Disconnecting prefrontal cortical neurons from the ventral midline thalamus: Loss of specificity due to progressive neural toxicity of an AAV-Cre in the rat thalamus

The thalamic reuniens (Re) and rhomboid (Rh) nuclei are bidirectionally connected with the medial prefrontal cortex (mPFC) and the hippocampus (Hip). Fiber-sparing N-methyl-D-aspartate lesions of the ReRh disrupt cognitive functions, including persistence of certain memories. Because such lesions irremediably damage neurons interconnecting the ReRh with the mPFC and the Hip, it is impossible to know if one or both pathways contribute to memory persistence. Addressing such an issue requires selective, pathway-restricted and direction-specific disconnections. A recent method associates a retrograde adeno-associated virus (AAV) expressing Cre recombinase with an anterograde AAV expressing a Cre-dependent caspase, making such disconnection feasible by caspase-triggered apoptosis when both constructs meet intracellularly. We injected an AAVrg-Cre-GFP into the ReRh and an AAV5-taCasp into the mPFC. As expected, part of mPFC neurons died, but massive neurotoxicity of the AAVrg-Cre-GFP was found in ReRh, contrasting with normal density of DAPI staining. Other stainings demonstrated increasing density of reactive astrocytes and microglia in the neurodegeneration site. Reducing the viral titer (by a 4-fold dilution) and injection volume (to half) attenuated toxicity substantially, still with evidence for partial disconnection between mPFC and ReRh. There is an imperative need to verify potential collateral damage inherent in this type of approach, which is likely to distort interpretation of experimental data. Therefore, controls allowing to distinguish collateral phenotypic effects from those linked to the desired disconnection is essential. It is also crucial to know for how long neurons expressing the Cre-GFP protein remain operational post-infection. •Combined AAV vectors to disconnect cortical neurons from reuniens & rhomboid targets.•AAV-Cre-GFP vectors may produce undesired neurotoxicity where injected in the brain.•The damaged region is colonized by microglia and reactive astrocytes.•DAPI staining is misleading in verifying integrity in AAVrg-Cre-GFP-infected tissue.•AAV-Cre-GFP toxicity can be attenuated/circumvented by titer/volume reduction.