Oxytocin in the periaqueductal grey regulates nociception in the rat

Studies have demonstrated that oxytocin (OXT) plays important roles in pain modulation in the central nervous system, and there are OXT receptors in the periaqueductal grey (PAG). The experiment was designed to investigate the effect of OXT in the PAG on antinociception. The results showed that (1) intra-PAG injection of OXT increased the pain threshold, whereas the local administration of the high specific OXT receptor antagonist, desGly-NH(2), d(CH(2))(5)[D-Tyr(2), Thr-sup-4]OVT decreased the pain threshold in a dose-dependent manner; (2) Pain stimulation could elevate OXT concentration in the PAG perfusion liquid. The data suggested that OXT in the PAG was involved in the antinociceptive process through the OXT receptor.     

Design of peptide oxytocin antagonists with strikingly higher affinities and selectivities for the human oxytocin receptor than atosiban.

The peptide oxytocin (OT) antagonist atosiban, approved for tocolytic use in Europe (under the tradename Tractocile), represents an important new therapeutic advance for the treatment of premature labor. This paper presents some new peptide OT antagonists which offer promise as superior tocolytics. The solid phase synthesis is reported of four pairs of L and D-2-naphthylalanine (L/D-2Nal) position-2 modified analogs of the following four oxytocin (OT) antagonists: des-9-glycinamide [1-(beta-mercapto-beta,beta-pentamethylene propionic acid), 2-O-methyltyrosine, 4-threonine]ornithine-vasotocin (desGly-NH(2),d(CH(2))(5)[Tyr(Me)(2),Thr(4)]OVT) (A); the Tyr-NH(2) (9) analog of (A), d(CH(2))(5)[Tyr(Me)(2),Thr(4),Tyr-NH(2) (9)]OVT (B); the Eda(9) analog of (A), d(CH(2))(5)[Tyr(Me)(2),Thr(4),Eda(9)]OVT (C); and the retro COCH(2)Ph(4-0H)(10) modified analog of (C), d(CH(2))(5)[Tyr(Me)(2),Thr(4),Eda(9)<-- COCH(2)Ph(4-0H)(10)]OVT (D). The eight new analogs of A-D are (1) desGly-NH(2),d(CH(2))(5)[D-2Nal(2),Thr(4)]OVT, (2) desGly-NH(2),d(CH(2))(5)[2-Nal(2),Thr(4)]OVT, (3) d(CH(2))(5)[D-2Nal(2),Thr(4),Tyr-NH(2) (9)]OVT, (4) d(CH(2))(5)[2Nal(2),Thr(4),Tyr-NH(2) (9)]OVT, (5) d(CH(2))(5)[D-2Nal(2),Thr(4),Eda(9)]OVT, (6) d(CH(2))(5)[2Nal(2),Thr(4),Eda(9)]OVT, (7) d(CH(2))(5)[D-2Nal(2),Thr(4),Eda(9)<-- COCH(2)Ph(4-0H)(10)]OVT, (8) d(CH(2))(5)[2Nal(2),Thr(4),Eda(9)<-- COCH(2)Ph(4-OH)(10)]OVT. Peptides 1-8 were evaluated for agonistic and antagonistic activities in in vitro and in vivo rat bioassays, in rat OT receptor (rOTR) binding assays and in human OT receptor (hOTR) and human vasopressin (VP) vasopressor (V(1a)) receptor (hV(1a)R) binding assays. Also reported are the hOTR and hV(1a)R affinity data for atosiban and for B. None of the eight peptides exhibit oxytocic or vasopressor agonism. Peptides 1-8 exhibit weak antidiuretic agonism (activities in the range 0.014-0.21 U/mg). Peptides 1-6 exhibit potent in vitro (no Mg(2+)) OT antagonism (anti-OT pA(2) values range from 7.63 to 8.08). Peptides 7 and 8 are weaker OT antagonists. Peptides 1-6 are all OT antagonists in vivo (estimated in vivo anti-OT pA(2) values in the range 6.94-7.23). Peptides 1-8 exhibit vasopressor antagonism, anti-V(1a) pA(2) values in the range 5.1-7.65. Peptides 1-8 exhibit high affinities for the rOTR (K(i) values = 0.3-7.8 nM). Peptides 1-4 and B exhibit surprisingly very high affinities for the hOTR; their K(i) values are 0.17, 0.29, 0.07, 0.14 and 0.59 nM, respectively. Peptides 1-4 and B exhibit respectively 449, 263, 1091, 546 and 129 times greater affinity for the hOTR than atosiban (K(i) = 76.4 nM). Peptides 1-4 exhibit high affinities for the hV(1a)R (K(i)s = 1.1 nM, 1.3 nM, 0.19 nM and 0.54 nM, all higher than the hV1(a)R affinities exhibited by atosiban (K(i) = 5.1 nM) and by B (K(i) = 5.26 nM). Because of their strikingly higher affinities for the hOTR than atosiban, peptides 1-4 and B exhibit gains in anti hOT/anti hV(1a) receptor selectivity compared with atosiban of 93, 64, 39, 56 and 127, respectively. These OT antagonists are thus promising candidates for development as potential new tocolytic agents.     

Design of oxytocin antagonists, which are more selective than atosiban.

We report the solid phase synthesis of four pairs of L- and D-thienylalanine (Thi/D-Thi) position two modified analogues of the following four oxytocin (OT) antagonists: des-9-glycinamide [1-(beta-mercapto-beta,beta-pentamethylene propionic acid), 2-O-methyltyrosine, 4-threonine]ornithine-vasotocin (desGly(NH2)9,d (CH2)5[Tyr(Me)2,Thr4]OVT) (A); the Tyr-(NH2)9 analogue of (A), d(CH2)5[Tyr(Me)2,Thr4,Tyr-(NH2)9]OVT (B); the Eda9 analogue (where Eda = ethylenediamine) of (A), d(CH2)5[Tyr(Me)2, Thr4, Eda9]OVT (C); and the retro Tyr10 modified analogue of (C), d(CH2)5[Tyr(Me)2, Thr4, Eda9<--Tyr10]OVT (D). The eight new analogues of A-D are (1) desGly(NH2),d(CH2)5[Thi2,Thr4]OVT, (2) desGly(NH2),d(CH2)5[D-Thi2,Thr4]OVT, (3) d(CH2)5[Thi2, Thr4,Tyr-(NH2)9]OVT, (4) d(CH2)5[D-Thi2,Thr4,Tyr-(NH2)9]OVT (5) d(CH2)5[Thi2,Thr4Eda9]OVT, (6) d(CH2)5[D-Thi2,Thr4,Eda9]OVT, (7) d(CH2) [Thi2,Thr4,Eda9<--Tyr10]OVT, (8) d(CH2),[D-Thi2,Thr4,Eda9<--Tyr10]OVT. We also report the synthesis of (C). Peptides 1-8 and C were evaluated for agonistic and antagonistic activities in in vitro and in vivo OT assays, in in vivo vasopressor (V1a receptor) assays and in in vivo antidiuretic (V2 receptor) assays. None of the eight peptides nor C exhibit oxytocic or vasopressor agonism. Peptides 1-8 are extremely weak V2 agonists (antidiuretic activities range from < 0.0005 to 0.20 U/mg). Peptide C is a weak mixed V2 agonist/antagonist. Peptides 1-8 and C exhibit potent in intro (no Mg2+) OT antagonism (anti-OT pA2 values range from 7.76 to 8.05). Peptides 1-8 are all OT antagonists in vivo (estimated in vivo anti-OT pA2 values range from 6.54-7.19). With anti-V1a pA2 values of approximately 5-5.80, peptides 1-8 exhibit marked reductions in anti-V1a potencies relative to those of the parent peptides A-D (anti-V1a pA2 range from 6.48 to 7.10) and to l-deamino[D-Tyr(Et)2, Thr4]OVT (Atosiban, trade name Tractocile) (anti-V1a pA2-6.14). Atosiban has recently been approved in Europe for clinical use for the prevention of premature labour (Pharm. J. 264(7-100): 871). Peptides 1-8 exhibit striking gains in in vitro anti-OT/anti-V1a selectivities with respect to the parent peptides A, B, C and D and to Atosiban. Peptides 1-8 exhibit anti-OT (in vitro)/anti-V1a selectivities of 450, 525, 550, 450, approximately 1080, 116, 355, 227 respectively. The corresponding values for A-D and Atosiban are 30, 4.2, 4.3, 2.6 and 37. With the exception of peptide 6, the remaining seven peptides exhibit 3-18-fold gains in anti-OT (in vivo)/anti-V1a selectivity with respect to Atosiban, peptides 1-8 exhibit anti-OT (in vivo)/anti-V1a selectivities of 22, approximately 82, approximately 82, 147, approximately 83, 11, 31 and 42. By comparison, Atosiban exhibits an anti-OT (in vivo)/anti-V1a selectivity = 8. With an estimated in vivo anti-OT pA2 value = 7.19+/-0.06, peptide 4 is equipotent with Atosiban (pA2 = 7.05+/-0.05). However, with its significantly reduced anti-vasopressor potency, pA2 = approximately 5, it is approximately 18 times more selective for OT receptors with respect to VP V1a receptors than Atosiban. Since we have shown that V1a antagonism could be an unwanted side-effect in tocolytics, peptide 4 and some of the OT antagonists reported here have advantages over Atosiban and thus may be suitable candidates for evaluation as potential tocolytic agents for the treatment of preterm labour.     

Oxytocin maintains as well as initiates female sexual behavior: effects of a highly selective oxytocin antagonist

In previous studies, central administration of the oxytocin (OT) antagonist d(CH2)5[Tyr(Me)2, Thr4, Tyr-NH(9)2]OVT (OTA1) blocked receptive and proceptive components of female sexual behavior (FSB) and increased male-directed agonistic behavior when given before progesterone (P) treatment in estradiol-primed female rats but not when given shortly before behavioral testing 4-6 h after P. Because the considerable V(1a) antagonist potency of OTA1 may have contributed to these results, we tested the effects of the far more selective OT antagonist desGly-NH2, d(CH2)5[d-Tyr2, Thr4]OVT (OTA2). In ovariectomized, estradiol benzoate-primed (1 microg x 2 days sc) rats, icv infusion of OTA2 (1 microg) prior to P injection (250 microg sc) significantly suppressed lordosis and hops and darts and trended toward significantly increasing male-directed kicks during testing at 4 and 6 h. Infusion of OTA2 3 h and 40 min after P did not alter behavior at 4 and 6 h after P but significantly decreased lordosis as well as hops and darts and increased male-directed kicks 8-12 h after P. These results provide further evidence that central OT receptor activation shortly after P treatment contributes to the subsequent onset and early expression of FSB and demonstrate, for the first time, that OT receptor activation at later time points also contributes to maintaining FSB. The FSB-stimulating effect of central OT appears to persist for several hours.     

Mitochondrial Fragmentation Is Involved in Methamphetamine-Induced Cell Death in Rat Hippocampal Neural Progenitor Cells

Methamphetamine (METH) induces neurodegeneration through damage and apoptosis of dopaminergic nerve terminals and striatal cells, presumably via cross-talk between the endoplasmic reticulum and mitochondria-dependent death cascades. However, the effects of METH on neural progenitor cells (NPC), an important reservoir for replacing neurons and glia during development and injury, remain elusive. Using a rat hippocampal NPC (rhNPC) culture, we characterized the METH-induced mitochondrial fragmentation, apoptosis, and its related signaling mechanism through immunocytochemistry, flow cytometry, and Western blotting. We observed that METH induced rhNPC mitochondrial fragmentation, apoptosis, and inhibited cell proliferation. The mitochondrial fission protein dynamin-related protein 1 (Drp1) and reactive oxygen species (ROS), but not calcium (Ca²⁺) influx, were involved in the regulation of METH-induced mitochondrial fragmentation. Furthermore, our results indicated that dysregulation of ROS contributed to the oligomerization and translocation of Drp1, resulting in mitochondrial fragmentation in rhNPC. Taken together, our data demonstrate that METH-mediated ROS generation results in the dysregulation of Drp1, which leads to mitochondrial fragmentation and subsequent apoptosis in rhNPC. This provides a potential mechanism for METH-related neurodegenerative disorders, and also provides insight into therapeutic strategies for the neurodegenerative effects of METH.     

Attenuation of Cocaine and Methamphetamine Neurotoxicity by Coenzyme Q10

The neurotoxic effects of cocaine and methamphetamine (METH) were studied in mice brain with a primary objective to determine the neuroprotective potential of coenzyme Q₁₀ (CoQ₁₀) in drug addiction. Repeated treatment of cocaine or METH induced significant reduction in the striatal dopamine and CoQ₁₀ in mice. Cocaine or METH-treated mice exhibited increased thiobarbituric acid reactive substances (TBARs) in the striatum and cerebral cortex without any significant change in the cerebellum. Complex I immunoreactivity was inhibited in both cocaine and METH-treated mice, whereas tyrosine hydroxylase (TH) immunoreactivity was decreased in METH-treated mice and increased in cocaine-treated mice. Neither cocaine nor METH could induce significant change in α-synuclein expression at the doses and duration we have used in the present study. CoQ₁₀ treatment attenuated cocaine and METH-induced inhibition in the striatal ¹⁸F-DOPA uptake as determined by high-resolution microPET neuroimaging. Hence exogenous administration of CoQ₁₀ may provide neuroprotection in drug addiction.     

Evidence that paraventricular nucleus oxytocin neurons link hypothalamic leptin action to caudal brain stem nuclei controlling meal size

Hindbrain projections of oxytocin neurons in the parvocellular paraventricular nucleus (pPVN) are hypothesized to transmit leptin signaling from the hypothalamus to the nucleus of the solitary tract (NTS), where satiety signals from the gastrointestinal tract are received. Using immunocytochemistry, we found that an anorectic dose of leptin administered into the third ventricle (3V) increased twofold the number of pPVN oxytocin neurons that expressed Fos. Injections of fluorescent cholera toxin B into the NTS labeled a subset of pPVN oxytocin neurons that expressed Fos in response to 3V leptin. Moreover, 3V administration of an oxytocin receptor antagonist, [d-(CH2)5,Tyr(Me)2,Orn8]-vasotocin (OVT), attenuated the effect of leptin on food intake over a 0.5- to 4-h period (P < 0.05). Furthermore, to determine whether oxytocin contributes to leptin's potentiation of Fos activation within NTS neurons in response to CCK, we counted the number of Fos-positive neurons in the medial NTS (mNTS) after 3V administration of OVT before 3V leptin and intraperitoneal CCK-8 administration. OVT resulted in a significant 37% decrease (P < 0.05) in the potentiating effect of leptin on CCK activation of mNTS neuronal Fos expression. Furthermore, 4V OVT stimulated 2-h food intake by 43% (P < 0.01), whereas 3V OVT at the same dose was ineffective. These findings suggest that release of oxytocin from a descending pPVN-to-NTS pathway contributes to leptin's attenuation of food intake by a mechanism that involves the activation of pPVN oxytocin neurons by leptin, resulting in increased sensitivity of NTS neurons to satiety signals.     

Involvement of the oxytocin system in the nucleus accumbens in the regulation of juvenile social novelty-seeking behavior

Exploration of novel environments, stimuli, and conspecifics is highly adaptive during the juvenile period, as individuals transition from immaturity to adulthood. We recently showed that juvenile rats prefer to interact with a novel individual over a familiar cage mate. However, the neural mechanisms underlying this juvenile social novelty-seeking behavior remain largely unknown. One potential candidate is the oxytocin (OXT) system, given its involvement in various motivated social behaviors. Here, we show that administration of the specific oxytocin receptor antagonist desGly-NH₂,d(CH₂)₅-[Tyr(Me)²,Thr⁴]OVT reduces social novelty seeking-behavior in juvenile male rats when injected into the nucleus accumbens (10 ng/0.5 μl/side). The same drug dose was ineffective at altering social novelty-seeking behavior when administered into the lateral septum or basolateral amygdala. These results are the first to suggest the involvement of the OXT system in the nucleus accumbens in the regulation of juvenile social novelty-seeking behavior.     

Amphetamine and Methamphetamine Differentially Affect Dopamine Transporters in Vitro and in Vivo

The psychostimulants d-amphetamine (AMPH) and methamphetamine (METH) release excess dopamine (DA) into the synaptic clefts of dopaminergic neurons. Abnormal DA release is thought to occur by reverse transport through the DA transporter (DAT), and it is believed to underlie the severe behavioral effects of these drugs. Here we compare structurally similar AMPH and METH on DAT function in a heterologous expression system and in an animal model. In the in vitro expression system, DAT-mediated whole-cell currents were greater for METH stimulation than for AMPH. At the same voltage and concentration, METH released five times more DA than AMPH and did so at physiological membrane potentials. At maximally effective concentrations, METH released twice as much [Ca²⁺]_i from internal stores compared with AMPH. [Ca²⁺]_i responses to both drugs were independent of membrane voltage but inhibited by DAT antagonists. Intact phosphorylation sites in the N-terminal domain of DAT were required for the AMPH- and METH-induced increase in [Ca²⁺]_i and for the enhanced effects of METH on [Ca²⁺]_i elevation. Calmodulin-dependent protein kinase II and protein kinase C inhibitors alone or in combination also blocked AMPH- or METH-induced Ca²⁺ responses. Finally, in the rat nucleus accumbens, in vivo voltammetry showed that systemic application of METH inhibited DAT-mediated DA clearance more efficiently than AMPH, resulting in excess external DA. Together these data demonstrate that METH has a stronger effect on DAT-mediated cell physiology than AMPH, which may contribute to the euphoric and addictive properties of METH compared with AMPH.The dopamine transporter (DAT)³ is a main target for psychostimulants, such as d-amphetamine (AMPH), methamphetamine (METH), cocaine (COC), and methylphenidate (Ritalin®). DAT is the major clearance mechanism for synaptic dopamine (DA) (1) and thereby regulates the strength and duration of dopaminergic signaling. AMPH and METH are substrates for DAT and competitively inhibit DA uptake (2, 3) and release DA through reverse transport (4–9). AMPH- and METH-induced elevations in extracellular DA result in complex neurochemical changes and profound psychiatric effects (2, 10–16). Despite their structural and pharmacokinetic similarities, a recent National Institute on Drug Abuse report describes METH as a more potent stimulant than AMPH with longer lasting effects at comparable doses (17). Although the route of METH administration and its availability must contribute to the almost four times higher lifetime nonmedical use of METH compared with AMPH (18), there may also be differences in the mechanisms that underlie the actions of these two drugs on the dopamine transporter.Recent studies by Joyce et al. (19) have shown that compared with d-AMPH alone, the combination of d- and l-AMPH in Adderall® significantly prolonged the time course of extracellular DA in vivo. These experiments demonstrate that subtle structural features of AMPH, such as chirality, can affect its action on dopamine transporters. Here we investigate whether METH, a more lipophilic analog of AMPH, affects DAT differently than AMPH, particularly in regard to stimulated DA efflux.METH and AMPH have been reported as equally effective in increasing extracellular DA levels in rodent dorsal striatum (dSTR), nucleus accumbens (NAc) (10, 14, 20), striatal synaptosomes, and DAT-expressing cells in vitro (3, 6). John and Jones (21), however, have recently shown in mouse striatal and substantia nigra slices, that AMPH is a more potent inhibitor of DA uptake than METH. On the other hand, in synaptosomes METH inhibits DA uptake three times more effectively than AMPH (14), and in DAT-expressing COS-7 cells, METH releases DA more potently than AMPH (EC₅₀ = 0.2 μm for METH versus EC₅₀ = 1.7 μm for AMPH) (5). However, these differences do not hold up under all conditions. For example, in a study utilizing C6 cells, the disparity between AMPH and METH was not found (12).The variations in AMPH and METH data extend to animal models. AMPH- and METH-mediated behavior has been reported as similar (22), lower (20), or higher (23) for AMPH compared with METH. Furthermore, although the maximal locomotor activation response was less for METH than for AMPH at a lower dose (2 mg/kg, intraperitoneal), both drugs decreased locomotor activity at a higher dose (4 mg/kg) (20). In contrast, in the presence of a salient stimuli, METH is more potent in increasing the overall magnitude of locomotor activity in rats yet is equipotent with AMPH in the absence of these stimuli (23).The simultaneous regulation of DA uptake and efflux by DAT substrates such as AMPH and METH, as well as the voltage dependence of DAT (24), may confound the interpretation of existing data describing the action of these drugs. Our biophysical approaches allowed us to significantly decrease the contribution of DA uptake and more accurately determine DAT-mediated DA efflux with millisecond time resolution. We have thus exploited time-resolved, whole-cell voltage clamp in combination with in vitro and in vivo microamperometry and Ca²⁺ imaging to compare the impact of METH and AMPH on DAT function and determine the consequence of these interactions on cell physiology.We find that near the resting potential, METH is more effective than AMPH in stimulating DAT to release DA. In addition, at efficacious concentrations METH generates more current, greater DA efflux, and higher Ca²⁺ release from internal stores than AMPH. Both METH-induced or the lesser AMPH-induced increase in intracellular Ca²⁺ are independent of membrane potential. The additional Ca²⁺ response induced by METH requires intact phosphorylation sites in the N-terminal domain of DAT. Finally, our in vivo voltammetry data indicate that METH inhibits clearance of locally applied DA more effectively than AMPH in the rat nucleus accumbens, which plays an important role in reward and addiction, but not in the dorsal striatum, which is involved in a variety of cognitive functions. Taken together these data imply that AMPH and METH have distinguishable effects on DAT that can be shown both at the molecular level and in vivo, and are likely to be implicated in the relative euphoric and addictive properties of these two psychostimulants.     

The Metabotropic Glutamate 5 Receptor Modulates Extinction and Reinstatement of Methamphetamine-Seeking in Mice

Methamphetamine (METH) is a highly addictive psychostimulant with no therapeutics registered to assist addicts in discontinuing use. Glutamatergic dysfunction has been implicated in the development and maintenance of addiction. We sought to assess the involvement of the metabotropic glutamate 5 receptor (mGlu5) in behaviours relevant to METH addiction because this receptor has been implicated in the actions of other drugs of abuse, including alcohol, cocaine and opiates. mGlu5 knockout (KO) mice were tested in intravenous self-administration, conditioned place preference and locomotor sensitization. Self-administration of sucrose was used to assess the response of KO mice to a natural reward. Acquisition and maintenance of self-administration, as well as the motivation to self-administer METH was intact in mGlu5 KO mice. Importantly, mGlu5 KO mice required more extinction sessions to extinguish the operant response for METH, and exhibited an enhanced propensity to reinstate operant responding following exposure to drug-associated cues. This phenotype was not present when KO mice were tested in an equivalent paradigm assessing operant responding for sucrose. Development of conditioned place preference and locomotor sensitization were intact in KO mice; however, conditioned hyperactivity to the context previously paired with drug was elevated in KO mice. These data demonstrate a role for mGlu5 in the extinction and reinstatement of METH-seeking, and suggests a role for mGlu5 in regulating contextual salience.     

Methamphetamine induces autophagy as a pro-survival response against apoptotic endothelial cell death through the Kappa opioid receptor

Methamphetamine (METH) is a psychostimulant with high abuse potential and severe neurotoxicity. Recent studies in animal models have indicated that METH can impair the blood–brain barrier (BBB), suggesting that some of the neurotoxic effects resulting from METH abuse could be due to barrier disruption. We report here that while chronic exposure to METH disrupts barrier function of primary human brain microvascular endothelial cells (HBMECs) and human umbilical vein endothelial cells (HUVECs), an early pro-survival response is observed following acute exposure by induction of autophagic mechanisms. Acute METH exposure induces an early increase in Beclin1 and LC3 recruitment. This is mediated through inactivation of the protein kinase B (Akt)/mammalian target of rapamycin (mTOR)/p70S6K pathway, and upregulation of the ERK1/2. Blockade of Kappa opioid receptor (KOR), and treatment with autophagic inhibitors accelerated METH-induced apoptosis, suggesting that the early autophagic response is a survival mechanism for endothelial cells and is mediated through the kappa opioid receptor. Our studies indicate that kappa opioid receptor can be therapeutically exploited for attenuating METH-induced BBB dysfunction.     

The newly synthesized pool of dopamine determines the severity of methamphetamine-induced neurotoxicity

The neurotransmitter dopamine (DA) has long been implicated as a participant in the neurotoxicity caused by methamphetamine (METH), yet, its mechanism of action in this regard is not fully understood. Treatment of mice with the tyrosine hydroxylase (TH) inhibitor α-methyl- p -tyrosine (AMPT) lowers striatal cytoplasmic DA content by 55% and completely protects against METH-induced damage to DA nerve terminals. Reserpine, by disrupting vesicle amine storage, depletes striatal DA by more than 95% and accentuates METH-induced neurotoxicity. l -DOPA reverses the protective effect of AMPT against METH and enhances neurotoxicity in animals with intact TH. Inhibition of MAO-A by clorgyline increases pre-synaptic DA content and enhances METH striatal neurotoxicity. In all conditions of altered pre-synaptic DA homeostasis, increases or decreases in METH neurotoxicity paralleled changes in striatal microglial activation. Mice treated with AMPT, l -DOPA, or clorgyline + METH developed hyperthermia to the same extent as animals treated with METH alone, whereas mice treated with reserpine + METH were hypothermic, suggesting that the effects of alterations in cytoplasmic DA on METH neurotoxicity were not strictly mediated by changes in core body temperature. Taken together, the present data reinforce the notion that METH-induced release of DA from the newly synthesized pool of transmitter into the extracellular space plays an essential role in drug-induced striatal neurotoxicity and microglial activation. Subtle alterations in intracellular DA content can lead to significant enhancement of METH neurotoxicity. Our results also suggest that reactants derived from METH-induced oxidation of released DA may serve as neuronal signals that lead to microglial activation early in the neurotoxic process associated with METH.     

Differential neurochemical consequences of an escalating dose-binge regimen followed by single-day multiple-dose methamphetamine challenges

Chronic intake of methamphetamine (METH) causes tolerance to its behavioral and subjective effects. To better mimic human patterns of drug abuse, the present study used a rodent model that took into account various facets of human drug administration and measured METH-induced effects on brain monoamine levels. Adult male Sprague–Dawley rats were injected with METH or saline according to an escalating dose schedule for 2 weeks. This was followed by a challenge regimen of either saline or one of two doses of METH (3 × 10 mg/kg every 2 h or 6 × 5 mg/kg given every hour, both given within a single day). Both challenge doses of METH caused significant degrees of depletion of dopamine in the striatum and norepinephrine and serotonin in the striatum, cortex, and hippocampus. Animals pre-treated with METH showed significant attenuation of METH-induced striatal dopamine depletion but not consistent attenuation of norepinephrine and serotonin depletion. Unexpectedly, METH pre-treated animals that received the 3 × 10 mg/kg challenge showed less increases in tympanic temperatures than saline pre-treated rats whereas METH pre-treated animals that received the 6 × 5 mg/kg METH challenge showed comparable increases in temperatures to saline pre-treated rats. Therefore, pre-treatment-induced partial protection against monoamine depletion is probably not because of attenuated METH-induced hyperthermia in those rats.     

Role of matrix metalloproteinase and tissue inhibitor of MMP in methamphetamine-induced behavioral sensitization and reward: implications for dopamine receptor down-regulation and dopamine release

Matrix metalloproteinases (MMPs) and its inhibitors (TIMPs) function to remodel the pericellular environment. We have demonstrated that methamphetamine (METH)-induced behavioral sensitization and reward were markedly attenuated in MMP-2- and MMP-9 deficient [MMP-2-(-/-) and MMP-9-(-/-)] mice compared with those in wild-type mice, suggesting that METH-induced expression of MMP-2 and MMP-9 in the brain plays a role in the development of METH-induced sensitization and reward. In the present study, we investigated the changes in TIMP-2 expression in the brain after repeated METH treatment. Furthermore, we studied a role of MMP/TIMP system in METH-induced behavioral changes and dopamine neurotransmission. Repeated METH treatment induced behavioral sensitization, which was accompanied by an increase in TIMP-2 expression. Antisense TIMP-2 oligonucleotide (TIMP-AS) treatment enhanced the sensitization, which was associated with the potentiation of METH-induced dopamine release in the nucleus accumbens (NAc). On the other hand, MMP-2/-9 inhibitors blocked the METH-induced behavioral sensitization and conditioned place preference, a measure of the rewarding effect, and reduced the METH-increased dopamine release in the NAc. Dopamine receptor agonist-stimulated [(35)S]GTPgammaS binding was reduced in the frontal cortex of sensitized rats. TIMP-AS treatment potentiated, while MMP-2/-9 inhibitor attenuated, the reduction of dopamine D2 receptor agonist-stimulated [(35)S]GTPgammaS binding. Repeated METH treatment also reduced dopamine D2 receptor agonist-stimulated [(35)S]GTPgammaS binding in wild-type mice, but such changes were significantly attenuated in MMP-2-(-/-) and MMP-9-(-/-) mice. These results suggest that the MMP/TIMP system is involved in METH-induced behavioral sensitization and reward, by regulating dopamine release and receptor signaling.     

Effects of 2-Deoxy-d-Glucose on Methamphetamine-Induced Dopamine and Serotonin Neurotoxicity

Abstract: The mechanisms underlying the neurotoxic actions of methamphetamine (METH) and related substituted amphetamines are unknown. Previous studies with 2-deoxyglucose (2-DG) have suggested that METH-induced neurotoxicity may involve exhaustion of intracellular energy stores. However, because 2-DG also produces hypothermic effects, and because METH's neurotoxic actions are highly susceptible to thermoregulatory influence, previous findings with 2-DG are difficult to interpret. The present studies were undertaken to further examine the influence of 2-DG's glucoprivic and thermic effects in the context of METH-induced dopamine (DA) and serotonin (5-HT) neurotoxicity. 2-DG protected against METH-induced DA neurotoxicity in both rats and mice. In both species, 2-DG, alone or in combination with METH, produced hypothermic effects. METH's toxic effects on brain 5-HT neurons were either unaffected or exacerbated by 2-DG, depending on species, brain region, and dose of METH tested. These results indicate that different mechanisms may underlie METH-induced DA and 5-HT neurotoxicity, and suggest that, as compared with 5-HT neurons, DA neurons are more susceptible to temperature influence, whereas 5-HT neurons are more vulnerable than DA neurons to metabolic compromise. Additional studies are needed to further assess the role of energy stores in the neurotoxic effects of METH and related drugs.     

Evaluation of the effects of alpha-phenyl-N-tert-butyl nitrone pretreatment on the neurobehavioral effects of methamphetamine

A relationship between formation of reactive oxygen species (ROS) and energy depletion has been proposed to play an important role in mediating methamphetamine (METH)-induced neurotoxicity. To evaluate this relationship, we examined the effect of the spin-trap agent, alpha-phenyl-N-tert-butyl nitrone (PBN) on hyperthermia and self-injurious behavior (SIB) and striatal dopamine (DA) depletion produced by METH (4 injections of 4 mg/kg, 2 hr intervals, s.c.) in BALB/c mice. Repeated administration of METH induced hyperthermia, incidence of SIB and striatal DA depletion (84% after 3 days). Pretreatment with PBN (4 injections of 60 or 120 mg/kg, i.p.) reduced METH-induced hyperthermia, but did not significantly attenuate METH-induced SIB or the striatal DA depletion. On the other hand, pretreatment with high doses of PBN (4 injections of 180 or 240 mg/kg, i.p.) protected against METH-induced hyperthermia and SIB, and PBN (180 mg/kg) also completely protected against the acute striatal DA depletion 60 min after the last injection of the drug. However, the long-lasting striatal DA depletion was only attenuated by 52 or 56%, respectively. These results indicate that METH-induced hyperthermia contributes to, but is not solely responsible for METH-induced neurotoxicity, and supports a role for formation of ROS and other mechanisms in the generation of METH-induced striatal dopaminergic neurotoxicity. In addition, the difference in the efficacy of PBN to protect against the acute or long-lasting striatal DA depletion induced by METH may indicate that both ROS formation and other mechanisms are required for METH-induced neurotoxicity to develop.     

Propentophylline increases striatal dopamine release but dampens methamphetamine-induced dopamine dynamics: A microdialysis study

While there are currently no medications approved for methamphetamine (METH) addiction, it has been shown that propentofylline (PPF), an atypical methylxanthine, can suppress the rewarding effects of methamphetamine (METH) in mice. This experiment studied the interactions of PPF with METH in striatal dopaminergic transmission. Herein, the impact of PPF (10–40 mM, intrastriatally perfused (80 min) on the effect of METH (5 mg/kg, i.p.) on striatal dopamine (DA) release was evaluated using brain microdialysis in Sprague–Dawley adult rats. METH was injected at the 60 min time point of the 80 min PPF perfusion. The extracellular levels of DA and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were determined using high performance liquid chromatography with electrochemical detection (HPLC-ED). PPF induced a concentration-dependent increase in DA release beginning 30 min after the onset of PPF perfusion. DA peak levels evoked by 40 mM PPF were similar to those induced by 5 mg/kg METH i.p. Only the highest concentration of PPF decreased the METH-induced DA peak (circa 70%). The significant decreases in extracellular levels of DOPAC and HVA evoked by METH were partially blocked by 10 and 20 mM PPF. Although 40 mM of PPF also partially blocked the METH-induced DOPAC decrease, it completely blocked HVA depletion after a transient increase in HVA levels in METH-treated rats. Data indicates for the first time that while PPF increases presynaptic striatal DA dynamics it attenuates METH-induced striatal DA release and metabolism.     

The effects of methamphetamine on serotonin transporter activity: role of dopamine and hyperthermia

Multiple administrations of methamphetamine (METH) rapidly decreased serotonin (5HT) transporter (SERT) function in rat striatum and hippocampus. The purpose of this study was to identify the mechanisms/ factors contributing to this METH-induced decrease in SERT function. Multiple high-dose METH injections rapidly decreased 5HT uptake without altering binding of the 5HT transporter ligand paroxetine. Hyperthermia contributed to this deficit in transporter function in striatum and hippocampus, as prevention of METH-induced hyperthermia attenuated this decrease. A role for dopamine (DA) was suggested by findings that pretreatment with the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine, the D1 antagonist SCH-23390, or the D2 antagonist eticlopride attenuated the METH-induced decrease in striatal, but not hippocampal, SERT activity. These effects were independent of the ability of these DA-antagonizing drugs to prevent METH-induced hyperthermia. These results suggest that DA contributes to the decrease in SERT function caused by multiple METH injections in the striatum, but not hippocampus, and that hyperthermia facilitates these deficits in SERT function in both brain regions. In contrast, the response of SERT to a single administration of METH was DA and hyperthermia independent. These findings suggest that the mechanisms/ factors involved in decreasing SERT activity after a single administration of METH are distinct from that caused by multiple administrations.