Dopamine and Serotonin-Induced Modulation of GABAergic and Glutamatergic Transmission in the Striatum and Basal Forebrain

Catecholamine receptor-mediated modulation of glutamatergic or GABAergic transmission in the striatum as well as basal forebrain (BF) has been intensively studied during these two decades. In the striatum, activation of dopamine (DA) D2 receptors in GABAergic terminals inhibits GABA release onto cholinergic interneurons by selective blockade of N-type calcium channels. In the BF, glutamatergic transmission onto cholinergic projection neurons is inhibited via DA D1-like receptors by selective blockade of P/Q-type calcium channels. On the other hand, presynaptic inhibition of the GABA release onto cholinergic neurons mediated by D1-like receptors or 5-HT1B receptors is independent of calcium influx. In addition, the DA receptor-mediated calcium influx dependent presynaptic inhibition mentioned above decreases with postnatal development, with selective coupling between DA receptors and each subtype of calcium channels being unchanged. Furthermore, the precise origin of these GABAergic or glutamatergic inputs to postsynaptic neurons can be identified by recent optogenetic approaches. Thus, modulatory mechanisms in specific synaptic connections between certain types of neurons in the striatum and BF are being identified

Parallel decrease in ω-conotoxin-sensitive transmission and dopamine-induced inhibition at the striatal synapse of developing rats

Whole-cell patch-clamp recordings of GABAergic IPSCs were made from cholinergic interneurones in slices of striatum from developing rats aged 21–60 days postnatal. In addition, the Ca2+ channel subtypes involved in synaptic transmission, as well as dopamine (DA)-induced presynaptic inhibition, were investigated pharmacologically with development by bath application of Ca2+ channel blockers and DA receptor agonists. The IPSC amplitude was reduced by ω-conotoxin GVIA (ω-CgTX) or ω-agatoxin TK (ω-Aga-TK) across the whole age range, suggesting that multiple types of Ca2+ channels mediate transmission of the synapse. The IPSC fraction reduced by ω-CgTX significantly decreased, whereas that reduced by ω-Aga-TK remained unchanged with development. DA or quinpirole, a D2-like receptor agonist, presynaptically reduced the IPSC amplitude throughout development. The DA-induced inhibition decreased with age in parallel with the decrease in N-type Ca2+ channels. DA showed no further inhibition of IPSCs after the inhibitory effect of ω-CgTX had reached steady state throughout development. These results demonstrate that there is a functional link between presynaptic N-type Ca2+ channels and D2-like DA receptors at inhibitory synapses in the striatum. They also demonstrate that the suppression of GABAergic transmission by D2-like receptors is mediated by modulation of N-type Ca2+ channels and decreases in parallel with the developmental decline in the contribution of N-type Ca2+ channels to exocytosis

NCI-H295R, a Human Adrenal Cortex-Derived Cell Line, Expresses Purinergic Receptors Linked to Ca²⁺-Mobilization/Influx and Cortisol Secretion

<div><p>Purinergic receptor expression and involvement in steroidogenesis were examined in NCI-H295R (H295R), a human adrenal cortex cell line which expresses all the key enzymes necessary for steroidogenesis. mRNA/protein for multiple P1 (A_2A and A_2B), P2X (P2X₅ and P2X₇), and P2Y (P2Y₁, P2Y₂, P2Y₆, P2Y₁₂, P2Y₁₃, and P2Y₁₄) purinergic receptors were detected in H295R. 2MeS-ATP (10–1000 µM), a P2Y₁ agonist, induced glucocorticoid (GC) secretion in a dose-dependent manner, while other extracellular purine/pyrimidine agonists (1–1000 µM) had no distinct effect on GC secretion. Extracellular purines, even non-steroidogenic ones, induced Ca²⁺-mobilization in the cells, independently of the extracellular Ca²⁺ concentration. Increases in intracellular Ca²⁺ concentration induced by extracellular purine agonists were transient, except when induced by ATP or 2MeS-ATP. Angiotensin II (AngII: 100 nM) and dibutyryl-cyclic AMP (db-cAMP: 500 µM) induced both GC secretion and Ca²⁺-mobilization in the presence of extracellular Ca²⁺ (1.2 mM). GC secretion by AngII was reduced by nifedipine (10–100 µM); whereas the Ca²⁺ channel blocker did not inhibit GC secretion by 2MeS-ATP. Thapsigargin followed by extracellular Ca²⁺ exposure induced Ca²⁺-influx in H295R, and the cells expressed mRNA/protein of the component molecules for store-operated calcium entry (SOCE): transient receptor C (TRPC) channels, calcium release-activated calcium channel protein 1 (Orai-1), and the stromal interaction molecule 1 (STIM1). In P2Y₁-knockdown, 2MeS-ATP-induced GC secretion was significantly inhibited. These results suggest that H295R expresses a functional P2Y₁ purinergic receptor for intracellular Ca²⁺-mobilization, and that P2Y₁ is linked to SOCE-activation, leading to Ca²⁺-influx which might be necessary for glucocorticoid secretion.</p></div

Dopamine D2-like receptors selectively block N-type Ca2+ channels to reduce GABA release onto rat striatal cholinergic interneurones

The modulatory roles of dopamine (DA) in inhibitory transmission onto striatal large cholinergic interneurones were investigated in rat brain slices using patch-clamp recording.Pharmacologically isolated GABAA receptor-mediated IPSCs were recorded by focal stimulation within the striatum. Bath application of DA reversibly suppressed the amplitude of evoked IPSCs in a concentration-dependent manner (IC50, 10.0 μm).A D2-like receptor agonist, quinpirole (3–30 μm), also suppressed the IPSCs, whereas a D1-like receptor agonist, SKF 81297, did not affect IPSCs. Sulpiride, a D2-like receptor antagonist, blocked the DA-induced suppression of IPSCs (apparent dissociation constant (KB), 0.36 μm), while a D1-like receptor antagonist, SCH 23390 (10 μm), had no effect.DA (30 μm) reduced the frequency of spontaneous miniature IPSCs (mIPSCs) without changing their amplitude distribution, suggesting that GABA release was inhibited, whereas the sensitivity of postsynaptic GABAA receptors was not affected. The effect of DA on the frequency of mIPSCs was diminished when extracellular Ca2+ was replaced by Mg2+ (5 mm), indicating that DA affected the Ca2+ entry into the presynaptic terminal.An N-type Ca2+ channel selective blocker, ω-conotoxin GVIA (ω-CgTX, 3 μm), suppressed IPSCs by 65.4%, whereas a P/Q-type Ca2+ channel selective blocker, ω-agatoxin IVA (ω-Aga-IVA, 200 nm), suppressed IPSCs by 78.4%. Simultaneous application of both blockers suppressed IPSCs by 95.9%. Assuming a 3rd power relationship between Ca2+ concentration and transmitter release, the contribution of N-, P/Q- and other types of Ca2+ channels to presynaptic Ca2+ entry is estimated to be, respectively, 29.8, 40.0 and 34.5% at this synapse. After the application of ω-CgTX, DA (30 μm) no longer affected IPSCs. In contrast, ω-Aga-IVA did not alter the level of suppression by DA, suggesting that the action of DA was selective for N-type Ca2+ channels.A G protein alkylating agent, N-ethylmaleimide (NEM), significantly reduced the DA-induced suppression of IPSCs.These results suggest that presynaptic D2-like receptors are present on the terminals of GABAergic afferents to striatal cholinergic interneurones, and down-regulate GABA release by selectively blocking N-type Ca2+ channels through NEM-sensitive G proteins

Detection of mRNA/protein for purinergic receptors in H295R.

<p>I) mRNA, mRNA for A_2A (<b>A</b>), A_2B (<b>B</b>), P2X₅ (<b>C</b>), P2X₇ (<b>D</b>), P2Y₁ (<b>E</b>), P2Y₂ (<b>F</b>), P2Y₆ (<b>G</b>), P2Y₁₂ (<b>H</b>), P2Y₁₃(<b>I</b>), and P2Y₁₄(<b>J</b>) were identified. Each image shows the PCR product bands at three different annealing temperatures, depending upon the melting temperatures (Tm) of the primers. The PCR product amplified for GAPDH (annealing temperature of 58°C) was also loaded on the same gel for each target. The lanes in each gel image show: molecular markers (M), three PCR products for the mRNA target primers (products in high, middle, and low annealing temperatures are indicated), and one using a GAPDH primer (G). Each value in parentheses indicates the molecular weight (bp) for the expected PCR product. The numbers on the three lanes of the targets represent the annealing temperatures used in the individual procedures. II) protein Anti-human antibodies for purinergic receptors were used to confirm the protein expression of the purinergic receptors for which mRNA was detected by PCR in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071022#pone-0071022-g001" target="_blank">Figure 1</a>–I. The followings are the target proteins and their predicted molecular weights; a: A_2A (45 kDa), <b>b:</b> A_2B (45 kDa), <b>c:</b> P2X₅ (47 kDa), <b>d:</b> P2X₇ (69 kDa), <b>e:</b> P2Y₁ (42 kDa), <b>f:</b> P2Y₂ (42 kDa), <b>g:</b> P2Y₆ (36 kDa), <b>h:</b> P2Y₁₂ (39 kDa), <b>i:</b> P2Y₁₃, (41 kDa), <b>j:</b> P2Y₁₄, (39 kDa), <b>k:</b> GAPDH (37 kDa). Molecular sizes (kDa), estimated by pre-stained weight marker, are shown on the left and the right sides of the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071022#pone-0071022-g001" target="_blank">Figure 1</a>–II. Each arrow head indicates the signal band that is clear and nearest to the predicted size of the target protein.</p

Detection of mRNA/protein for the key molecules for store operated calcium entry (SOCE) in H295R.

<p>I) mRNA, Stromal interaction molecule 1 (STIM1), a Ca²⁺ sensor expressed on endoplasmic reticulum, calcium release-activated calcium channel protein 1 (Orai-1), a component for connection between STIM1 and Ca channels, and TRPC channels, a possible Ca channel for SOCE were examined. mRNA for STIM1 (<b>A</b>) Orai-1 (<b>B</b>), and subtypes of TRPC1 (<b>C</b>), C3 (<b>D</b>), C5 (<b>E</b>), and C6 (<b>F</b>) were identified in H295R. The lanes in each gel image show: molecular markers (M), three PCR products for the mRNA target primers (products in high, middle, and low annealing temperatures are indicated), and one using a GAPDH primer (G). Each value in parentheses indicates the molecular weight (bp) for the expected PCR product. The numbers on the three lanes of the targets represent the annealing temperatures used in the individual procedures. II) protein, Anti-human antibodies for STIM1, Orai-1, and TRIPC channels were used to confirm the expression of proteins for which mRNA was detected by PCR in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071022#pone-0071022-g008" target="_blank"><b>figure 8</b></a>–<b>I</b>. The followings are the target proteins and their predicted molecular weights; <b>a</b>–<b>1:</b> STIM1 (84 kDa), <b>a</b>–<b>2:</b> STIM1 with control peptide (0.2 µg/mL) in the primary antibody reaction, <b>b:</b> Orai-1 (52 kDa), <b>c</b>: TRPC1 (83 kDa), <b>d:</b> TRPC3 (97 kDa), <b>e:</b> TRPC5 (110 kDa), <b>f:</b> TRPC6 (100 kDa). Molecular sizes (kDa), estimated by pre-stained weight marker, are shown on the left side of each membrane image. Each arrow head indicates the signal band that is clear and nearest to the predicted size of the target protein.</p