Comparison of bNOS and chat immunohistochemistry in the laterodorsal tegmentum (LDT) and the pedunculopontine tegmentum (PPT) of the mouse from brain slices prepared for electrophysiology

Maxime Veleanu; Tina E Axen; Morten P Kristensen; Kristi A Kohlmeier

doi:10.1016/j.jneumeth.2016.01.020

Comparison of bNOS and chat immunohistochemistry in the laterodorsal tegmentum (LDT) and the pedunculopontine tegmentum (PPT) of the mouse from brain slices prepared for electrophysiology

Maxime Veleanu, Tina E Axen, Morten P Kristensen, Kristi A Kohlmeier

8 Citations (Scopus)

Abstract

BACKGROUND: Identification of cell phenotype from brain slices upon which in vitro electrophysiological recordings have been performed often relies on conducting post hoc immunohistochemistry on tissue that necessarily has not been ideally prepared for immunohistochemical procedures. In such studies, antibody labeling against neuronal nitric oxide synthase (bNOS) has been used to identify cholinergic neurons of the laterodorsal tegmental nucleus (LDT) and the pedunculopontine tegmental nuclei (PPT), two brainstem nuclei importantly involved in arousal. However, a widespread perception maintains that antibody staining for enzymes involved in synthesis or transport, of acetylcholine would be a more definitive marker and hence, preferable.

NEW METHOD: Colocalization of bNOS and CHAT in the LDT/PPT, and presence of parvalbumin (PV), was examined in non-ideally prepared mouse brain slices using currently available antibodies.

RESULTS: Using fluorescent-based immunohistochemistry in LDT/PPT slices prepared for in vitro recordings, a near 100% colocalization of bNOS and CHAT was observed.

COMPARISON WITH EXISTING METHOD: We confirm in the mouse, findings of near 100% colocalization of bNOS and CHAT in the LDT/PPT, and we expand upon data from rat studies using optimally prepared tissue, that for dendritic visualization, bNOS staining exceeded the quality of CHAT staining for visualization of a higher degree of detail of fine processes. PV is not highly present in the mouse LDT/PPT.

CONCLUSION: CHAT and bNOS are equally useful target proteins for immunofluorescent identification of cholinergic LDT/PPT cells in mouse brain slices prepared for in vitro recordings, however, antibody targeting of bNOS allows for a superior appreciation of structural detail.

Original language	English
Journal	Journal of Neuroscience Methods
Volume	263
Pages (from-to)	23-35
Number of pages	13
ISSN	0165-0270
DOIs	https://doi.org/10.1016/j.jneumeth.2016.01.020
Publication status	Published - 1 Apr 2016

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Journal Article
Research Support, Non-U.S. Gov't

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10.1016/j.jneumeth.2016.01.020

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Comparison of bNOS and chat immunohistochemistry in the laterodorsal tegmentum (LDT) and the pedunculopontine tegmentum (PPT) of the mouse from brain slices prepared for electrophysiology. / Veleanu, Maxime; Axen, Tina E; Kristensen, Morten P et al.
In: Journal of Neuroscience Methods, Vol. 263, 01.04.2016, p. 23-35.

Research output: Contribution to journal › Journal article › Research › peer-review

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title = "Comparison of bNOS and chat immunohistochemistry in the laterodorsal tegmentum (LDT) and the pedunculopontine tegmentum (PPT) of the mouse from brain slices prepared for electrophysiology",

abstract = "BACKGROUND: Identification of cell phenotype from brain slices upon which in vitro electrophysiological recordings have been performed often relies on conducting post hoc immunohistochemistry on tissue that necessarily has not been ideally prepared for immunohistochemical procedures. In such studies, antibody labeling against neuronal nitric oxide synthase (bNOS) has been used to identify cholinergic neurons of the laterodorsal tegmental nucleus (LDT) and the pedunculopontine tegmental nuclei (PPT), two brainstem nuclei importantly involved in arousal. However, a widespread perception maintains that antibody staining for enzymes involved in synthesis or transport, of acetylcholine would be a more definitive marker and hence, preferable.NEW METHOD: Colocalization of bNOS and CHAT in the LDT/PPT, and presence of parvalbumin (PV), was examined in non-ideally prepared mouse brain slices using currently available antibodies.RESULTS: Using fluorescent-based immunohistochemistry in LDT/PPT slices prepared for in vitro recordings, a near 100% colocalization of bNOS and CHAT was observed.COMPARISON WITH EXISTING METHOD: We confirm in the mouse, findings of near 100% colocalization of bNOS and CHAT in the LDT/PPT, and we expand upon data from rat studies using optimally prepared tissue, that for dendritic visualization, bNOS staining exceeded the quality of CHAT staining for visualization of a higher degree of detail of fine processes. PV is not highly present in the mouse LDT/PPT.CONCLUSION: CHAT and bNOS are equally useful target proteins for immunofluorescent identification of cholinergic LDT/PPT cells in mouse brain slices prepared for in vitro recordings, however, antibody targeting of bNOS allows for a superior appreciation of structural detail.",

keywords = "Journal Article, Research Support, Non-U.S. Gov't",

author = "Maxime Veleanu and Axen, {Tina E} and Kristensen, {Morten P} and Kohlmeier, {Kristi A}",

year = "2016",

month = apr,

day = "1",

doi = "10.1016/j.jneumeth.2016.01.020",

language = "English",

volume = "263",

pages = "23--35",

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T1 - Comparison of bNOS and chat immunohistochemistry in the laterodorsal tegmentum (LDT) and the pedunculopontine tegmentum (PPT) of the mouse from brain slices prepared for electrophysiology

AU - Veleanu, Maxime

AU - Axen, Tina E

AU - Kristensen, Morten P

AU - Kohlmeier, Kristi A

PY - 2016/4/1

Y1 - 2016/4/1

N2 - BACKGROUND: Identification of cell phenotype from brain slices upon which in vitro electrophysiological recordings have been performed often relies on conducting post hoc immunohistochemistry on tissue that necessarily has not been ideally prepared for immunohistochemical procedures. In such studies, antibody labeling against neuronal nitric oxide synthase (bNOS) has been used to identify cholinergic neurons of the laterodorsal tegmental nucleus (LDT) and the pedunculopontine tegmental nuclei (PPT), two brainstem nuclei importantly involved in arousal. However, a widespread perception maintains that antibody staining for enzymes involved in synthesis or transport, of acetylcholine would be a more definitive marker and hence, preferable.NEW METHOD: Colocalization of bNOS and CHAT in the LDT/PPT, and presence of parvalbumin (PV), was examined in non-ideally prepared mouse brain slices using currently available antibodies.RESULTS: Using fluorescent-based immunohistochemistry in LDT/PPT slices prepared for in vitro recordings, a near 100% colocalization of bNOS and CHAT was observed.COMPARISON WITH EXISTING METHOD: We confirm in the mouse, findings of near 100% colocalization of bNOS and CHAT in the LDT/PPT, and we expand upon data from rat studies using optimally prepared tissue, that for dendritic visualization, bNOS staining exceeded the quality of CHAT staining for visualization of a higher degree of detail of fine processes. PV is not highly present in the mouse LDT/PPT.CONCLUSION: CHAT and bNOS are equally useful target proteins for immunofluorescent identification of cholinergic LDT/PPT cells in mouse brain slices prepared for in vitro recordings, however, antibody targeting of bNOS allows for a superior appreciation of structural detail.

AB - BACKGROUND: Identification of cell phenotype from brain slices upon which in vitro electrophysiological recordings have been performed often relies on conducting post hoc immunohistochemistry on tissue that necessarily has not been ideally prepared for immunohistochemical procedures. In such studies, antibody labeling against neuronal nitric oxide synthase (bNOS) has been used to identify cholinergic neurons of the laterodorsal tegmental nucleus (LDT) and the pedunculopontine tegmental nuclei (PPT), two brainstem nuclei importantly involved in arousal. However, a widespread perception maintains that antibody staining for enzymes involved in synthesis or transport, of acetylcholine would be a more definitive marker and hence, preferable.NEW METHOD: Colocalization of bNOS and CHAT in the LDT/PPT, and presence of parvalbumin (PV), was examined in non-ideally prepared mouse brain slices using currently available antibodies.RESULTS: Using fluorescent-based immunohistochemistry in LDT/PPT slices prepared for in vitro recordings, a near 100% colocalization of bNOS and CHAT was observed.COMPARISON WITH EXISTING METHOD: We confirm in the mouse, findings of near 100% colocalization of bNOS and CHAT in the LDT/PPT, and we expand upon data from rat studies using optimally prepared tissue, that for dendritic visualization, bNOS staining exceeded the quality of CHAT staining for visualization of a higher degree of detail of fine processes. PV is not highly present in the mouse LDT/PPT.CONCLUSION: CHAT and bNOS are equally useful target proteins for immunofluorescent identification of cholinergic LDT/PPT cells in mouse brain slices prepared for in vitro recordings, however, antibody targeting of bNOS allows for a superior appreciation of structural detail.

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1016/j.jneumeth.2016.01.020

DO - 10.1016/j.jneumeth.2016.01.020

M3 - Journal article

C2 - 26820905

SN - 0165-0270

VL - 263

SP - 23

EP - 35

JO - Journal of Neuroscience Methods

JF - Journal of Neuroscience Methods

ER -

Comparison of bNOS and chat immunohistochemistry in the laterodorsal tegmentum (LDT) and the pedunculopontine tegmentum (PPT) of the mouse from brain slices prepared for electrophysiology

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