CaMKIIα promoter-controlled circuit manipulations target both pyramidal cells and inhibitory interneurons in cortical networks

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Key findings

• The CaMKIIα promoter delivered by AAV is not restricted to excitatory principal neurons: it drives reporter expression in at least five distinct, largely non-overlapping classes of cortical GABAergic interneuron. Five to six weeks after injecting AAV5-CaMKIIα-ChR2(H134R)-mCherry-WPRE-hGH into mouse hippocampal CA1, medial prefrontal cortex and basolateral amygdala, mCherry appears in parvalbumin, somatostatin, neuronal nitric oxide synthase, neuropeptide Y and cholecystokinin-expressing GABAergic cells — populations that together make up around 60% of the whole cortical inhibitory cell population.
• The fraction of interneurons captured by a CaMKIIα-driven AAV varies sharply with brain region and interneuron class, so leakage cannot be assumed uniform. With AAV5-CaMKIIα-ChR2(H134R)-mCherry, mCherry reaches as high as 88% of parvalbumin-immunostained interneurons in medial prefrontal cortex and basolateral amygdala against only 5% in hippocampus; somatostatin interneurons show 67% in basolateral amygdala against 3% in hippocampus; nNOS-expressing cells show 65% in basolateral amygdala, 27% in medial prefrontal cortex and 8% in hippocampus; NPY-expressing cells show 74% in basolateral amygdala, 56% in prefrontal cortex and 24% in hippocampus. Cholecystokinin-positive cells are the exception, labelled at a similar rate in all three areas (25%, 26% and 26%).
• Diluting the virus reduces interneuron labelling but does not abolish it where labelling is strongest. A tenfold dilution of AAV5-CaMKIIα-ChR2(H134R)-mCherry-WPRE-hGH in saline significantly lowers overall interneuron labelling (7 ± 38% versus 27 ± 48%, median ± interquartile range, p = 0.009, Mann-Whitney test on pooled data from all regions) and eliminates it in hippocampus, yet a considerable fraction of prefrontal cortex and basolateral amygdala interneurons still express mCherry.
• Interneuron labelling by the CaMKIIα promoter is independent of AAV serotype and transgene cargo. AAV9-CaMKIIα-hChR2(E123T/T159C)-mCherry and AAV9-CaMKIIα-ArchT(PV2527)-GFP label interneurons at rates statistically indistinguishable from AAV5-CaMKIIα-ChR2(H134R)-mCherry (33 ± 36% and 35 ± 20% versus 27 ± 48%, median ± interquartile range, p = 0.44 and 0.98, Mann-Whitney test), reaching as high as 77% of somatostatin interneurons in basolateral amygdala.
• AAV delivery amplifies CaMKIIα promoter leakage into GABAergic cells well beyond what a transgenic line shows, so transgenic data cannot be used to justify viral specificity. CaMKIIα-GFP transgenic mice contain significantly fewer GFP-expressing inhibitory cells than virus-injected cortex (1.5 ± 11.5% versus 27 ± 48%, median ± interquartile range, p < 0.001), with the highest ratio in basolateral amygdala somatostatin interneurons (34%), about 10% to 14% of nNOS- and NPY-expressing cells, and virtually no parvalbumin interneurons labelled in any region examined.
• Channelrhodopsin-2 expressed from the CaMKIIα promoter is functionally sufficient to drive interneuron firing, not merely present. With synaptic transmission blocked by the ionotropic glutamate receptor antagonist kynurenic acid and the chloride channel blocker picrotoxin, blue light evokes spikes in most interneurons recorded as well as in pyramidal neurons, including fast-spiking parvalbumin interneurons, somatostatin interneurons showing firing accommodation, and late-spiking NPY-expressing neurogliaform cells in hippocampus and medial prefrontal cortex. Across all recorded neurons, spike number varies linearly with the area of the light-evoked membrane voltage change, and some neurons show substantial subthreshold ChR2-mediated voltage responses without firing.
• Reporter immunodetection systematically underestimates functional opsin expression, so a negative reporter signal does not establish that an interneuron is unaffected by optogenetic stimulation. Parvalbumin and somatostatin interneurons in hippocampus and somatostatin interneurons in medial prefrontal cortex show no obvious mCherry even after immunostaining enhancement, yet spike readily on blue light delivery. Although the area of ChR2-evoked voltage responses differs significantly between interneurons with and without mCherry signal, firing can still be evoked in the mCherry-negative cells. For contrast, parvalbumin interneurons in medial prefrontal cortex show both large light-evoked responses and strong mCherry immunoreactivity, and mCherry content in basolateral amygdala somatostatin interneurons varies between cells.

Abstract

A key assumption in studies of cortical functions is that excitatory principal neurons, but not inhibitory cells express calcium/calmodulin-dependent protein kinase II subunit α (CaMKIIα) resulting in a widespread use of CaMKIIα promoter-driven protein expression for principal cell manipulation and monitoring their activities. Using neuroanatomical and electrophysiological methods we demonstrate that in addition to pyramidal neurons, multiple types of cortical GABAegic cells are targeted by adeno-associated viral vectors (AAV) driven by the CaMKIIα promoter in both male and female mice. We tested the AAV5 and AAV9 serotype of viruses with either Channelrhodopsin 2 (ChR2)-mCherry or Archaerhodopsin-T-green fluorescent protein (GFP) constructs, with different dilutions. We show that in all cases, the reporter proteins can visualize a large fraction of different interneuron types, including parvalbumin (PV), somatostatin (SST), neuronal nitric oxide synthase (nNOS), neuropeptide Y (NPY), and cholecystokinin (CCK)-containing GABAergic cells, which altogether cover around 60% of the whole inhibitory cell population in cortical structures. Importantly, the expression of the excitatory opsin Channelrhodopsin 2 in the interneurons effectively drive spiking of infected GABAergic cells even if the immunodetectability of reporter proteins is ambiguous. Thus, our results challenge the use of CaMKIIα promoter-driven protein expression as a selective tool in targeting cortical glutamatergic neurons using viral vectors.