title: A fourth generation of neuroanatomical tracing techniques: exploiting the offspring of genetic engineering.
abstract: The first three generations of neuroanatomical tract-tracing methods include, respectively, techniques exploiting degeneration, retrograde cellular transport and anterograde cellular transport. This paper reviews the most recent development in third-generation tracing, i.e., neurochemical fingerprinting based on BDA tracing, and continues with an emerging tracing technique called here 'selective fluorescent protein expression' that in our view belongs to an entirely new 'fourth-generation' class. Tracing techniques in this class lean on gene expression technology designed to 'label' projections exclusively originating from neurons expressing a very specific molecular phenotype. Genetically engineered mice that express cre-recombinase in a neurochemically specific neuronal population receive into a brain locus of interest an injection of an adeno-associated virus (AAV) carrying a double-floxed promoter-eYFP DNA sequence. After transfection this sequence is expressed only in neurons metabolizing recombinase protein. These particular neurons promptly start manufacturing the fluorescent protein which then accumulates and labels to full detail all the neuronal processes, including fibers and terminal arborizations. All other neurons remain optically 'dark'. The AAV is not replicated by the neurons, prohibiting intracerebral spread of 'infection'. The essence is that the fiber projections of discrete subpopulations of neurochemically specific neurons can be traced in full detail. One condition is that the transgenic mouse strain is recombinase-perfect. We illustrate selective fluorescent protein expression in parvalbumin-cre (PV-cre) mice and choline acetyltransferase-cre (ChAT-cre) mice. In addition we compare this novel tracing technique with observations in brains of native PV mice and ChAT-GFP mice. We include a note on tracing techniques using viruses.
authors.affiliation: Department of Anatomy and Neurosciences, Neuroscience Campus Amsterdam, Vrije University Medical Center, Amsterdam, The Netherlands. Electronic address: fg.wouterlood@vumc.nl.
authors.forename: Floris G
authors.initials: FG
authors.lastname: Wouterlood
authors.affiliation: Department of Integrative Neurophysiology, Center for Neurogenomics and Cognitive Research (CNCR), Neuroscience Campus Amsterdam, VU University, Amsterdam, The Netherlands; Department of Brain and Cognitive Sciences, McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA, USA.
authors.forename: Bernard
authors.initials: B
authors.lastname: Bloem
authors.affiliation: Department of Integrative Neurophysiology, Center for Neurogenomics and Cognitive Research (CNCR), Neuroscience Campus Amsterdam, VU University, Amsterdam, The Netherlands.
authors.forename: Huibert D
authors.initials: HD
authors.lastname: Mansvelder
authors.affiliation: Department of Integrative Neurophysiology, Center for Neurogenomics and Cognitive Research (CNCR), Neuroscience Campus Amsterdam, VU University, Amsterdam, The Netherlands.
authors.forename: Antonio
authors.initials: A
authors.lastname: Luchicchi
authors.affiliation: Bioengineering Department, James E. Clark Center, Stanford University, Stanford, CA, USA.
authors.forename: Karl
authors.initials: K
authors.lastname: Deisseroth
mesh_terms: D000818:Animals; D001921:Brain / Q000033:anatomy & histology / Q000378:metabolism / Q000821:virology; D008822:Mice, Transgenic; D056347:Neuroanatomical Tract-Tracing Techniques / Q000379:methods*; D009474:Neurons / Q000166:cytology / Q000378:metabolism / Q000821:virology
keywords: Anterograde tracing; Cell type specific innervation; Cre-mice; Green fluorescent protein; Laser scanning; Neurochemical fingerprinting; Selective expression of fluorescent proteins; Tracing methods; Transgenic mice; Virus tracing