The findings of this study provide fundamentally novel insight into the molecular basis by which cocaine induces dendritic spine plasticity in the NAc. The data establish that downregulation of Rac1 activity induced by repeated cocaine is both necessary and sufficient for the cocaine-mediated increase in thin dendritic spines on NAc medium spiny neurons. The transient reduction in Rac1 signaling enhances actin turnover, as evidenced by the decrease in the inactive (phosphorylated) form of cofilin, which leads to more coflin activity and more thin spines. Our results also directly relate such downregulation of Rac1 signaling, and enhanced cofilin activity, in the NAc to greater sensitivity to the rewarding effects of cocaine. Indeed, the use of Rac1-pa, which allowed the novel manipulation of an intracellular signaling protein in real time in vivo, made it possible to examine the structural and behavioral consequences of the highly transient cocaine-induced decrease in Rac1 activity selectively within the adult NAc.
While our findings are in agreement with previous results demonstrating the importance of the cofilin pathway and actin dynamics in cocaine-induced structural plasticity, the precise details of cocaine regulation of cofilin phosphorylation are more complicated16–18
. For example, Shen et al.17
reported no change in p-cofilin, but an increase in total cofilin, in response to a cocaine challenge in rats withdrawing from prior chronic cocaine exposure. This contrasts with our data showing a transient decrease in p-coflin, and no change in total cofilin, in response to a cocaine challenge. These apparent discrepancies may be due to several factors that are known to be critical for drug-induced plasticity, such as the use of rats vs. mice, the withdrawal times after the course of chronic cocaine (24 hours in our experiments and 3 weeks in Shen et al.), or methodological differences in measuring cofilin (total NAc extracts in our study and crude postsynaptic density fractions in Shen et al.). Importantly, however, the net effect—an increase in cofilin and actin dynamics—is the same in both studies. Moreover, the finding that inhibiting actin turnover by local injection of latrunculin into NAc blocks cocaine-induced increases in spine density and locomotor sensitization18
, along with the report of RhoA GTPase downregulation by chronic cocaine36
, further support the role of enhanced actin dynamics in mediating structural and behavioral plasticity to chronic cocaine.
Previous reports have suggested that Rac1 activity promotes spine development, while inhibition of Rac1 reduces spine number, in other neural systems37
. However, the role of Rac1 in the regulation of actin dynamics and spine morphology is far more complicated and depends on many factors such as age and neuronal type, and may even vary between in vivo
vs. in vitro
systems22, 23, 25
. Here, we demonstrate that decreased Rac1 signaling in the NAc in vivo
increases spine formation, particularly of more immature, thin spines through a cofilin-mediated mechanism. Cofilin activity has been shown previously to increase actin depolymerization, nucleation, and branching, ultimately leading to thinner spines and new cellular protrusions38
. However, it should be noted that the changes in Rac1 and cofilin activity observed here may not occur exclusively at the spine, but could instead occur throughout the entire neuron, including the soma where Rac1 has been shown to regulate gene transcription39
Cocaine-induced behavioral and synaptic plasticity has been strongly associated with adaptations in excitatory glutamatergic transmission in the NAc6, 40–43
. For example, at early withdrawal time points after the last cocaine exposure, including those examined here, there is an increase in thin (more highly plastic) spines and synaptic depression17, 44
, perhaps representing an increased pool of silent synapses45
. The role of Rac1 signaling in mediating silent synapse formation, which has not yet been investigated directly, now warrants examination. It will also be important in future studies to determine whether the influence of Rac1 on cocaine regulation of spine plasticity of NAc medium spiny neurons is selective for various subtypes of these neurons, which play distinct roles in the addiction process35
Recently, cocaine has been reported to induce kalirin-7, another Rho GEF, and loss of kalirin-7 in knockout mice blocks cocaine’s induction of NAc spines and cocaine reward46
. However, how these findings on kalirin-7 relate to Rac1 is unknown, since kalirin-7 induction would be expected to increase Rac1 activity and we show here that downregulation of Rac1, not activation, induces spines and cocaine reward. It is possible that the paradoxical effects seen in kalirin-7 knockout mice are mediated via loss of kalirin-7 in other brain regions or earlier in development, or perhaps mediated via actions of kalirin-7 on Rho GTPases other than Rac1.
While repeated administration of opiate drugs of abuse, like psychostimulants, causes sensitized behavioral responses to the drugs, opiates decrease dendritic spine density on NAc medium spiny neurons in striking contrast to the induction seen with psychostimulants5
. Virtually nothing is known about the effect of opiate drugs on activity of the Rac1-cofilin pathway in the NAc, and the role of actin dynamics in mediating opiate-induced addictive behaviors remains unexplored. Moreover, while several prior studies that blocked cocaine-induced increases in spine density, through a variety of pharmacological and molecular manipulations, concomitantly observed blunted behavioral effects of cocaine10, 13, 18
, several studies have seen the opposite9, 11, 12, 16
. These findings highlight the need for further research to carefully delineate the likely complex and time-dependent role of spine plasticity of NAc medium spiny neurons in mediating distinct aspects of behavioral adaptations to cocaine. One key consideration is that most studies that have examined drug-induced morphological plasticity in the NAc have relied, as we do in the present investigation, on the use of investigator-administered drug. An important subject for future studies is to determine whether the molecular changes seen using such non-contingent drug administration are the same as those that occur with drug self-administration paradigms, highlighting the critical importance of investigating Rac1 signaling in these additional models of drug addiction47–49
Together, our data support a scheme whereby repeated administration of cocaine leads to downregulation of the Rac1 GEF Tiam1 and upregulation of the Rac GAP RacGAP1, thereby priming NAc medium spiny neurons for transient reductions in Rac1 activity in response to a subsequent cocaine challenge. This cascade (Supplemental Fig. 1
) may be a mechanism by which chronic cocaine exposure induces long-term changes in plasticity in NAc, and provides new directions for the development of novel therapies for cocaine addiction.