian rhythms.

Gender difference in tooth autotransplantation with complete root formation: a retrospective survey.

Gender-related risk factors in the survival of transplanted teeth with complete root formation have not yet been identified. The purpose of this study was to investigate gender differences in tooth autotransplantation at dental clinics. We asked participating dentists to provide information on transplantations they had undertaken from 1 January 1990 to 1931 December 2010. The data were screened to exclude patients who underwent more than one transplantation, smokers or those whose smoking habits were unknown, patients under 30 or who were 70 years old and over, cases where the transplanted teeth had incomplete root formation or multiple roots and those with fewer than 20 present teeth post-operation. We analysed 73 teeth of 73 males (mean age, 47.2 years) and 106 teeth of 106 females (mean age, 45.3 years) in this study. The cumulative survival rate and mean survival time were calculated using the Kaplan-Meier method. The cumulative survival rate for males was 88.3% at the 5-year mark, 64.8% at 10 years and 48.6% at 15 years; for females, it was 97.2% at the 5-year mark, 85.9% at 10 years and 85.9% at 15 years. A log-rank test indicated the difference between males and females to be significant (P = 0.011). There was also a significant difference in the main causes for the loss of transplanted teeth: males lost more transplanted teeth due to attachment loss than females (P < 0.05). These results indicate that males require more attention during the autotransplantation process, particularly at the stage of pre-operation evaluation and that of follow-up maintenance.

AM-36 modulates the neutrophil inflammatory response and reduces breakdown of the blood brain barrier after endothelin-1 induced focal brain ischaemia.

BACKGROUND AND PURPOSE: Following transient focal stroke, rapid accumulation and activation of neutrophils in the ischaemic region is deleterious due to release of reactive oxygen species and myeloperoxidase (MPO). The purpose of this study was to examine whether AM-36, both a Na+ channel blocker and an antioxidant, afforded neuroprotection by modulating neutrophil accumulation into brain, following endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAo) in conscious rats. EXPERIMENTAL APPROACH: AM-36 was administered at 3 and 24 h after ET-1-induced MCAo. Functional recovery was determined using grid-walking and cylinder tests. Image analysis of brain sections was used to determine infarct volume. The effect of AM-36 on neutrophil infiltration and their interaction with macrophages was examined in rats at 48 h following MCAo by both an MPO assay and double-label immunofluorescence. Blood brain barrier (BBB) breakdown was measured by the area stained by intravenous Evans Blue. KEY RESULTS: AM-36 reduced functional deficits in both tests such that no difference existed from pre-ischaemic values at 48 h. Neutrophil infiltration, assessed by MPO activity, and infarct volume were significantly reduced following AM-36 administration by 54 and 60% respectively. Similarly, immunofluorescence revealed that AM-36 reduced neutrophil infiltration by approximately 50% in selected brain regions, when compared to controls, and also modulated macrophage phagocytosis of neutrophils. Breakdown of the BBB was significantly reduced by 60% following AM-36 treatment. CONCLUSIONS AND IMPLICATIONS: These findings suggest that AM-36 can directly modulate the neutrophil inflammatory response and reduce BBB breakdown following MCAo.

Drebrin A regulates hippocampal LTP and hippocampus-dependent fear learning in adult mice.

Structural plasticity of dendritic spines, which underlies higher brain functions including learning and memory, is dynamically regulated by the actin cytoskeleton and its associated proteins. Drebrin A is an F-actin-binding protein preferentially expressed in the brain and localized in the dendritic spines of mature neurons. Isoform conversion from drebrin E to drebrin A and accumulation of the latter in dendritic spines occurs during synapse maturation. We have previously demonstrated that drebrin A plays a pivotal role in spine morphogenesis and plasticity. However, it is unclear whether drebrin A plays a specific role in processes required for structural plasticity, and whether drebrin E can substitute in this role. To answer these questions, we analyzed mutant mice (named DAKO mice), in which isoform conversion from drebrin E to drebrin A is disrupted. In DAKO mouse brain, drebrin E continues to be expressed throughout life instead of drebrin A. Electrophysiological studies using hippocampal slices revealed that long-term potentiation of CA1 synapses was impaired in adult DAKO mice, but not in adolescents. In parallel with this age-dependent impairment, DAKO mice exhibited impaired hippocampus-dependent fear learning in an age-dependent manner; the impairment was evident in adult mice, but not in adolescents. In addition, histological investigation revealed that the spine length of the apical dendrite of CA1 pyramidal cells was significantly longer in adult DAKO mice than in wild-type mice. Our data indicate that the roles of drebrin E and drebrin A in brain function are different from each other, that the isoform conversion of drebrin is critical, and that drebrin A is indispensable for normal synaptic plasticity and hippocampus-dependent fear memory in the adult brain.

Molecular cloning and expression of human neurochondrin-1 and -2.

Human neurochondrins have been cloned from a brain cDNA library. The human neurochondrin-1 and -2 predict leucine-rich (15.8 and 15.9%) proteins of 729 and 712 amino acid residues, with molecular weights of 78.9 and 77.2 kDa, respectively. The deduced amino acid sequence indicates 98% identity among human, mouse and rat species. Northern analysis indicates that about 4 kb human neurochondrin mRNAs are abundant in the fetal and the adult brain.

Allopregnanolone increases mature excitatory synapses along dendrites via protein kinase A signaling.

Allopregnanolone (APα; 5α-pregnan-3α-ol-20-one) is synthesized in both the periphery and central nervous system and is known to be a potent positive allosteric modulator of the GABAA receptor. Because APα was suggested to improve the symptoms of depression and Alzheimer's disease (AD), which involve synaptic dysfunction and loss, we examined whether APα affects excitatory synapses. Drebrin, which is an actin-binding protein, forms a unique stable actin structure in dendritic spines, and drebrin levels correlate positively with cognitive levels in AD and mild cognitive impairment. We investigated whether APα increases excitatory synapse density along dendrites of mature hippocampal neurons using drebrin-imaging-based evaluation of mature synapses. We prepared primary cultures of hippocampal neurons and either transfected them with GFP or immunostained them against drebrin. Morphological analysis of GFP-transfected neurons revealed that a 24-h exposure to 0.3 or 1 µM APα significantly increased dendritic spine density without any morphological changes to spines. Drebrin cluster density was also increased by 0.3 and 1 µM APα. The protein kinase A (PKA) inhibitor H-89 inhibited the APα-induced increase in drebrin cluster density. These data demonstrate that APα increases mature excitatory synapses via activation of PKA. Therefore, the PKA-cAMP response element-binding protein (CREB) signaling pathway is likely to be involved in the APα-induced increase of mature excitatory synapses. Another possibility is that the PKA-dependent increase in AMPA receptors at dendritic spines mediates the APα function. In conclusion, our study indicates that APα may improve neuropsychiatric disorder outcomes via increasing the numbers of mature excitatory synapses.

The effects of epinephrine on norepinephrine release in essential hypertension.

The effects of endogenous epinephrine (E), released by glucagon injection, and exogenously infused E on plasma norepinephrine (NE) and cardiovascular responses before and after beta-blockade were studied in patients with essential hypertension and in age-matched normotensive controls. The resting plasma NE and E levels were significantly higher in the borderline hypertensive subjects (NE: 251 +/- 21 pg/ml [SEM], p less than 0.005; E: 57 +/- 5, p less than 0.05, n = 18) than in controls (NE: 129 +/- 12; E: 39 +/- 5, n = 18). An intravenous injection of glucagon (1.0 mg) induced a transient rise of both plasma catecholamine levels and blood pressure in every subject studied. Plasma E levels rose transiently and returned to the basal levels by 20 minutes after the injection, whereas plasma NE levels showed a more prolonged rise over 20 minutes. beta-Blockade with propranolol did not affect the plasma E response to glucagon, but inhibited the prolonged rise of plasma NE levels. An intravenous infusion of exogenous E (1.25-1.50 micrograms/min) for 30 minutes caused an apparent rise of both plasma NE levels and blood pressure, which lasted more than 60 minutes after stopping the E infusion. Propranolol did not affect the time course of plasma E but again inhibited the prolonged rise of both plasma NE levels and blood pressure. No significant differences could be observed in the cardiovascular or plasma NE responses to glucagon or to E infusion between normal and hypertensive subjects. These findings lend support to the view that plasma E can act physiologically as a sustained stimulator of presynaptic beta-adrenergic receptors, which leads to an enhanced NE release from peripheral sympathetic nerve terminals and a rise of blood pressure in humans.

Identification of cDNAs encoding two novel nuclear proteins, IMUP-1 and IMUP-2, upregulated in SV40-immortalized human fibroblasts.

Using a model system of young, senescent and SV40-immortalized WI-38 fibroblasts, we identified two mRNAs upregulated in immortalized cells (imup-1, immortalization-upregulated protein 1, and imup-2). Compared to normal tissues, both genes were more frequently expressed in cancer cells. The open reading frame of imup-1 spans 321bp, coding for a 10.9 kDa protein of 106 amino acids, while an insertion of 59bp in the otherwise identical mRNA of imup-2 leads to a frameshift, resulting in an 8.5 kDa protein of 85 amino acids. Database searches identified these genes on chromosome 19, which could account for the cloned imup-1 and imup-2 transcripts by alternative splicing. Southern blot analysis of digested genomic DNA confirmed that both transcripts are derived from a single locus. The expressed proteins IMUP-1 and IMUP-2 share 46 identical N-terminal amino acids, whereas the C-termini are unrelated. Green fluorescent protein-fusions of both IMUP-1 and IMUP-2 accumulated in the nucleus of HeLa cells. The C-terminus of IMUP-1 contains a bipartite nuclear localization signal, the deletion of which impaired nuclear translocation. In-vitro translated proteins bound to poly(rG), but did not interact with single-stranded DNA or double-stranded DNA. The nuclear localization of IMUP-1 and IMUP-2 as well as the upregulation of both underlying mRNAs in immortalized cells suggest a function in immortalization.

Role of N-linked oligosaccharides attached to human renin expressed in COS cells.

One or both of two putative N-glycosylation sites (at asparagine-5 and -75) of human renin was eliminated by amino acid replacement of the asparagine residue with an alanine residue using site-directed mutagenesis. The three glycosylation-deficient renins (Asn-5, Asn-75, Asn-5 and -75 mutants) were expressed in COS cells and secreted into the conditioned media. The secreted amounts of the three mutants were different from one another, although the mutant and wild-type renins had practically the same specific activity. An Asn-5 and -75 mutant which did not contain any glycosylation sites was unstable in the medium, suggesting that the N-linked oligosaccharides play an important role in stabilization of human renin.

Characterization of monoclonal antibodies against human prorenin profragment and identification of active prorenins in plasma.

Monoclonal antibodies were raised against a synthetic peptide (43 amino acid residues) that corresponds to the complete profragment of human prorenin. Seven monoclonal antibodies were chosen for further characterization. Two antibodies, 2-X-C1 and 4-X-E1, reacted with the middle region and C-terminus of the profragment and were isotyped IgG1. The affinity constants of these antibodies against the human profragment were 7.6 x 10(8) and 3.0 x 10(7) M-1, respectively. Immunoaffinity columns containing the antibodies 2-X-C1 and 4-X-E1, respectively, were used for the characterization of active prorenin in human plasma. This active prorenin strongly bound to the 4-X-E1 column and eluted as two separate peaks which corresponded to fully and partially active prorenin, respectively. The partially active prorenin had higher activity with a small substrate, tridecapeptide, than with a large one, angiotensinogen, although the fully active prorenin had the same renin activity irrespective of the size of the substrate. These data suggest that new forms of prorenin, active prorenin, exist in human plasma and that their active sites are completely or partially exposed to the substrates. Moreover, the active prorenin in plasma was found not only in human but also in all tested mammalians. Cross-reactivity among the profragments of mammalian plasma prorenins can be explained by conservation of the amino acid sequence (epitope) of the combining site.

Site-directed mutagenesis of human prorenin. Substitution of three arginine residues in the propeptide with glutamine residues yields active prorenin.

Human prorenin is an inactive zymogen comprising 43 amino acid residues at the amino terminus of human renin. The aim of this work was to determine why prorenin is inactive at neutral pH. Eighteen different mutant prorenins, in which positively charged residues in the propeptide were substituted with either glutamine (Gln) or lysine (Lys) residues by site-directed mutagenesis, were expressed in COS-7 cells and characterized. By replacing each of the three arginine (Arg) residues (Arg10P, Arg15P, and Arg20P) with Gln residues, partially active prorenins were produced, which exhibited significant but not full renin activity without trypsin activation. The effect of double or triple amino acid substitutions on the appearance of active prorenin was cumulative, the activity reaching about 80% in a mutant in which all the three Arg residues were replaced by Gln residues. In contrast, mutant prorenins with Lys residues substituted for the Arg residues were inactive. These results clearly indicate that the positive charges of the three Arg residues are essential for maintenance of the human prorenin in an inactive form.

Isolation and characterization of recombinant human prorenin in Chinese hamster ovary cells.

Recombinant human prorenin (rh-prorenin) was purified from supernatants of Chinese hamster ovary (CHO) cell line transfected with the cDNA for rh-prorenin by employing a simple two-step procedure which consisted of ammonium sulfate precipitation and immunoaffinity chromatography using a monoclonal antibody specific for the profragment of human prorenin. About 100-fold purification with 35% recovery was achieved after the two steps. Purified rh-prorenin migrated as a single protein band with apparent molecular weights of 46,000-47,000 and about 50,000 on SDS-PAGE and gel filtration (HPLC), respectively, although it consisted of multiple components (pI values, 5.6-6.4) that could be resolved by isoelectric focusing (IEF). The treatment of rh-prorenin with endo-beta-N-acetylglucosaminidase converted the rather broad protein band to a sharp band on SDS-PAGE and reduced the number of multiple pI peaks on IEF. Amino-terminal sequence analysis of both the purified rh-prorenin and rh-renin revealed Leu-Pro-Thr-Asp- and Leu-Thr-Leu-Gly-, respectively, which agreed with those predicted from the base sequences of their cDNA. These data suggested that microheterogeneity of rh-prorenin is due to the carbohydrate moiety, but not to the protein moiety. Purified rh-prorenin was almost inactive, but was cleaved at the carboxyl end of a dibasic pair Lys-2-Arg-1 by trypsin and converted to active renin. However, at the early stage during trypsin activation, new intermediate forms between rh-prorenin and rh-renin were formed, suggesting multiple activation steps of rh-prorenin in addition to the one step activation.

Fos expression in neurons immunoreactive for neuronal nitric oxide synthase in the rat paraventricular nucleus after intraperitoneal injection of interleukin-1 beta.

Double immunostaining for Fos and neuronal nitric oxide synthase (nNOS) was used to examine whether nNOS-immunoreactive neurons in the paraventricular hypothalamic nucleus (PVN) are activated to express Fos immunoreactivity by intraperitoneal injection of interleukin-1 beta (IL-1 beta) in the rat. Quantitative analysis revealed that some nNOS-positive PVN neurons are activated by IL-1 beta (4 microg/kg, i.p.) administration, but the majority of the IL-1 beta-activated PVN neurons do not express nNOS and are distributed mainly in the parvocellular part of the PVN.

Methamphetamine-induced Fos expression in the substantia nigra pars reticulata in rats with a unilateral 6-OHDA lesion of the nigrostriatal fibers.

In rats with a unilateral 6-hydroxydopamine (6-OHDA)-induced lesion in the nigrostriatal fibers, methamphetamine (3 mg/kg, i.p.) induced Fos-like immunoreactivity (FLI), which was inhibited by pretreatment with N-methyl-D-aspartate antagonist MK-801 (1 mg/kg, i.p.), not only in the medial striatum contralateral to the lesion but also in the substantia nigra pars reticulata (SNr) ipsilateral to the lesion. Thus, hemispheric asymmetries in FLI were induced by methamphetamine in the medial striatum and the SNr in the 6-OHDA model of turning which may be related to the altered function of glutamatergic transmission.

Cardiovascular and respiratory actions of pituitary adenylate cyclase-activating polypeptides.

Effects of pituitary adenylate cyclase-activating polypeptide (PACAP38) and PACAP27 on the cardiovascular and respiratory systems were examined and compared to those of vasoactive intestinal polypeptide (VIP) in anesthetized beagle dogs. Intravenous PACAP27 and PACAP38 produced a decrease in mean arterial blood pressure (MBP), and an increase in both femoral arterial blood flow (ABF) and in frequency of respiration (FR) with a dose-dependent relationship between 10 and 300 pmol/kg. PACAP27 produced a dose-dependent increase in heart rate (HR) between 10 and 300 pmol/kg while PACAP38 induced tachycardia which was not dose-dependent. Administration of 300 pmol/kg PACAP38 and PACAP27 produced extreme hypertension after transient hypotension. PACAP38 produced severe bradycardia after transient tachycardia. The cardiovascular actions of PACAP38 were persistent compared to those of PACAP27. Intravenous injection of 10-300 pmol/kg VIP brought about hypotension, tachycardia and an increase in ABF and FR with a dose-dependent relationship. VIP, at 2000 pmol/kg, did not produce the biphasic response obtained by a large dose of PACAP38. The present studies demonstrate that PACAP partially possesses VIP-like cardiovascular and respiratory actions and that the C-terminal 11 amino acid residues of PACAP38 are presumably responsible for a prolongation of its actions.

Cardiovascular and sympathetic effects of proadrenomedullin NH2-terminal 20 peptide in conscious rats.

Proadrenomedullin NH2-terminal 20 peptide (PAMP) and adrenomedullin (AM), which are derived from the same gene, are novel vasodilative peptides and have been shown to exhibit hypotensive action in anesthetized animals. To avoid the modification via anesthesia, we investigated the effects of intravenously administered PAMP on mean arterial pressure, heart rate (HR), and renal sympathetic nerve activity (RSNA) relative to those of AM in conscious unrestrained rats. We also examined whether the arterial baroreceptor reflex was altered with the two peptides. Intravenous injection of rat PAMP (rPAMP) (10, 20 and 50 nmol/kg) and rat AM (rAM) (0.3, 1.0 and 3.0 nmol/kg) similarly elicited dose-related hypotension accompanied by increases in HR and RSNA. However, the responses to rPAMP were less potent in magnitude and shorter in duration than those to rAM. Moreover, rAM facilitated baroreflex control, whereas rPAMP attenuated it. These findings indicate that although PAMP, as well as AM, may play an important role as a circulating hormone in the systemic circulation of conscious rats, the two peptides derived from an identical origin might have different mechanisms responsible for their cardiovascular and RSNA actions.

Increase in norepinephrine but not nitric oxide metabolite levels in the hypothalamic paraventricular nucleus region in response to air jet and swing rotation in freely moving conscious rats.

Chronically instrumented, conscious rats were used to examine whether mild exteroceptive stress produces differential neurochemical changes in the hypothalamic paraventricular nucleus (PVN) region. We constructed systems for stress experiment of air jet and swing rotation that were conducted on freely moving conscious rats in a computer-controlled home cage. Concentration of extracellular norepinephrine (NE) and nitric oxide metabolites (NO(X)(-)), nitrite (NO(2)(-)) and nitrate (NO(3)(-)), in the PVN region was then measured by high-performance liquid chromatography with the respective detector; blood pressure (BP) and heart rate (HR) were also measured. Both stressors increased NE concentration in the PVN region as well as BP and HR. Neither stressor altered NO(X)(-) in the PVN region. Cardiovascular and NE changes showed reproducibility in intensity-dependent manner in response to repeated stressors. This finding demonstrated that exteroceptive stress produced different effects on the neurochemical mediators, NE and NO, in the PVN region.

GABA(A) and GABA(B) receptors modulating basal and footshock-induced nitric oxide releases in rat prefrontal cortex.

Using an in vivo brain microdialysis technique, we measured extracellular levels of nitric oxide (NO) metabolites (NO(x)(-)) in the medial prefrontal cortex (mPFC) upon perfusion of gamma-aminobutyric acid (GABA) receptor antagonists as well as agonists, and also examined the effects of GABA receptor agonists on mild intermittent footshock-induced NO releases in the mPFC in conscious rats. Perfusion of either bicuculline methiodide, a GABA(A) receptor antagonist, or saclofen, a GABA(B) receptor antagonist, through a microdialysis probe resulted in dose-dependent increases in NO(x)(-) levels. Higher-dose perfusion of either muscimol (50 microM), a GABA(A) receptor agonist, or baclofen (250 microM), a GABA(B) receptor agonist resulted in a significant decrease in NO(x)(-) levels. The elevated levels of NO(x)(-) after mild intermittent footshock were attenuated by perfusion of either muscimol (10 microM) or baclofen (50 microM), either of which alone did not affect basal NO(x)(-) levels. These findings are likely to provide helpful clues to our understanding of the inhibitory modulation of basal and footshock-induced NO metabolites releases by GABA(A) and GABA(B) receptors in the mPFC.

Methamphetamine induces fos expression in the striatum and the substantia nigra pars reticulata in a rat model of Parkinson's disease.

In rats with a unilateral 6-hydroxydopamine (6-OHDA) lesion in the nigrostriatal pathway, methamphetamine (3 mg/kg, i.p.) induced Fos-like immunoreactivity (FLI) not only in the striatum on the intact side but also in the substantia nigra pars reticulata (SNr) on the lesioned side. The methamphetamine-induced hyperexpression of FLI in the SNr on the lesioned side was suppressed by pretreatment with either dopamine D1 receptor antagonist SCH-23390 (0.5 mg/kg, i.p.), D2 receptor antagonist raclopride (2 mg/kg, i.p.) or N-methyl-d-aspartate receptor antagonist MK-801 (1 mg/kg, i.p.), which was concomitant with inhibition of the methamphetamine-induced rotational behavior of each antagonist. However, the hyperexpression of FLI in the SNr was not suppressed by intrastriatal grafts of fetal ventral mesencephalon which could suppress the methamphetamine-induced rotation completely. These results indicate that opposite hemispheric asymmetries in FLI are induced by methamphetamine in the striatum and the SNr in the 6-OHDA rats. It is suggested that the FLIs in the two discrete sites are activated independently by different mechanisms, and furthermore, different neuronal pathways are involved in the methamphetamine-induced rotation and Fos expression in the SNr of 6-OHDA rats.