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

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 GABAA receptor (GABAAR)-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 GABAAR synaptic strength, glutamatergic plasticity and CRF neuromodulation possibly through AMPAR and potassium channel trafficking and eCB signaling within the LHb.

(which was not certified by peer review) is the author/funder.This article is a US Government work.It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted December 7, 2023.; https://doi.org/10.1101/2023.12.06.570160 doi: bioRxiv preprint hippocampal CA1 neurons 17 .Similarly, transient recruitment of GluA2-lacking calcium permeable AMPARs (CP-AMPARs) through phosphorylation coordinated by AKAP150/PKA/CaN is required along with NMDARs not only for the induction of inputspecific LTP and LTD but also for homeostatic plasticity (synaptic scaling) in the hippocampus 9,18,19 .
In spite of the increasing in-depth mechanistic insights into the role of AKAP150 complex in hippocampal-related learning and memory processes, less is known about the normal and pathological roles of AKAP150 complex-dependent signaling in neural processes within reward-related brain circuits that could contribute to reward-related behaviors as well as in the development of neurological and neuropsychiatric illnesses.This is an important area of psychiatric research as human studies of polymorphisms of AKAP5 also indicate that individuals carrying AKAP5 polymorphisms show altered emotional processing and behavioral responses including aggression, expression of anger and impulsivity associated with alterations in the function in limbic regions [20][21][22] .
Moreover, copy number variations in AKAP5 have been found in DNA samples of schizophrenia patients but not in control subjects 23 , suggesting the possible involvement of AKAP5 in the pathogenesis of schizophrenia, a neurodevelopmental disorder also linked to reward circuit dysfunction and high rates of addiction [24][25][26] .
105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder.This article is a US Government work.It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted December 7, 2023.; https://doi.org/10.1101/2023.12.06.570160 doi: bioRxiv preprint Here, we attempted to address the potential impact of AKAP150-anchored PKA on the lateral habenula (LHb); an anti-reward brain region hub that regulates midbrain monoaminergic centers and is involved in reward/motivation, mood regulation and decision-making.Accumulating evidence indicate that LHb hyperactivity plays an instrumental role in pathophysiology of depression and possibly other mood disorders and substance use disorders, thus LHb is gaining interest as a potential target for neuromodulation and antidepressants [35][36][37][38] .LHb neurons are excited by aversive and unpleasant events or the absence of expected reward, and inhibited by unexpected reward, encoding behavioral avoidance and reward prediction errors through suppression of VTA dopamine (DA) and dorsal raphe nucleus (DRN) serotonin systems 35, 39 .The majority of LHb neurons are believed to be glutamatergic and long-range projecting, although local glutamatergic and GABAergic connections within the LHb are reported [40][41][42][43] .LHb neurons receive glutamatergic, GABAergic and co-releasing glutamate/GABA inputs from the basal ganglia and diverse limbic areas including medial prefrontal cortex (mPFC), entopeduncular nucleus (EP), lateral preoptic area (LPO), lateral hypothalamus (LH), ventral pallidum (VP), medial and lateral septum, central amygdala (CeA), bed nucleus of stria terminals (BNST) as well as receiving reciprocal inputs from the VTA and periaqueductal gray (PAG).LHb projects to the substantia nigra, VTA, rostromedial tegmental area (RMTg), DRN, locus coeruleus (LC) and PAG 35,44 .The majority of the glutamatergic output of LHb exerts a potent feedforward inhibitory influence on monoaminergic systems including VTA DA neuronal activity by excitation of GABAergic interneurons and of GABAergic neurons of the RMTg [45][46][47][48] .
105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder.This article is a US Government work.It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted December 7, 2023.; https://doi.org/10.1101/2023.12.06.570160 doi: bioRxiv preprint LHb hyperactivity is found to be a common finding associated with anhedonia, lack of motivation and social withdrawal which reflect some of the core features of reward deficits seen in clinical depression 36,[49][50][51] .In general, LHb dysfunction can mediate negative affective states, social deficits, risky decision-making and impulsivity (as shown in patients with depression, schizophrenia, Parkinson's disease and attention-deficit hyperactivity disorder, ADHD) 36,[49][50][51][52][53][54][55][56][57] .Given the known postsynaptic PKA-mediated control of LHb synaptic function and intrinsic excitability by neuromodulatory actions of CRF-CRFR1 signaling 58 , here we investigated the potential impact of AKAP150-anchored PKA on LHb synaptic and neuronal function and CRF neuromodulation within the LHb using whole-cell patch clamp recordings and AKAP150PKA knockin mouse model (hereafter referred to as PKA mice) 14 .The PKA mice have an internal deletion of ten amino acids within the PKA-RII subunit binding domain near the AKAP C terminus.For AKAP150 complex-related studies, they are advantageous compared to AKAP150 knockout mice as the mutation only affects AKAP150-anchoring of PKA without disrupting other AKAP150 interactions 14 .We found that the genetic disruption of PKA anchoring to AKAP150 significantly altered both AMPAR-and GABAAR-mediated synaptic transmission and impaired the induction of an eCB-mediated LTD in LHb neurons.Moreover, we observed that PKA mutation enhanced LHb intrinsic excitability which then blunted the excitatory effects of CRF on LHb neuronal activity.Given the significant and multifaceted impact of AKAP150 anchoring of PKA in regulation of glutamatergic transmission and plasticity and neuronal excitability of LHb as well as alteration of CRF regulation of LHb excitability, our data suggest novel roles for AKAP150 complex in normal LHb function and potential 105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder.This article is a US Government work.It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted December 7, 2023.; https://doi.org/10.1101/2023.12.06.570160 doi: bioRxiv preprint contributions of defective AKAP150-mediated PKA anchoring to aberrant LHb activity, dysregulation of CRF neuromodulation within LHb circuits, and hence mood dysregulation.

Animals
All experiments were carried out using 5-7wk old male WT (C57/Bl6) and ΔPKA mice in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and were approved by the Uniformed Services University and the University of Colorado (Denver) Institutional Animal Care and Use Committees.Mice were group housed in standard cages under a 12hr/12hr light-dark cycle with standard laboratory lighting conditions (lights on, 0600-1800), with ad libitum access to food and water.All procedures were conducted beginning 2-4hr after the start of the light-cycle.
All efforts were made to minimize animal suffering and reduce the number of animals used throughout this study.
Sagittal slices containing LHb were cut at 220µm using a vibratome (Leica; Wetzler, Germany) and incubated in above prepared ACSF at 34°C for at least 1 hour prior to 105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder.This article is a US Government work.It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted December 7, 2023.; https://doi.org/10.1101/2023.12.06.570160 doi: bioRxiv preprint electrophysiological experiments.Slices were then transferred to a recording chamber and perfused with ascorbic-acid free ACSF at 28 °C.

Electrophysiology
All whole-cell recordings were performed on LHb-containing slices using patch pipettes (3-6 MOhms) and a patch amplifier (MultiClamp 700B) under infrared-differential interference contrast microscopy.Data acquisition and analysis were carried out using DigiData 1440A, pCLAMP 10 (Molecular Devices), Clampfit, Origin 2016 (OriginLab), Mini Analysis 6.0.3 (Synaptosoft Inc.) and GraphPad Prism 10.Signals were filtered at 3 kHz and digitized at 10 kHz.In all of our recordings, the cell input resistance and series resistance were monitored through the experiment and if these values changed by more than 10%, data were not included.
(which was not certified by peer review) is the author/funder.This article is a US Government work.It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted December 7, 2023.; https://doi.org/10.1101/2023.12.06.570160 doi: bioRxiv preprint neurons were voltage-clamped at −70 mV and recorded over 10 sweeps, each lasting 50 seconds.
In some experiments, electrically-evoked AMPAR-mediated EPSCs were isolated and recorded using ACSF containing picrotoxin (100 μM).The patch pipettes were filled with cesium-gluconate based solution as described above for mEPSC recordings.Cells were voltage-clamped at -70 mV, except during LTD protocol.Paired AMPAR-mediated EPSCs were stimulated at 0.1 Hz (100 ms) using a bipolar stainless steel stimulating electrode placed ~200-400 µm from the recording site in stria medularis in LHb slices.The stimulation intensity was adjusted so that the amplitude of synaptic responses ranged about ~50% of maximum response.LTD was induced using low frequency stimulation, LFS, 1 Hz for 15 min while LHb neurons were voltageclamped at -40 mV.
To assess LHb intrinsic excitability and membrane properties, LHb slices were perfused with ascorbic-free ACSF and patched with potassium gluconate-based internal solution (130 mM K-gluconate, 15 mM KCl, 4 mM ATP-Na + , 0.3 mM GTP-Na + , 1 mM EGTA, and 5 mM HEPES, pH adjusted to 7.28 with KOH, osmolarity adjusted to 275 to 280 mOsm).LHb intrinsic excitability experiments were performed with fast-synaptic transmission blockade by adding DNQX (10µM), picrotoxin (100 µM), and D-APV (50 µM) to the ACSF.LHb neurons were given increasingly depolarizing current steps at +10pA intervals ranging from +10pA to +100pA, allowing us to measure AP generation in response to membrane depolarization (5 sec duration).Current injections were separated by a 20s interstimulus interval and neurons were kept at ~-65 to -70 mV with manual direct current injection between pulses.Resting membrane potential (RMP) was assessed immediately after achieving whole-cell patch configuration in current clamp mode.Input resistance (Rin) was measured during a -50pA step (5s duration) and calculated by dividing the steady-state voltage response by the current-pulse amplitude (-50pA) and presented as MOhms (MΩ).The number of APs induced by depolarization at each intensity was counted and averaged for each experimental group.As previously described 58 AP number, AP threshold, fast and medium after-hyperpolarization amplitudes (fAHP and mAHP), AP halfwidth, AP amplitude were assessed using Clampfit and measured at the current step that was sufficient to generate the first AP/s.

Drugs
For all drug experiments a within-subjects experimental design was employed.Stock solutions for CRF were prepared in distilled water and diluted (1:1000) to final concentration in ACSF of 250nM.Baseline recordings were first performed (depolarization-induced AP/mIPSC/mEPSC) for each neuron and then CRF (250 nM Tocris-1151) was added to the slice by the perfusate and response tested following 30-45min of CRF application.
The brains were dissected and placed in 4% PFA for 24 h and then cryoprotected by submersion in 20% sucrose for 3 d, frozen on dry ice, and stored at-70°C until sectioned.Sections of the LHb were cut using a cryostat (Leica CM1900) and mounted on slides.Serial coronal sections (20 m) of the midbrain containing the LHb (from -2.64 to -4.36 mm caudal to bregma (Paxinos and Watson, 2007) were fixed in 4% PFA for 5 min, washed in 1x PBS, and then blocked in 10% normal goal serum (NGS) containing 0.3% Triton X-100 in 1x PBS for 1 h.Sections were incubated in goat anti-AKAP150 antibody (1:500, Santa Cruz Sc-6445) in carrier solution (5 % NGS in 0.1% Triton X-100 in 1x PBS) overnight at room temperature.After rinsing in 1x PBS, sections were incubated for 2 h in Alexa Fluor® 488 labeled chicken anti-goat IgG (diluted 1:200).
Finally, sections were rinsed in 1x PBS, dried, and cover slipped with Prolong mounting medium containing DAPI to permit visualization of nuclei.Background staining was assessed by omission of primary antibody in the immunolabeling procedure (negative control).Brain tissue sections of mouse with previously established presence of AKAP150 immunoreactive neurons (hippocampus, VTA) were also processed as positive control tissues.Images were captured using a Leica DMRXA Fluorescence microscope.

Data analysis
Values are presented as means ± SEM.Statistical significance was determined using unpaired or paired two tailed Student's t-test, two-way ANOVA or repeated-measures ANOVA (RM-ANOVA) with Bonferroni post hoc analysis.The threshold for significance was set at *P < 0.05 for all analyses.The peak values of the evoked paired EPSCs were measured relative to the same baseline.A stable baseline value was considered in each sweep of paired pulses starting at 20-50ms right before the emergence of the EPSC current using p-Clamp 10 software.The paired pulse ratio (PPR) was calculated as the amplitude of the second EPSP divided by the amplitude of the first EPSC.The inverse square of the coefficient of variation (CV=SD/mean) was also used as the second measure for identifying the presynaptic expression of plasticity.For calculating significance of EPSC amplitude changes after LTD induction protocol, amplitudes of EPSCs to the first pulse were used.Mini Analysis software was used to detect and measure mIPSCs and mEPSCs using preset detection parameters of mIPSCs and mEPSCs with an amplitude cutoff of 5 pA.The Kolmogorov-Smirnov test (KS test) was performed for the statistical analyses of cumulative probability plots of mEPSCs and mIPSCs.All statistical analyses were performed using GraphPad Prism 10.

AKAP150 expression in the LHb.
Figure 1 depicts a representative 40x image of LHb of a young adult male mouse taken at AP location (-1.34 relative to bregma).We observed wide expression of AKAP150 in the LHb at three AP locations (-1.06, -1.34 and -1.46) in 4 wild type (WT) mice.

Effects of AKAP-PKA mutation on synaptic transmission and glutamatergic LTD in LHb neurons
To examine the effects of genetic disruption of PKA anchoring to AKAP150 on AMPAR and GABAAR-mediated synaptic transmission, we recorded either mEPSCs (Figure 2) or mIPSCs (Figure 3) from LHb neurons from WT and and PKA mice with deficiency in PKA-anchoring to AKAP150 14 .PKA mutation significantly decreased the average amplitude (inset in Figure 2B), frequency (inset in Figure 2C) and charge transfer (inset in Figure 2D) of mEPSCs and correspondingly shifted the cumulative probability curves of mEPSC amplitude (to the left indicative of smaller amplitude, Figure 2B), inter-event 105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder.This article is a US Government work.It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted December 7, 2023.; https://doi.org/10.1101/2023.12.06.570160 doi: bioRxiv preprint interval (IEI, to the right indicative of lower frequency, Figure 2C) and charge transfer (to the left indicative of lower charge transfer, Figure 2D) without altering mEPSC tau decay (Figure 2, unpaired Student's t-tests, Kolmogorov-Smirnov tests, p<0.05, p<0.01, p<0.001, p<0.0001), suggesting both pre-and postsynaptic suppression of glutamatergic transmission in LHb neurons.Only the cumulative probability curve of mIPSC amplitude (Figure 3B) was significantly shifted to the right by this genetic AKAP-PKA disruption, which may indicate an increase in postsynaptic GABAAR function at a subset of GABAergic synapses onto LHb neurons (Figure 3, Kolmogorov-Smirnov tests, p<0.0001).All other mIPSC properties were not significantly different between PKA and WT.
Previously, it has been reported that LFS can induce a retrograde presynaptic eCB-mediated LTD of the AMPAR-mediated electrically-evoked EPSCs in LHb neurons by postsynaptic activation of group I metabotropic glutamate receptors (mGluR-LTD) 60 or through calcium permeable AMPARs (CP-AMPARs) that further activate NMDARs 61,62 .Here, we also used an identical LFS protocol to induce a presynaptic LTD and assessed PPRs and 1/CV 2 values as the two main indicators of presynaptic expression of synaptic plasticity.As shown in Figure 4, the LTD protocol strongly induced eCB-LTD onto LHb neurons in WT mice (Figure 4A and 4C) which was associated with significant increases in PPRs (Figure 4D) and corresponding decreases in 1/CV 2 (Figure 4E) suggesting the expression of eCB-mediated LTD.On the other hand, LHb neurons from PKA mice (Figure 4B

Effects of AKAP-PKA mutation on LHb intrinsic excitability
Given that PKA regulation of various ion channels in the neuronal membrane requires anchoring of PKA by AKAP150 63 , it was possible that the genetic disruption of PKA-AKAP association in PKA mice could also impact intrinsic plasticity through changes in trafficking and/or function of a number of voltage-gated channels.Consistently, we observed that LHb neurons of PKA mice exhibited significantly higher intrinsic excitability in the absence of synaptic transmission compared to those from WT mice (Figure 5A).Furthermore, PKA mutation-induced intrinsic plasticity was associated with lower amplitude of mAHPs (Figure 5D), and shorter AP half widths (Figure 5G) suggesting that the genetic disruption of AKAP150-PKA anchoring modified intrinsic active and passive neuronal membrane properties, which could also influence synaptic conductance (Figure 5 A-G, intrinsic excitability: F (1, 209) = 21.22,2-way ANOVA; mAHPs and AP half width: unpaired Student's t test, p<0.05, p<0.01, p<0.0001).

Effects of AKAP-PKA mutation on CRF neuromodulation within the LHb
Previously, we demonstrated that the LHb is a highly CRF-responsive brain region with PKA-dependent regulation of LHb synaptic inhibition and intrinsic excitability.We showed that CRF acting through postsynaptic CRFR1 and PKA signaling increases LHb excitability through PKA-dependent suppression of small conductance potassium SK channel activity, as well as presynaptic GABA release via retrograde eCB-CB1 receptor (CB1R) signaling in rat LHb neurons without any significant alterations in glutamatergic transmission 58 .In contrast to our earlier findings in rat LHb where exogenous CRF did not alter mEPSCs, CRF bath application significantly decreased the average frequency of mEPSCs (inset in Figure 6C) and correspondingly shifted the cumulative probability curves of mEPSC IEI to the right (Figure 6C) (Figure 6, paired Student's t-tests, Kolmogorov-Smirnov tests, p<0.05, p<0.0001), suggesting of CRF-induced suppression of presynaptic glutamate release in mouse LHb neurons.This diminishing effect of CRF on presynaptic glutamate release was absent in LHb neurons of PKA mice and we even detected a slight but significant leftward shift in the cumulative probability curves of mEPSC IEI (Figure 7C) that may suggest an unmasked potentiating effects of CRF on presynaptic glutamate release upon defective AKAP150-PKA associations (Figure 7, Kolmogorov-Smirnov tests, p<0.05).
In contrast, we observed similar effects of the PKA mutation on GABAergic transmission in mouse LHb and rat LHb; exogenous CRF significantly diminished the average frequency of mIPSCs (inset in Figure 8C) and resulted in a significant shift in the cumulative probability curves of mIPSC IEI to the right (Figure 8C) (Figure 8, paired Student's t-tests, Kolmogorov-Smirnov tests, p<0.05, p<0.0001), indicating CRFinduced suppression of presynaptic GABA release onto mouse LHb neurons.This diminishing effect of CRF on presynaptic GABAergic transmission remained intact in LHb neurons of PKA mice as evident with a smaller but still significant rightward shift in the cumulative probability curves of mIPSC IEI (Figure 9C).However, CRF additionally decreased the average amplitude (inset in Figure 9B) and charger transfer (inset in Figure 9D) of mIPSCs as well as shifted their corresponding cumulative probability curves of mIPSC amplitude (Figure 9B) and charge transfer (Figure 9D) to the left.These results may indicate that CRF alters postsynaptic function and/or trafficking of GABAARs upon disruption of AKAP150-PKA association, potentially favoring AKAP150-dependent regulation of anchored PKC and/or CaN phosphatase activity, both of which can negatively regulate postsynaptic GABAARs 29,64 , downstream of CRF-CRFR-PKC signaling 65,66 (Figure 9, paired Student's t-tests, Kolmogorov-Smirnov tests, p<0.05, p<0.0001).
Similar to our earlier findings in rat LHb 58 , CRF bath application exerted similar effects on intrinsic excitability and intrinsic membrane properties of male mouse LHb.
We found that CRF significantly increased LHb intrinsic excitability (with blocked fast AMPAR, NMDAR and GABAAR-mediated transmission) (Figure 10A-B) coincident with higher input resistance (Figure 10C), reduced levels of mAHPs (Figure 10E), lower AP threshold (Figure 10F) and smaller AP amplitudes (Figure 10G) in LHb neurons.The only exception was that CRF-induced increases in fAHPs were not observed in mouse LHb (Figure10D) (Figure 10 A

Discussion
Most of our understanding of the role of AKAP150 in the brain relates to hippocampal studies but emerging evidence also suggests novel roles for AKAP150 in reward-related brain regions critical for control of mood, motivation, reward, and stress responses 8, 15, 27-31, 33, 34 .Here, we uncovered a multifaceted essential regulatory role of AKAP150 in synaptic function, neuronal activity and CRF neuromodulatory actions within the LHb using the PKA knockin mouse model with deficiency of AKAP150 anchoring of PKA.
We found that in LHb neurons AKAP150-anchored PKA is required for postsynaptic regulation of AMPAR trafficking and/or function at glutamatergic synapses and for the expression of glutamatergic eCB-LTD.In contrast, AKAP150-PKA signaling may provide an inhibitory feedback mechanism for postsynaptic trafficking and/or function of GABAARs or regulate gene expression programs that indirectly control inhibitory synaptic strength 67 .Moreover, we found that defects in AKAP150-PKA mediated expression, trafficking, and/or gating of potassium channels that regulate LHb excitability could blunt CRF neuromodulatory effects within the LHb.
Postsynaptic AMPAR and GABAAR trafficking and function is necessary for maintaining basal synaptic transmission as well as induction and expression of synaptic plasticity which can be altered through phosphorylation-dephosphorylation processes that require AKAP150 63 .There are four subunits of AMPARs (GluR1-GluR4) of which LHb neurons express high levels of GluA1-containing rectifying AMPARs that lack GluA2 (also called calcium permeable, CP-AMPARs with fast kinetics, high conductance and strong inward rectification) but also express low levels of both GluA2containing AMPARs (with slower kinetics, low conductance and impermeability to calcium) and NMDARs at their glutamatergic synapses 68,69 .AKAP150-anchored PKA is shown to phosphorylate Ser-845 on the GluA1 subunit of AMPARs to increase membrane trafficking of AMPARs at glutamatergic synapses, while AKAP150-anchored protein kinase C (PKC), through phosphorylation of Ser-831 on GluA1 results in emergence of CP AMPARs at synapses 70  synapse that is required for hippocampal LTD induction 9 .Interestingly, LFS can also induce eCB-mediated LTD in the LHb through increased activity of CP-AMPAR (as a major source of calcium) that further engages NMDARs to trigger eCB-LTD 61,62 .Given that the majority of AMPARs in the LHb are CP-AMPARs, a reduced level of CP-AMPARs in LHb neurons of PKA mice could result in lower levels of depolarization and postsynaptic calcium needed for eCB production, thereby deficits in induction and expression of eCB-LTD.Moreover, since basal PKA phosphorylation of L-type calcium channels (LTCC) is necessary for depolarization-induced activation of LTCCs, the PKA mutation could further diminish Ca 2+ influx through LTCC as an unopposed CaN activity can dephosphorylate LTCCs 14 .This in addition to the presence of fewer CP-AMPARs in LHb neurons of PKA mice might result in further reduction in calcium influx, defective eCB production, and hence impaired eCB-LTD in LHb neurons.
GABAARs at GABAergic synapses onto LHb neurons are mainly composed of a combination of the 1-3, 1 and 1-2 subunits 75 .There is less known about PKAdependent regulation of GABAARs in the LHb.Our previous study in VTA DA neurons suggests that activation of dopamine D2 receptors results in PKA inhibition that promotes AKAP150-CaN-mediated internalization of GABAAR receptors and the expression of LTD at GABAergic synapses onto VTA DA neurons 29 .The expression of an inhibitory mGluR-dependent postsynaptic LTD at GABAergic synapses onto LHb neurons requires a PKC-dependent phosphorylation of the 2 receptor subunits of GABAARs, reducing GABAAR single-channel conductance 60 .This also excludes the possibility that a biased AKAP150-PKC signaling in the absence of AKAP-PKA association in PKA mice could promote the basal increase in the conductance of GABAARs in LHb neurons.Therefore, it is still an open question which AKAP150 associations with other binding partners could promote forward trafficking of GABAARs in LHb neurons.
In addition to alterations of synaptic transmission and LTD by PKA mutation, we observed a significant increase in LHb intrinsic excitability associated with higher input resistance and lower amplitude of mAHPs, mimicking the effects of exogenous CRF (as we observed in both mouse and rat LHb) and after a severe early like stress (i.e., maternal deprivation) 58 .However, the diminishing effects of exogenous CRF and maternal deprivation on mAHPs and the resultant hyperexcitability were due to the PKA-dependent decrease in the function and/or abundance of SK channels 58 , a mechanism that is less likely to underlie PKA mutation-induced LHb intrinsic plasticity.
Afterhyperopolarizations including fAHPs and mAHPs are mediated by diverse types of potassium channels that repolarize the membrane to regulate and limit excessive neuronal excitability.In addition to SK channels, voltage gated K + channel 7 (Kv7, also known as M currents) contribute to mAHP in neurons 76 .Therefore, it is possible that genetic disruption of AKAP150 anchoring of PKA in PKA favors AKAP150-anchored PKC and the resultant inhibition of M-type mAHPs 12 to increase LHb intrinsic excitability in PKA mice, which could saturate and occlude the excitatory actions of CRF on LHb intrinsic excitability.Consistent with this interpretation, it has been shown that activation of LHb M channels reduces LHb neuronal activity and blocks the anxiety-like phenotype in alcohol-withdrawn rats 77 .Given that that the majority of synaptic inputs to the LHb corelease glutamate and GABA 41 , our observation of CRF-induced suppression of both presynaptic glutamate and GABA release in mouse LHb is not surprising as CB1R are expressed on presynaptic terminals in the LHb where CB1R activation by eCBs can 105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder.This article is a US Government work.It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted December 7, 2023.; https://doi.org/10.1101/2023.12.06.570160 doi: bioRxiv preprint reduce the probability of presynaptic glutamate and GABA release onto LHb neurons at distinct synaptic inputs to the LHb (e.g., LPO) although the effect on presynaptic GABA release is assumed to be predominantly larger 78 .PKA mutation to some extent reduced the suppressing effects of CRF on presynaptic GABA release but also unmasked a small potentiating effect of CRF on presynaptic glutamate release.This could be due to decreased depolarization and/ or calcium influx from the fewer CP-AMPARs available at the synapse as well as the less effective influx of calcium from hypofunctional LTCC in PKA mice, which could in turn lead to dysregulation of eCB production that not only prevented the expression of eCB-LTD but also blunted the inhibitory effects of CRF on synaptic transmission by shifting excitation/inhibition balance to more excitation.Therefore, we assume that disruption of AKAP150 Overall, our study highlights significant and multifaceted impacts of AKAP150 anchoring of PKA in regulation of glutamatergic transmission and plasticity, and neuronal excitability of LHb neurons.Moreover, defects in AKAP150-mediated PKA anchoring under pathological processes could favor other, yet to be discovered, AKAP150 interactions in LHb neurons that promote LHb hyperactivity and dysregulate 105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder.This article is a US Government work.It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted December 7, 2023.; https://doi.org/10.1101/2023.12.06.570160 doi: bioRxiv preprint CRF neuromodulation within the LHb, reminiscent of the effects of a severe early life stress 58 and alcohol withdrawal 79,80 .Future studies are needed to increase our understanding of cell-type and input-specific neuroplasticity and neuromodulation of LHb activity through alterations in LHb AKAP150 complex interactions in neurological and neuropsychiatric illnesses.Figure 1 105 and is also made available for use under a CC0 license.
. In the LHb, it has been shown that cocaine can induce synaptic potentiation and hyperexcitability in LHb neurons projecting to RMTg (LHb →RMTg neurons) through Ser-845 phosphorylation of GluA1 that increases trafficking of GluA1 AMPARs in LHb →RMTg neurons71 .Additionally, phosphorylation of Ser-831 on GluA1 subunit of AMPARs by β-calcium/calmodulin-dependent kinase type II (CaMKIIβ) in the LHb is shown to promote GluA1 AMPAR insertion into synapses and glutamatergic potentiation, resulting in LHb hyperactivity and behavioral depression72 .Given that the knockin mutation in PKA mice leads to reductions in postsynaptic PKA localization in dendritic spines14 , our observation of lower levels of mEPSC amplitude and charge transfer in PKA mice are most likely due to decreased PKA-dependent Ser-845 phosphorylation of GluA1 rectifying AMPARs by the genetic disruption of AKAP150 anchoring of PKA to GluA1.Note that the decrease in frequency of mEPSC in PKA mice is likely postsynaptic, related to an increase in the number of silent synapses following the loss of AMPARs at the synapse73 rather than a change in presynaptic glutamate release.Whether, upregulation of CaMKII-regulated AKAP79/150 depalmitoylation that is important in AKAP150 removal from dendritic spine and structural LTD74 is favored in PKA mice is an open question.Interestingly, disruption of PKA anchoring in PKA mice is shown to impair an NMDAR-dependent LTD induced by prolonged low-frequency stimulation (LFS; 1 Hz, 15 min similar to the LTD protocol in LHb) in CA1 hippocampal neurons of 2-week old mice due to decreased S845 phosphorylation of CP-AMPARs in PKA mice that prevents the AKAP150-PKA-dependent transient recruitment of CP-AMPARs to the anchoring of PKA seems to promote LHb hyperexcitability through synaptic and intrinsic mechanisms that may relate to the lack of AKAP150-dependent PKA-mediated signaling as well as favoring unopposed non-PKA-mediated AKAP150 interactions.These concepts are briefly summarized in Fig 11, which depicts in schematic form representative GABAergic and glutamatergic terminals innervating a spine and dendritic shaft of an LHb neuron.Sites in this synaptic complex where AKAP-mediated signaling plays a potential role in synaptic function and plasticity are indicated.

Figure 1 :
Figure 1: AKAP 150 is expressed in the LHb.Example of a brain section stained with

Figure 6 .
Figure 6.CRF decreased presynaptic glutamate release in the LHb of WT mice.(A)

Figure 7 .
Figure 7. CRF slightly potentiated presynaptic glutamate release in the LHb of PKA

Figure 8 .Figure 9 .
Figure 8. CRF significantly suppressed presynaptic GABA release in the LHb of WT

Figure 10 :
Figure 10: Genetic disruption of AKAP150-anchored PKA occluded the effects of CRF

Figure 9 105
Figure 9 105 and is also made available for use under a CC0 license.(whichwas not certified by peer review) is the author/funder.This article is a US Government work.It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license.(whichwas not certified by peer review) is the author/funder.This article is a US Government work.It is not subject to copyright under 17 USC ) 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 PKA mice (n=7 cells/6 mice).