Activation of InsP₃ receptors is sufficient for inducing graded intrinsic plasticity in rat hippocampal pyramidal neurons

Abstract

The synaptic plasticity literature has focused on establishing necessity and sufficiency as two essential and distinct features in causally relating a signaling molecule to plasticity induction, an approach that has been surprisingly lacking in the intrinsic plasticity literature. In this study, we complemented the recently established necessity of inositol trisphosphate (InsP3) receptors (InsP3R) in a form of intrinsic plasticity by asking if InsP3R activation was sufficient to induce intrinsic plasticity in hippocampal neurons. Specifically, incorporation of d-myo-InsP3 in the recording pipette reduced input resistance, maximal impedance amplitude, and temporal summation but increased resonance frequency, resonance strength, sag ratio, and impedance phase lead. Strikingly, the magnitude of plasticity in all these measurements was dependent on InsP3 concentration, emphasizing the graded dependence of such plasticity on InsP3R activation. Mechanistically, we found that this InsP3-induced plasticity depended on hyperpolarization-activated cyclic nucleotide-gated channels. Moreover, this calcium-dependent form of plasticity was critically reliant on the release of calcium through InsP3Rs, the influx of calcium through N-methyl-d-aspartate receptors and voltage-gated calcium channels, and on the protein kinase A pathway. Our results delineate a causal role for InsP3Rs in graded adaptation of neuronal response dynamics, revealing novel regulatory roles for the endoplasmic reticulum in neural coding and homeostasis.

Keywords: HCN channels; endoplasmic reticulum; hippocampus; inositol trisphosphate receptors; intrinsic plasticity.

Fig. 1.

Inclusion of inositol trisphosphate (InsP₃) in the recording pipette was sufficient to induce plasticity in intrinsic response dynamics of a hippocampal pyramidal neuron. A and B: schematic diagram depicting the whole cell current-clamp recording setup (A) and experimental protocol (B, top) used to study InsP₃-induced plasticity of intrinsic response dynamics (IRD). Whole cell current-clamp recording was performed with 10 μM d-myo-InsP₃ in the recording pipette. The color code for all traces (B, top) and the chirp stimulus (B, bottom) are also depicted. V_m, membrane potential. C: voltage response of a representative neuron to the pulse-current injections (VI in B), at the beginning (blue) and after 45 min (orange) of the experiment. Input resistance (R_in) values obtained from these traces are also shown. D: time course of an estimate of input resistance, R̄_in (computed from steady-state voltage response of the neuron to the hyperpolarizing pulse part of the chirp stimulus shown in B). E: voltage response of the neuron to the chirp stimulus at the beginning (blue; 0–5 min average) and the end (orange; 40–45 min average) of the experiment. F: impedance amplitude profiles obtained from traces shown in E, also depicting the measurements obtained from these profiles: resonance frequency (f_R), maximal impedance amplitude (|Z|_max), and resonance strength (Q). G: time course of f_R. H: impedance phase profiles obtained from traces shown in E, also depicting the values of total inductive phase (Φ_L) obtained from these profiles. I: voltage response of the neuron to the injection of a sequence of 5 alpha currents (20 Hz) at the beginning (blue) and end (orange) of the experiment. The values of summation ratio (S_α) are shown, and the red arrow indicates an increase in the rebound potential after 45 min. All traces and analyses presented are from the same neuron.

Fig. 2.

InsP₃-induced plasticity in intrinsic response dynamics was graded, with the gradation dependent on InsP₃ concentration ([InsP₃]) in the recording pipette. A: population plots of R_in measured at the beginning (blue) and the end (orange) of experiments where different [InsP₃] were included in the recording pipette. The color codes for [InsP₃] are as follows: cyan, 10 nM (n = 8); green, 100 nM (n = 6); red, 1 μM (n = 6); and black, 10 μM (n = 6). B: time courses of normalized R̄_in when experiments were performed with various [InsP₃] in the recording pipette. C: population plots of f_R measured at the beginning (blue) and the end (orange) of experiments where different [InsP₃] were included in the recording pipette. D: time courses of normalized f_R when experiments were performed with various [InsP₃] in the recording pipette. E: %change in R_in and f_R (at the end of the experiment, with reference to the beginning) plotted as a function of [InsP₃] in the recording pipette. For A–E, data are means ± SE and P values (when presented) are from paired Student's t-tests. F: summary plot of %change in various measurements (from their respective baseline values) after 45 min into the recording with various [InsP₃]. Data are medians and quartiles. *P < 0.05, Mann-Whitney test.

Fig. 3.

InsP₃-induced plasticity was abolished in the presence of ZD7288. Experiments depicted were performed with 10 μM InsP₃ and 20 μM ZD7288 (n = 8) in the recording pipette. A: voltage response of a representative neuron to the pulse-current injections at the beginning (blue) and after 45 min (orange) of the experiment. R_in values obtained from these traces are also shown. B: population plots of R_in measured at the beginning (blue) and the end (orange) of experiments performed in the presence of ZD7288. C: time course of normalized R̄_in in the presence (green) and absence (black; control) of ZD7288. D: voltage response of the neuron to the injection of a sequence of 5 alpha currents (20 Hz) at the beginning (blue) and end (orange) of the experiment. The S_α values are shown. E: voltage response of the neuron to the chirp stimulus at the beginning (blue; 0–5 min average) and the end (orange; 40–45 min average) of the experiment. F and G: impedance amplitude (F) and phase (G) profiles obtained from traces shown in E, also depicting the measurements obtained from these profiles: f_R, |Z|_max, Q, and Φ_L. H: population plots of f_R measured at the beginning (blue) and the end (orange) of experiments performed in the presence of ZD7288. I: time course of f_R in the absence (black; top) and presence of ZD7288 (green; bottom). For B and C and for H and I, data are means ± SE and P values (when presented) are from paired Student's t-tests. J: summary plot of %change in various measurements (from their respective baseline values) after 45 min into the recording in the presence of ZD7288. Data are medians and quartiles. *P < 0.05, Mann-Whitney test.

Fig. 4.

InsP₃-induced plasticity was abolished in presence of BAPTA. Experiments depicted were performed with 10 μM InsP₃ and 20 mM BAPTA (n = 5) in the recording pipette. A: population plots of R_in measured at the beginning (blue) and the end (orange) of experiments performed in the presence of BAPTA. B: time course of normalized R̄_in in the presence (green) and absence (black; control) of BAPTA. C: population plots of f_R measured at the beginning (blue) and the end (orange) of experiments performed in the presence of BAPTA. D: time course of normalized f_R in the absence (black; top) and presence of BAPTA (green; bottom). For A–D, data are means ± SE and P values (when presented) are from paired Student's t-tests. E: summary plot of %change in various measurements (from their respective baseline values) after 45 min into the recording in the presence of BAPTA. Data are medians and quartiles. *P < 0.05, Mann-Whitney test.

Fig. 5.

InsP₃ receptors (InsP₃R), NMDA receptors (NMDAR), and voltage-gated calcium channels (VGCCs) contribute as calcium sources for InsP₃-induced plasticity. All experiments depicted were performed with 10 μM InsP₃ in the recording pipette. A and B: time courses of normalized R̄_in (A) and f_R (B) in the presence (green) and absence of 1 mg/ml heparin (black; control) in the recording pipette. C and D: summary plots of values of R_in (C) and f_R (D) at the beginning (blue) and at the end (orange) of experiments when various channels/receptors were blocked using specific pharmacological agents. For A–D, data are means ± SE. *P < 0.05, paired Student's t-test. E and F: summary plots of InsP₃-induced changes (%) in R_in (E) and f_R (F) obtained when various channels/receptors were blocked using specific pharmacological agents (green) compared with the control (black) where no other pharmacological agent was employed. Data are medians and quartiles. *P < 0.05, Mann-Whitney test. Pharmacological agents indicated in C–F are defined as follows: InsP₃R, 1 mg/ml heparin in recording pipette (n = 6); AMPAR+GABAR, 10 μM (+)bicuculline, 10 μM picrotoxin, 10 μM 6-cyano-7-nitroquinoxaline-2,3-dione, and 2 μM CGP55485 in extracellular recording solution (n = 5); NMDAR, 50 μM 2-amino-5-phosphonovaleric acid (d,l-APV) in extracellular recording solution (n = 5); T Ca²⁺ (T-type calcium channels), 50 μM NiCl₂ in extracellular recording solution (n = 5); L Ca²⁺ (L-type calcium channel), 10 μM nimodipine in extracellular recording solution (n = 5); T+L Ca²⁺, 50 μM NiCl₂ and 10 μM nimodipine in extracellular recording solution (n = 5). See text for definitions.

Fig. 6.

InsP₃-induced plasticity was dependent on the PKA signaling pathway. All experiments depicted were performed with 10 μM InsP₃ in the recording pipette. A: voltage response of a representative neuron to the pulse-current injections at the beginning (blue) and after 45 min (orange) of an experiment where 20 μM PKA inhibitor (PKAi) peptide was included in the recording pipette. R_in values obtained from these traces are also shown. B: population plots of R_in measured at the beginning (blue) and the end (orange) of experiments performed in the presence of PKAi peptide (n = 6). C: voltage response of the neuron to the chirp stimulus at the beginning (blue; 0–5 min average) and the end (orange; 40–45 min average) of an experiment where 20 μM PKAi peptide was included in the recording pipette. f_R values obtained from these traces are also shown. D: population plots of f_R measured at the beginning (blue) and the end (orange) of experiments performed in the presence of PKAi peptide. E–H: same as A–D, but the experiments were performed in the presence of 500 nM KT5720 in the recording solution, instead of PKAi. For A–H, data are means ± SE and P values correspond to paired Student's t-test. I and J: time courses of normalized R̄_in (I) and f_R (J) with 10 μM InsP₃ and 20 μM PKAi peptide (n = 6) in the recording pipette (green), 10 μM InsP₃ in the recording pipette and 500 nM KT5720 (n = 6) in the bath (purple), or only 10 μM InsP₃ (black; control) in the recording pipette. K: summary plots of %change in R_in and f_R obtained from experiments performed with the 2 different PKA inhibitors and their comparison with the control experiments. Color codes are the same as in I and J. Data are medians and quartiles. *P < 0.05, Mann-Whitney test.