Group I mGluR agonist DHPG facilitates spontaneous firing and induces excitatory inward current in cerebellar MLIs.

<p>(A) Puff-application of DHPG (arrowhead) increased the firing rate of a MLI under control conditions (upper). Time course of the firing rate (lower). The average firing rate calculated for 1-s bins was plotted at each time point. (B) A non-competitive mGluR1 antagonist CPCCOEt (30 µM) blocked the DHPG-induced firing facilitation (upper). Time course of the firing rate with the antagonist (lower). (C) Mean effects of DHPG on MLI firing. Facilitation was significantly blocked by CPCCOEt (*<i>p</i><0.05, <i>n</i> = 5). (D) Puff-application of DHPG (arrowhead) induced slow inward currents under control conditions. The inward current was blocked by CPCCOEt (100 µM) and a competitive mGluR1 antagonist LY367385 (100 µM). Average traces obtained from three continuous current events are shown. (E) Time courses of the effects of the mGluR1 antagonists CPCCOEt (black circles, 100 µM, <i>n</i> = 5) and LY367385 (gray circles, 100 µM, <i>n</i> = 4). Black and gray bars indicate the time periods of CPCCOEt and LY367385 application, respectively.</p

GABA_B receptor activation has no effect on the DHPG-induced inward current in MLIs.

<p>(A) Application of the GABA_B receptor agonist baclofen (3 µM) did not alter the DHPG-induced inward current. Averaged traces obtained from three continuous current events are shown. (B) Time course of the effect of baclofen on the DHPG-induced inward current (<i>n</i> = 6). The amplitude of the DHPG-evoked current is expressed as a percentage of the averaged control over the 4-min period before baclofen application.</p

Effects of tyrosine kinase inhibitors on DHPG-induced inward current in MLIs.

<p>(A) Representative examples of the DHPG-induced inward current. Averaged traces were obtained from three continuous current events. Genistein (30 µM, left), a genistein-inactive analogue genistin (30 µM, center), and AG490 (30 µM, right) were bath-applied for 15 min. (B) Time courses of the effects of the tyrosine kinase inhibitors, genistein and AG490, and genistin on the DHPG-induced inward current. The DHPG-current was suppressed by genistein (black circles, <i>n</i> = 4) and AG490 (gray circles, <i>n</i> = 5), but not by genistin (white circles, <i>n</i> = 4). A black bar indicates the time period of drug application.</p

Induction of mGluR1-mediated inward current requires activation of TRPC1 but not TRPC3 in MLIs.

<p>(A) Nonselective TRPC channel blockers SKF96365 (30 µM) and 2-APB (100 µM) reduced the DHPG-induced inward current. (B) Time courses of the effects of SKF96365 (black circles, 30 µM, <i>n</i> = 4) and 2-APB (gray circles, 100 µM, <i>n</i> = 5) on the DHPG-induced inward current. A black bar indicates the time of drug application. (C) A TRPC3 blocker Pyr3 (30 µM) suppressed the DHPG-induced inward current in PCs (right) but not in MLIs (left). (D) Time courses of the effects of Pyr3 on the DHPG-induced inward current in MLIs (white circles, <i>n</i> = 5) and in PCs (black circles, <i>n</i> = 6). (E) Dialysis of an anti-TRPC1 antibody (1∶200 dilution) into MLIs through patch pipettes markedly reduced the DHPG-induced inward current (left), while dialysis of the antibody preincubated with its antigenic peptide did not (right). The current traces were obtained at the indicated time points (2 and 12 min) after establishing whole-cell configuration. (F) Time course of the effect of the anti-TRPC1 antibody on the DHPG-induced inward current (black circles, <i>n</i> = 6) compared to control experiments using the antibody preincubated with its antigenic peptide (white circles, n = 5).</p

Activation of G proteins and PLC is required for induction of a component of the mGluR1-mediated inward current in MLIs.

<p>(A) Dialysis of a nonhydrolyzable G protein inactivator GDPβS (1 mM) into MLIs through patch pipettes decreased the DHPG-induced inward current gradually but did not inhibit the current completely. (B) Time courses of the effect of GDPβS on the DHPG-induced inward current. GDPβS reduced the current amplitude in a dose-dependent manner (gray circles, 300 µM, <i>n</i> = 5; black circles, 1 mM, <i>n</i> = 7). (C) The G protein-independent component of the DHPG-induced inward current was blocked by the nonselective TRPC channel blocker 2-APB (100 µM, <i>n</i> = 5). The DHPG-current was recorded by whole-cell recordings with patch pipettes containing 1 mM GDPβS. The gray line indicates the average percent value obtained during 12–16 min (59%). (D) A minor component of the mGluR1-mediated inward current was dependent on PLC activation. Infusion of a PLC inhibitor U73122 (black circles, 5 µM, <i>n</i> = 5) decreased the DHPG-induced inward current moderately, while U73343, an inactive analog of U73122, at the same concentration did not alter the current amplitude (white circles, <i>n</i> = 5).</p

Effects of Src tyrosine kinase inhibitor and MEK inhibitors on the DHPG-induced inward current in MLIs.

<p>(A) Representative traces under control conditions and in the presence of the Src tyrosine kinase inhibitor PP2 (30 µM) and the inactive PP2 analog PP3 (10 µM) are shown. Average traces were obtained from three continuous current events. (B) Time courses of the effects of PP2 and PP3 on the DHPG-induced inward current. The DHPG-current was suppressed by PP2 (black circles, <i>n</i> = 5) but not by PP3 (white circles, <i>n</i> = 4). A black bar indicates the time period of drug application. (C) The G protein-independent component of the DHPG-induced inward current was blocked by the Src tyrosine kinase inhibitor PP2 (30 µM). The DHPG-induced currents were recorded by whole-cell recordings with patch pipettes containing 1 mM GDPβS. The gray line indicates the average percent value during 12–16 min (63%). (D) MEK inhibitors PD98059 (10 µM) and SL327 (10 µM) reduced the DHPG-induced inward current. Averaged traces obtained from three continuous current events are shown. (E) Time courses of the effects of PD98059 (black circles, 10 µM, <i>n</i> = 5) and SL327 (gray circles, 10 µM, <i>n</i> = 4) on the DHPG-induced inward current (<i>n</i> = 5). A black bar indicates the time point for application of the drugs. (F) Schematic diagram of signaling pathways required for the induction of mGluR1-mediated inward current in MLIs. The activation of mGluR1 opens TRPC1 channels through both G protein-dependent and G-protein-independent Src–ERK1/2 signaling pathways. DAG, diacylglycerol.</p

NPS stimulates glutamatergic input of BLA interneurons.

<p>NPS stimulates glutamatergic synaptic activity dependent on action potential propagation in BLA interneurons. (A) Examples of glutamatergic sEPSCs recorded in a BLA interneuron before and (B) during action of NPS. (C) Cumulative amplitude and (D) inter-event interval histograms obtained from the same neuron shown in (A, B) before addition of NPS and after a steady-state effect had been reached. (E) sEPSC amplitude and (F) frequency pooled during control conditions and after addition of NPS demonstrates a significant increase in sEPSC frequency. (G) mEPSCs recorded in the presence of TTX were unchanged in amplitude as well as (H) frequency by addition of NPS. (I) Representative traces of EPSCs recorded before and after addition of 200 nM NPS. (J) Time course of mean paired-pulse ratio of eEPSCs showing lack of NPS effects on paired-pulse facilitation in BLA interneurons. **, p<0.01. Bar, time of NPS addition.</p

NPS effects in BLA interneurons.

<p>Excitatory synaptic transmission is indispensable for the NPS action. (A) In the continuous presence of TTX to block synaptic transmission, application of NPS (as indicated by bar) produces no effect on holding current and thus shows no direct postsynaptic induction of an inward current in BLA interneurons nor (B) intercalated cells. (C) Under current-clamp conditions, NPS application induces a depolarizing response associated with increased spike activity triggered upon depolarizing current injections in BLA interneurons. (D, E) Block of excitatory synaptic transmission by NBQX and AP5 abolishes the effect of NPS on (D) sIPSCs amplitude as well as (E) frequency recorded from BLA projection neurons.</p

Probe location in the EPN.

<p>(A) Injection cannula were stereotactically implanted at a 10° angle, aiming at the dorsal portion of the EPN (B) Histological verification of probe location in a cresyl violet stained coronal section from a representative animal. A small lesion indicates the injection site, which is located in the dorsal EPN. LA, lateral amygdala; BLA, basolateral amygdala; EPN, endopiriform nucleus; PC, piriform cortex; III, third ventricle (C) Summary of injection sites in individual animals of all three test groups. Illustrations modified from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002695#pone.0002695-Paxinos1" target="_blank">[37]</a>.</p

Behavioral NPS effects.

<p>A selective reduction of contextual fear responding was observed upon NPS administration. (A) Injection of 0.1 nmole NPS to the endopiriform nucleus (n = 14), resulted in significantly reduced freezing behavior during contextual fear memory retrieval, as compared to vehicle injected controls (n = 12). At a dose of 0.01 nmole, NPS had no such effect (n = 12). Time course analysis of conditioned freezing behavior revealed a continuous reduction during the contextual retrieval session. For better comparison the 2-min context exposure was dissected into eigth intervals of either 10 s 1,3,5,7) or 20 s (2,4,6,8) length, analogous to those in cued retrieval. (B) In cued fear memory retrieval, a somewhat reduced freezing response was observed in the 0.1 nmole group, but this changes failed to reach significance level. The reduction was mostly observed during inter-stimulus-intervals and hence may have resulted from the animals' reduced fear response to the background context, whereas the responses during tone exposure were almost identical between groups. 1,3,5,7, conditioned stimluli (CS+, 10 s), 2,4,6,8 inter-stimulus-intervals (20 s). (C, D) A similar specific reduction during contextual versus cued memory retrieval was observed for a second defensive behavior, risk-assessment. (E, F) No change of anxiety-related behavior was apparent in an elevated plus maze. All values are means±SEM; *, p<0.05; compared to vehicle, **, p<0.01.</p