Essential function of alpha-calcium/calmodulin-dependent protein kinase II in neurotransmitter release at a glutamatergic central synapse

Abstract

A significant fraction of the total calciumcalmodulin-dependent protein kinase II (CaMKII) activity in neurons is associated with synaptic connections and is present in nerve terminals, thus suggesting a role for CaMKII in neurotransmitter release. To determine whether CaMKII regulates neurotransmitter release, we generated and analyzed knockout mice in which the dominant alpha-isoform of CaMKII was specifically deleted from the presynaptic side of the CA3-CA1 hippocampal synapse. Conditional CA3 alpha-CaMKII knockout mice exhibited an unchanged basal probability of neurotransmitter release at CA3-CA1 synapses but showed a significant enhancement in the activity-dependent increase in probability of release during repetitive presynaptic stimulation, as was shown with the analysis of unitary synaptic currents. These data indicate that alpha-CaMKII serves as a negative activity-dependent regulator of neurotransmitter release at hippocampal synapses and maintains synapses in an optimal range of release probabilities necessary for normal synaptic operation.

Figure 1

Generation of fα-CaMKII recombinant mice. (a Top) Region of the wild-type α-CaMKII locus containing the targeted exon 2. (Middle) α-CaMKII targeting vector with a loxP site inserted into intron 1 and an LFNT cassette containing neo-tk and recombinase recognition sites inserted into intron 2. (Bottom) fα-CaMKII locus. Cre/loxP mediated recombination at the fα-CaMKII locus will excise 4 kb of sequence that includes exon 2. Open triangle, loxP; open oval, FRT; neo, neomycin resistance gene; tk, thymidine kinase gene; DT-A, diphtheria toxin gene. Restriction enzymes: B, BamHI; R1, EcoRI; Sp, SpeI; N, NcoI; X, XhoI; Sm, SmaI. (b) Tail DNA digested with BamHI and screened with a 3′ probe external to the recombined locus. +/+, wild type; +/f, heterozygous; f/f, homozygous fα-CaMKII.

Figure 2

Expression studies of CA3 α-CaMKII KO mice. (a–d) Dark field images of sagittal brain sections from 4-mo-old wild-type (a), α-CaMKII floxed (c), CA3 α-CaMKII KO (b), and global α-CaMKII KO (d) mice hybridized with an α-CaMKII-specific probe. Deletion of the fα-CaMKII gene was specific to hippocampal CA3. Cx, cortex; St, striatum; Th, thalamus; Cb, cerebellum. Arrow in b indicates the location of hippocampal CA3. (e–h) Dark-field higher magnification images of the hippocampus from α-CaMKII floxed (e), global α-CaMKII KO (f), and 1- (g) and 3-mo-old (h) CA3 α-CaMKII KO mice after hybridization with a probe specific for α-CaMKII. These images show that gene knockout is highly specific to the CA3 region of the hippocampus, indicated with double arrows. DG, dentate gyrus. (i–l) Confocal images from 3-mo-old floxed (i and k) and 4-mo-old CA3 α-CaMKII KO (j and l) mice after immunohistochemical staining with anti-α-CaMKII antibody. Lower-magnification sagittal images on top, followed by high-magnification images of hippocampal CA3 show that the α-CaMKII protein is absent from the vast majority of pyramidal cell bodies (stratum pyramidale; indicated with arrows) and dendrites (strata radiatum, oriens) in the CA3 region, whereas axons projecting from dentate granule cells to the stratum lucidum are strongly stained. H, hippocampus; sp, stratum pyramidale; sr, stratum radiatum; so, stratum oriens; sl, stratum lucidum; sl-m., stratum lacunosum-moleculare; GL, granule layer; ML, molecular layer. (m and n) Dark-field images of the hippocampus from 4-mo-old floxed (m) and CA3 α-CaMKII KO mice (n) after hybridization with a probe specific for β-CaMKII. No compensatory increase in β-CaMKII expression was observed in hippocampal regions.

Figure 3

Synaptic facilitation and depression during trains of repetitive presynaptic stimulation in control and CA3 α-CaMKII KO mice. (A) Synaptic responses during high-frequency stimulation. After baseline recording, 300 pulses were delivered at 70-ms intervals (14 Hz, first arrow). Normalized successive EPSCs are plotted as a function of stimulus number for control and KO mice (means ± SEM). Bath solution contained 50 μM NMDA receptor antagonist (d-2-amino-5-phosphonopentanoic acid, D-APV) to prevent long-term potentiation. (B) First 120 EPSCs from A at an expanded time scale. (C) The recovery phase from synaptic depression from A. The amplitude of the EPSC was normalized by the mean size of the last 30 EPSCs during 14-Hz stimulation. (D) Representative traces from control and mutant neurons for stimulation frequency 20 Hz are shown. (E–H) Response to stimulus trains (30 EPSCs) of different frequency. The presynaptic inputs were stimulated at 20 Hz (E), 10 Hz (F), 5 Hz (G), and 1 Hz (H). Values of the EPSC amplitude were normalized to the first EPSC of the stimulation train. (I) Summary graphs compare frequency dependence of synaptic facilitation in control and KO mice.

Figure 4

Synaptic release probability at hippocampal CA3-CA1 synapses in control and CA3 α-CaMKII KO mice. (A) Comparison of synaptic efficacy using synaptic input–output curves. The extracellular field potential amplitude is plotted as a function of stimulation intensity. (B) Examples (average of 10 EPSCs) of PPF in control (Upper) and mutant (Lower) synapses. (C) PPF is plotted as a function of the interstimulus interval. Pairs of EPSCs were elicited 10–15 times for each interval tested. The values of PPF were calculated and averaged for each interstimulus interval. To calculate PPF, the EPSC for the second pulse was normalized to the EPSC induced by the first pulse. (D–F) Rate of MK-801 blockade is not altered in KO mice. (D) Synaptic responses were blocked by bath application of 20 μM 6-cyano-7-nitroquinoxaline-2,3-dione at a holding potential of −70 mV. (E) The progressive block by MK-801 (40 μM) of the NMDA receptor EPSC recorded from the same cell as in D at a holding potential of −40 mV. MK-801 was applied to the slice in the absence of presynaptic stimulation for 10 min. To measure rate of MK-801 block, the Schaffer collateral input was stimulated at 0.1 Hz frequency. EPSC amplitudes were normalized by the first EPSC. (F) Averaged data from control (open circles) and KO (filled circles) mice. Error bars show SEM. (G) An example of the intensity threshold test in a slice from control mouse. Mean EPSCs are shown as a function of stimulation current intensity (20 individual responses were averaged to obtain each point). Note the sharp threshold for the appearance of the EPSC, which indicates stimulation of a single presynaptic input. (H) Superimposed successive CA1 EPSCs recorded in response to minimal paired stimulation (50-ms interval, Upper) of a single CA3 neuron. (Lower) Traces represent average of successes only (potency) for first (Left) and second (Right) EPSCs. Potency value for first EPSC = 6.3 pA; for second EPSC, =6.2 pA. (I) Representative traces of unitary EPSCs recorded in response to short trains (15 stimuli, 50-ms interpulse interval) of presynaptic stimulation in slices from control (Upper) and CA3 α-CaMKII KO (Lower) mice. (J) Estimates of release probability at unitary synapses during repetitive stimulation in slices from control (open circles) and KO (filled circles) mice. Trains of unitary EPSCs were elicited 8–12 times for each cell tested. All unitary responses were classified as 1 (successes) or 0 (failures of synaptic transmission). The obtained parameters were then averaged for each individual experiment to provide an estimate of probability of success (the situation when release occurred).