Structural basis of Arc oligomerization.

Arc N-lobe (a.a. 207-278) and C-lobe (a.a. 278-370), which together form the Arc GAG, are each composed of four alpha helices surrounding a hydrophobic core (Zhang et al., 2015). Arc N-lobe helix α4 is contiguous with Arc C-lobe helix α1’, and these are separated by a putative hinge region. Hydrophobic amino acids that form the core of Arc N-lobe in lower-vertebrates are replaced in higher vertebrates creating a pocket that accommodates aromatic side chains of bound ligands including TARPγ2 and CaMKII (Zhang et al., 2015). The positions of phosphorylation sites ArcS260 and ArcT278 can be visualized in the structure of Arc N-lobe in complex with either TARPγ2 or CaMKII (Figure 2A, ​,2B2B and S7). ArcS260 is at the C-terminus of helix α3 while ArcT278 is at the putative hinge region between the lobes. Both phosphorylation sites are distant from the Arc N-lobe hydrophobic pocket and bound ligand, but close to surfaces between adjacent N-lobe protomers in the crystals (Figure 2A and ​and2B).2B). Accordingly, we considered the possibility that inter-protomer interactions identified from the crystal structure might be physiologically relevant and modified by phosphorylation. We noted that Arc N-lobe interaction surfaces are identical in two independent complex structures that display different symmetry groups, are similar to Arc C-lobe (Figure 2C), and are similar to HIV C-lobe interactions important for retrovirus capsid assembly (Figure 2D) (Pornillos et al., 2009; Pornillos et al., 2011). The combined buried area of N-lobe and C-lobe putative interaction surfaces is greater than 960 Ų and comparable to the combined areas of retroviral capsids (Pornillos et al., 2009; Pornillos et al., 2011). Moreover, the degree of “fit” of interaction surfaces assessed by a calculated shape correlation (SC) (Lawrence and Colman, 1993) (SC of 1 indicates precise meshing while 0 indicates uncorrelated topography) is 0.717 for Arc N-lobe and 0.697 for Arc C-lobe. These SCs are comparable to physiologically relevant interaction surfaces (Rouvinski et al., 2015). Surfaces that mediate inter-protomer associations of the rat Arc N-lobe and C-lobe are evolutionarily conserved (Figures 3A and ​and4A4A).

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Figure 2

Comparison of dimers of Arc N-lobe, C-lobe and HIV C-lobe deduced from crystal structures.

Dimers are generated by application of crystal symmetry to the protomer present in the asymmetric unit. Red dashes indicate dimer interface of N-lobe or C-lobe. (A, B) Structures of complex crystals of Arc N-lobe with TARPγ2 peptide (PDB ID, 4X3H) or CamKII fragment (PDB ID, 4X3I). TARPγ2 peptide and CamKII fragment are colored purple. 260S is indicated in red ball. Unstructured 278T is displayed as a pink ball. Ubiquitination sites K268 and K269 are show in green. (C) Structure of Arc C-Lobe crystal (PDB ID, 4X3X). 278T is indicated in red ball. (D) HIV capsid CTD dimer (PDB ID, 1A43). In each case, the monomers that comprise the dimer are colored differently. Helix labels in parentheses follow references for Arc (Zhang et al., 2015) and HIV capsid domain (Pornillos et al., 2009).

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Figure 3

Arc N-lobe and oligomerization.

(A) The sequence alignment of Arc N-lobe (PDB ID, 4X3H) from typical species based on their 3D structural superposition with HIV capsid CTD (PDB ID, 1A43). Hs, Homo sapiens, NP_056008.1; Rn, Rattus norvegicus, NP_062234.1; Gga, Gallus gallus, NP_989763.1; Aca, Anolis carolinensis, XP_003223977.1; Xtr, Xenopus (Silurana) tropicalis, XP_002934511.1; Dme, Drosophila melanogaster, Arc1, Q7K1U0; Dr2, Danio rerio, XP_001919627; _N refer to N-lobe. Secondary structures of Arc N-lobe and HIV capsid CTD (PDB 1A43) are shown on the bottom in red and on the top in blue, respectively. The cyan solid circles (monomer A) and red stars (monomer B) below the sequence alignment mark amino acids that are predicted to mediate dimerization of Arc N-lobe. The green up triangles indicate hydrophobic pocket of Arc N-lobe, and the pair of aromatic residues supporting beta strand formation of N-lobe are highlighted with orange right deltoids. Blue down triangles indicate residues in the HIV C-lobe dimer interface. The phosphorylation sites S260 and T278 are shown in bold black with box. (B-C) magnified view of the interaction between two subunits of Arc N-lobe in grey (monomer A) and brown (monomer B), separately (PDB ID, 4X3H). The residues important for interaction are shown as a stick representation. (D-E) Polymerization status of various full-length Arc proteins in solution (10uM) examined by dynamic light scattering (DLS) with increasing temperature (1°C/min) from 20°C to 30°C and assayed after an additional 20 min at 30°C. (D) Mutation of predicted dimerization sites prevents oligomerization. (E) Phosphomimic mutations of S260 prevent oligomerization.

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Figure 4

Arc C-Lobe and oligomerization.

(A) The sequence alignment of Arc C-lobe (PDB ID, 4X3X) for typical species based on their 3D structural superposition with HIV capsid CTD (PDB ID, 1A43). Hs, Homo sapiens, NP_056008.1; Rn, Rattus norvegicus, NP_062234.1; Gga, Gallus gallus, NP_989763.1; Aca, Anolis carolinensis, XP_003223977.1; Xtr, Xenopus (Silurana) tropicalis, XP_002934511.1; Dme, Drosophila melanogaster, Arcl, Q7K1U0; Dr2, Danio rerio, XP_001919627; _C refer to C-lobe. Secondary structures of Arc C-lobe and HIV capsid CTD (PDB 1A43) are shown on the bottom in red and on the top in blue, respectively. The cyan solid circles (monomer A) and red stars (monomer B) below the sequence alignment mark amino acids that mediate inter-subunit interactions of Arc C-lobe. The blue down triangles indicate residues in the HIV C-lobe dimer interface. The phosphorylation site T278 is shown in bold black with box. (B) Detailed view of the interaction between two subunits of Arc C-lobe in grey (monomer A) and brown (monomer B), separately (PDB ID, 4X3X). The residues important for interaction are shown as a stick representation. (C, D) Polymerization status of various full-length Arc proteins examined in solution (10uM) with increasing temperature (1°C/min) from 20°C to 30°C. (C) Mutations in the putative hinge region. After an additional 6 minutes at 30°C the mutant ArcT278D peaks ~1276 KDa (hexamer of tetramers) calculated based on gyration radius, while WT Arc peaks ~2458 KDa (dodecamer of tetramers). (D) Mutation of the C-lobe interface blocks oligomerization of full-length Arc.

We monitored oligomerization using dynamic light scattering (DLS) in various conditions of incubation temperature and protein concentration. As reported (Byers et al., 2015; Myrum et al., 2015; Pastuzyn et al., 2018), full-length bacterially expressed Arc protein (isolated at 4°C) oligomerizes in solution (Figure 3D) and is temperature dependent. HIV GAG shows similar temperature-dependent oligomerization (Barklis et al., 2009) that is relevant to viral capsid assembly (Serriere et al., 2013). At 20°C, a 10 μM solution of Arc had a calculated mass of 210 kDa indicating Arc behaves as a stable tetramer. Arc tetramer formation is dependent on the N-terminal half of Arc that includes basic, α-helical regions thought to associate with phospholipid membranes (Myrum et al., 2015) since isolated Arc GAG domain remains monomeric at 20°C or 30°C even at 200μM (Figure S2A). Upon shifting the solution to higher temperature and maintaining for an additional 20 min, full-length Arc exhibited an increase in estimated radius indicating higher order oligomerization. Higher incubation temperature (up to 37°C) or higher initial concentration of full-length Arc resulted larger oligomers. In all conditions a single peak of oligomeric state was observed, confirming that the protein solution was homogeneous and not irregular misfolded aggregates (Figure 3D and S2B). During incubation, the mean molecular weight species increased steadily in solution without stabilized plateau (Figure S2B). Oligomerization at 30°C was reversible upon return to 20°C until the average molecular weight reached ~800 kDa (an tetramer of Arc tetramers) (Figure S2C), which supports the notion that oligomers assemble in a reversible, temperature-dependent process. Based on these results, experiments were performed at 30°C with an initial protein concentration of 0.5 mg/ml in the remainder of the study, where the estimated radius of the 12 nm peak corresponds to ~1860 kDa (an octomer of Arc tetramers) at the end of a 20 min incubation period.

Next, we examined the N-lobe structure to identify specific amino acids that mediate inter-protomer interactions and test their effect on oligomerization of full-length recombinant Arc. Intermolecular associations of Arc N-lobe mediated by the first two α-helices of monomer A packed against the last two α-helices of monomer B buries areas of 421 Ų per protomer (Figure 3B). The aromatic side-chain of F256 at the center of the bundle forms a small hydrophobic core, whereas polar residues at the periphery participate in hydrophilic interactions. Hydrogen bond interactions, predicted based on distances < 4 Å, occur between side chains of contiguous monomers K257-Q241, K257-N244 and W253-Y237 (Figure 3B). K257 also bonds with E270 of the same monomer and is within 4.6 Å of S260 (Figure 3C). Analysis of charge proximity suggests an important role for the long flexible basic side chain of K257 that can function as a bridge between monomers. Consistent with this analysis full-length Arc mutants ArcK257A and ArcQ241A formed tetramers at 20°C but failed to oligomerize at 30°C (Figure 3D).

Arc C-lobe protomers associate in the crystal structure by interactions between α2 of protomer A and α3 of protomer B and exhibit the same symmetry as Arc N-lobe homophilic interactions (Figure 2C). The Arc C-lobe buried surface area 546Ų compares to 922Ų of HIV C-lobe interface and 612Ų of RSV C-lobe interface. Like Arc N-lobe, basic amino acid side chains form a bridge between C-lobe protomers. Arc R335 (monomer B) hydrogen bonds with E320 (monomer A) while R338 and H339 (monomer A) hydrogen bond with D289 and Y313 (monomer B)(Figure 4B). Point mutants of all of these sites were generated but only Arc R335E expressed. DLS demonstrated ArcR335E behaves like WT at 20°C but fails to oligomerize at 30°C (Figure 4C). These findings indicate that interactions mediated by both the Arc N-lobe and Arc C-lobe are required for high order oligomerization of full-length Arc.

The interaction surfaces for both Arc N-lobe and C-lobe that mediate oligomer assembly are asymmetric in that they involve different surfaces of protomer A and protomer B (Figure 2A, ​,2B,2B, and ​and2C).2C). This creates flexibility for oligomer assembly with four different possible configurations of each full-length Arc molecule within an oligomer (Figure S2D and S2E) and may underlie the variable size of Arc oligomers. This is distinct from retroviral capsid assembly, which is largely mediated by symmetric interactions between lobes (Figure 2D) and creates oligomers of fixed size (Serriere et al., 2013).

Phosphomimic mutations of Arc disrupt high order oligomerization

We examined a panel of full-length Arc point mutants to assess the effect of negative charge at phosphorylation sites. Phosphomimetic mutants ArcS260D, ArcS260D,T278D and ArcS260D,T278A formed tetramers at 20°C but did not oligomerize at 30°C (Figure 3E). By contrast, ArcS260A,T278A and ArcS260A displayed temperature-dependent oligomerization. ArcS260 is 4.61Å from the basic residue of K257 (Figure 3C) and the added volume of S260 phosphate will reduce the distance to K257 for salt bridge formation. These observations indicate that addition of negative charge at ArcS260 prevents temperature-dependent oligomerization and supports a model in which phosphorylation of S260 promotes an intramolecular salt bridge with nearby K257 that competes with intermolecular interactions needed for high order oligomerization.

ArcT278D formed tetramers at 20°C and high order oligomers at 30°C although the size was less than WT Arc (Figure 4D). ArcT278 is within an extended alpha helical region that spans Arc N-lobe α4 to the Arc C-lobe α1’ (Figure 3A and Figure 4A) but is not sufficiently constrained to be resolved in either crystal structure. Contiguous G277 is predicted to confer flexibility between Arc N- and C-lobes. The demonstrated importance of flexibility between retroviral GAG N-lobe and C-lobe domains for capsid assembly (Pornillos et al., 2009; Pornillos et al., 2011) suggested that flexibility at ArcG277 may be important for oligomerization. Consistent with this model, ArcG277D formed tetramers at 20°C but failed to form high order oligomers at 30°C (Figure 4D). The proximity of ArcT278 to the putative hinge suggests that if phosphorylation of ArcT278 occurs in vivo it may inhibit or slow oligomerization by reducing necessary flexibility between lobes.

Interaction surfaces essential for high order oligomerization of Arc are required for its function to depress synaptic strength.

We compared the structure-function relationship for Arc oligomerization and synaptic depression in Purkinje neurons. Parallel fiber inputs to Purkinje neurons exhibit activity-dependent depression that is induced by postsynaptic activation of metabotropic glutamate receptors (mGluR-LTD)(Linden, 2012). Arc is expressed at low basal levels in Purkinje cells and does not contribute to the early phase of LTD but is transcriptionally induced and is required for the protein synthesis-dependent late phase of LTD (Smith-Hicks et al., 2010). Consistent with these studies, whole-cell voltage-clamp recordings made from mouse Purkinje cells expressing WT Arc transgene in cultures revealed reduced miniature excitatory post-synaptic current (mEPSC) amplitudes (Figure 5A). Moreover, Arc transgene expression occluded chemical LTD evoked by treatment of neurons with PDA (Figure 5A). The CaMKII inhibitor KN-93 did not alter effects of Arc on baseline synaptic strength or occlusion of PDA (Figure 5A).

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Figure 5

Arc oligomerization, synaptic function and regulation by CaMKII.

Whole-cell voltage-clamp recordings from mouse Purkinje cells expressing Arc transgenes in culture. (A) Basal mEPSC amplitude was driven low and chemical LTD evoked by PDA was occluded in Arc-transfected Purkinje cells. This effect was not blocked by pretreatment with the CaMKII inhibitor KN-93. N= 10 cells/group. (B) Phosphomimic mutant at the site S260 and C-lobe mutant R335E did not reduce the basal amplitude of mEPSCs and did not occlude subsequent PDA-evoked chemical LTD. ArcS260A mimics WT Arc by reducing mEPSC and occluding chemical LTD. N= 10 cells/group. The scale bars for the insets on (A) and (B) are both 500 msec, 20 pA. (C) Purkinje cells in primary cerebellar culture were transfected with Arc transgene or an empty vector control plasmid and examined using a standard protocol that induces dendrite-specific LTD in Purkinje cells treated with the control plasmid (n=8; or untransfected cells, not shown). In Arc expressing cells (n=7) LTD was occluded in the paired pathway that received glutamate/depolarization conjunction as indicated by the horizontal bars (paired pathway), while LTP was induced in the control pathway. LTP was abolished by pretreatment with the CamKII inhibitor KN-93 (n=8). Scale bars = 2 sec, 50 pA. (D) LTD induced by glutamate/depolarizing pairing (indicated by horizontal bars) was occluded by both WT, ArcS260A and ArcS260,T278A. LTP of control pathway was abolished in neurons expressing de-phosphorylation mimic mutants at ArcS260A. Arc plasmid (n=7); Arc S260A plasmid (n=8); Arc S260A,T278A plasmid (n=6). Scale bars = 2 sec, 60 pA.

We next examined Arc point mutants in Purkinje neurons. Mutant Arc proteins that do not form high order oligomers including ArcS260D, ArcS260D/T278D and ArcR335E lacked WT Arc function and did not reduce the amplitude of mEPSCs or occlude PDA-induced LTD (Figure 5B). By contrast, mutant Arc proteins that do form high order oligomers including ArcS260A, ArcS260A/T278A and ArcT278D behaved like WT Arc to reduce the amplitude of spontaneous mEPSCs and occlude the effect of PDA treatment (Figure 5B). As controls for these studies we confirmed that mutations of Arc that disrupt high order oligomerization do not disrupt binding of full-length Arc to TARPγ2 peptide (Figure S3A). Additionally, we examined the subcellular localization of WT and mutant Arc transgenes in neurons co-transfected with the interacting vesicle protein endophilin3 as well as GFP-Rab5, GFP-Rab7 and GFP-Rab11 to mark specific vesicle populations. WT Arc, ArcS260D/T2278D and ArcS260A/T2278A showed similar co-IP with endophilin3 transgene from HEK293 cells (not shown), and co-localization in neuronal dendrites on punctate structures (presumed endosomes) with endophilin3, Rab5, Rab7 and Rab11 transgenes (Figure S3B–D). Thus, mutations that inactivate Arc synaptic function do not necessarily prevent its association with vesicles. These findings are consistent with a model in which the N-terminal half of Arc contributes importantly to oligomerization, mediates association with endophilin and dynamin (Chowdhury et al., 2006), is basic and palmitoylated to enhance association with phospholipids (Barylko et al., 2018; Myrum et al., 2015), but individual interactions of N-lobe and C-lobe are required for high order oligomerization and Arc’s canonical synaptic function.

Input-specific reversal of Arc by CaMKII activation.

To examine input-specific effects of Arc we examined the late phase of cerebellar LTD using a well-established protocol in which two iontophoresis electrodes are positioned to stimulate separate dendritic branches of cultured Purkinje neurons in an alternating fashion. Following acquisition of baseline responses to test pulses of glutamate, test pulses are halted at the “control” electrode and LTD is induced by applying glutamate/depolarization conjunctive stimulation at the “paired” electrode. This results in input-specific induction of LTD (Linden, 2012; Wang and Linden, 2000). In Purkinje neurons expressing Arc transgene glutamate/depolarization conjunctive stimulation at the “paired” electrode at t= 0 min resulted in an unexpected, rapid potentiation of the “control” pathway (Figure 5C). Potentiation was evident within 5 min and subsequent bouts of conjunctive stimulation at 1= 10 and 20 min evoked a saturating potentiation that persisted to the end of the recording at t=90 min. We termed this phenomenon Arc-dependent heterosynaptic LTP (Arc-hLTP). Arc-hLTP required both glutamate stimulation of the paired pathway and somatic depolarization since either manipulation alone failed to evoke potentiation of the control pathway (not shown).

Combined depolarization and glutamate stimulation results in a cell-wide rise of Ca²⁺ (Linden, 2012), and we considered the possibility that this stimulus activates CaMKII and acutely reverses the synaptic action of Arc. Consistent with this mechanism, bath application of the CaMKII inhibitor KN93 (5 μM) prevented Arc-hLTP without changing the amplitude of the response in the paired pathway (Figure 5C). We noted that activation of PKC by PDA did not result in reversal of Arc action (Figure 5A) and experiments monitored the effect of the PI3Kinase inhibitor Wortmannin (1 μM) and found no effect on Arc-hLTP (not shown). We repeated the experiment in Purkinje neurons expressing ArcS260A or ArcS260A/T278A. Purkinje neurons expressing either mutant failed to show Arc-hLTP of the control pathway (Figure 5D). These observations support the model that CaMKII oligomerization-disrupting phosphorylation of ArcS260 acutely reverses Arc’s synaptic weakening function in Purkinje neurons.

Arc phosphorylation controls the magnitude of mGluR-LTD at the Schaffer collateral-CA1 synapse.

To examine the role of Arc GAG domain phosphorylation in neural plasticity in vivo, we generated a knock-in murine model (Figure S4). Arc^S260A/T278A KI mice were backcrossed at least five generations to C57BL/6J. Homozygous Arc^S260A/T278A KI mice (Arc 260/278 AA/AA) are viable, produce offspring according to Mendelian genetics, show normal brain morphology, and reach the same body weight at adulthood as wild-type littermates. The basal expression of Arc protein and mRNA transcript in Arc 260/278 AA/AA was comparable to WT mice, and there was no significant difference in the expression of PSD95, TARPγ2, NMDAR subunits, AMPAR subunits, or mGluR subunits (Figure S4).

Electrophysiological recordings of the Schaffer-CA1 synapse were performed using acute hippocampal slices and field recordings. Recordings of the baseline relationship between the presynaptic fiber volley amplitude and the slope of the evoked postsynaptic potential revealed similar synaptic strength (Figure 6A). An assessment of the paired pulse ratio suggests similar presynaptic release probability (Figure 6B). We next examined theta burst induced LTP and found that it was not different comparing WT and Arc 260/278 AA/AA slices (Figure 6C). By contrast, mGluR-LTD evoked by bath application of (S)-3,5-dihydroxyphenylglycine (DHPG) revealed enhanced magnitude of depression in Arc 260/278 AA/AA slices (Figure 6D). The maximum stable synaptic depression in WT slices was 18% 20 min after induction and 30% 20 min after induction in Arc 260/278 AA/AA slices. We considered the possibility that Arc protein induced by DHPG might impact subsequent TBS induced LTP, however, when the baseline was adjusted to compensate for enhanced mGluR-LTD in Arc 260/278 AA/AA slices there was no difference compared to LTP in WT slices (Figure 6D and S5). We conclude that genetic interruption of Arc GAG domain phosphorylation has the effect of selectively increasing the magnitude of mGluR-LTD.

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Figure 6

Enhanced DHPG-induced LTD in Arc 260/278 AA/AA mutant mice.

(A) The input-output ratio is not altered in Arc 260/278 AA/AA mutant mice. For input-output ratio: WT n = 86, I/O =2.70 ± 0.15 ms⁻¹; Mutant n=81, I/O = 2.64 ± 0.14 ms⁻¹. P>0.05 with two-tailed student’s t-test. (B) Paired pulse facilitation is not altered in Arc 260/278 AA/AA mutant mice. WT n=13; Mutant n = 10. P>0.05 with two-way repeated measures ANOVA with Bonferoni post-hoc test. (C) Theta-burst stimulation (TBS) induced LTP is not altered in Arc 260/278 AA/AA mutant mice. n=5 for each group. P>0.05 with two-tailed student’s t-test. (D) DHPG-induced LTD is significantly enhanced in Arc 260/278 AA/AA mutant mice. The TBS-induced LTP following DHPG-LTD is significantly reduced in Arc 260/278 AA/AA mutant mice when normalized to original baseline, but not to last 6 min of LTD (see supplement figure S5). WT n=10; mutant n=8. * P<0.05 with two-tailed student’s t-test.

Phosphorylation of Arc GAG domain is essential for learning and memory.

Arc 260/278 AA/AA and littermate WT mice were trained in a delayed fear conditioning paradigm, in which presentation of an auditory cue (conditioned stimulus, CS) in a training context was repeatedly paired with an intrinsically aversive stimulus (mild footshock, US). Arc 260/278 AA/AA mice showed deficits in acquisition of fear to context as judged by development of freezing response during intertrial intervals (Figure 7A–B). The learning deficit in Arc 260/278 AA/AA mice was also observed as a low acquisition of freezing to CS (Figure 7C). This learning deficit could not be explained by changes in sensitivity to the US as both genotypes demonstrated comparable levels of motor activation and body displacement by the footshock (Figure 7A insert). The role of anxiety as a non-cognitive factor in this deficit was also unlikely as Arc 260/278 AA/AA mice were not different from controls avoiding anxiogenic areas in plus maze and open field (Figure S6). Testing for long-term contextual memory revealed that Arc 260/278 AA/AA mice demonstrated significantly lower levels of freezing responses than their WT littermates (Figure 7D–E and ​and7G).7G). Similarly, long-term memory of the CS when tested in a new context was significantly impaired in Arc 260/278 AA/AA mice (Figure 7F and ​and7H7H–I). The new context did not support generalization of fear from the previous context (Figure 7G) allowing for testing the acquisition of second-order conditioning (Gewirtz and Davis, 2000; Helmstetter and Fanselow, 1989; Rizley and Rescorla, 1972). This phenomenon represents the association of the CS with a new context resulting in acquirement of freezing responses to the context that was not associated with the US. Presentation of the CS in the new context increased freezing to this context in WT mice (Figure 7F and ​and7J).7J). However, Arc 260/278 AA/AA mice did not show consistent freezing responses in this situation (Figure 7F and ​and7J).7J). As second-order conditioning is supported by strong US-CS associations (Helmstetter and Fanselow, 1989), the deficits of Arc 260/278 AA/AA mice acquiring second-order freezing response might be a result of their primary deficits in the first-order conditioning. Similarly, the impairments observed in Arc 260/278 AA/AA mice in long-term fear memory are likely consequences of their principal deficits in ability to quickly acquire the conditional associations during learning phases. Testing of Arc 260/278 AA/AA mice in the Morris water maze that requires many more trials than classical fear conditioning revealed only marginal deficits in spatial memory (Figure S6). Thus, Arc 260/278 AA/AA genetic perturbation is sufficient to deteriorate fast acquisition of new memories. This effect was observed for acquisition of hippocampus-dependent contextual memories as well as for acquisition of amygdala-dependent cued memory (Phillips and LeDoux, 1992) indicating that the functional role of CaMKII-dependent phosphorylation of Arc is not limited to hippocampus-specific neuronal circuit.

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Figure 7

Arc 260/278 AA/AA mutant mice are deficient in acquisition of fear memory.

(A) Time course of freezing to context and conditioned stimulus, CS (shaded areas), observed in Arc 260/278 AA/AA mutant mice and littermate control WT mice during training in a delayed fear conditioning (Session 1). Arrows indicate presentation of unconditioned stimulus, US (a foot-shock) at the end of each CS. Insert shows equal sensitivity to a foot-shock in both genotypes (ANOVA, F(1, 19)=0.1, P>0.9). (B) Acquisition of fear to context is delayed in Arc 260/278 AA/AA mice (ANOVA, effect of genotype F(1,19)=5.63, P<0.028; genotype × intertrial interval (ITI) interaction F(3,57)=4.23, P<0.009). Pre-training basal levels (BL) of freezing were low in both genotypes. (C) Acquisition of fear to a conditioned stimulus (CS) is impaired in Arc 260/278 AA/AA mice (ANOVA, effect of genotype F(1,19)=7.60, P<0.013; genotype × inter-trial interval (ITI) interaction F(2,38)=3.98, P<0.027). (D) Time course of freezing to the training context after a 24-hr delay (Session 2). (E) Long-term contextual fear memory was impaired in Arc 260/278 AA/AA mice (ANOVA, effect of genotype F(1,19)=5.05, P<0.037; genotype × time block interaction, P>0.7). Average levels of freezing in Session 2 are shown. (F) Time course of freezing after a 28-hr delay tested in a new context before and after presentation of the CS (shown as shaded areas) (Session 3). (G) Freezing (time %) in the 1^st 2 min of each session (S1, S2, S3) indicated no generalization of fear to a new context in either genotype. Initial levels of freezing in Session1 and Session 3 were comparable (P>0.8, NS) but dramatically lower than in Session 2 (ANOVA, effect of genotype F(1,19)=4.29, P<0.052, genotype × session interaction F(2,38)=5.22), P<0.009). (H) Long-term fear memory to the CS was impaired in Arc 260/278 AA/AA mice (ANOVA, effect of genotype F(1,19)=6.77, P<0.018; genotype × inter-trial interval (ITI) interaction, P>0.11). (I) Average levels of freezing to the CS were lower in Arc 260/278 AA/AA mice than in their WT littermates. (J) Acquisition of secondary fear to the new context was impaired in Arc 260/278 AA/AA mice (ANOVA, effect of genotype F(1,19)=5.34, P<0.032, genotype × session interaction F(3,57)=4.49), P<0.007). ITI - intertrial intervals in Session 3. Single and double asterisks in panels B-C, G, and J indicate significant deficits in Arc 260/278 AA/AA mice at p levels 0.05 (*) or 0.001 (**) (LSD post-hoc tests applied to a significant interaction in ANOVA). Pound signs in panels D-E and H-J indicate significant differences between genotypes (ANOVA, main effect of genotype, P<0.05). Numbers of cases are shown next to the labels.

We thank Biogen for support during the initial studies and Jason Shepherd for helping to initiate electrophysiological studies. We are thankful to Tatiana Melnikova, Catherine LaCourse and Hye Won Chi for help in behavioral studies. Research support was provided by NARSAD (W.Z., P.F.W.), R01 NS095901 (D.J.L.), R01 MH053608 (D.J.L., P.F.W.), P50 MH100024 (D.J.L, R.L.H., P.F.W.).