Table 2.

Selected recent advances in neurotechnologies for structural analysis of circuit architecture in rodent models.

A. Viral Vectors and Vector-Based Labeling Methods
Tool	Summary	Citation
rAAV2-retro	A recombinant AAV2 variant for retrograde targeting of projection neurons that can be used for both functional and tracing studies. Efficient retrograde transduction was observed in many cortical and subcortical regions.	[26]
PHP.eB and PHP.S	AAV9-based vectors for efficient CNS (PHP.eB) or PNS (PHP.S) transduction after peripheral (intravenous or retro-orbital) virus administration. Can be used with a titratable inducer vector for controlled sparseness of multicolor labels that preserves color diversity; inducers involve use of tetracycline-controlled transactivator (tTA) or Cre-dependent Flp-based constructs.	[29,57]
TRIO	Tracing the Relationship Between Input and Output; A combinatorial two-vector system that maps the input-output relationship of a population of neurons. In this method, canine adenovirus-2 (CAV-2) [25] is used to deliver a Flp recombinase transgene to axons in a specific projection terminal field for retrograde transduction of the cell bodies; Flp-dependent RVdG [24] component vectors are later delivered to the cell bodies for monosynaptic retrograde tracing of inputs. Cre-dependent Flp can be used for cell type-specific targeting using a Cre driver line (cTRIO). Cannot be used for functional studies due to lethality of RVdG.	[6]
INTERSECT	INTronic Recombinase Sites Enabling Combinatory Targeting; A two-component system that allows for functional projection targeting using Flp- and Cre-dependent viral vectors via axonal targeting in a downstream region using replication incompetent herpes simplex virus (HSV) [27] carrying a Cre-dependent Flp recombinase transgene. Either Cre- and Flp-ON and -OFF strategies can be used.	[5]
MAP-Seq	Multiplexed Analysis of Projections by Sequencing; allows for parallel mapping of single neuron axonal arbors via recovery of RNA barcodes in from terminal fields after delivery of AAV viral barcode libraries to the cell body. Does not distinguish fibers of passage, so downstream regions must be chosen carefully for RNA recovery and sequencing.	[56]
mGRASP	Mammalian GFP Reconstitution Across Synaptic Partners; A method for fluorescently labeling synaptic connections that employs AAV-mediated delivery of synapse-targeted split GFP fragments in genetically defined pre- and post-synaptic neuronal partners. Cre-ON and Cre-OFF strategies can be used for studying microcircuits.	[54,55]
B. Large Volume Imaging Modalities
Light Sheet Microscopy (LSM)	Originally developed over 100 years ago, LSM illuminates the sample with a thin sheet of light and detects the emitted fluorescent signal with an orthogonally arranged detection objective. Variants include CLARITY optimized LSM (COLM) for use in cleared tissue [76], SPED (Spherical Aberration-assisted Extended Depth of Field) LSM that improves scan speed via extended depth of field [68], and an adaptive LSM that integrates multiple fields of view with 10 degrees of freedom that are autonomously adjusted in real time for improved spatial resolution and image quality [11].	[10,11,68]
High-Speed Volumetric STP Tomography	High-speed Volumetric Serial Two-Photon Tomography; A high speed imaging platform based on Serial Two-Photon Tomography (STP) [13] that creates 3D reconstructions of neuronal axonal arbors via the integration of fast volumetric 2-photon microscopy and a vibrating microtome to image bright, sparsely labelled neurons in cleared samples embedded in gelatin. Includes computational tools for the registration and visualization of large (up to 100 TB) data sets, although labeling must be sufficiently sparse to prevent neurite reconstruction errors when axons from different neurons are closely positioned.	[12]
C. Tissue Clearing Methods
CLARITY	Hydrogel-based clearing method that utilizes 4% SDS for lipid removal after sample has been embedded in an acrylamide-bisacrylamide gel and cross-linked with formaldehyde. Clearing can be accelerated with electrophoresis at the expense of tissue integrity. Compatible with immunolabeling and endogenous fluorescence. The EDC-CLARITY variant is compatible with HCR (hybridization chain reaction) probes for bulk RNA labeling.	[75,76,86]
PACT PACT-deCAL	Passive CLARITY Technique; A passive CLARITY-based clearing method for rapid clearing of thick sections that employs 8% SDS as the detergent. Compatible with immunolabeling, endogenous fluorescence, smFISH (single molecule), and smHCR probes for single and bulk RNA labeling. Produces reversible expansion of tissue and can be used with RIMS (Reflective Index Matching Solution), a non-viscous mounting medium that decreases the refractive index of the sample for better optical access. PACT-deCAL uses EDTA/EGTA to decalcify samples for bone clearing.	[71,78,85,167]
PARS	Perfusion Assisted Agent Release In Situ; An active CLARITY-based clearing method that involves intracranial and/or transcardial perfusion of reagents for whole body clearing.	[71,78]
SWITCH	System-Wide Control of Interaction Time and Kinetics of Chemicals; A fixation and clearing method that exploits the pH dependence of glutaraldehyde-tissue gel formation for uniform fixation prior to delipidation with SDS. This method provides added tissue integrity for multiplexed immunolabeling. Not compatible with smFISH or smHCR probes.	[84]
uDISCO	A whole-body clearing method based on 3DISCO; it utilizes dehydration with tert-butanol followed by delipidation with diphenyl ether for fast sample clearing. Maintenance of endogenous fluorescence is improved relative to 3DISCO and other solvent based methods, whereby fluorescence deteriorates within several days after clearing. Shrinks tissues by approximately 40% for faster LSM imaging.	[79]
Sca/eS	An improved version of Sca/eA2 [168] that achieves tissue transparency via partial delipidation and hyperhydration via urea, sorbitol, glycerol, and Triton X-100. Preserves endogenous fluorescence and limits expansion better than most other methods, although large0 sample clearing can take several weeks. A simplified protocol Sca/eSQ can be used in thick (<500 micron) sections.	[72]
CUBIC	Clear, Unobstructed, Brain/Body Imaging Cocktails and Computational Analysis; A clearing method based on Sca/eA2 that uses urea, aminoalcohols, TRITON X-100, and high sucrose concentrations. Maintains endogenous fluorescence, can be perfused for whole body clearing, produces reversible tissue expansion, and exhibits superior decolorization (i.e. loss of the heme chromophore) relative to other techniques.	[73,74]
Tissue Expansion Methods for High Resolution Microscopy
ExM	Expansion Microscopy; A tissue expansion technology whereby the fixed and permeabilized sample is embedded in a superabsorbent hydrogel containing sodium acrylate and acrylamide, cross-linked with N-N′-methylenebisacrylamide, and digested with a protease to produce a 4.5-fold sample expansion. Newer variants display improved protein retention (proExM) and are compatible with immunolabeling, smFISH, and smHCR (ExFISH).	[80–82]
ePACT	Expansion PACT; Variant of the PACT tissue clearing method that utilizes a superabsorbent hydrogel and enzymatic digestion to increase sample size up to 5-fold for high resolution imaging with preserved endogenous fluorescence.	[78]
MAP	Magnified Analysis of Proteome; a hydrogel-based clearing method that expands tissue without the use of enzymatic digestion via treatment with high acrylamide concentrations (up to 20%) prior to SDS treatment. Compatible with immunolabeling but not RNA detection.	[83]