Regional Cellular Environment Shapes Phenotypic Variations of Hippocampal and Neocortical Chandelier Cells.

Different cortical regions processing distinct information, such as the hippocampus and the neocortex, share common cellular components and circuit motifs but form unique networks by modifying these cardinal units. Cortical circuits include diverse types of GABAergic interneurons (INs) that shape activity of excitatory principal neurons (PNs). Canonical IN types conserved across distinct cortical regions have been defined by their morphological, electrophysiological, and neurochemical properties. However, it remains largely unknown whether canonical IN types undergo specific modifications in distinct cortical regions and display "regional variants." It is also poorly understood whether such phenotypic variations are shaped by early specification or regional cellular environment. The chandelier cell (ChC) is a highly stereotyped IN type that innervates axon initial segments of PNs and thus serves as a good model with which to address this issue. Here, we show that Cadherin-6 (Cdh6), a homophilic cell adhesion molecule, is a reliable marker of ChCs and Cdh6-CreER mice (both sexes) provide genetic access to hippocampal ChCs (h-ChCs). We demonstrate that, compared with neocortical ChCs (nc-ChCs), h-ChCs cover twice as much area and innervate twice as many PNs. Interestingly, a subclass of h-ChCs exhibits calretinin (CR) expression, which is not found in nc-ChCs. Furthermore, we find that h-ChCs appear to be born earlier than nc-ChCs. Surprisingly, despite the difference in temporal origins, ChCs display host-region-dependent axonal/synaptic organization and CR expression when transplanted heterotopically. These results suggest that local cellular environment plays a critical role in shaping terminal phenotypes of regional IN variants in the hippocampus and the neocortex.SIGNIFICANCE STATEMENT Canonical interneuron (IN) types conserved across distinct cortical regions such as the hippocampus and the neocortex are defined by morphology, physiology, and gene expression. However, it remains unknown whether they display phenotypic variations in different cortical regions. In addition, it is unclear whether terminal phenotypes of regional IN variants belonging to a canonical IN type are determined intrinsically or extrinsically. Our results provide evidence of striking differences in axonal/synaptic organization and calretinin expression between hippocampal chandelier cells (ChCs) and neocortical ChCs. They also reveal that local cellular environment in distinct cortical regions regulates these terminal phenotypes. Therefore, our study suggests that local cortical environment shapes the phenotypes of regional IN variants, which may be required for unique circuit operations in distinct cortical regions.

Wild-type neural progenitors divide and differentiate normally in an amyloid-rich environment.

Adult neurogenesis is modulated by a balance of extrinsic signals and intrinsic responses that maintain production of new granule cells in the hippocampus. Disorders that disrupt the proliferative niche can impair this process, and alterations in adult neurogenesis have been described in human autopsy tissue and transgenic mouse models of Alzheimer's disease. Because exogenous application of aggregated Aß peptide is neurotoxic in vitro and extracellular Aß deposits are the main pathological feature recapitulated by mouse models, cell-extrinsic effects of Aß accumulation were thought to underlie the breakdown of hippocampal neurogenesis observed in Alzheimer's models. We tested this hypothesis using a bigenic mouse in which transgenic expression of APP was restricted to mature projection neurons. These mice allowed us to examine how wild-type neural progenitor cells responded to high levels of Aß released from neighboring granule neurons. We find that the proliferation, determination, and survival of hippocampal adult-born granule neurons are unaffected in the APP bigenic mice, despite abundant amyloid pathology and robust neuroinflammation. Our findings suggest that Aß accumulation is insufficient to impair adult hippocampal neurogenesis, and that factors other than amyloid pathology may account for the neurogenic deficits observed in transgenic models with more widespread APP expression.

Strain background influences neurotoxicity and behavioral abnormalities in mice expressing the tetracycline transactivator.

The tet-off system has been widely used to create transgenic models of neurological disorders including Alzheimer's, Parkinson's, Huntington's, and prion disease. The utility of this system lies in the assumption that the tetracycline transactivator (TTA) acts as an inert control element and does not contribute to phenotypes under study. Here we report that neuronal expression of TTA can affect hippocampal cytoarchitecture and behavior in a strain-dependent manner. While studying neurodegeneration in two tet-off Alzheimer's disease models, we unexpectedly discovered neuronal loss within the dentate gyrus of single transgenic TTA controls. Granule neurons appeared most sensitive to TTA exposure during postnatal development, and doxycycline treatment during this period was neuroprotective. TTA-induced degeneration could be rescued by moving the transgene onto a congenic C57BL/6J background and recurred on reintroduction of either CBA or C3H/He backgrounds. Quantitative trait analysis of B6C3 F2 TTA mice identified a region on Chromosome 14 that contains a major modifier of the neurodegenerative phenotype. Although B6 mice were resistant to degeneration, they were not ideal for cognitive testing. F1 offspring of TTA C57BL/6J and 129X1/SvJ, FVB/NJ, or DBA/1J showed improved spatial learning, but TTA expression caused subtle differences in contextual fear conditioning on two of these backgrounds, indicating that strain and genotype can interact independently under different behavioral settings. All model systems have limitations that should be recognized and mitigated where possible; our findings stress the importance of mapping the effects caused by TTA alone when working with tet-off models.

Intersectional monosynaptic tracing for dissecting subtype-specific organization of GABAergic interneuron inputs.

Functionally and anatomically distinct cortical substructures, such as areas or layers, contain different principal neuron (PN) subtypes that generate output signals representing particular information. Various types of cortical inhibitory interneurons (INs) differentially but coordinately regulate PN activity. Despite a potential determinant for functional specialization of PN subtypes, the spatial organization of IN subtypes that innervate defined PN subtypes remains unknown. Here we develop a genetic strategy combining a recombinase-based intersectional labeling method and rabies viral monosynaptic tracing, which enables subtype-specific visualization of cortical IN ensembles sending inputs to defined PN subtypes. Our approach reveals not only cardinal but also underrepresented connections between broad, non-overlapping IN subtypes and PNs. Furthermore, we demonstrate that distinct PN subtypes defined by areal or laminar positions display different organization of input IN subtypes. Our genetic strategy will facilitate understanding of the wiring and developmental principles of cortical inhibitory circuits at unparalleled levels.

Brain-wide distribution of reporter expression in five transgenic tetracycline-transactivator mouse lines.

The spatial pattern of transgene expression in tetracycline-controlled mouse models is governed primarily by the driver line used to introduce the tetracycline-controlled transactivator (tTA). Detailed maps showing where each tTA driver activates expression are therefore essential for designing and using tet-regulated models, particularly in brain research where cell type and regional specificity determine the circuits affected by conditional gene expression. We have compiled a comprehensive online repository of serial microscopic images showing brain-wide reporter expression for five commonly used tTA driver lines. We have spatially registered all images to a common three-dimensional mouse brain anatomical reference atlas for direct comparison of spatial distribution across lines. The high-resolution images and associated metadata are shared via the web page of the EU Human Brain Project. Images can be inspected using an interactive viewing tool that includes an optional overlay feature providing anatomical delineations and reference atlas coordinates. Interactive viewing is supplemented by semi-quantitative analyses of expression levels within anatomical subregions for each tTA driver line.

Humanized Tau Mice with Regionalized Amyloid Exhibit Behavioral Deficits but No Pathological Interaction.

Alzheimer's disease (AD) researchers have struggled for decades to draw a causal link between extracellular Aß aggregation and intraneuronal accumulation of microtubule-associated protein tau. The amyloid cascade hypothesis posits that Aß deposition promotes tau hyperphosphorylation, tangle formation, cell loss, vascular damage, and dementia. While the genetics of familial AD and the pathological staging of sporadic disease support this sequence of events, attempts to examine the molecular mechanism in transgenic animal models have largely relied on models of other inherited tauopathies as the basis for testing the interaction with Aß. In an effort to more accurately model the relationship between Aß and wild-type tau in AD, we intercrossed mice that overproduce human Aß with a tau substitution model in which all 6 isoforms of the human protein are expressed in animals lacking murine tau. We selected an amyloid model in which pathology was biased towards the entorhinal region so that we could further examine whether the anticipated changes in tau phosphorylation occurred at the site of Aß deposition or in synaptically connected regions. We found that Aß and tau had independent effects on locomotion, learning, and memory, but found no behavioral evidence for an interaction between the two transgenes. Moreover, we saw no indication of amyloid-induced changes in the phosphorylation or aggregation of human tau either within the entorhinal area or elsewhere. These findings suggest that robust amyloid pathology within the medial temporal lobe has little effect on the metabolism of wild type human tau in this model.

Transgene expression in the Nop-tTA driver line is not inherently restricted to the entorhinal cortex.

The entorhinal cortex (EC) plays a central role in episodic memory and is among the earliest sites of neurodegeneration and neurofibrillary tangle formation in Alzheimer's disease. Given its importance in memory and dementia, the ability to selectively modulate gene expression or neuronal function in the EC is of widespread interest. To this end, several recent studies have taken advantage of a transgenic line in which the tetracycline transactivator (tTA) was placed under control of the neuropsin (Nop) promoter to limit transgene expression within the medial EC and pre-/parasubiculum. Although the utility of this driver is contingent on its spatial specificity, no detailed neuroanatomical analysis of its expression has yet been conducted. We therefore undertook a systematic analysis of Nop-tTA expression using a lacZ reporter and have made the complete set of histological sections available through the Rodent Brain Workbench tTA atlas, www.rbwb.org . Our findings confirm that the highest density of tTA expression is found in the EC and pre-/parasubiculum, but also reveal considerable expression in several other cortical areas. Promiscuous transgene expression may account for the appearance of pathological protein aggregates outside of the EC in mouse models of Alzheimer's disease using this driver, as we find considerable overlap between sites of delayed amyloid deposition and regions with sparse ß-galactosidase reporter labeling. While different tet-responsive lines can display individual expression characteristics, our results suggest caution when designing experiments that depend on precise localization of gene products controlled by the Nop-tTA or other spatially restrictive transgenic drivers.

Impaired Recall of Positional Memory following Chemogenetic Disruption of Place Field Stability.

The neural network of the temporal lobe is thought to provide a cognitive map of our surroundings. Functional analysis of this network has been hampered by coarse tools that often result in collateral damage to other circuits. We developed a chemogenetic system to temporally control electrical input into the hippocampus. When entorhinal input to the perforant path was acutely silenced, hippocampal firing patterns became destabilized and underwent extensive remapping. We also found that spatial memory acquired prior to neural silencing was impaired by loss of input through the perforant path. Together, our experiments show that manipulation of entorhinal activity destabilizes spatial coding and disrupts spatial memory. Moreover, we introduce a chemogenetic model for non-invasive neuronal silencing that offers multiple advantages over existing strategies in this setting.

Specificity and efficiency of reporter expression in adult neural progenitors vary substantially among nestin-CreER(T2) lines.

Transgenic lines expressing a controllable form of Cre recombinase have become valuable tools for manipulating gene expression in adult neural progenitors and their progeny. Neural progenitors express several proteins that distinguish them from mature neurons, and the promoters for these genes have been co-opted to produce selective transgene expression within this population. To date, nine CreER(T2) transgenic lines have been designed using the nestin promoter; however, only a subset are capable of eliciting expression within both neurogenic zones of the adult brain. Here we compare three such nestin-CreER(T2) lines to evaluate specificity of expression and efficiency of recombination. Each line was examined by using three different Cre reporter strains that varied in sensitivity. We found that all three nestin-CreER(T2) strains induced reporter expression within the main neurogenic areas, albeit to varying degrees depending on the reporter. Unexpectedly, we found that two of the three lines induced substantial reporter expression outside of neurogenic areas. These lines produced strong labeling in cerebellar granule neurons, with additional expression in the cortex, hippocampus, striatum, and thalamus. Reporter expression in the third nestin-CreER(T2) line was considerably more specific, but was also less efficient, labeling a smaller percentage of the target population than the other two drivers. Our findings suggest that each nestin-CreER(T2) line may best serve different experimental needs, depending on whether specificity or efficiency is of greatest concern. Our study further demonstrates that each new pair of driver and responder lines should be evaluated independently, as both components can significantly influence the resulting expression pattern.