AKAP150-anchored PKA regulates synaptic transmission and plasticity, neuronal excitability and CRF neuromodulation in the mouse lateral habenula

Abstract

The scaffolding A-kinase anchoring protein 150 (AKAP150) is critically involved in kinase and phosphatase regulation of synaptic transmission/plasticity, and neuronal excitability. Emerging evidence also suggests that AKAP150 signaling may play a key role in brain's processing of rewarding/aversive experiences, however its role in the lateral habenula (LHb, as an important brain reward circuitry) is completely unknown. Using whole cell patch clamp recordings in LHb of male wildtype and ΔPKA knockin mice (with deficiency in AKAP-anchoring of PKA), here we show that the genetic disruption of PKA anchoring to AKAP150 significantly reduces AMPA receptor-mediated glutamatergic transmission and prevents the induction of presynaptic endocannabinoid-mediated long-term depression in LHb neurons. Moreover, ΔPKA mutation potentiates GABA_A receptor-mediated inhibitory transmission while increasing LHb intrinsic excitability through suppression of medium afterhyperpolarizations. ΔPKA mutation-induced suppression of medium afterhyperpolarizations also blunts the synaptic and neuroexcitatory actions of the stress neuromodulator, corticotropin releasing factor (CRF), in mouse LHb. Altogether, our data suggest that AKAP150 complex signaling plays a critical role in regulation of AMPA and GABA_A receptor synaptic strength, glutamatergic plasticity and CRF neuromodulation possibly through AMPA receptor and potassium channel trafficking and endocannabinoid signaling within the LHb.

Fig. 1. AKAP150 is expressed in the LHb.

Example of a brain section stained with antibody to AKAP150 (green), showing the expression of AKAP150 in the mouse LHb. Scale bar, 20 μm.

Fig. 2. Genetic disruption of AKAP150-anchored PKA depressed glutamatergic transmission in LHb neurons.

a Sample AMPAR-mediated mEPSC traces from WT (left) and ΔPKA mice (calibration bars: 20 pA/1 s). Average bar graphs of mEPSC and cumulative probability plots of (b) amplitude, (c) frequency (inter-event interval), (d) charge transfer and (e) τ decay for all mEPSCs in WT (filled symbols, n = 26 cells from 9 mice) and ΔPKA (open symbols, n = 15 cells from 8 mice). Two-tailed unpaired Student’s t-tests and Kolmogorov-Smirnov tests, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. In this and all the following figures, the average data are presented as mean ± SEM.

Fig. 3. Genetic disruption of AKAP150-anchored PKA potentiated GABAergic transmission in LHb neurons.

a Sample GABA_AR-mediated mIPSC traces from WT (left) and ΔPKA mice (calibration bars: 20 pA/1 s). Average bar graphs of mIPSC and cumulative probability plots of (b) amplitude, (c) frequency (inter-event interval), (d) charge transfer and (e) τ decay for all mIPSCs in WT (filled symbols, n = 20 cells from 6 mice) and ΔPKA (open symbols n = 22 cells from 7 mice). Two-tailed Kolmogorov-Smirnov tests, ****p < 0.0001.

Fig. 4. Genetic disruption of AKAP150-anchored PKA impaired eCB-LTD induction in LHb neurons.

a, b Single experiments showing induction of LTD recorded in LHb neurons from WT (a) and ΔPKA (b) mice. At the arrow, LTD was induced using LFS while cells were depolarized at −40 mV. Insets: averaged EPSCs before (black, labeled as 1) and 25 min after LFS (red, labeled as 2). In this and all figures, ten consecutive traces from each condition were averaged for illustration as inset. Calibration: 100–200 pA, 10 ms. c Average LTD experiments with corresponding PPRs (d, f) and 1/CV² (e, g) in LHb neurons recorded from WT (filled symbols, n = 6 cells from 6 mice) and ΔPKA (open symbols n = 5 cells from 5 mice). Mixed-effects ANOVA and two-tailed unpaired Student’s t-test, *p < 0.05, *p < 0.01).

Fig. 5. Genetic disruption of AKAP150-anchored PKA increased LHb intrinsic excitability.

All recordings in this graph were performed with fast synaptic transmission blocked. a–g AP recordings in response to depolarizing current steps and corresponding measurements of Rin, fAHP, mAHP, AP threshold, AP amplitude and AP half-width in LHb neurons from WT (black filled symbols, n = 12 cells from 7 mice) and ΔPKA (black open symbols, n = 10 cells from 6 mice). 2-way ANOVA and two-tailed unpaired Student’s t test, *p < 0.05, *p < 0.01, ****p < 0.0001).

Fig. 6. CRF decreased presynaptic glutamate release in the LHb of WT mice.

a Sample AMPAR-mediated mEPSC traces from WT mouse before (black) and after CRF application (red, 250 nM) (calibration bars: 20 pA/1 s). Average bar graphs of mEPSC and cumulative probability plots of (b) amplitude, (c) frequency (inter-event interval), (d) charge transfer and (e) τ decay for all mEPSCs in WT mice before (black filled symbols) and after CRF (red filled symbols) (n = 6 cells from 4 mice). Two-tailed paired Student’s t-tests and Kolmogorov-Smirnov tests, *p < 0.05, ****p < 0.0001.

Fig. 7. CRF slightly potentiated presynaptic glutamate release in the LHb of ΔPKA mice.

a Sample AMPAR-mediated mEPSC traces from ΔPKA mouse before (black) and after CRF application (red, 250 nM) (calibration bars: 20 pA/1 s). Average bar graphs of mEPSC and cumulative probability plots of (b) amplitude, (c) frequency (inter-event interval), (d) charge transfer and (e) τ decay for all mEPSCs in ΔPKA mice before (black open symbols) and after CRF (red open symbols) (n = 5 cells from 5 mice). Two-tailed Kolmogorov-Smirnov tests, *p < 0.05.

Fig. 8. CRF significantly suppressed presynaptic GABA release in the LHb of WT mice.

a Sample GABA_AR-mediated mIPSC traces from WT mouse before (black) and after CRF application (red, 250 nM) (calibration bars: 20 pA/1 s). Average bar graphs of mIPSC and cumulative probability plots of (b) amplitude, (c) frequency (inter-event interval), (d) charge transfer and (e) τ decay for all mIPSCs in WT mice before (black filled symbols) and after CRF (red filled symbols) (n = 3 cells from 3 mice). Two-tailed paired Student’s t-tests and Kolmogorov-Smirnov tests, *p < 0.05, ****p < 0.0001.

Fig. 9. CRF suppressed presynaptic GABA release (to a lesser extent than that of WT) but also postsynaptically depressed GABAergic transmission in the LHb of ΔPKA mice.

a Sample GABA_AR-mediated mIPSC traces from ΔPKA mouse before (black) and after CRF application (red, 250 nM) (calibration bars: 20 pA/1 s). Average bar graphs of mIPSC and cumulative probability plots of (b) amplitude, (c) frequency (inter-event interval), (d) charge transfer and (e) τ decay for all mIPSCs in ΔPKA mice before (black open symbols) and after CRF (red open symbols) (n = 5 cells from 5 mice). Two-tailed paired Student’s t-tests and Kolmogorov-Smirnov tests, *p < 0.05, ****p < 0.0001.

Fig. 10. Genetic disruption of AKAP150-anchored PKA occluded the effects of CRF on LHb excitability.

All recordings in this graph were performed with fast synaptic transmission blocked. a–h AP recordings in response to depolarizing current steps with representative AP traces (in response to a 40 pA current step) and corresponding measurements of Rin, fAHP, mAHP, AP threshold, AP amplitude and AP half-width before (baseline, black filled symbols), after CRF (250 nM, red filled symbols) bath application in LHb neurons from WT mice (n = 8 cells/7 mice). i–p AP recordings in response to depolarizing current steps with representative AP traces (in response to a 40 pA current step) and corresponding measurements of Rin, fAHP, mAHP, AP threshold, AP amplitude and AP half-width before (baseline, black open symbols), after CRF (250 nM, red open symbols) bath application in LHb neurons from ΔPKA mice (n = 7 cells from 6 mice). 2-way repeated-measures ANOVA and two-tailed paired Student’s t-tests, *p < 0.05, **p < 0.01.