Adeno-associated virus (AAV)-mediated suppression of Ca2+/calmodulin kinase IV activity in the nucleus accumbens modulates emotional behaviour in mice

Abstract

Background: Calcium/calmodulin-dependent protein kinase IV (CaMKIV) controls activity-dependent gene transcription by regulating the activity of the cyclic AMP response element binding protein (CREB). This signaling pathway is involved in gating emotional responses in the CNS but previous studies did not address the potential roles of CaMKIV in discrete brain regions. In the present study, we aimed at specifically dissecting the role of CaMKIV in the nucleus accumbens of adult mice.

Results: We used recombinant adeno-associated virus (rAAV)-mediated gene transfer of a dominant-negative CaMKIV variant (rAAV-dnCaMKIV) to inhibit endogenous CaMKIV in the nucleus accumbens. rAAV-dnCaMKIV treated animals were subjected to a battery of tests including, prepulse inhibition of the acoustic startle response, open field, social interaction and anxiety-related behaviour. We found that basal locomotor activity in the open field, and prepulse inhibition or startle performance were unaltered in mice infected with rAAV-dnCaMKIV in the nucleus accumbens. However, anxiogenic effects were revealed in social interaction testing and the light/dark emergence test.

Conclusion: Our findings suggest a modulatory role of CaMKIV in the nucleus accumbens in anxiety-like behaviour but not sensorimotor gating.

Figure 1

Characterization of rAAV-mediated transgene expression. A) Schematic illustration of the rAAV-dnCaMKIV expression cassette, for nomenclature see Methods section. B) Immunoblot analysis of neuronal activity-dependent induction of c-Fos expression in uninfected cultured mouse hippocampal neurons or in cultured mouse hippocampal neurons infected with rAAV-dnCaMKIV or rAAV-hrGFP. The neurons were treated for 4 hr with the GABA_A receptor bicuculline (50 μM) to induce action potential bursting [2, 19], or were left untreated. Expression of c-Fos, hrGFP, Flag-tagged dnCaMKIV, and calmodulin (loading control) was analyzed. C) Immunohistochemical analysis of dnCaMKIV expression (using antibodies to the Flag-tag) and expression of the neuronal marker NeuN in the NAc of animals infected in the NAc with rAAV-dnCaMKIV. The overlay of representative photomicrographs (10× objective) of Flag-immunohistochemistry (green) and NeuN (red) is shown. ac, anterior commissure. D) Photomicrograph (100× objective) showing overlay of Flag-immunoreactivity (green) and Hoechst stain (blue). Arrows indicate the cytosolic localization of Flag-tagged dnCaMKIV in two representative neurons; the nuclei in those neurons are indicated with asterisks. E) Schematic diagrams showing the approximate extension of transgene expression (gray shading) in rAAV-dnCaMKIV injected mice. Numbers, distance from bregma [42].

Figure 2

Anxiety-related behaviours observed in the dark/light box. Expression of dnCaMKIV in the NAc increased anxiogenic behaviour during dark/light emergence test performance. Time spent in the lit compartment, emergence frequency and rearing were significantly reduced in dnCaMKIV animals compared to rAAV-empty infused controls (EMPTY) (dnCaMKIV: n = 11, EMPTY: n = 11; p < 0.05 is indicated by asterisks). Means ± S.E.M. are shown.

Figure 3

Social interaction testing. Interaction with an unknown social partner revealed an increase in anxiety-related behaviour in rAAV-dnCAMKIV treated animals. The total amount of social behaviours was decreased in those mice, mainly due to a significant decrease in approaching and following the social partner. Social avoidance/anxiety-related behaviour (stretched-attend posture and evade upon contact) was increased in animals expressing dnCaMKIV compared to EMPTY (dnCaMKIV: n = 11, EMPTY: n = 11; p < 0.05 is indicated by asterisks). Means ± S.E.M. are shown.