Viral delivery of shRNA to amygdala neurons leads to neurotoxicity and deficits in Pavlovian fear conditioning

Highlights

• Viral delivery of shRNA to amygdala rat neurons in vivo impaired fear conditioning.

• These findings were independent of the shRNA sequence used.

• The use of shRNAs in behavioral neuroscience warrants careful study design.

• Potential methods to alleviate shRNA induced toxicity are discussed.

Abstract

The use of viral vector technology to deliver short hairpin RNAs (shRNAs) to cells of the nervous system of many model organisms has been widely utilized by neuroscientists to study the influence of genes on behavior. However, there have been numerous reports that delivering shRNAs to the nervous system can lead to neurotoxicity. Here we report the results of a series of experiments where adeno-associated viruses (AAV), that were engineered to express shRNAs designed to target known plasticity associated genes (i.e. Arc, Egr1 and GluN2A) or control shRNAs that were designed not to target any rat gene product for depletion, were delivered to the rat basal and lateral nuclei of the amygdala (BLA), and auditory Pavlovian fear conditioning was examined. In our first set of experiments we found that animals that received AAV (3.16E13–1E13 GC/mL; 1 μl/side), designed to knockdown Arc (shArc), or control shRNAs targeting either luciferase (shLuc), or nothing (shCntrl), exhibited impaired fear conditioning compared to animals that received viruses that did not express shRNAs. Notably, animals that received shArc did not exhibit differences in fear conditioning compared to animals that received control shRNAs despite gene knockdown of Arc. Viruses designed to harbor shRNAs did not induce obvious morphological changes to the cells/tissue of the BLA at any dose of virus tested, but at the highest dose of shRNA virus examined (3.16E13 GC/mL; 1 μl/side), a significant increase in microglia activation occurred as measured by an increase in IBA1 immunoreactivity. In our final set of experiments we infused viruses into the BLA at a titer of (1.60E+12 GC/mL; 1 μl/side), designed to express shArc, shLuc, shCntrl or shRNAs designed to target Egr1 (shEgr1), or GluN2A (shGluN2A), or no shRNA, and found that all groups exhibited impaired fear conditioning compared to the group which received a virus that did not express an shRNA. The shEgr1 and shGluN2A groups exhibited gene knockdown of Egr1 and GluN2A compared to the other groups examined respectively, but Arc was not knocked down in the shArc group under these conditions. Differences in fear conditioning among the shLuc, shCntrl, shArc and shEgr1 groups were not detected under these circumstances; however, the shGluN2A group exhibited significantly impaired fear conditioning compared to most of the groups, indicating that gene specific deficits in fear conditioning could be observed utilizing viral mediated delivery of shRNA. Collectively, these data indicate that viral mediated shRNA expression was toxic to neurons in vivo, under all viral titers examined and this toxicity in some cases may be masking gene specific changes in learning. Therefore, the use of this technology in behavioral neuroscience warrants a heightened level of careful consideration and potential methods to alleviate shRNA induced toxicity are discussed.

Introduction

The ability to genetically modify organisms provides the means to determine how individual genes contribute to the functioning of the nervous system. Currently, mouse and rat gene knockouts/ins and transgenics are the main systems used to investigate the role and function of individual mammalian genes in behavioral neuroscience (Capecchi, 2005). One technology to study gene function that offers alternatives to the above mentioned technology is to use viruses to deliver transgenes of interest to specific cells or regions of the nervous system. If the virus is designed to contain a gene that codes for a short hairpin RNA (shRNA), specific gene products (i.e. mRNAs) can be targeted for degradation via the RNA interference (RNAi) pathway (Hommel, Sears, Georgescu, Simmons, & DiLeone, 2003). This technology can essentially be used to knockout genes in particular tissues/cells quickly, before or after behavioral training relatively easily.

Intriguingly, there are numerous reports that in vivo use of shRNAs can be problematic. For example, there have been studies that report that viral delivery of shRNAs to the mouse and rat brain is associated with neural toxicity. AAV mediated delivery of shRNA to the striatum of mice has been reported to induce neural toxicity, neuronal cell loss, increased inflammation as measured by an increase in microglia activation, motor disturbances and early demise (Martin et al., 2011, McBride et al., 2008). These findings were caused by the expression of all shRNAs examined, even shRNAs that were not designed to target any gene product for depletion. Similar neurotoxicity has been reported to be induced by viral delivery of shRNA to the rat substantia nigra (Khodr et al., 2011, Ulusoy et al., 2009), cerebellum (Boudreau, Martins, & Davidson, 2009), and red nucleus (Ehlert, Eggers, Niclou, & Verhaagen, 2010). However, despite these previous findings that viral mediated delivery of shRNA may cause neural toxicity when administered to the central nervous system of mice and rats, it remains a common tool to examine the role of genes in behavior.

In this series of experiments, we were specifically interested in determining if robust learning and memory deficits could be created when viral mediated delivery of shRNA technology was used to target the mRNAs for the Activity regulated cytoskeletal (Arc) gene, Early Growth Response 1 (Egr1) gene, and the GluN2A gene – a subunit of the N-Methyl-D-aspartate receptor (NMDAR). Because these genes have previously been shown to be critical for amygdala dependent Pavlovian fear conditioning (Jones et al., 2001, Maddox et al., 2011, Plath et al., 2006, Ploski et al., 2008, Walker and Davis, 2008), we reasoned that if this technology was effective, it should be able to knockdown these genes within the amygdala and impair Pavlovian fear conditioning. Therefore, we designed adeno-associated viruses that harbored short-hairpin RNAs (shRNA) designed to target the mRNAs for each of these genes. These viruses, along with control viruses which harbor shRNA genes that are not designed to target any rat mRNA for degradation, were bilaterally infused into the rat basal and lateral amygdala and subsequently these animals were auditory fear conditioned and fear memory was assessed. We found that the viruses designed to express control shRNAs and shRNAs designed to target known plasticity associated genes (i.e. Arc, Egr1 and GluN2A) were toxic and auditory Pavlovian fear conditioning was significantly impaired. Our results indicate that the impairments in Pavlovian fear conditioning were due to the viruses harboring shRNA genes and were not due to surgery, the virus itself or viral mediated GFP expression and these results were dose dependent. Unfortunately, even when significantly lower doses of shRNA harboring viruses were infused into the amygdala, impairments in fear conditioning were observed, which likely prevented the detection of gene specific deficits in fear learning. Here we report our findings and discuss the implications of these results.