Search

A barrier to advancing engineered adeno-associated viral vectors (AAVs) for precision access to cell subtypes is a lack of high-throughput, high-resolution assays to characterize in vivo transduction profiles. In this study, we developed an ultrasensitive, sequential fluorescence in situ hybridization (USeqFISH) method for spatial transcriptomic profiling of endogenous and viral RNA with a short barcode in intact tissue volumes by integrating hydrogel-based tissue clearing, enhanced signal amplification and multiplexing using sequential labeling. Using USeqFISH, we investigated the transduction and cell subtype tropisms across mouse brain regions of six systemic AAVs, including AAV-PHP.AX, a new variant that transduces robustly and efficiently across neurons and astrocytes. Here we reveal distinct cell subtype biases of each AAV variant, including a bias of AAV-PHP.N toward excitatory neurons. USeqFISH also enables profiling of pooled regulatory cargos, as we show for a 13-variant pool of microRNA target sites in AAV genomes. Lastly, we demonstrate potential applications of USeqFISH for in situ AAV profiling and multimodal single-cell analysis in non-human primates.

Recombinant adeno-associated viruses (AAVs) are commonly used gene delivery vehicles for neuroscience research. They have two engineerable features: the capsid (outer protein shell) and cargo (encapsulated genome). These features can be modified to enhance cell type or tissue tropism and control transgene expression, respectively. Several engineered AAV capsids with unique tropisms have been identified, including variants with enhanced central nervous system transduction, cell type specificity, and retrograde transport in neurons. Pairing these AAVs with modern gene regulatory elements and state-of-the-art reporter, sensor, and effector cargo enables highly specific transgene expression for anatomical and functional analyses of brain cells and circuits. Here, we discuss recent advances that provide a comprehensive (capsid and cargo) AAV toolkit for genetic access to molecularly defined brain cell types.

Adeno-associated viruses (AAVs) can enable robust and safe gene delivery to the mammalian central nervous system (CNS). While the scientific community has developed numerous neurotropic AAV variants for systemic gene-transfer to the rodent brain, there are few AAVs that efficiently access the CNS of higher order primates. We describe here AAV.CAP-Mac, an engineered AAV variant that enables systemic, brain-wide gene delivery in infants of two Old World primate species—the rhesus macaque (Macaca mulatta) and the green monkey (Chlorocebus sabaeus). We identified CAP-Mac using a multi-species selection strategy, initially screening our library in the adult common marmoset (Callithrix jacchus) and narrowing our pool of test-variants for another round of selection in infant macaques. In individual characterization, CAP-Mac robustly transduces human neurons in vitro and Old World primate neurons in vivo, where it targets all lobes of cortex, the cerebellum, and multiple subcortical regions of disease relevance. We use CAP-Mac for Brainbow-like multicolor labeling of macaque neurons throughout the brain, enabling morphological reconstruction of both medium spiny neurons and cortical pyramidal cells. Because of its broad distribution throughout the brain and high neuronal efficiency in infant Old World primates compared to AAV9, CAP-Mac shows promise for researchers and clinicians alike to unlock novel, noninvasive access to the brain for efficient gene transfer.

Genetic intervention is increasingly being explored as a therapeutic option for debilitating disorders of the central nervous system. The safety and efficacy of gene therapies rely upon expressing a transgene in affected cells while minimizing off-target expression. Here we show organ-specific targeting of adeno-associated virus (AAV) capsids after intravenous delivery, which we achieved by employing a Cre-transgenic-based screening platform and sequential engineering of AAV-PHP.eB between the surface-exposed AA452 and AA460 of VP3. From this selection, we identified capsid variants that were enriched in the brain and targeted away from the liver in C57BL/6J mice. This tropism extends to marmoset (Callithrix jacchus), enabling robust, non-invasive gene delivery to the marmoset brain after intravenous administration. Notably, the capsids identified result in distinct transgene expression profiles within the brain, with one exhibiting high specificity to neurons. The ability to cross the blood–brain barrier with neuronal specificity in rodents and non-human primates enables new avenues for basic research and therapeutic possibilities unattainable with naturally occurring serotypes.

Extracellular electrophysiology and functional MRI are complementary techniques that provide information about cellular and network-level neural activity, respectively. However, electrodes for electrophysiology are typically made from metals, which cause significant susceptibility artifacts on MR images. Previous work has demonstrated that insulated carbon fiber bundle electrodes reduce the volume of magnetic susceptibility artifacts and can be used to record local field potentials (LFP), but the relatively large diameter of the probes make them unsuitable for multi- and single-unit recordings. Although single carbon fiber electrodes have recently been used to record single-unit activity, these probes require modifications in order to aid insertion and the use of these probes in fMRI has yet to be validated. Therefore, there is a need for a single-carbon fiber electrode design that (1) minimizes the volume of the susceptibility artifact, (2) can record from a wide frequency band that includes LFP and multi- and single-unit recording, and (3) is practical to insert without additional modifications. Here, we demonstrate that carbon-fiber electrodes made from single carbon monofilaments (35 μm in diameter) meet all of these criteria. Carbon monofilament electrodes modified with the conductive polymer poly(3,4-ethylenedioxythiophene) (PEDOT) have lower impedances and higher signal-to-noise ratio recordings than platinum-iridium electrodes, a current gold standard for chronic single-unit recording. Furthermore, these probes distort a significantly smaller volume of voxels compared to tungsten and platinum-iridium electrodes in agarose phantom and in vivo MR images, leading to higher contrast-to-noise ratio in regions proximal to the electrode implantation site during fMRI. Collectively, this work establishes that carbon monofilaments are a practical choice for combined electrophysiology-fMRI experiments.

article i nfo Human ventral temporal cortex (VTC) plays a pivotal role in high-level vision. An under-studied macroanatomical feature of VTC is the mid-fusiform sulcus (MFS), as hallow longitudinal sulcus separating the lateral and medial fusiform gyrus (FG). Here, we quantified the morphological features of the MFS in 69 subjects (ages 7-40), and in- vestigated its relationship to both cytoarchitectonic and functional divisions of VTC with four main findings. First, despite being a minor sulcus, we found that the MFS is a stable macroanatomical structure present in all 138 hemi- spheres with morphological characteristics developed by age 7. Second, the MFS is the locus of a lateral-medial cytoarchitectonic transition within the posterior FG serving as the boundary between cytoarchitectonic regions FG1 and FG2. Third, the MFS predicts a lateral-medial functional transition in eccentricity bias representations in children, adolescents, and adults. Fourth, the anterior tip of the MFS predicts the location of a face-selective re- gion, mFus-faces/FFA-2. These findings are the fi rst to illustrate that am acroanatomical landmark identifies both cytoarchitectonic and functional divisions of high-levelsensory cortex in humans and have important implications for understanding functional and struc tural organization in the human brain.