Viral vector‐mediated Cre recombinase expression in substantia nigra induces lesions of the nigrostriatal pathway associated with perturbations of dopamine‐related behaviors and hallmarks of programmed cell death

Abstract

Cre/loxP recombination is a widely used approach to study gene function in vivo, using mice models expressing the Cre recombinase under the control of specific promoters or through viral delivery of Cre‐expressing constructs. A profuse literature on transgenic mouse lines points out the deleterious effects of Cre expression in various cell types and tissues, presumably by acting on illegitimate loxP‐like sites present in the genome. However, most studies reporting the consequences of Cre‐lox gene invalidation often omit adequate controls to exclude the potential toxic effects of Cre, compromising the interpretation of data. In this study, we report the anatomical, neurochemical, and behavioral consequences in mice of adeno‐associated virus (AAV)‐mediated Cre expression in the dopaminergic nuclei substantia nigra, at commonly used viral titers (3 × 109 genome copies/0.3 μL or 2 × 109 genome copies/0.6 μL). We found that injecting AAV‐eGFP‐Cre into the SN engendered drastic and reproducible modifications of behavior, with increased basal locomotor activity as well as impaired locomotor response to cocaine compared to AAV‐eGFP‐injected controls. Cre expression in the SN induced a massive decrease in neuronal populations of both pars compacta and pars reticulata and dopamine depletion in the nigrostriatal pathway. This anatomical injury was associated with typical features of programmed cell death, including an increase in DNA break markers, evidence of apoptosis, and disrupted macroautophagy. These observations underscore the need for careful control of Cre toxicity in the brain and the reassessment of previous studies. In addition, our findings suggest that Cre‐mediated ablation may constitute an efficient tool to explore the function of specific cell populations and areas in the brain, and the impact of neurodegeneration in these populations.

Activation of Preoptic GABAergic or Glutamatergic Neurons Modulates Sleep-Wake Architecture, but Not Anesthetic State Transitions

“…This is important because we show that activation of glutamatergic neurons in the MnPO caused a sustained decrease in body temperature, indicating that we activated at least a proportion of the glutamatergic cells that also promote sleep. Contrasting our results with those obtained in control mice, it seems unlikely that tissue damage caused by the injection procedure or the vector itself [13] selectively lesioned the sleep-promoting cells, sparing a neuronal group that causes hypothermia. The fact that VLPO GABAergic neurons did not increase sleep during the light period is not inconsistent with prior studies.…”

“…Post hoc analysis revealed no differences in the amount of wake-like episodes between vehicle and CNO (p > 0.9999). To rule out any non-specific effect of CNO, an adverse effect from the vector injection (i.e., mechanical and/or inflammatory tissue damage), or Cre-associated toxicity [13] on anesthetic induction/recovery time, we conducted additional anesthesia experiments using the following groups of Vgat-Cre and Vglut2-Cre mice: (1) mice injected with the vector but without expression of hM3Dq receptors ( Figure S1; Vgat-Cre, n = 7 and Vglut2-Cre, n = 6); (2) sham operated Vgat-Cre (n = 6) and Vglut2-Cre (n = 5); and (3) naive mice (n = 5 in each mouse line; the results from both groups are shown in Figure S2). Relative to vehicle, 1.0 mg/kg CNO administration did not alter anesthetic state transitions in any of the three control groups.…”

The mammalian target of rapamycin (mTOR) kinase mediates haloperidol-induced cataleptic behavior

“…We next assessed the striatal motor functions in mTOR mutant and control mice 2 weeks after AAV-Cre-GFP or AAV-GFP-control injections. As Cre-recombinase injection in the brain may affect behaviors 26 , we have included an additional control group for Cre: WT [C57BL/6] mice injected with AAV-Cre-GFP (Cre-control) or AAV-GFP (GFP-control) in all of our longitudinal behavioral analyses.…”

Old neurochemical markers, new functional directions?

“…Despite these issues, postmortem measurement of neurotransmitters remains a convenient and precise method with specific advantages for addressing many questions. Significantly, this approach confers the potential to quantify multiple neurotransmitters and metabolites across the CNS and subterritories of specific brain regions [see for instance the publication of one of the authors (Pifl, Schingnitz, & Hornykiewicz, )], permitting the assessment of neurotransmitter systems in different conditions (Rezai Amin et al, ) and between individuals in distinct cohorts (Dellu‐Hagedorn, Rivalan, Fitoussi, & Deurwaerdere, ; Schepisi et al, ). Here Hörtnagl et al () show that these values can be determined with anatomical precision and permit elucidation of potential relationships between pairs of brain regions that could uncover unsuspected information on functional organization.…”

“…(b) Among the heterologous controls, adjacent striatal territories, together with corticostriatal projections, could play a role in regulating the biochemical activity of DA terminals, due to the ascending striatonigrostriatal loops from the shell of the nucleus accumbens to the putamen (Adapted from Haruno & Kawato, 2006). metabolites across the CNS and subterritories of specific brain regions [see for instance the publication of one of the authors (Pifl, Schingnitz, & Hornykiewicz, 1991)], permitting the assessment of neurotransmitter systems in different conditions (Rezai Amin et al, 2019) and between individuals in distinct cohorts (Dellu-Hagedorn, Rivalan, Fitoussi, & Deurwaerdere, 2018;Schepisi et al, 2014). Here Hörtnagl et al (2019) show that these values can be determined with anatomical precision and permit elucidation of potential relationships between pairs of brain regions that could uncover unsuspected information on functional organization.…”