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

Abstract

Numerous studies of hippocampal synaptic function in learning and memory have established the functional significance of the scaffolding A-kinase anchoring protein 150 (AKAP150) in kinase and phosphatase regulation of synaptic receptor and ion channel trafficking/function and hence synaptic transmission/plasticity, and neuronal excitability. Emerging evidence also suggests that AKAP150 signaling may play a critical role in brain's processing of rewarding/aversive experiences. Here we focused on an unexplored role of AKAP150 in the lateral habenula (LHb), a diencephalic brain region that integrates and relays negative reward signals from forebrain striatal and limbic structures to midbrain monoaminergic centers. LHb aberrant activity (specifically hyperactivity) is also linked to depression. Using whole cell patch clamp recordings in LHb of male wildtype (WT) and ΔPKA knockin mice (with deficiency in AKAP-anchoring of PKA), we found that the genetic disruption of PKA anchoring to AKAP150 significantly reduced AMPA receptor (AMPAR)-mediated glutamatergic transmission and prevented the induction of presynaptic endocannabinoid (eCB)-mediated long-term depression (LTD) in LHb neurons. Moreover, ΔPKA mutation potentiated GABA_A receptor (GABA_AR)-mediated inhibitory transmission postsynaptically while increasing LHb intrinsic neuronal excitability through suppression of medium afterhyperpolarizations (mAHPs). Given that LHb is a highly stress-responsive brain region, we further tested the effects of corticotropin releasing factor (CRF) stress neuromodulator on synaptic transmission and intrinsic excitability of LHb neurons in WT and ΔPKA mice. As in our earlier study in rat LHb, CRF significantly suppressed GABAergic transmission onto LHb neurons and increased intrinsic excitability by diminishing small-conductance potassium (SK) channel-mediated mAHPs. ΔPKA mutation-induced suppression of mAHPs also blunted the synaptic and neuroexcitatory actions of CRF in mouse LHb. Altogether, our data suggest that AKAP150 complex signaling plays a critical role in regulation of AMPAR and GABA_AR synaptic strength, glutamatergic plasticity and CRF neuromodulation possibly through AMPAR and potassium channel trafficking and eCB signaling within the LHb.

Figure 1:. AKAP 150 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, 20μm.

Figure 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/1s). 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) and ΔPKA (open symbols) mice (n=15–26 cells from 8–9 mice/group).

Figure 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/1s). 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) and ΔPKA (open symbols) mice (n=15–26 cells from 8–9 mice/group).

Figure 4:

Genetic disruption of AKAP150-anchored PKA impaired eCB-LTD induction in LHb neurons. (A) and (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 −40mV. 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 and F) and 1/CV² (E and G) in LHb neurons recorded from WT (filled symbols) and ΔPKA (open symbols) neurons (n=5–6 cells/5–6 mice/group).

Figure 5:

Genetic disruption of AKAP150-anchored PKA increased LHb intrinsic excitability and occluded the effects of CRF on LHb 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) and ΔPKA (black open symbols) (n=10–12 cells/6–7 mice/group).

Figure 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, 250nM) (calibration bars: 20 pA/1s). 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).

Figure 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, 250nM) (calibration bars: 20 pA/1s). 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).

Figure 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, 250nM) (calibration bars: 20 pA/1s). 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).

Figure 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, 250nM) (calibration bars: 20 pA/1s). 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).

Figure 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 40pA current step) and corresponding measurements of Rin, fAHP, mAHP, AP threshold, AP amplitude and AP half width before (baseline, black filled symbols), after CRF (250nM, 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 40pA current step) and corresponding measurements of Rin, fAHP, mAHP, AP threshold, AP amplitude and AP half width before (baseline, black open symbols), after CRF (250nM, red open symbols) bath application in LHb neurons from ΔPKA mice (n=7 cells/6 mice).

Figure 11.

Schematic proposed model illustrating regulatory roles of AKAP150-anchored PKA signaling in synaptic function, LTD and intrinsic excitability of LHb neurons. This model predicts that AKAP150-PKA association is necessary for maintenance of AMPARs at glutamatergic synapses and expression of LTD by LFS. We assume that the sources of increased depolarization and calcium influx for eCB production during LTD induction may arise from CP AMPARs and/or LTCC both regulated by AKAP150-PKA complex. Therefore, disruption of AKAP150 anchoring of PKA to CP AMPARs in LHb neurons in ΔPKA mice results in lower number of CP AMPARs at glutamatergic synapses. The reduced influx of calcium from fewer available CP AMPARs at the synapse as well as hypofunctional LTCC by the genetic disruption of PKA-AKAP150 association can impair eCB production and thus the expression of eCB-LTD. Our model also predicts that defective AKAP150-PKA association may reduce trafficking and/or function of potassium channels mediating mAHPs (e.g., M currents, not shown). The model also shows the known effects of CRF-CRFR1-PKA signaling in the LHb which results in eCB production as well as promote LHb hyperexcitability through modulation of trafficking or conductance of SK potassium channels. Thus, we assume that under pathological conditions AKAP150-PKA dysregulation of CP AMPARs, LTCC, M currents and eCB signaling could promote LHb hyperexcitability and blunt CRF neuromodulatory actions. While our model may indicate the existence of inhibitory effects of AKAP150-PKA on GABA_AR feedforward trafficking, it is still unclear which AKAP150 partners may contribute to regulation of GABA_ARs in LHb neurons. The ? indicates such unresolved questions.