Rats that differentially respond to cocaine differ in their dopaminergic storage capacity of the nucleus accumbens

Cocaine (COC) inhibits the re-uptake of dopamine. However, the dopamine response to COC also depends on dopamine inside storage vesicles. The aim of this study was to investigate whether rats that differentially respond to COC differ in their dopaminergic storage capacity of the nucleus accumbens. Total and vesicular levels of accumbal dopamine as well as accumbal vesicular monoamine transporter-2 levels were established in high (HR) and low responders (LR) to novelty rats. Moreover, the effects of reserpine (RES) on the COC-induced increase of extracellular accumbal dopamine were investigated. HR displayed higher accumbal levels of total and vesicular dopamine than LR. Moreover, HR displayed more accumbal vesicular monoamine transporters-2 than LR. COC increased extracellular accumbal dopamine more strongly in HR than in LR. A low dose of RES prevented the COC-induced increase of accumbal dopamine in LR, but not in HR. A higher dose of RES was required to inhibit the COC-induced increase of accumbal dopamine in HR. These data demonstrate that HR were marked by a larger accumbal dopaminergic storage pool than LR. It is hypothesized that HR are more sensitive to COC than LR, because COC can release more dopamine from accumbal storage vesicles in HR than in LR.     

3-Methoxytyramine Formation Following Monoamine Oxidase Inhibition Is a Poor Index of Dendritic Dopamine Release in the Substantia Nigra

Abstract: This study was undertaken, using microdialysis, to compare the extracellular concentration of 3-methoxytyramine and dopamine in dialysate from the striatum and substantia nigra, after pargyline (75 mg/kg), after pargyline plus amphetamine (3 mg/kg), and after pargyline plus reserpine (5 mg/kg) administration. Treatment with pargyline alone increased the extracellular dopamine concentration by 70% in the striatum and by 140% in the substantia nigra and induced in both regions a time-dependent accumulation of 3-methoxytyramine. The addition of d-amphetamine to pargyline increased the extracellular dopamine concentration, compared with pargyline-treated controls, to the same extent in both the substantia nigra (maximally by 360%) and the striatum (maximally by 400%), but the concomitant increase of 3-methoxytyramine accumulation in the dialysate was relatively smaller in the substantia nigra compared with the striatum. Reserpine treatment decreased the extracellular dopamine concentration in both regions below the detection level (<10% of basal value). When pargyline was added to reserpine, the striatal extracellular dopamine concentration increased to 50% of pargyline-treated controls and the striatal 3-methoxytyramine accumulation was less than in pargyline-treated controls. However, in the substantia nigra, the addition of pargyline to reserpine resulted in dopamine concentrations as high as after pargyline only and the 3-methoxytyramine accumulation was not changed compared with pargyline-treated controls. In summary, our results indicate that dopamine in the substantia nigra is released from reserpine-sensitive storage sites and that pargyline-induced 3-methoxytyramine accumulation is a poor indicator of the local dopamine release. The latter observation may be explained by the fact that the dopamine-metabolizing enzyme, catechol-O-methyltransferase, is located inter alia in the dopamine-containing cell bodies/dendrites in the substantia nigra, in contrast to the situation in the terminals in the striatum where catechol-O-methyltransferase is located only in nondopaminergic cells.     

Individual differences in the effect of social defeat on anhedonia and histone acetylation in the rat hippocampus

Major depression is a growing problem worldwide with variation in symptoms and response to treatment. Individual differences in response to stress may contribute to such observed individual variation in behavior and pathology. Therefore, we investigated depressive-like behavior following exposure to repeated social defeat in a rat model of individual differences in response to novelty. Rats are known to exhibit either high locomotor activity and sustained exploration (high responders, HR) or low activity with minimal exploration (low responders, LR) in a novel environment. We measured anhedonia using the sucrose preference test in HR and LR rats following exposure to social defeat stress or in basal, non-defeated conditions. We then compared histone acetylation in the hippocampus in HR and LR defeat and non-defeated rats and measured mRNA levels of histone deacetylases (HDAC) 3, 4, 5, and Creb binding protein (CBP). We found that basally, HR rats consumed more sucrose solution than LR rats, but reduced consumption after exposure to defeat. LR rats' preference was unaffected by social defeat. We found that HR rats had higher levels of histone acetylation on H3K14 and H2B than LR rats in non-stress conditions. Following defeat, this acetylation pattern changed differentially, with HR rats decreasing acetylation of H3K14 and H2B and LR's increasing acetylation of H3K14. Acetylation on histone H4 decreased following defeat with no individual variation. Basal differences in CBP expression levels may underlie the observed acetylation pattern; however we found no significant effects of defeat in levels of HDACs 3, 4, 5 in the hippocampus.     

Biochemistry of Somatodendritic Dopamine Release in Substantia Nigra: An In Vivo Comparison with Striatal Dopamine Release

Abstract: The somatodendritic release of dopamine in substantia nigra previously has been suggested to be nonvesicular in nature and thus to differ from the classical, exocytotic release of dopamine described for the dopaminergic nerve terminal in striatum. We have compared the effects of reserpine, a compound that disrupts vesicular sequestration of monoamines, on the storage and release of dopamine in substantia nigra and striatum of rats. Reserpine administration (5 mg/kg, i.p.) significantly decreased the tissue level of dopamine in substantia nigra pars reticulata, substantia nigra pars compacta, and striatum. In these brain areas, reserpine-induced reductions in tissue dopamine level occurred within 2 h and persisted at 24 h postdrug. In vivo measurements using microdialysis revealed that reserpine administration rapidly decreased the extracellular dopamine concentration to nondetectable levels in substantia nigra as well as in striatum. In both structures, it was observed that reserpine treatment significantly attenuated the release of dopamine evoked by a high dose of amphetamine (10 mg/kg, i.p.) given 2 h later. In contrast, dopamine efflux in response to a low dose of amphetamine (2 mg/kg, i.p.) was not altered by reserpine pretreatment either in substantia nigra or in striatum. The present data suggest the existence, both at the somatodendritic and at the nerve terminal level, of a vesicular pool of dopamine that is the primary site of transmitter storage and that can be displaced by high but not low doses of amphetamine. The physiological release of dopamine in substantia nigra and in striatum is dependent on the integrity of this vesicular store.     

Transport of 5-hydroxytryptamine in membrane vesicles from rat basophilic leukemia cells

Rat basophilic leukemia (RBL) cells were grown as tumors. Membrane vesicles were isolated from them and serotonin transport was measured. Two types of transport were identified. One was sensitive to imipramine and sodium-dependent, while the other was sensitive to reserpine and ATP-dependent. The transport systems exhibit different affinities for serotonin (sodium-dependent, Km 0.22 microM; ATP-dependent, Km 2.6 microM) and are different in their substrate specificity, the former being much more specific. The 5-hydroxytryptamine transport by the reserpine-sensitive system was strongly inhibited by other biogenic amines, like norepinephrine, epinephrine and dopamine, whereas that by the imipramine-sensitive system was not. Upon Ficoll gradient centrifugation the two transport systems were separated. The reserpine-sensitive activity was found much further into the gradient than the imipramine-sensitive one. The latter co-migrated with the receptor of IgE, which is located in the plasma membrane. Characterization of latter showed that in addition to the dependence of 5-hydroxytryptamine influx on external sodium it was also absolutely dependent on external chloride and was strongly stimulated by internal potassium. On the other hand, efflux required external potassium. An alternative potassium independent way of loss of labelled 5-hydroxytryptamine was by exchange. A small but consistent stimulation of influx was observed in the presence of valinomycin, indicating that the process is electrogenic. The reserpine-sensitive system could also be driven in the absence of ATP. This required the imposition of pH gradient (acid inside) and was stimulated by an artificially imposed membrane potential (positive inside).     

A single exposure to novelty differentially affects the accumbal dopaminergic system of apomorphine-susceptible and apomorphine-unsusceptible rats

Individual differences in responses to mild, acute stressors in laboratory animals have commonly been observed in behavioural tests and at the level of hypothalamic-pituitary-adrenal axis responses. These differences are associated with dopamine transmission in the nucleus accumbens. Although the effect of mild stressors on dopamine transmission has been studied with microdialysis, it has not been studied at the level of the catecholaminergic network in the nucleus accumbens. In this study we have used microdialysis to measure extracellular concentrations of dopamine in vivo and immunocytochemistry for the enzyme tyrosine hydroxylase to assess the effect of a single exposure to novelty on the neurochemistry of the nucleus acc umbens in apomorphine-susceptible and apomorphine-unsusceptible rats. These rats are a valid animal model for studying individual differences in responses to environmental stressors and drugs of abuse. We demonstrated that a mild stressor like novelty increased the extracellular concentration of dopamine in the nucleus accumbens in apomorphine-susceptible rats to a larger and longer-lasting degree than in apomorphine-unsusceptible rats. Furthermore we demonstrated that novelty increased the tyrosine hydroxylase-immunoreactive fibre network in the nucleus accumbens shell of apomorphine-susceptible rats, which are rats that are particularly reactive to stressors, but not in the shell of apomorphine-unsusceptible rats, which are rats that are relatively stress-resistant. In conclusion, we have shown that the accumbal dopaminergic system of apomorphine-susceptible rats is more sensitive to an environmental stressor than that of apomorphine-unsusceptible rats. Combined with the fact that these animals also differ in their sensitivity to drugs of abuse, which are known to affect the dopaminergic system, these data provide a solid basis for further studying the differences in the dopaminergic responsiveness to drugs of abuse between apomorphine-susceptible and apomorphine-unsusceptible rats.     

Loaded dopamine is preferentially stored in the halo portion of PC12 cell dense core vesicles

Large dense core vesicles in rat pheochromocytoma cells are morphologically distinct from dense core vesicles in mast and chromaffin cells in that the dense core occupies a much smaller fraction of the vesicular volume, allowing for a much larger vesicular clear space, or halo. In this work, we present evidence indicating that upon treatment with L-DOPA the majority of the dopamine loaded into these vesicles is preferentially compartmentalized into the halo portion of the vesicle. Amperometry was used to monitor release of loaded neurotransmitter from cells in both isotonic and hypertonic extracellular conditions, with the latter condition causing inhibition of dense core dissociation. In combination with this we have used transmission electron microscopy to determine the morphological characteristics of dense core vesicles before and after treatment with L-DOPA in solutions of varied osmolarity. The results provide a more complete understanding of the complex interaction of molecules within dense core vesicles, suggesting that newly loaded dopamine is located in the halo of the vesicle. This finding has fundamental significance for studies of neurotransmitter release from dense core vesicles, as the core appears to have a function involving more than simple storage of neurotransmitter and associated molecules, and the often overlooked vesicular halo appears to be an important storage compartment for neurotransmitter.     

Effects of repeated morphine on locomotion, place preference and dopamine in heterozygous glial cell line-derived neurotrophic factor knockout mice

Glial cell line-derived neurotrophic factor (GDNF) has been shown to be involved in the maintenance of striatal dopaminergic neurons. Neurotrophic factors are crucial for the plasticity of central nervous system and may be involved in long-term responses to drug exposure. To study the effects of reduced GDNF on dopaminergic behaviour related to addiction, we compared the effects of morphine on locomotor activity, conditioned place preference (CPP) and extracellular accumbal dopamine in heterozygous GDNF knockout mice (GDNF+/-) with those in their wild-type (Wt) littermates. When morphine 30 mg/kg was administered daily for 4 days, tolerance developed towards its locomotor stimulatory action only in the GDNF+/- mice. A morphine 5 mg/kg challenge dose stimulated locomotor activity only in the GDNF+/- mice withdrawn for 96 h from repeated morphine treatment, whereas clear and similar sensitization of the locomotor response was seen after a 10 mg/kg challenge dose in mice of both genotypes. Morphine-induced CPP developed initially similarly in Wt and GDNF+/- mice, but it lasted longer in the Wt mice. The small challenge dose of morphine increased accumbal dopamine output slightly more in the GDNF+/- mice than in the Wt mice, but doubling the challenge dose caused a dose-dependent response only in the Wt mice. In addition, repeated morphine treatment counteracted the increase in the accumbal extracellular dopamine concentration we previously found in drug-naive GDNF+/- mice. Thus, reduced endogenous GDNF level alters the dopaminergic behavioural effects to repeatedly administered morphine, emphasizing the involvement of GDNF in the neuroplastic changes related to long-term effects of drugs of abuse.     

Mitochondrial damage by nitric oxide is potentiated by dopamine in PC12 cells

Mitochondrial damage in PC12 cells, a model for dopaminergic cells, was examined in terms of the contribution of oxidative stress, nitric oxide (*NO), and dopamine to impairment of mitochondrial respiratory control (RC). A kinetic analysis suggested that the oxidative deamination of dopamine catalyzed by monoamine oxidase (MAO) was not a significant source of hydrogen peroxide, because of constrains imposed by the low cytosolic level of dopamine. *NO induced irreversible damage of mitochondrial complex I in PC12 cells: this damage followed a sigmoid response on *NO concentration with a well-defined threshold level. Dopamine did not elicit damage of mitochondria in PC12 cells; however, the amine potentiated the effects of *NO at or near the threshold level, thus leading to irreversible impairment of mitochondrial respiration. This synergism between *NO and dopamine was not observed at *NO concentrations below the threshold level. Depletion of dopamine from the storage vesicles by reserpine protected mitochondria from *NO damage. Dopamine oxidation by *NO increased with pH, and occurred at modest levels at pH 5.5. In spite of this, calculations showed that the oxidation of dopamine in the storage vesicles (pH 5.5) was higher than that in the cytosol (pH 7.4), due to the higher dopamine concentration in the storage vesicles (millimolar range) compared to that in the cytosol (micromolar range). It is suggested that storage vesicles may be the cellular sites where the potential for dopamine oxidation by *NO is higher.These data provide further support to the hypothesis that dopamine renders dopaminergic cells more susceptible to the mitochondrial damaging effects of *NO. In the early stages of Parkinson's disease, *NO production increases until reaching a point near the threshold level that induces neuronal damage. Dopamine stored in dopaminergic cells may cause these cells to be more susceptible to the deleterious effects of *NO, which involve irreversible impairment of mitochondrial respiration.     

Synthesis,subcellular distribution and turnover of dopamine beta-hydroxylase in organ cultures of sympathetic ganglia and adrenal medullae

The synthesis, subcellular distribution and turnover of dopamine beta-hydroxylase was studied in organ cultures of rat adrenal medullae and superior cervical ganglia. After exposure to [3H]leucine for 1 or 3 h, the tissues were homogenized at various time intervals and the amount of labelled dopamine beta-hydroxylase in different subcellular fractions (cytosol, soluble and membrane-bound fraction of catecholamine storage vesicles) was determined by immunoprecipitation and subsequent electrophoresis. In cultured adrenal medullae, induction of dopamine beta-hydroxylase initiated in vivo by administration of reserpine affected both soluble and membrane-bound pools of dopamine beta-hydroxylase to a similar extent after pulse-labelling for 1 or 3 h. The half-lives of dopamine beta-hydroxylase, which amounted to 6 h for the cytosol, 7.5 h for the soluble vesicular and 32 h for the membrane-bound vesicular pools were not altered by pretreatment with reserpine. In superior cervical ganglia the half-lives of the soluble pools were 2-3 times longer than in the adrenal medulla, whereas the half-life of the membrane-bound fraction was the same as in the adrenal medulla. In both organs the most heavily labelled fraction (both after a pulse of 1 or 3 h) was always that of the vesicular membrane, suggesting that newly-synthesized dopamine beta-hydroxylase is immediately incorporated into the storage vesicles and not via release into the cytosol from the site of synthesis. The fact that the half-life of membrane-bound dopamine beta-hydroxylase is markedly longer than that of the two soluble pools suggests that the single pools are not only independently supplied by newly-synthesized DBH but there is also no appreciable subsequent exchange between soluble and membrane-bound pools.     

Differences in Nicotine-Induced Dopamine Release and Nicotine Pharmacokinetics Between Lewis and Fischer 344 Rats

Studies have shown a greater preference for the self-administration of drugs such as nicotine and cocaine in the Lewis rat strain than in the Fischer 344 strain. We examined some factors that could contribute to such a difference. The baseline level of extracellular dopamine in nucleus accumbens shell was about 3-times higher in Fischer rats than in Lewis rats (3.18 ± 0.26 vs. 1.09 ± 0.14 pg/sample). Nicotine (50-100 g/kg)-induced release of dopamine, expressed in absolute terms, was similar in the two strains. Dopamine release expressed in relative terms (as percent of baseline), however, was significantly greater in Lewis rats than in Fischer rats at 30 min after the first nicotine injection. We suggest that the relative increase is of more influence than the absolute level for determining preference; a lower physiological extracellular dopamine level thus represent a risk factor for increased preference. Amphetamine-induced dopamine release expressed in relative terms was not greater in the Lewis strain. In the initial time period of the microdialysis experiments, a sharper peak in nicotine-induced accumbal dopamine release in Lewis and a less but more sustained release in Fischer rats was observed. This release pattern paralleled the faster clearance of nicotine from blood of Lewis compared to Fischer rats. In tissue slices the electrically induced dopamine release was highest in the nucleus accumbens and lowest in the ventral tegmentum. A significant effect of nicotine was lowering the electrically induced release of dopamine in frontal cortex slices from Fischer brain and increasing this dopamine release in the ventral tegmentum of Lewis brain slices indicating that the ventral tegmentum, an area controlling dopamine release in the accumbens, is more responsive to nicotine in the Lewis rat. Nicotine levels tended to be more sustained in Fischer rats in different brain regions, although the difference in nicotine levels between the strains was not significant at any time period. Several factors contribute to nicotine preference, including the endogenous dopamine level, and the sensitivity of ventral tegmentum neurons to nicotine-induced dopamine release. Strain differences in pharmacokinetics of nicotine may also play a role.     

GZ‐793A,a lobelane analog,interacts with the vesicular monoamine transporter‐2 to inhibit the effect of methamphetamine

(R)‐3‐[2,6‐cis‐Di(4‐methoxyphenethyl)piperidin‐1‐yl]propane‐1,2‐diol (GZ‐793A) inhibits methamphetamine‐evoked dopamine release from striatal slices and methamphetamine self‐administration in rats. GZ‐793A potently and selectively inhibits dopamine uptake at the vesicular monoamine transporter‐2 (VMAT2). This study determined GZ‐793A's ability to evoke [³H]dopamine release and inhibit methamphetamine‐evoked [³H]dopamine release from isolated striatal synaptic vesicles. Results show GZ‐793A concentration‐dependent [³H]dopamine release; nonlinear regression revealed a two‐site model of interaction with VMAT2 (High‐ and Low‐EC₅₀ = 15.5 nM and 29.3 μM, respectively). Tetrabenazine and reserpine completely inhibited GZ‐793A‐evoked [³H]dopamine release, however, only at the High‐affinity site. Low concentrations of GZ‐793A that interact with the extravesicular dopamine uptake site and the High‐affinity intravesicular DA release site also inhibited methamphetamine‐evoked [³H]dopamine release from synaptic vesicles. A rightward shift in the methamphetamine concentration‐response was evident with increasing concentrations of GZ‐793A, and the Schild regression slope was 0.49 ± 0.08, consistent with surmountable allosteric inhibition. These results support a hypothetical model of GZ‐793A interaction at more than one site on the VMAT2 protein, which explains its potent inhibition of dopamine uptake, dopamine release via a High‐affinity tetrabenazine‐ and reserpine‐sensitive site, dopamine release via a Low‐affinity tetrabenazine‐ and reserpine‐insensitive site, and a low‐affinity interaction with the dihydrotetrabenazine binding site on VMAT2. GZ‐793A inhibition of the effects of methamphetamine supports its potential as a therapeutic agent for the treatment of methamphetamine abuse.

     

Reserpine pretreatment prevents increases in extracellular striatal dopamine following L-DOPA administration in rats with nigrostriatal denervation

The influence of L-DOPA and reserpine on extracellular dopamine (DA) levels in the striatum of intact and dopaminergic denervated rats was studied using the brain microdialysis technique. In intact rats, reserpine (5 mg/kg s.c.) reduced extracellular DA levels to 4% of basal values. L-DOPA (50 mg/kg i.p.) had no effect on extracellular DA levels in reserpine-pretreated rats. In rats with 6-hydroxydopamine-induced lesion of the nigrostriatal dopaminergic system, basal levels of extracellular DA were low but markedly increased by L-DOPA (50 mg/kg i.p.). In 6-hydroxydopamine-lesioned rats, pretreatment with reserpine (5 mg/kg s.c.) diminished L-DOPA (50 mg/kg i.p.)-induced increases in extracellular DA levels to 16% of those obtained in denervated animals not pretreated with reserpine (p<0.01). These results suggest that in the intact striatum, extracellular DA stems mainly from vesicular storage sites and that in the striatum with dopaminergic denervation, a large part of the L-DOPA-derived extracellular DA is also derived from a vesicular pool that is released by an exocytosis mechanism.     

Ir gene-controlled response to haptenated hen ovomucoid: isotypic specificity and dominant nonresponsiveness

We have examined the isotypic pattern of the response of mice to the Ir gene-controlled antigen, dinitrophenyl-ovomucoid (DNP-OM). H-2 kappa mice are high responders (HR); (H-2b,d mice are low responders (LR). The isotype patterns of HR and LR strains differ both quantitatively and qualitatively. In the primary response to doses of 20-100 micrograms DNP-OM, HR strains produce IgM and IgG antibodies, whereas LR strains produce only IgM. Background genes modify the kinetics of the IgGl primary response in HR strains, but no background was found which allowed an IgG response in a LR strain. In secondary responses, priming with 0.2 microgram DNP-OM increases secondary responses in HR strains, and decreases them in LR strains. Control of this response maps to I-A, and is not altered by the bm 12 I-Ab mutation. The LR phenotype is dominant in (HR X LR)F1 mice.     

Selective activation of cholinergic interneurons enhances accumbal phasic dopamine release: setting the tone for reward processing

Dopamine plays a critical role in motor control, addiction, and reward-seeking behaviors, and its release dynamics have traditionally been linked to changes in midbrain dopamine neuron activity. Here, we report that selective endogenous cholinergic activation achieved via in vitro optogenetic stimulation of nucleus accumbens, a terminal field of dopaminergic neurons, elicits real-time dopamine release. This mechanism occurs via direct actions on dopamine terminals, does not require changes in neuron firing within the midbrain, and is dependent on glutamatergic receptor activity. More importantly, we demonstrate that in vivo selective activation of cholinergic interneurons is sufficient to elicit dopamine release in the nucleus accumbens. Therefore, the control of accumbal extracellular dopamine levels by endogenous cholinergic activity results from a complex convergence of neurotransmitter/neuromodulator systems that may ultimately synergize to drive motivated behavior.     

Cyclic AMP-Dependent Modulation of Vesicular Monoamine Transport in Pheochromocytoma Cells

Abstract: Cyclic AMP (cAMP) is well known to enhance tyrosine hydroxylase activity in PC12 cells. We were able to demonstrate, however, that the cellular dopamine level in PC12 was lowered by dibutyryl cAMP. Furthermore, the decrease in the cellular level of dopamine was accompanied by about a 10-fold increase in the medium. The aim of this work was to elucidate the effect of cAMP on catecholamine transport. Dibutyryl cAMP did not induce exocytotic release of norepinephrine but rather inhibited its uptake. As with forskolin and cholera toxin, physiological signaling molecules such as vasoactive intestinal polypeptide (VIP) and AMP, for which PC12 cells are known to have receptors linked to activation of adenylate cyclase, also inhibited norepinephrine uptake. The inhibitory effects of dibutyryl cAMP, VIP, and AMP were dose dependent, and EC₅₀ values were estimated to be 100 µ M , 10 n M , and 1.0 µ M , respectively. The inhibition profile of dibutyryl cAMP over the time course of norepinephrine uptake was biphasic: Inhibition became clearly detectable after the cytosolic pool of norepinephrine had been saturated. This profile is similar to that of reserpine. Nomifensine, however, inhibited uptake at a rather constant rate throughout the entire time course. The ATP-dependent serotonin uptake by digitonin-permeabilized cells was lowered to ∼50% that of the control by dibutyryl cAMP treatment before permeabilization, indicating inhibition of vesicular monoamine transport. This effect was also dependent on a dibutyryl cAMP concentration with an EC₅₀ of ≤100 µ M . These results suggest that cAMP may be capable of elevating extracellular dopamine levels in the nervous system by inhibiting its translocation into storage vesicles while enhancing its synthesis in the cytosol. Moreover, endogenous neurotransmitters such as VIP, AMP, and adenosine may act as intrinsic antidepressants via the cAMP pathway.     

Solubilization and reconstitution of the catecholamine transporter from bovine chromaffin granules.

The catecholamine transporter from bovine chromaffin granules has been solubilized by using low concentrations of sodium cholate in the presence of phospholipids. The functional solubilized protein has been incorporated into liposomes after removal of the detergent either by gel filtration or by dialysis. Reserpine-sensitive accumulation against a concentration gradient is achieved by artifically imposing a pH gradient across the membrane. In the reconstituted system adenosine 5'-triphosphate (ATP) serves as an energy source only at higher detergent concentrations. The proton-translocating adenosine triphosphatase (ATPase) is solubilized in parallel with the increasing efficiency of ATP as an energy source. Several criteria are proposed to distinguish between carrier-mediated (reserpine sensitive) and unmediated transport in the reconstituted system. The reserpine-sensitive process shows affinity and ss presented in this communication provide further support for the contention that concentrative uptake in biogenic amine storage vesicles is driven by a transmembrane pH gradient, which, in the native system, is generated by a proton-translocating ATPase. Moreover, the assays described provide a tool for the isolation and purification of the transport protein.     

Theory for the impact of basal turnover on dopamine clearance kinetics in the rat striatum after medial forebrain bundle stimulation and pressure ejection

Although microdialysis measurements suggest that extracellular dopamine concentrations in the rat striatum are in the low nanomolar range, some recent voltammetry studies suggest that the concentration may be considerably higher, perhaps in the micromolar range. The presence of such high dopamine levels in the extracellular space has to be rationalized with the rapid, linear clearance of extracellular dopamine observed after electrical stimulation of the medial forebrain bundle. Kinetic analysis of dopamine clearance after evoked release suggests that the basal extracellular dopamine concentration is below the K(M) of dopamine uptake, which is near 0.2 microm. However, dopamine clearance after pressure ejection of dopamine into the rat striatum is slow and non-linear, which may alternatively be a sign that basal dopamine release is only slightly slower than the maximal velocity of dopamine uptake, Vmax. A high basal extracellular dopamine concentration would exist if basal dopamine release were only slightly slower than the Vmax of uptake. This report introduces a new kinetic analysis of dopamine uptake that sheds light on the possible source of the different clearance rates observed following evoked dopamine release and dopamine pressure ejection. Furthermore, the analysis rationalizes the rapid dopamine clearance after evoked release with the possibility that basal extracellular dopamine levels are above the K(M) of the transporter.     

The influence of heterogeneity on the analysis of sleep-wake architecture in the single-prolonged stress rats

Objective: To observe the influence of heterogeneity on sleep-wake architecture in single-prolonged stress(SPS) animal model. Methods: SPS rats were subdivided into low responders(LR) and high responders(HR) based on their freezing responses to a novel environment. Sleeping time(ST), awakening numbers(AN), brief awakening numbers(b AN) and frequency distribution of sleep bouts were used as observing indicators, single factor variance analysis combined with Dunnett t test were used to compare the differences between control, exposure, LR and HR groups. Results: We found sleeping time was increased only in HR group. Moreover, awakening numbers and brief awakening number increased in exposure group and HR group during the light phase, but not in LR group. The number of sleep bouts for the ranges of 40-80 s increased obviously in HR group, but not in exposure and LR group. In addition, there were significant correlation between sleep-related parameters and freezing in HR group, but not in LR group. Conclusion: Heterogeneity existed in SPS model in view of different sleep-wake architectures of SPS rats. Rats in HR group exactly mimicked the freezing response and sleep disorders of PTSD. So HR rats were more appropriate to be used as PTSD-like models, especially when studying sleep disorder in PTSD.     

Sources contributing to the average extracellular concentration of dopamine in the nucleus accumbens

Mesolimbic dopamine neurons fire in both tonic and phasic modes resulting in detectable extracellular levels of dopamine in the nucleus accumbens (NAc). In the past, different techniques have targeted dopamine levels in the NAc to establish a basal concentration. In this study, we used in vivo fast scan cyclic voltammetry (FSCV) in the NAc of awake, freely moving rats. The experiments were primarily designed to capture changes in dopamine caused by phasic firing - that is, the measurement of dopamine 'transients'. These FSCV measurements revealed for the first time that spontaneous dopamine transients constitute a major component of extracellular dopamine levels in the NAc. A series of experiments were designed to probe regulation of extracellular dopamine. Lidocaine was infused into the ventral tegmental area, the site of dopamine cell bodies, to arrest neuronal firing. While there was virtually no instantaneous change in dopamine concentration, longer sampling revealed a decrease in dopamine transients and a time-averaged decrease in the extracellular level. Dopamine transporter inhibition using intravenous GBR12909 injections increased extracellular dopamine levels changing both frequency and size of dopamine transients in the NAc. To further unmask the mechanics governing extracellular dopamine levels we used intravenous injection of the vesicular monoamine transporter (VMAT2) inhibitor, tetrabenazine, to deplete dopamine storage and increase cytoplasmic dopamine in the nerve terminals. Tetrabenazine almost abolished phasic dopamine release but increased extracellular dopamine to ～500?nM, presumably by inducing reverse transport by dopamine transporter (DAT). Taken together, data presented here show that average extracellular dopamine in the NAc is low (20-30?nM) and largely arises from phasic dopamine transients.