Optimized CLARITY technique detects reduced parvalbumin density in a genetic model of schizophrenia.
BACKGROUND
Novel tissue clearing technologies have, for the first time, made it possible to study intact tissue samples. This approach provides a tool for further clarifying findings from animal models of schizophrenia by studying parvalbumin-positive (PV+) interneuron density from a 3D perspective.


METHODS
This study has developed an optimised CLARITY protocol, including an improved electrophoretic tissue clearing (ETC) chamber, an evaluation of antibody diffusion into cleared tissue slices, and a computational method for detecting PV+ interneurons in 3D.


RESULTS
A reduced PV+ interneuron density was found in both prelimbic and motor cortex regions of the Df(h15q13)/+ mice, while no changes were observed in the Df(h22q11)/+ mice.


METHODS
The developed ETC chamber enables tissue clearing of variable tissue sizes while minimizing the resistance. It was found that a high concentration of primary and secondary antibodies were necessary for sufficient antibody staining of PV+ interneurons. Additionally, the developed computational method showed improved detection rates of interneurons compared to non-processed image stacks.


CONCLUSIONS
Our optimization of the CLARITY technology and automated 3D counting of cells were found to be useful for quantification of PV+ interneuron density. The results may provide insight into understanding the pathophysiology underlying the phenotype observed in Df(h15q13)/+ mice.
Department of Synaptic transmission, H. Lundbeck A/S, Ottiliavej 7, 2500 Valby, Denmark. Electronic address: joak@lundbeck.com.
Joakim
J
Bastrup
Department of Synaptic transmission, H. Lundbeck A/S, Ottiliavej 7, 2500 Valby, Denmark. Electronic address: pehj@lundbeck.com.
Peter H
PH
Larsen
D000818:Animals; D002452:Cell Count / Q000295:instrumentation* / Q000379:methods; D002478:Cells, Cultured; D004586:Electrophoresis / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D019544:Equipment Failure Analysis; D007118:Immunoassay / Q000295:instrumentation / Q000379:methods; D007395:Interneurons / Q000378:metabolism* / Q000473:pathology*; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D010320:Parvalbumins / Q000378:metabolism*; D012559:Schizophrenia / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*
D010320:Parvalbumins
3D analysis; CLARITY; PARVALBUMIN; Schizophrenia