Functional and phosphoproteomic analysis of ß-adrenergic receptor signaling at excitatory synapses in the CA1 region of the ventral hippocampus.

Activation of ß-adrenergic receptors (ß-ARs) not only enhances learning and memory but also facilitates the induction of long-term potentiation (LTP), a form of synaptic plasticity involved in memory formation. To identify the mechanisms underlying ß-AR-dependent forms of LTP we examined the effects of the ß-AR agonist isoproterenol on LTP induction at excitatory synapses onto CA1 pyramidal cells in the ventral hippocampus. LTP induction at these synapses is inhibited by activation of SK-type K+ channels, suggesting that ß-AR activation might facilitate LTP induction by inhibiting SK channels. However, although the SK channel blocker apamin enhanced LTP induction, it did not fully mimic the effects of isoproterenol. We therefore searched for potential alternative mechanisms using liquid chromatography-tandem mass spectrometry to determine how ß-AR activation regulates phosphorylation of postsynaptic density (PSD) proteins. Strikingly, ß-AR activation regulated hundreds of phosphorylation sites in PSD proteins that have diverse roles in dendritic spine structure and function. Moreover, within the core scaffold machinery of the PSD, ß-AR activation increased phosphorylation at several sites previously shown to be phosphorylated after LTP induction. Together, our results suggest that ß-AR activation recruits a diverse set of signaling pathways that likely act in a concerted fashion to regulate LTP induction.

Novel Ca2+-dependent mechanisms regulate spontaneous release at excitatory synapses onto CA1 pyramidal cells.

Although long thought to simply be a source of synaptic noise, spontaneous, action potential-independent release of neurotransmitter from presynaptic terminals has multiple roles in synaptic function. We explored whether and to what extent the two predominantly proposed mechanisms for explaining spontaneous release, stochastic activation of voltage-gated Ca2+ channels (VGCCs) or activation of Ca2+-sensing receptors (CaSRs) by extracellular Ca2+, played a role in the sensitivity of spontaneous release to the level of extracellular Ca2+ concentration at excitatory synapses at CA1 pyramidal cells of the adult male mouse hippocampus. Blocking VGCCs with Cd2+ had no effect on spontaneous release, ruling out stochastic activation of VGCCs. Although divalent cation agonists of CaSRs, Co2+ and Mg2+, dramatically enhanced miniature excitatory postsynaptic current (mEPSC) frequency, potent positive and negative allosteric modulators of CaSRs had no effect. Moreover, immunoblot analysis of hippocampal lysates failed to detect CaSR expression, ruling out the CaSR. Instead, the increase in mEPSC frequency induced by Co2+ and Mg2+ was mimicked by lowering postsynaptic Ca2+ levels with BAPTA. Together, our results suggest that a reduction in intracellular Ca2+ may trigger a homeostatic-like compensatory response that upregulates spontaneous transmission at excitatory synapses onto CA1 pyramidal cells in the adult hippocampus. NEW & NOTEWORTHY We show that the predominant theories for explaining the regulation of spontaneous, action potential-independent synaptic release do not explain the sensitivity of this type of synaptic transmission to external Ca2+ concentration at excitatory synapses onto hippocampal CA1 pyramidal cells. In addition, our data indicate that intracellular Ca2+ levels in CA1 pyramidal cells regulate spontaneous release, suggesting that excitatory synapses onto CA1 pyramidal cells may express a novel, rapid form of homeostatic plasticity.

Differential Regulation of NMDA Receptor-Mediated Transmission by SK Channels Underlies Dorsal-Ventral Differences in Dynamics of Schaffer Collateral Synaptic Function.

Behavioral, physiological, and anatomical evidence indicates that the dorsal and ventral zones of the hippocampus have distinct roles in cognition. How the unique functions of these zones might depend on differences in synaptic and neuronal function arising from the strikingly different gene expression profiles exhibited by dorsal and ventral CA1 pyramidal cells is unclear. To begin to address this question, we investigated the mechanisms underlying differences in synaptic transmission and plasticity at dorsal and ventral Schaffer collateral (SC) synapses in the mouse hippocampus. We find that, although basal synaptic transmission is similar, SC synapses in the dorsal and ventral hippocampus exhibit markedly different responses to θ frequency patterns of stimulation. In contrast to dorsal hippocampus, θ frequency stimulation fails to elicit postsynaptic complex-spike bursting and does not induce LTP at ventral SC synapses. Moreover, EPSP-spike coupling, a process that strongly influences information transfer at synapses, is weaker in ventral pyramidal cells. Our results indicate that all these differences in postsynaptic function are due to an enhanced activation of SK-type K+ channels that suppresses NMDAR-dependent EPSP amplification at ventral SC synapses. Consistent with this, mRNA levels for the SK3 subunit of SK channels are significantly higher in ventral CA1 pyramidal cells. Together, our findings indicate that a dorsal-ventral difference in SK channel regulation of NMDAR activation has a profound effect on the transmission, processing, and storage of information at SC synapses and thus likely contributes to the distinct roles of the dorsal and ventral hippocampus in different behaviors.SIGNIFICANCE STATEMENT Differences in short- and long-term plasticity at Schaffer collateral (SC) synapses in the dorsal and ventral hippocampus likely contribute importantly to the distinct roles of these regions in cognition and behavior. Although dorsal and ventral CA1 pyramidal cells exhibit markedly different gene expression profiles, how these differences influence plasticity at SC synapses is unclear. Here we report that increased mRNA levels for the SK3 subunit of SK-type K+ channels in ventral pyramidal cells is associated with an enhanced activation of SK channels that strongly suppresses NMDAR activation at ventral SC synapses. This leads to striking differences in multiple aspects of synaptic transmission at dorsal and ventral SC synapses and underlies the reduced ability of ventral SC synapses to undergo LTP.

Basal levels of AMPA receptor GluA1 subunit phosphorylation at threonine 840 and serine 845 in hippocampal neurons.

Dephosphorylation of AMPA receptor (AMPAR) GluA1 subunits at two sites, serine 845 (S845) and threonine 840 (T840), is thought to be involved in NMDA receptor-dependent forms of long-term depression (LTD). Importantly, the notion that dephosphorylation of these sites contributes to LTD assumes that a significant fraction of GluA1 subunits are basally phosphorylated at these sites. To examine this question, we used immunoprecipitation/depletion assays to estimate the proportion of GluA1 subunits basally phosphorylated at S845 and T840. Although dephosphorylation of S845 is thought to have a key role in LTD, our results indicate that few GluA1 subunits in hippocampal neurons are phosphorylated at this site. In contrast, ∼50% of GluA1 subunits are basally phosphorylated at T840, suggesting that dephosphorylation of this site can contribute to the down-regulation of AMPAR-mediated synaptic transmission in LTD.

Ionotropic NMDA receptor signaling is required for the induction of long-term depression in the mouse hippocampal CA1 region.

Previous studies have provided strong support for the notion that NMDAR-mediated increases in postsynaptic Ca(2+) have a crucial role in the induction of long-term depression (LTD). This view has recently been challenged, however, by findings suggesting that LTD induction is instead attributable to an ion channel-independent, metabotropic form of NMDAR signaling. Thus, to explore the role of ionotropic versus metabotropic NMDAR signaling in LTD, we examined the effects of varying extracellular Ca(2+) levels or blocking NMDAR channel ion fluxes with MK-801 on LTD and NMDAR signaling in the mouse hippocampal CA1 region. We find that the induction of LTD in the adult hippocampus is highly sensitive to extracellular Ca(2+) levels and that MK-801 blocks NMDAR-dependent LTD in the hippocampus of both adult and immature mice. Moreover, MK-801 inhibits NMDAR-mediated activation of p38-MAPK and dephosphorylation of AMPAR GluA1 subunits at sites implicated in LTD. Thus, our results indicate that the induction of LTD in the hippocampal CA1 region is dependent on ionotropic, rather than metabotropic, NMDAR signaling.

Cyclothiazide-induced persistent increase in respiratory-related activity in vitro.

Hypoglossal (XII) motoneurons (MNs) innervate the genioglossus muscle of the tongue, which plays an important role in maintaining upper airway patency, particularly during sleep, and modulating upper airway resistance. Discovering methods for inducing long-term increases in genioglossal motoneuronal excitability to AMPA-mediated drive may help in the development of therapeutics for upper airway motor disorders such as obstructive sleep apnoea. We show that the diuretic, anti-hypertensive, AMPA receptor modulator cyclothiazide (CTZ) induces a profound and long-lasting increase in the amplitude of respiratory-related XII nerve activity in rhythmically active neonatal rat medullary slices. Treatment of the slice with CTZ (90 µM) for 1 h increased the integrated XII ( XII) nerve burst amplitude to 262 ± 23% of pre-treatment control at 1 h post-treatment;much of this increase lasted at least 12 h. The amount of CTZ-induced facilitation (CIF) was dependent upon both CTZ dose and exposure time and was accompanied by a long-lasting increase in endogenous AMPA-mediated drive currents to XII MNs. CIF, however, is not a form of plasticity and does not depend on AMPA or NMDA receptor activation for its induction. Nor does it depend on coincident protein kinase A or C activity. Rather, measurement of mEPSCs along with mass spectrometric analysis of CTZ-treated slices indicates that the cause is prolonged bioavailability of CTZ. These results illustrate a latent residual capacity for potentiating AMPA-mediated inspiratory drive to XII MNs that might be applied to the treatment of upper airway motor deficits.

Protein kinase G-dependent mechanisms modulate hypoglossal motoneuronal excitability and long-term facilitation.

Since protein kinase-dependent modulation of motoneuronal excitability contributes to adaptive changes in breathing, we hypothesized that cGMP-dependent pathways activating protein kinase G (PKG) modulate motoneuronal inspiratory drive currents and long-term plasticity. In a medullary slice preparation from neonatal rat (postnatal days 0-4) generating spontaneous respiratory-related rhythm, hypoglossal (XII) motoneuronal inspiratory drive currents and respiratory-related XII nerve activity were recorded. Focal application of a PKG activator, 8-bromoguanosine-3',5'-cyclomonophosphate (8-Br-cGMP), to voltage-clamped XII motoneurones decreased inspiratory drive currents. In the presence of tetrodotoxin (TTX), 8-Br-cGMP decreased the exogenous postsynaptic inward currents induced by focal application of AMPA. Intracellular dialysis of XII motoneurones with an inhibitory peptide to PKG (PKGI) increased endogenous inspiratory-drive currents and exogenous AMPA-induced currents. Application of 8-Br-cGMP with PKGI had no further effect on spontaneous or evoked currents, confirming that the observed effects were induced by PKG. However, PKG differentially increased longer-term plasticity. Three 3 min applications (separated by 5 min) of the α(1)-adrenergic agonist phenylephrine (PE) in combination with 8-Br-cGMP yielded greater in vitro long-term facilitation than PE alone. These data indicate the presence of a cGMP/PKG-dependent signalling pathway in XII motoneurones that modulates inspiratory drive currents and plasticity of XII motoneurones, possibly contributing to their adaptation during physiological challenges, such as sleep and exercise.