Journal: Frontiers in Neuroscience

Article Title: TRPC1/5-Ca V 3 Complex Mediates Leptin-Induced Excitability in Hypothalamic Neurons

doi: 10.3389/fnins.2021.679078

Figure Lengend Snippet: Ca V 3 co-precipitation with TRPC5. Immunoprecipitation of brain and hypothalamus (Hyt) extracts with Ca V 3.1 (A) or Ca V 3.2 (B) and TRPC5 in the same samples show their mutual interaction. (C) IP of brain and Hyt samples using Ca V 3.1 (left) or Ca V 3.2 (right) and TRPC5 show their interaction with TRPC1. IgG was used as IP negative control in all cases, ( n = 3 for all panels).

Article Snippet: Samples were then processed by addition of the primary antibody and incubation for 1 h at 4°C (antibodies: α1H (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, United States, RRID:AB_2259537 ), α1G [Millipore, Cat. #MABN464); TRPC5 (Alomone, RRID:AB_2040241 )], TRPC1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, United States, RRID:AB_2207905 ), and IgG (Cat. #20008−1−100 and #20009−1−100, Alpha Diagnostics, San Antonio, TX) and overnight incubation, followed by incubation for 1 h with protein A/G agarose beads (Biovision, Mountain View, CA) on a shaking plate at 4°C.

Techniques: Immunoprecipitation, Negative Control

Journal: Frontiers in Neuroscience

Article Title: TRPC1/5-Ca V 3 Complex Mediates Leptin-Induced Excitability in Hypothalamic Neurons

doi: 10.3389/fnins.2021.679078

Figure Lengend Snippet: Ca 2+ influx via TRPC1/5 depolarizes and recruits T-type channels. (A) Cartoon depicting T-type calcium channels co-exist in a complex with TRPC1/5. Membrane depolarization induced by TRPC channel-mediated cation influx recruits T-type channel activity. Chelation of Ca 2+ influx with a fast chelator that binds Ca 2+ closer to the mouth’s pore (BAPTA-AM, K ON = 6 × 10 8 ) is represented in orange. Chelation of Ca 2+ influx with a slower chelator (EGTA-AM, K ON = 1.5 × 10 6 ) is represented in light orange. (B) Resting membrane potential (RMP) from control ( n = 24) and leptin-treated neurons in the absence ( n = 11) or presence of 2APB ( n = 5). Values for control and leptin-treated neurons were reproduced from (A) for comparison. Cultured hypothalamic neurons were incubated with either 10 μM BAPTA-AM ( n = 5) or EGTA-AM ( n = 4) for 30 min at 37°C prior to whole-cell current clamp to assess leptin-mediated effects on the RMP. # Significantly different from others, P < 0.05, ANOVA, F ( 2 , 37 ) = 8.927. *Significantly different from control, P < 0.05, t -test. BAPTA: t = 4.743, df = 8. EGTA: t = 2.579, df = 6. (C) Example traces of APs elicited by a 20 pA/s ramp protocol under control conditions (top trace), and after the addition of 100 nM leptin in the presence of BAPTA (middle) or EGTA (bottom). (D) Rheobase, AP number, and slope values elicited by 20 pA/s ramps for each treatment ( n = 4–24). *Significantly different from control, P < 0.05, t -test. Rheobase: t = 3.285 (df = 40, control), t = 0.4757 (df = 12, BAPTA incubation), t = 2.019 (df = 6, EGTA incubation). AP number: t = 3.065 (df = 39, control), t = 0.044 (df = 11, BAPTA), t = 3.376 (df = 6, EGTA). Slope: t = 6.495 (df = 30, control), t = 1.245 (df = 9, BAPTA incubation), t = 2.446 (df = 6, EGTA incubation). (E) Rheobase values elicited by 40 pA/s ramps for each treatment ( n = 8–23). *Significantly different from others, P < 0.05, ANOVA, F ( 2 , 36 ) = 0.2637 for control neurons, F ( 2 , 32 ) = 12.36 for leptin-treated neurons. (F) T-type channel availability (SSI) and open probability (SSA) as function of voltage. SSI parameters: V 50 = –65.4 ± 1.0 mV, k = 7.1 ± 0.5 ( n = 21). SSA parameters: V 50 = –41.8 ± 0.7 mV, k = 5.2 ± 0.3 ( n = 22). Black circles indicate the availability and the open probability of T-type channels) at the indicated RMP. Red circles indicate steady sate properties at depolarized RMP after leptin (100 nM) incubation. Window current, underneath SSA and SSI curves is indicated in turquoise. Window currents were calculated for each recording neuron from the SSA and SSI curves fitted with the Boltzmann function ( n = 18, see section “Materials and Methods”); SEM is indicated in gray. The steady-state current was normalized to its maximum value (Normalized Isst). Steady-state current at the RMP (empty black box plot) increased after incubation with leptin (empty red box plot).

Article Snippet: Samples were then processed by addition of the primary antibody and incubation for 1 h at 4°C (antibodies: α1H (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, United States, RRID:AB_2259537 ), α1G [Millipore, Cat. #MABN464); TRPC5 (Alomone, RRID:AB_2040241 )], TRPC1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, United States, RRID:AB_2207905 ), and IgG (Cat. #20008−1−100 and #20009−1−100, Alpha Diagnostics, San Antonio, TX) and overnight incubation, followed by incubation for 1 h with protein A/G agarose beads (Biovision, Mountain View, CA) on a shaking plate at 4°C.

Techniques: Membrane, Activity Assay, Control, Comparison, Cell Culture, Incubation