TY  - JOUR
AB  - BACKGROUND: Activity-dependent markers such as c-Fos, a rapid indicator of neuronal activation, and GAD67, an enzyme essential for GABA synthesis in inhibitory neurons, are extensively employed to elucidate neural circuit dynamics. Given that many studies span extended periods with multiple experimental groups, it is crucial to ensure long-term storage of non-frozen brain tissue does not compromise immunodetection. NEW METHOD: Here, we evaluated the impact of storage duration on the immunodetection of c-Fos and GAD67 in rat brains. Intact brains, fixed in paraformaldehyde, were stored at 4 °C in phosphate-buffered saline with sodium azide to prevent bacterial growth. Brains were assessed at two storage durations - short (1.5 months) and prolonged (10 months). Brain sections were immunostained for c-Fos and GAD67 and imaged by confocal microscopy. RESULTS: We observed robust c-Fos immunoreactivity across multiple regions of the mPFC, hippocampus, and neocortex, with no significant differences attributable to storage duration. Additionally, quantifications of total GAD67-positive cells and cells co-labeled for c-Fos/ GAD67 confirmed that immunodetection of inhibitory neurons remains intact when whole brains are stored for up to 10 months. In contrast, prolonged storage of brain slices strongly reduced c-Fos, but increased GAD67 staining. COMPARISON WITH EXISTING METHOD(S): The stability of c-Fos and GAD67 in tissue stored long-term at 4 °C remains untested. CONCLUSIONS: These findings underscore that whereas intact brains can be safely stored for prolonged periods at 4°C without compromising antigenicity, brain slices are highly susceptible to storage-induced deterioration - insights with implications for planning and interpreting immunohistochemical studies in neuroscience.
AU  - Dimitrov, S.*
AU  - Shan, X.*
AU  - Born, J.
AU  - Inostroza, M.*
C1  - 75838
C2  - 58135
CY  - Radarweg 29, 1043 Nx Amsterdam, Netherlands
TI  - Impact of tissue storage time on immunodetection of c-Fos and GAD67 in the rat brain.
JO  - J. Neurosci. Methods
VL  - 425
PB  - Elsevier
PY  - 2025
SN  - 0165-0270
ER  - 

TY  - JOUR
AB  - Background: Dense and unbiased cellular-resolution representations of extended volumetric central nervous system soft-tissue anatomy are difficult to obtain, even in experimental post-mortem settings. Interestingly, X-ray phase-contrast computed tomography (X-PCI-CT), an emerging soft-tissue-sensitive volumetric imaging technique, can provide multiscale organ- to cellular-level morphological visualizations of neuroanatomical structure.New Method: Here, we tested different nervous-tissue fixation procedures, conventionally used for transmission electron microscopy, to better establish X-PCI-CT-specific sample-preparation protocols. Extracted rat spinal medullas were alternatively fixed with a standard paraformaldehyde-only aldehyde-based protocol, or in combination with glutaraldehyde. Some specimens were additionally post-fixed with osmium tetroxide. Multiscale X-PCI-CT datasets were collected at several synchrotron radiation facilities, using state-of-the-art setups with effective image voxel sizes of 3.0(3) to 0.3(3) mu m(3), and compared to high-field magnetic resonance imaging, histology and vascular fluorescence microscopy data.Results: Multiscale X-PCI-CT of aldehyde-fixed spinal cord specimens resulted in dense histology-like volumetric representations and quantifications of extended deep spinal micro-vascular networks and of intra-medullary cell populations. Osmium post-fixation increased intra-medullary contrast between white and gray-matter tissues, and enhanced delineation of intra-medullary cellular structure, e.g. axon fibers and motor neuron perikarya.Comparison with Existing Methods: Volumetric X-PCI-CT provides complementary contrast and higher spatial resolution compared to 9.4T MRI. X-PCI-CT's advantage over planar histology is the volumetric nature of the cellular-level data obtained, using samples much larger than those fit for volumetric vascular fluorescence microscopy.Conclusions: Deliberately choosing (post-)fixation protocols tailored for optimal nervous-tissue structural preservation is of paramount importance in achieving effective and targeted neuroimaging via the X-PCI-CT technique.
AU  - Barbone, G.E.*
AU  - Bravin, A.*
AU  - Mittone, A.*
AU  - Kraiger, M.
AU  - Hrabě de Angelis, M.
AU  - Bossi, M.*
AU  - Ballarini, E.*
AU  - Rodriguez-Menendez, V.*
AU  - Ceresa, C.*
AU  - Cavaletti, G.*
AU  - Coan, P.*
C1  - 59015
C2  - 47653
CY  - Radarweg 29, 1043 Nx Amsterdam, Netherlands
TI  - Establishing sample-preparation protocols for X-ray phase-contrast CT of rodent spinal cords: Aldehyde fixations and osmium impregnation.
JO  - J. Neurosci. Methods
VL  - 339
PB  - Elsevier
PY  - 2020
SN  - 0165-0270
ER  - 

TY  - JOUR
AB  - BACKGROUND: Generation and phenotyping of mutant mouse models continues to increase along with the search for the most efficient phenotyping tests. Here we asked if a combination of different locomotor tests is necessary for comprehensive locomotor phenotyping, or if a large data set from an automated gait analysis with the CatWalk system would suffice. NEW METHOD: First we endeavored to meaningfully reduce the large CatWalk data set by Principal Component Analysis (PCA) to decide on the most relevant parameters. We analyzed the influence of sex, body weight, genetic background and age. Then a combination of different locomotor tests was analyzed to investigate the possibility of redundancy between tests. RESULT: The extracted 10 components describe 80% of the total variance in the CatWalk, characterizing different aspects of gait. With these, effects of CatWalk version, sex, body weight, age and genetic background were detected. In addition, the PCA on a combination of locomotor tests suggests that these are independent without significant redundancy in their locomotor measures. COMPARISON WITH EXISTING METHODS: The PCA has permitted the refinement of the highly dimensional CatWalk (and other tests) data set for the extraction of individual component scores and subsequent analysis. CONCLUSION: The outcome of the PCA suggests the possibility to focus on measures of the front and hind paws, and one measure of coordination in future experiments to detect phenotypic differences. Furthermore, although the CatWalk is sensitive for detecting locomotor phenotypes pertaining to gait, it is necessary to include other tests for comprehensive locomotor phenotyping.
AU  - Zimprich, A.
AU  - Östereicher, M.A.
AU  - Becker, L.
AU  - Dirscherl, P.
AU  - Ernst, L.
AU  - Fuchs, H.
AU  - Gailus-Durner, V.
AU  - Garrett, L.
AU  - Giesert, F.
AU  - Glasl, L.
AU  - Hummel, A.M.
AU  - Rozman, J.
AU  - Hrabě de Angelis, M.
AU  - Vogt-Weisenhorn, D.
AU  - Wurst, W.
AU  - Hölter, S.M.
C1  - 51095
C2  - 42774
TI  - Analysis of locomotor behavior in the German Mouse Clinic.
JO  - J. Neurosci. Methods
PY  - 2017
SN  - 0165-0270
ER  - 

TY  - JOUR
AB  - Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson's disease. LRRK2 kinase activity is required for toxicity in neuronal cell cultures suggesting that selective kinase inhibitors may prevent neurodegeneration in patients. Directly monitoring LRRK2 activity in cells would be advantageous for the development of small molecule LRRK2 inhibitors. Here, we demonstrate that a monoclonal anti-LRRK2 antibody directed against the activation segment binds less efficiently to native LRRK2 protein in the presence of ATP-competitive LRRK2 inhibitors. Since kinase inhibitors prevent autophosphorylation and refolding of the activation segment, we hypothesize that the antibody preferentially binds to the active conformation of LRRK2 under native conditions.
AU  - Gillardon, F.*
AU  - Kremmer, E.
AU  - Froehlich, T.*
AU  - Ueffing, M.
AU  - Hengerer, B.*
AU  - Gloeckner, C.J.
C1  - 22920
C2  - 30958
SP  - 62-68
TI  - ATP-competitive LRRK2 inhibitors interfere with monoclonal antibody binding to the kinase domain of LRRK2 under native conditions. A method to directly monitor the active conformation of LRRK2?
JO  - J. Neurosci. Methods
VL  - 214
IS  - 1
PB  - Elsevier Science
PY  - 2013
SN  - 0165-0270
ER  - 

TY  - JOUR
AB  - The value of avian models in peripheral nerve research recently became substantiated by the immunobiological similarity of avian inflammatory demyelinating polyradiculoneuropathy to human Guillain-Barré syndrome providing an alternative animal model for experimental autoimmune neuritis. As electrophysiologic evaluation of nerve roots is essential part of the diagnosis of polyradiculoneuropathies in humans, it would be favourable to have similar research methods available for juvenile chickens. Hence, this study was performed (1) to establish a tool-set that allows for reproducible evaluation of the tibial/sciatic nerve and its nerve roots, (2) to achieve age-matched reference values, and (3) to trace the kinetics of peripheral nerve maturation within chickens. Nine chickens underwent serial electrodiagnostic examinations between the age of 6 and 15 weeks. Several methods of sensory and motor nerve fiber stimulation of the tibial/sciatic nerve were tested and modified or established. Ultimately, scalp-recorded somatosensory evoked potentials, compound muscle action potentials elicited by tibial/sciatic nerve electrical as well as spinal magnetic stimulation and motor nerve conduction velocity were available for tibial/sciatic nerve and nerve root evaluation in chickens. Base values were obtained for all investigations and parameters. Results indicated that the maturation of the nerve fibers is incomplete up to the age of 15 weeks. The methods tested here provide an excellent tool-set for quantitative tibial/sciatic nerve and nerve root assessment in avian polyradiculoneuropathies, especially within the scope of longitudinal monitoring of the disease course.
AU  - Bader, S.R.*
AU  - Fischer, A.*
AU  - Emrich, D.*
AU  - Jütting, U.
AU  - Weyh, T.*
AU  - Kaspers, B.*
AU  - Matiasek, K.
C1  - 6341
C2  - 28499
SP  - 342-349
TI  - Evaluation of lumbosacral nerve root conduction in chickens by electrophysiological testing including high-resolution spinal magnetic stimulation.
JO  - J. Neurosci. Methods
VL  - 194
IS  - 2
PB  - Elsevier
PY  - 2011
SN  - 0165-0270
ER  - 

TY  - JOUR
AB  - Striking inconsistencies between the results of morphometric and electrophysiologic examinations of the regenerating nerve were observed in a previous study featuring the bridging of a 14mm gap in the rat sciatic nerve. To shed light on this dichotomy, seven further rats were subjected to permanent sciatic nerve transection and assessed electrophysiologically, histologically and by retrograde axonal tracing at various postoperative intervals (1h to 8 weeks). The results of the histological examinations and retrograde tracing revealed that in spite of the fact that compound muscle action potentials could be recorded in the gastrocnemius muscle, no reinnervation of the gastrocnemius muscle, either physiological or aberrant, had actually taken place. Furthermore, it was established that the electrical activity recorded in the gastrocnemius muscle after stimulation of the proximal or distal stump is generated by surrounding hind limb muscles unaffected by denervation. These are stimulated either directly, or indirectly due to spreading of the impulse. It is therefore strongly recommended that caution should be exercised when interpreting recordings from the gastrocnemius muscle after stimulation of a regenerating sciatic nerve in laboratory rodents.
AU  - Rupp, A.*
AU  - Dornseifer, U.*
AU  - Fischer, A.*
AU  - Schmahl, W.*
AU  - Rodenacker, K.
AU  - Jütting, U.
AU  - Gais, P.
AU  - Biemer, E.*
AU  - Papadopulos, N. *
AU  - Matiasek, K.*
C1  - 744
C2  - 24745
SP  - 266-277
TI  - Electrophysiologic assessment of sciatic nerve regeneration in the rat: Surrounding limb muscles feature strongly in recordings from the gastrocnemius muscle.
JO  - J. Neurosci. Methods
VL  - 166
IS  - 2
PB  - Elsevier
PY  - 2007
SN  - 0165-0270
ER  - 

TY  - JOUR
AU  - Schneider, I.
AU  - Tirsch, W.S.
AU  - Faus-Kessler, T.
AU  - Becker, L.
AU  - Kling, E.
AU  - Busse, R.-L. A.
AU  - Bender, A.*
AU  - Feddersen, B.*
AU  - Tritschler, J.
AU  - Fuchs, H.
AU  - Gailus-Durner, V.
AU  - Englmeier, K.-H.
AU  - Hrabě de Angelis, M.
AU  - Klopstock, T.
C1  - 5247
C2  - 23874
SP  - 82-90
TI  - Systematic, standardized and comprehensive neurological phenotyping of inbred mice strains in the Germany Mouse Clinic.
JO  - J. Neurosci. Methods
VL  - 157
IS  - 1
PY  - 2006
SN  - 0165-0270
ER  - 

TY  - JOUR
AU  - Lipinski, H.-G.
C1  - 18712
C2  - 11815
TI  - Brain Tissue Slice Thickness Monitored by Ion Profile Measurement.
JO  - J. Neurosci. Methods
PY  - 1991
SN  - 0165-0270
ER  -