Dopamine D1 and D2 Receptors and Their Signal System Present in Coated Vesicles Prepared from Bovine Striatal Tissue

Abstract: Coated vesicles (CVs) isolated from bovine striatal tissue were examined to determine whether they are associated with dopamine signal systems consisting of dopamine D₁ and D₂ receptors, G proteins, and adenylate cyclase. Dopamine receptors in CVs were characterized by a dopamine D₁ receptor antagonist, [³H]SCH 23390, and a dopamine D₂ receptor antagonist, [³H]-spiroperidol. The bindings of both ligands were specifically saturable and reversible with a dissociation constant ( K _D) of 0.65 and 0.5 n M , respectively. Dopaminergic antagonists and agonists inhibited the specific bindings of [³H]SCH 23390 and [³H]spiroperidol in a stereoselective and concentration-dependent manner with an appropriate rank order potency for dopamine D₁ or D₂ receptors. The regulations of the agonist binding by guanyl-5-ylimidodiphosphate were observed. ADP ribosylation of the CVs with [³²P]NAD demonstrated predominant labeling of bands of M_r 47,000–52,000, 42,000–45,000, and 40,000-39,000, which corresponded to the known molecular weights of the α subunits of G_s and G_i proteins. The presence of α and β subunits of G proteins in the CVs was also confirmed by immunoblotting assay. Adenylate cyclase activity, which was stimulated by SKF 38393 and inhibited by dopamine D₂ receptor agonists, was present in the CVs. These findings suggest that the dopamine D₁ and D₂ receptors in the CVs couple with adenylate cyclase via G_s/G_i protein.     

The Synthesis, Storage, and Release of Propionylcholine by the Electric Organ of Torpedo marmorata

Abstract: Little is known about the specificity of the mechanisms involved in the synthesis and release of acetylcholine for the acetyl moiety. To test this, blocks of tissue from the electric organ of Torpedo were incubated with either [1-¹⁴C]acetate or [1-¹⁴C]propionate, and the synthesis, storage, and release of [1-¹⁴C]acetylcholine and [¹⁴C]propionylcholine were compared. To obtain equivalent amounts of the two labeled choline esters, a 50-fold higher concentration of propionate than of acetate was needed. Following subcellular fractionation, similar proportions of [¹⁴C]acetylcholine and [¹⁴C]propionylcholine were recovered with synaptosomes and with synaptic vesicles. Furthermore, both labeled choline esters were protected to a similar extent from degradation during homogenization of tissue in physiological medium, indicating that the two choline esters were equally well incorporated into synaptic vesicles. Yet depolarization of tissue blocks by 50 m M KCI released much less [¹⁴C]propionylcholinc than [¹⁴C]acetylcholine. During field stimulation of the tissue blocks, the difference between the releasibility of the two choline esters was less marked, but acetylcholine was still released in preference to propionylcholine. Evidence for specificity of the release mechanism was also obtained when the release of the two choline esters in response to field stimulation was compared in tissue blocks preincubated with both [³H]choline and [¹⁴C]propionate.     

Pre-synaptic nicotinic and D2 receptors functionally interact on dopaminergic nerve endings of rat and mouse nucleus accumbens

The existence of pre-synaptic auto- and hetero receptors which modulate neurotransmitter release is well documented. Emerging evidence show that in some cases these pre-synaptic receptors may also cross-talk with each other. The aim of the present work was to investigate whether acetylcholine receptors (nAChRs) and dopamine (DA) autoreceptors, which are both able to modulate DA release, functionally interact on the same nerve endings. We used rat and mouse nucleus accumbens synaptosomes pre-labeled with [³H]DA and exposed to nicotinic and dopaminergic receptor ligands. Both nicotinic agonists and 4-aminopyridine (4-AP) provoked [³H]DA release which was inhibited by quinpirole and blocked by sulpiride and raclopride. Both the inhibitory effect of quinpirole and the stimulatory effect of (−)nicotine did not change when the nAChRs or the DA receptors were desensitized. (−)Nicotine and 4-AP were able to stimulate [³H]DA overflow also in mouse synaptosomes and this overflow was partially inhibited by quinpirole. In the β₂ knockout mice quinpirole was still able to inhibit the [³H]DA overflow elicited by 4-AP. To conclude: in rat and mouse the (−)nicotine evoked-release can be modulated by D₂/D₃ autoreceptors present on the DA terminals and nAChRs function is independent from D₂/D₃ autoreceptors which themselves may function independently from the activation of nAChRs.     

Dopaminergic Regulation of Septohippocampal Cholinergic Neurons

Abstract: The extent to which acetylcholine (ACh) release in the hippocampus is regulated by dopaminergic mechanisms was assessed using in vivo microdialysis in freely moving rats. Systemic administration of the dopamine (DA) receptor agonist apomorphine (1.0 mg/kg) or the specific D₁ agonist CY 208–243 (1.0 mg/kg) increased microdialysate concentrations of ACh in the hippocampus. The D₂ receptor agonist quinpirole (0.5 mg/kg) produced a small but statistically significant decrease in hippocampal ACh release. d -Amphetamine (2.0 mg/kg) increased ACh release, an effect that was blocked by the D₁ receptor antagonist SCH 23390 (0.3 mg/kg) but not by the D₂ antagonist raclopride (1.0 mg/kg). These findings suggest that endogenous DA stimulates septo-hippocampal cholinergic neurons primarily via actions at D₁ receptors. In addition, these results are similar to previous findings regarding the dopaminergic regulation of cortical ACh release, and suggest that the anatomical continuum formed by basal forebrain cholinergic neurons that project to the cortex and hippocampus acts as a functional unit, at least with respect to its regulation by DA.     

Chronic Dopamine D2 Receptor Activation Does Not Affect Survival and Differentiation of Cultured Dopaminergic Neurons: Morphological and Neurochemical Observations

Abstract: Primary cultures of rat ventral mesencephalon were used to elucidate the role of chronic stimulation of dopamine (DA) D₂ autoreceptors in the development of fetal dopaminergic neurons in vitro. Cultured dopaminergic neurons, as visualized by tyrosine hydroxylase immunocytochemistry, became more differentiated in the course of cultivation time and exhibited specific high-affinity uptake for [³H]DA. In rat striatal tissue, activation of D₂ receptors has been shown to inhibit the release of DA. Previously accumulated [³H]DA was released from the cultures upon depolarization in a Ca²⁺-dependent manner. K⁺-evoked [³H]DA release could be inhibited by the selective D₂ receptor agonists LY 171555 and N0437 in a concentration-dependent manner. The inhibitory effects of LY 171555 and N0437 were antagonized by the selective DA D₂ receptor antagonist sulpiride. These observations are indicative for the expression of functional D₂ receptors in the cultures. Daily treatment of these cultures for 7 days with LY 171555 or sulpiride did not lead to any change in protein content, the number of tyrosine hydroxylase-immunoreactive neurons, or the uptake capacity for [³_H]DA. Our data demonstrate that chronic stimulation of DA D₂ receptors does not impair survival or differentiation of cultured fetal dopaminergic neurons.     

Dopamine Agonist-Mediated Inhibition of Acetylcholine Release in Rat Striatum Is Modified by Thyroid Hormone Status

Abstract: K⁺-evoked acetyl[³H]choline ([³H]ACh) release was inhibited in a concentration-dependent manner by apomorphine and the D₂ agonist quinpirole in striatal slices prepared from euthyroid and hypothyroid rats. However, there was a significant increase in the maximum inhibition observed with both agonists in the hypothyroid compared with the euthyroid group, which paralleled the increased D₂ agonist sensitivity reported for stereotyped behavior. The D₂ antagonist raclopride decreased, and the D, antagonist SCH 23390 increased, the inhibition of [³H]ACh release by apomorphine, confirming an inhibitory role for D₂ receptors and an opposing role for D₁ receptors. Because there is no difference in D₁ or D₂ receptor concentration between the euthyroid and hypothyroid groups, it is suggested that thyroid hormone modulation of D₂ receptor sensitivity affects a receptor-mediated event. Following intrastriatal injection of pertussis toxin (PTX), apomorphine no longer inhibited [³H]ACh release. In fact, increased [³H]- ACh release was observed, an effect reduced by SCH 23390, providing evidence that D₁ receptors enhance [³H]- ACh release, and confirming that a PTX-sensitive G protein mediates the D₂ response. As it has been reported that thyroid hormones modulate G protein expression, this mechanism may underlie their effect on dopamine agonist- mediated inhibition of ACh.     

Chimeric D2/D3 Dopamine Receptors Efficiently Inhibit Adenylyl Cyclase in HEK 293 Cells

Abstract: Despite a high degree of sequence homology, the dopamine D₂ and D₃ receptors have substantially different second messenger coupling properties. We have used chimeric D₂/D₃ receptors to investigate the contribution of the intracellular loops to the signaling properties of these receptors. In HEK 293 cells, D₂ receptors inhibit prostaglandin E₁-stimulated cyclic AMP levels by >90%, whereas D₃ receptors inhibit cyclic AMP accumulation by only 20%. In chimeras that have the second or third intracellular loop, or both loops simultaneously, switched between the D₂ and D₃ receptors, the maximal inhibition of adenylyl cyclase is 60–90%. In addition, the potency of quinpirole to inhibit adenylyl cyclase activity at some of the chimeras is altered compared with the wild-type receptors. It appears that the intracellular loops of the D₃ receptor are capable of interacting with G proteins, as when these loops are expressed in the D₂ receptor, the chimeras inhibit adenylyl cyclase similarly to the wild-type D₂ receptor. Our data suggest that the overall conformation of the D₃ receptor may be such that it interacts with G proteins only weakly, but when the intracellular loops are expressed in another context or the D₃ receptor structure is altered by the introduction of D₂ receptor sequence, this constraint may be lifted.     

A Synaptosomal Preparation from the Guinea Pig Ileum Myenteric Plexus

Abstract: Our interest in investigating the presynaptic modulation of acetylcholine release led to the development of a synaptosomal preparation from the guinea pig ileum myenteric plexus-longitudinal muscle. A crude synaptosomal fraction (P₂) was obtained by homogenization and differential centrifugation. The preparation exhibited a specific uptake system for choline and for nor-adrenaline (NA), but not for 5-hydroxytryptamine (5-HT). Synaptosomes were isolated from this P₂ fraction by an isoosmotic density gradient prepared from sucrose and metrizamide. The resultant synaptosomal fraction was enriched about sevenfold in both choline uptake and in choline acetyltransferase (ChAT). Choline was transported by a high-affinity system with a K_m of 6.5 × 10⁻⁷ M and a V_max of 41 pmol/mg protein/min. Electron microscopy confirmed the synaptosomal nature of the gradient fraction. Some synaptosomal profiles contained only small, translucent vesicles whereas others also contained large (approx. 100 nm diameter) electron-opaque vesicles. The crude synaptosomal fraction synthesized acetylcholine (ACh) from exogenous choline and it released the synthesized ACh in a calcium-dependent manner.     

Opposing Actions of D1 and D2-Dopamine Receptors on Arachidonic Acid Release and Cyclic AMP Production in Striatal Neurons

Abstract: D₁-and D₂-dopamine receptors exert important physiological actions on striatal neurons, but the intracellular second messenger pathways activated by these receptors are still incompletely understood. Using primary cultures of rat striatal cells, we have examined the effects of activating D₁ or D₂ receptors on arachidonic acid (AA) release and cyclic AMP accumulation. In striatal neurons labeled by incubation with [³H]AA, D₂-receptor stimulation enhanced release of [³H]AA produced by application of the Ca²⁺ ionophore A23187 or of the purinergic agonist ATP. By contrast, D₁-receptor stimulation inhibited [³H]AA release. This inhibitory effect of D₁ receptors was accompanied by stimulation of adenylyl cyclase activity, measured as accumulation of cyclic AMP, and was mimicked by application of the adenylyl cyclase activator forskolin. The results indicate the existence of a novel signaling pathway for D₂ and D₁ receptors in striatum, potentiation and inhibition, respectively, of Ca²⁺-evoked AA release.     

Dormancy-associated embryonic mRNAs and proteins in imbibing Avena fatua caryopses

The mechanisms controlling seed dormancy maintenance and release are not understood. To characterize the molecular events accompanying dormancy release, two-dimensional gel electrophoresis was used to monitor changes in soluble proteins and in vitro translation products of embryonic mRNA populations during imbibition of dormant and nondormant (after-ripened) Avena fatua L. caryopses. No differences were observed between in vitro translation products of mRNA extracted from dry dormant and nondormant embryos. However, the expression patterns of several imbibition- and germination-associated mRNAs were temporally modulated during the first 24 h of imbibition. Two dormancy-associated mRNAs, represented by polypeptides D₁ and D₂, were differentially overexpressed in dormant embryos after 3 h of imbibition. mRNA levels for D₁ and D₂ were about 8- and 3-fold higher, respectively, in dormant embryos than in nondormant embryos after 3 h of imbibition. Overexpression of D₁ continued through 12 h of imbibition, while expression of both mRNAs fell to low and equivalent amounts in dormant and nondormant embryos after 24 h. Similar dormancy-associated changes in two soluble proteins were observed during imbibition. The results demonstrate that steady-state levels of specific mRNAs and proteins change during early imbibition of dormant and nondormant A. fatua embryos and indicate that these changes may be associated with differential gene expression responsible for the maintenance of dormancy.     

Epidermal Growth Factor Promotes Uncoupling from Adenylyl Cyclase of the Rat D2S Receptor Expressed in GH4C1 Cells

Abstract: In anterior pituitary cells or when transfected into host cell lines, the D₂ dopamine receptor inhibits adenylyl cyclase and activates potassium channels. The GH-3 pituitary tumor cell line, which lacks functional D₂ receptors, responds to epidermal growth factor (EGF) by expressing a D₂ receptor that, paradoxically, couples to potassium channel activation but poorly inhibits adenylyl cyclase; this was correlated with a pronounced increase in α subunit of the G protein G₁₃. In this study we have investigated the effects of EGF on the transduction mechanisms of D₂ receptors in GH4C1 cells transfected and permanently overexpressing the rat short D₂ receptor. Activation of D₂ receptors in these cells resulted in both inhibition of adenylyl cyclase and opening of potassium channels and inhibition of prolactin release by both cyclic AMP-dependent and independent mechanisms. Exposure of the transfected GH4C1 cells to EGF caused a dramatic decrease in the coupling efficiency of the D₂ receptor to inhibit cyclic AMP-dependent responses, leaving its activity toward potassium channels unchanged. The EGF treatment led to the concomitant increase in the membrane content of G₁₃ protein. These results suggest that the transmembrane signaling specificity of G protein-coupled receptors can be modulated by the relative amounts of different G proteins at the cell membrane.     

Effects of Cerebral Ischemia on Regional Dopamine Release and D1 and D2 Receptors

Abstract: To expand on the nature of regional cerebral vulnerability to ischemia, the release of dopamine (DA) and dopaminergic (D₁ and D₂) receptors were investigated in Mongolian gerbils subjected to bilateral carotid artery occlusion (15 min) alone or with reflow (1–2 h). Extracellular cortical and striatal content of DA and its metabolites was measured by microdialysis using HPLC with electrochemical detection. The kinetic properties of D₁ and/or D₂ receptor binding sites were determined in cortical and striatal membranes with the use of radiolabeled ligands (¹²⁵I-SCH23982 and [³H]YM-09151-2, respectively). The ischemic release of DA from the striatum was greater (400-fold over preischemic level) than that from the cortex (12-fold over preischemic content). The affinity for the D₁-receptor ligand was lower ( K _D= 1.248 ± 0.047 n M ) after ischemia than that for sham controls ( K _D= 0.928 ± 0.032 n M, p < 0.001). The number of binding sites for D₂ receptors decreased in striatum ( B _max= 428 ± 18.4 fmol/mg of protein) after ischemia compared with sham controls ( B _max= 510 ± 25.2 fmol/mg of protein, p < 0.05). D₁ or D₂ binding sites were not changed either in the ischemic cortex or postischemic striatum and cortex. The findings strongly suggest that the ischemic release of DA from striatum is associated with early transient changes in D₁- and D₂-mediated DA neurotransmission.     

Repeated Reserpine Increases Striatal Dopamine Receptor and Guanine Nucleotide Binding Protein RNA

Abstract: In the present study the effects of repeated administration of reserpine on striatal dopamine receptor and guanine nucleotide binding protein mRNAs were determined. Twenty-four hours after seven consecutive daily injections of reserpine—a treatment that is known to produce functional sensitization of D₁ and D₂ dopamine receptors—the level of striatal D₁ dopamine receptor mRNA was unchanged. However, the level of mRNA for the G protein G_sα was increased by 127%. After extended reserpine treatment for 14 days, levels of both striatal D₁ DA receptor and G_sα mRNAs were elevated by 99 and 78%, respectively. Seven days of reserpine treatment also increased levels of mRNA of the striatal D₂ dopamine receptor and of G proteins G_i2α and G_oα by 200, 79, and 32%, respectively. After 14 days of reserpine treatment the level of striatal D₂ dopamine receptor mRNA was increased by twofold. In contrast, levels of the mRNAs coding for the G proteins G_i2α and G_oα were unchanged. These data suggest that dopamine receptors and their respective G proteins play important roles in the development of sensitization of striatal dopamine receptors during repeated reserpine treatment. Furthermore, the persistent increase in level of striatal G_sα mRNA suggests that this G protein is necessary to maintain supersensitivity of the striatal D₁ dopamine receptor system following long-term dopamine depletion.     

The Effect of pH on Glycolysis and Phosphofructokinase Activity in Cultured Cells and Synaptosomes

Abstract: Several G_i-linked neurotransmitter receptors, including dopamine D₂ receptors, act synergistically with Ca²⁺-mobilizing stimuli to potentiate release of arachidonic acid (AA) from membrane phospholipids. In brain, AA and its metabolites are thought to act as intracellular second messengers, suggesting that receptor-dependent potentiation of AA release may participate in neuronal transmembrane signaling. To study the molecular mechanisms underlying this modulatory response, we have now used Chinese hamster ovary cells transfected with rat D₂-receptor cDNA, CHO(D₂). Two antisense oligodeoxynucleotides corresponding to distinct cDNA sequences of cytosolic, AA-specific phospholipase A₂ (cPLA₂) were synthesized and added to cultures of CHO(D₂) cells. Incubation with antisense oligodeoxynucleotides inhibited D₂ receptor-dependent release of AA but had no effect on D₂-receptor binding or D₂ inhibition of cyclic AMP accumulation. In addition, pharmacological experiments showed that D₂ receptor-dependent AA release was prevented by nonselective phospholipase inhibitors (such as mepacrine) but not by inhibitors of membrane-bound, non-AA-specific PLA₂ (such as p -bromophenacyl bromide). cPLA₂ is expressed in brain tissue. The results, showing that cPLA₂ participates in receptor-dependent potentiation of AA release in CHO(D₂) cells, suggest that this phospholipase may serve a similar signaling function in brain.     

Zinc Allosterically Modulates Antagonist Binding to Cloned D1 and D2 Dopamine Receptors

Abstract: Cations of various size and charge were used as atomic scale probes of D₁ and D₂ dopamine receptors. Those cations that perturbed the binding of D₁- and D₂-selective dopamine receptor antagonists were identified by screening at 5 m M cation. Pseudo-noble-gas-configuration d-transition metals, such as zinc, exerted a complete inhibition of specific binding, whereas most other cations had little or no effect. The nature of zinc's actions was characterized by measuring the radioligand binding properties of [³H]SCH-23390 and [³H]methylspiperone to cloned D_1A and D_2L dopamine receptors in either the presence or absence of Zn²⁺. Zinc exerts a low-affinity, dose-dependent, EDTA-reversible inhibition of the binding of subtype-specific antagonists primarily by decreasing the ligands' affinity for their receptors. The mechanism of zinc inhibition appears to be allosteric modulation of the dopamine receptor proteins because zinc increases the dissociation constant ( K _D) of ligand binding, Schild-type plots of zinc inhibition reach a plateau, and zinc accelerates antagonist dissociation rates. Here we demonstrate the effect of zinc on the binding of D₁- and D₂-selective antagonists to cloned dopamine receptors and show that the inhibition by zinc is through a dose-dependent, reversible, allosteric, two-state modulation of dopamine receptors.     

Dopamine D2-Receptor Isoforms Expressed in AtT20 Cells Inhibit Q-Type High-Voltage-Activated Ca2+ Channels via a Membrane-Delimited Pathway

Abstract : Dopamine D₂ receptors both acutely and chronically inhibit high-voltage-activated Ca²⁺ channels (HVA-CCs). Two alternatively spliced isoforms, D_2L (long) and D_2S (short), are expressed at high levels in rat pituitary intermediate lobe melanotropes but are lacking in anterior lobe corticotropes. We stably transfected D_2L and D_2S into corticotrope-derived AtT20 cells. Both isoforms coupled to inhibition of Q-type calcium channels through pertussis toxin-sensitive G proteins. Thus, we have created a model system in which to study the kinetics of D₂-receptor regulation of Ca²⁺ channels. Rapid inhibition of HVA-CCs was characterized using a novel fluorescence video imaging technique for the measurement of millisecond kinetic events. We measured the time elapsed (lag time) between the arrival of depolarizing isotonic 66 m M K⁺, sensed by fluorescence from included carboxy-X-rhodamine (CXR), and the beginning of increased intracellular Ca²⁺ levels (sensed by changes in indo 1 fluorescence ratio). The lag time averaged 350-550 ms, with no significant differences among cell types. Addition of the D₂-agonist quinpirole (250 μ M ) to the K⁺/CXR solution significantly increased the lag times for D₂-expressing cells but did not alter the lag time for AtT20 controls. The increased lag times for D_2L - and D_2S-transfected cells suggest that at least a fraction of the Ca²⁺ channels was inhibited within the initial 350-550 ms. As this inhibition time is too fast for a multistep second messenger pathway, we conclude that inhibition occurs via a membrane-delimited diffusion mechanism.     

The effect of ergosterol, ergocalciferol and cholecalciferol on calcium-controlled peroxidase secretion by sugarbeet cells

Provitamin D₂ (ergosterol), vitamins D₂ and D₃ reduced the calcium-mediated peroxidase secretion in three types of sugarbeet cells in suspension cultures. Vitamin D₂ was the most effective in habituated non-organogenic cells which were the less sensitive to calcium; provitamin D₂ was the most effective in habituated organogenic cells, while normal non-organogenic cells were equally sensitive to the three types of vitamin D. The calcium ionophore A23187 slightly restricted peroxidase release in all cases, except in the habituated organogenic cells in the presence of calcium where it exerted a promotive effect. The inhibiting effect of vitamin D₂ was not counteracted by the ionophore except in this habituated organogenic cell line.     

Effects of the Dopamine D3 Antagonist PD 58491 and Its Interaction with the Dopamine D3 Agonist PD 128907 on Brain Dopamine Synthesis in Rat

Abstract: The dopamine (DA) D₃ receptor antagonist PD 58491 {3-[4-[1-[4-[2-[4-(3-diethylaminopropoxy)phenyl]-benzoimidazol-1-yl-butyl]-1 H -benzoimidazol-2-yl]-phenoxy]propyl]diethylamine} bound with high affinity and selectivity to recombinant human DA D₃ versus D_2L and D_4.2 receptors transfected into Chinese hamster ovary cells: K _i values of 19.5 n M versus 2,362 and >3,000 n M , respectively. In contrast, the putative DA D₃ receptor antagonist (+)-AJ76 displayed low affinity and selectivity for D₃ versus D_2L and D_4.2 receptors (91 n M vs. 253 and 193 n M , respectively). In vitro, PD 58491 (1 n M −1µ M ) exhibited D₃ receptor antagonist activity, reversing the quinpirole (10 n M )-induced stimulation of [³H]thymidine uptake in D₃ CHOpro-5 cells, but did not have any significant intrinsic activity by itself in this assay. PD 58491 did not decrease the γ-butyrolactone-induced increase in DA synthesis ( l -3,4-dihydroxyphenylalanine accumulation) in rat striatum, indicating that the compound possessed no in vivo DA D₂/D₃ receptor agonist action at DA autoreceptors. PD 58491 (3–30 mg/kg, i.p.) generally did not alter DA or serotonin synthesis in either the striatum or mesolimbic region of rat brain. The D₃-preferring agonist PD 128907 decreased DA synthesis in striatum and mesolimbic regions, and this effect was attenuated by pretreatment with PD 58491. These findings support the hypothesis that DA D₃ autoreceptors may in part modulate the synthesis and release of DA in striatum and mesolimbic regions.     

Activation of post-synaptic dopamine D₁ receptors promotes the release of tissue plasminogen activator in the nucleus accumbens via PKA signaling

We have previously demonstrated that tissue plasminogen activator (tPA) plays an important role through the conversion of plasminogen to plasmin in the release of dopamine in the nucleus accumbens (NAc) evoked by depolarization or the systemic administration of drugs of abuse such as morphine and nicotine. In the present study, we examined the mechanisms by which drugs of abuse increase extracellular tPA activity in the NAc in vivo using in situ zymography. The dopamine D₁ receptor (D₁R) agonist SKF38393, but not D₂ receptor agonist quinpirole, significantly increased extracellular tPA activity in the NAc. The effect of SKF38393 was blocked by pre-treatment with the dopamine D₁R antagonist SCH23390. Microinjection of Rp-cAMPs, a protein kinase A inhibitor, into the NAc completely blocked the effect of SKF38393. Systemic administration of morphine and methamphetamine increased extracellular tPA activity in the NAc, and these effects were completely blocked by pre-treatment with SCH23390 and raclopride. The results suggest that activation of post-synaptic dopamine D₁Rs in the NAc leads to an increase in extracellular tPA activity via protein kinase A signaling. Furthermore, dopamine D₂ receptors are also involved in the release of tPA induced by morphine and methamphetamine.     

Prediction, from temperature, of eyeing, hatching and 'swim-up' times for salmonid embryos

SUMMARY. 1. The literature contains a number of curves relating time (days) required for median hatch (=D₂) to water temperature (T,°C) for the eggs of several salmonid fishes. There are relatively few data on the relationships between time to median eyed (= D₁), days) or time to median swim-up (= D₃, days) and temperature.
2. From published data, over most of the range 0-9.5°C, approximate relationships are D,=0.5D₂ for Atlantic salmon (Salmo salar L.) and D3 = 1.7D₂ for eight species of Salmo and Oncorhynchus.
3. Field and hatchery tests suggest that these are useful empirical models for approximate prediction of D₁ and D₃ from D₂ for most salmonids.