N/OFQ-induced KATP channel activation is mediated by PI3K, PKA and PKC.

<p><i>A-E</i>: Representative recordings showing the effect of N/OFQ in the presence of specific inhibitors for PI3K (wortmannin; Wort), PKA (H89) or PKC (calphostin C; CalC), as indicated. Scale bars in <i>A</i> apply to all traces. <i>F</i>: Grouped data showing that kinase inhibitors attenuate the N/OFQ effect. Combined application of H89 and CalC did not have an additive effect. Numbers in bars represent the number of cells examined in each group. * p<0.05, *** p<0.001 vs. N/OFQ.</p

Alterations in basic membrane properties of Hu97/18

<p>SPNs. (A–C): Membrane capacitance (A), membrane resistance (B), and membrane time constant (C) did not differ between Hu18/18 and Hu97/18 SPNs. Membrane capacitance measurement was pooled from the experiments with potassium-based and caesium-based internal solutions (Hu18/18 n = 46, Hu97/18 n = 44), while membrane resistance and tau were measured with potassium-based internal solution only (Hu18/18 n = 21, Hu97/18 n = 17). (D – J): Cells were patch-clamped with potassium-based solution, and membrane voltage changes in response to the injected current were recorded and analysed. (D) Representative I–V response of a Hu18/18 SPN to current injection (50pA increments from -200pA, 1 s each). (E) I–V curves showed no difference between Hu97/18 and Hu18/18 SPNs. (F) Rheobase and (G) rheobase frequency were not different between the genotypes. (H) Hu97/18 had a lower resting membrane potential than Hu18/18 SPNs (p = 0.03). (I) Action potential (AP) threshold was more depolarized in Hu97/18 SPNs (p = 0.02). (J) The change in membrane voltage from resting potential to AP threshold was not different between Hu18/18 and Hu97/18 SPNs. For the experiments in D–J, Hu18/18 n = 11 and Hu97/18 n = 12. *p<0.05 (unpaired Student’s t-test).</p

Whole-cell properties of CA1 pyramidal neurons from Hu18/18 and Hu97/18 mice at 9 months of age.

<p>(A–D) Whole-cell patch recordings from CA1 pyramidal neurons revealed no difference in membrane properties including membrane capacitance (Cm, Hu18/18 n = 14, Hu97/18 n = 11), membrane resistance (Rm, Hu18/18 n = 14, Hu97/18 n = 11), membrane tau (ôm, Hu18/18 n = 14, Hu97/18 n = 11) or resting membrane potential (Em, Hu18/18 n = 14, Hu97/18 n = 13). I–V plots (E, Hu18/18 n = 14, Hu97/18 n = 13), mEPSC frequency (F, Hu18/18 n = 13, Hu97/18 n = 13) and mEPSC amplitude (G, Hu18/18 n = 13, Hu97/18 n = 13) were also similar between genotypes.</p

N/OFQ inhibits orexin neurons via NOP receptor activation in the rat.

<p><i>A</i>: N/OFQ hyperpolarizes orexin neurons. <i>B</i>: Representative voltage clamp trace showing a TTX-insensitive outward current induced by N/OFQ application. <i>C</i>: N/OFQ-induced effect is significantly attenuated by UFP-101. However, the effect persists in the presence of naloxone (E), indicating a specific activation of NOP receptors. D: N/OFQ is ineffective on orexin neurons patched with GDPβS in the recording pipette. E: Grouped data is shown. Numbers in bars represent the number of cells examined in each group. ** p<0.01, *** p<0.005 vs. N/OFQ.</p

sEPSC amplitude and frequency change in Hu97/18 SPNs.

<p>Cells were patch-clamped with a potassium-based internal solution at V_h = -70 mV and sEPSCs were recorded. (A) Representative sEPSC traces for Hu18/18 (top) and Hu97/18 SPNs (bottom). (B) The average sEPSC amplitude of Hu18/18 and Hu97/18 cells (difference did not reach significance, p = 0.12). (C) Cumulative probability showed a decrease in sEPSC amplitude in Hu97/18 SPNs (significant genotype and amplitude interaction, p<0.0001). (D) Amplitude distribution analysis showed a significant increase in the percentage of small events (<10pA) and a trend towards a decrease in big events (>15pA) in Hu97/18 SPNs. (E) There was no difference in sEPSC decay time between Hu18/18 and Hu97/18 SPNs. (F) Cumulative probability showed an increase in sEPSC inter-event intervals in Hu97/18 SPNs (significant genotype and inter-event intervals interaction, p<0.0001). (G) The average sEPSC frequency was decreased in Hu97/18 SPNs (p = 0.038). For all experiments, Hu18/18 n = 20 and Hu97/18 n = 17. *P<0.05 (two-way ANOVA with Bonferroni correction, D; unpaired Student’s t-test, G).</p

N/OFQ does not consistently modulate excitatory transmission to orexin neurons.

<p><i>A</i>: mEPSC traces from a representative orexin neuron before (control: CTL) and during N/OFQ (0.3 µM, 5 min) application, showing a lack of effect. <i>B-C</i>: Cumulative frequency (freq.) graphs for mEPSC interevent interval (<i>C</i>) and amplitude (<i>D</i>) for the cell shown in <i>A</i>. Solid and broken lines represent CTL and N/OFQ conditions, respectively. <i>D-E</i>: Grouped data (n = 8) of mEPSC interevent interval (<i>D</i>) and amplitude (<i>E</i>) in control and N/OFQ.</p

Subtle changes in membrane properties and unaltered sEPSC characteristics in Hu97/18 SPNs at 6 months of age.

<p>(A) At 6 months of age, action potential threshold was more depolarized in Hu97/18 SPNs. (B) No significant change in resting membrane potential was observed. sEPSC amplitude (C) and frequency (D) were unaffected in Hu97/18 SPNs at 6 months of age. For these experiments, Hu18/18 n = 13 and Hu97/18 n = 16. *p<0.05 (unpaired Student’s t-test).</p

Long-term potentiation (LTP) is impaired in 9-month old Hu97/18 CA1.

<p>(A) Representative fEPSP traces from hippocampal slices of 9-month old Hu18/18 and Hu97/18 mice in response to two 0.1 ms pulses separated by 100 ms. fEPSPs were recorded in CA1 stratum radiatum during stimulation of the Schaffer collateral pathway. (B) Paired pulse facilitation (PPF) was measured by dividing the slope of the second response to that of the first and was expressed as percent increase. PPF was significantly lower in Hu97/18 CA1 (Hu18/18 n = 8, Hu97/18 n = 6). (C) fEPSPs were normalized to a 10-minute baseline period prior to high-frequency stimulation (HFS; 100 Hz for 1 s×3, 10 s inter-train interval). LTP was easily obtained in hippocampal slices from Hu18/18 mice (n = 6) but showed severe impairment in slices from Hu97/18 mice (n = 6). Representative traces before (black) and 50–60 minutes after (grey) HFS are shown. ***p<0.0001, t-test of average % above baseline 50–60 minutes post-HFS. (D–F) Paired pulse facilitation (D,E) and LTP (F) graphs as above but conducted in slices from 3-month old animals. n = 6 for each genotype for both (E) and (F).</p

N/OFQ activates KATP channels in orexin neurons.

<p><i>A</i>: Current responses to voltage-ramps before (control; CTL) and during N/OFQ application in 2.5 mM extracellular potassium. B: Subtraction of currents recorded in two conditions (N/OFQ - CTL) reveals an N/OFQ-induced current (2.5 mM K⁺). Increasing the extracellular potassium concentration to 10 mM shifted the current-voltage relationship rightward. <i>C</i>. Grouped data showing that the reversal potential of the N/OFQ current is dependent on the concentration of extracellular potassium. *** p<0.005. <i>D-F</i>: N/OFQ responses are attenuated by BaCl₂ (<i>D</i>) and glibenclamide (Glib) (<i>E</i>) but not tertiapin Q (TQ) (<i>F</i>). Grouped data are shown in (G). *** p<0.005, n.s. non-significant vs. N/OFQ. Numbers in bars represent the number of cells examined in each group. <i>H</i>: Once N/OFQ current is induced, it is persistent and retains Ba⁺⁺ sensitivity even after a prolonged washout.</p

No changes in membrane properties and sEPSC characteristics in Hu97/18 SPNs at 3 months of age.

<p>At 3 months of age, action potential threshold (A) and resting membrane potential (B) were unaltered in Hu97/18 (n = 10), in comparison to Hu18/18 SPNs (n = 11). Likewise, sEPSC amplitude (C) and frequency (D) were unaffected in Hu97/18 (n = 8) when compared to Hu18/18 SPNs (n = 8).</p