CaMKII controls neuromodulation via neuropeptide gene expression and axonal targeting of neuropeptide vesicles

Abstract

Ca 2+ /calmodulin-dependent kinase II (CaMKII) regulates synaptic plasticity in multiple ways, supposedly including the secretion of neuromodulators like brain-derived neurotrophic factor (BDNF). Here, we show that neuromodulator secretion is indeed reduced in mouse α-and βCaMKII-deficient (αβCaMKII double-knockout [DKO]) hippocampal neurons. However, this was not due to reduced secretion efficiency or neuromodulator vesicle transport but to 40% reduced neuromodulator levels at synapses and 50% reduced delivery of new neuromodulator vesicles to axons. αβCaMKII depletion drastically reduced neuromodulator expression. Blocking BDNF secretion or BDNF scavenging in wild-type neurons produced a similar reduction. Reduced neuromodulator expression in αβCaMKII DKO neurons was restored by active βCaMKII but not inactive βCaMKII or αCaMKII, and by CaMKII downstream effectors that promote cAMP-response element binding protein (CREB) phosphorylation. These data indicate that CaMKII regulates neuromodulation in a feedback loop coupling neuromodulator secretion to βCaMKII-and CREB-dependent neuromodulator expression and axonal targeting, but CaMKIIs are dispensable for the secretion process itself.

Dynamin controls neuropeptide secretion by organizing dense-core vesicle fusion sites

“…Evoked DCV exocytosis was quantified during two bursts of eight trains of 50 action potentials (APs) at 50 Hz ( Fig. 2, A to C ) ( 8 , 39 ). Dynamin 3 KO cultures were used as littermate controls since DCV exocytosis in these neurons was similar to that in WT neurons ( Fig.…”

“…Dynamin 2 WT was extract from a complementary DNA library with primers forward 5′-CTACCGGACTCAGATCTCGAATGGGCAACCGCGGGATGG-3′ and reverse 5′-GTACCGTCGACTGCAGAATTCTAGTCGAGCAGGGACGGC-3′ and cloned into the plasmids pSyn(pr)Dynamin2-IRES2-NLSmCherry, pSyn(pr)Dynamin2(K44A)-IRES2-NLSmCherry, and pSyn(pr)Dynamin2(E560K)-IRES2-NLSmCherry. pSyn(pr)Synapsin-mCherry, pSyn(pr)NPY-pHluorin, pSyn(pr)BDNF-pHluorin, pSyn(pr)iCre-eGFP, and pSyn(pr)SNAP25-IRES2-eGFP were previously described ( 6 – 8 , 15 , 17 , 39 ). pSyn(pr)αSNAP-IRES2-NLSmCherry and pSyn(pr)NSF-IRES2-eGFP were made by substituting the cytomegalovirus promoter with synapsin promoter in plasmids previously described ( 61 ).…”

“…Neurons at DIV 14 to 15 were treated with tetrodotoxin (TTX; Sigma-Aldrich, 10 μM) overnight and then placed in the imaging chamber containing Tyrode’s solution [2 mM CaCl 2 , 2.5 mM KCl, 119 mM NaCl, 2 mM MgCl 2 , 30 mM glucose, and 25 mM Hepes (pH 7.4)]. Following procedures previously described in ( 39 ), experiments were performed at RT with superfusion of Tyrode’s buffer unless otherwise specified. Images were acquired on an Axiovert II microscope (Zeiss, Oberkochen, Germany) with a 40× oil objective [numerical aperture (NA) = 1.3] for SV fusion, DCV fusion, and trafficking or on a Nikon Eclipse Ti microscope with 63× oil objective (NA = 1.4) for axonal targeting.…”

“…For SypHy experiments, the imaging protocol included 10 s of baseline recording, 5 s of acid wash in Tyrode’s solution [2 mM CaCl 2 , 2.5 mM KCl, 119 mM NaCl, 2 mM MgCl 2 , 30 mM glucose, and 25 mM MES (pH 5.5)], and allowed to recover for 15 s. As previously described ( 39 ), electrical field stimulation was then applied using a A-385 stimulus isolator (WPI) controlled by a Master 8 (AMPI), delivering 1-ms 30-mA pulses for 5 s at 40 Hz, followed by 1 min of recovery time, an additional 5 s of Tyrode’s (pH 5.5) superfusion, and a final 5-s perfusion with modified Tyrode’s solution containing NH 4 Cl [2 mM CaCl 2 , 2.5 mM KCl, 119 mM NaCl, 2 mM MgCl 2 , 30 mM glucose, 25 mM Hepes, and 50 mM NH 4 Cl (pH 7.4)] delivered by gravity flow through a capillary placed above the cell.…”

“…For DCV fusion, neurons were imaged for 30 s as baseline, stimulated with electrical field stimulation for eight pulses of 1 s at 50 Hz separated by 0.5 s, allowed to rest for 30 s after which the stimulation was repeated. After an additional 30 s of recovery, Tyrode’s solution containing NH 4 Cl was superfused for 5 s, as previously described ( 39 ).…”