Confocal Laser Scanning: of Instrument, Computer Processing, and Men

The mouse is a useful and popular model for studying of visual cortical function. To facilitate the translation of results from mice to primates, it is important to establish the extent of cortical organization equivalence between species and to identify possible differences. We focused on the different types of interneurons as defined by calcium binding protein (CBP) expression in the layers of primary visual cortex (V1) in mouse and rhesus macaque. CBPs parvalbumin (PV), calbindin (CB), and calretinin (CR) provide a standard, largely non-overlapping, labelling scheme in macaque, with preserved corresponding morphologies in mouse, despite a slightly higher overlap. Other protein markers, that are relevant in mouse, are not preserved in macaque. We fluorescently tagged CBPs in V1 of both species, using antibodies raised against preserved aminoacid sequences. Our data demonstrate important similarities between the expression patterns of interneuron classes in the different layers between rodents and primates. However, in macaque, expression of parvalbumin and calbindin is more abundant, calretinin expression is lower, and the laminar distribution of interneuron populations is more differentiated. Our results reveal an integrated view of interneuron types that provides a basis for translating results from rodents to primates, and suggest a reconciliation of previous results.

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“…We adapted an objective method ( Wouterlood et al. 2008 ; Beliën and Wouterlood 2012 ; Kooijmans et al. 2014 ) to count cell bodies ( Fig.…”

Section: Methodsmentioning

confidence: 99%

A Quantitative Comparison of Inhibitory Interneuron Size and Distribution between Mouse and Macaque V1, Using Calcium-Binding Proteins

Kooijmans

Sierhuis

Self

et al. 2020

Cerebral Cortex Communications

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“…After threshold analysis, we conducted voxel matrix analysis (SCIL_Image, TNO, Delft, The Netherlands). Scripts were used to instruct the software to determine whether conspicuous pairs of boutons and 3D objects fulfilled criteria to be counted as “contacts” (Beliën & Wouterlood, ). Briefly, structure A defined by its voxel matrix (matrix A, e.g., a bouton recorded in one channel of the confocal instrument) was considered to be in apposition with structure B (e.g., a dendrite recorded in a complementary channel in the confocal instrument and defined by voxel matrix B) if a minimum of 100 voxels overlap was reported by the software of the involved voxel matrices (A) and (B).…”

Section: Methodsmentioning

confidence: 99%

“…Briefly, structure A defined by its voxel matrix (matrix A, e.g., a bouton recorded in one channel of the confocal instrument) was considered to be in apposition with structure B (e.g., a dendrite recorded in a complementary channel in the confocal instrument and defined by voxel matrix B) if a minimum of 100 voxels overlap was reported by the software of the involved voxel matrices (A) and (B). This criterion is extensively discussed in Beliën and Wouterlood () and in Wouterlood and Beliën (). Also for colocalization of fluorescence signal in structures, a quantitative criterion was applied: “colocalization” was reported by the software if a minimum of 200 voxels of the voxel matrix of a 3D object (e.g., a bouton) recorded in one channel in the confocal instrument coincided with the voxel matrix belonging to a 3D object scanned in a complementary channel (e.g., an aggregate of VGAT signal).…”

Section: Methodsmentioning

confidence: 99%

“…Appropriate isodensity envelope thresholds were calculated by processing the images with the public‐domain program ImageJ (Rasband, 1997–), exploiting the plugin “3D object counter” (Fabrice Cordelières, Institut Curie, Orsay, France; see Bolte & Cordelières, ; procedure explained and discussed by Beliën and Wouterlood, ; Wouterlood & Beliën, ). Output consisted of thresholds defining the isodensity envelopes of boutons and 3D objects in their respective voxel matrices.…”

Section: Methodsmentioning

confidence: 99%

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Mesencephalic dopamine neurons interfacing the shell of nucleus accumbens and the dorsolateral striatum in the rat

Wouterlood

Engel

Daal

et al. 2018

J of Neuroscience Research

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Parallel corticostriatonigral circuits have been proposed that separately process motor, cognitive, and emotional‐motivational information. Functional integration requires that interactions exist between neurons participating in these circuits. This makes it imperative to study the complex anatomical substrate underlying corticostriatonigral circuits. It has previously been proposed that dopaminergic neurons in the ventral mesencephalon may play a role in this circuit interaction. Therefore, we studied in rats convergence of basal ganglia circuits by depositing an anterograde neuroanatomical tracer into the ventral striatum together with a retrograde fluorescent tracer ipsilaterally in the dorsolateral striatum. In the mesencephalon, using confocal microscopy, we looked for possible appositions of anterogradely labeled fibers and retrogradely labeled neurons, “enhancing” the latter via intracellular injection of Lucifer Yellow. Tyrosine hydroxylase (TH) immunofluorescence served to identify dopaminergic neurons. In neurophysiological experiments, we combined orthodromic stimulation in the medial ventral striatum with recording from ventral mesencephalic neurons characterized by antidromic stimulation from the dorsal striatum. We observed terminal fields of anterogradely labeled fibers that overlap populations of retrogradely labeled nigrostriatal cell bodies in the substantia nigra pars compacta and lateral ventral tegmental area (VTA), with numerous close appositions between boutons of anterogradely labeled fibers and nigrostriatal, TH‐immunopositive neurons. Neurophysiological stimulation in the medial ventral striatum caused inhibition of dopaminergic nigrostriatal neurons projecting to the ventrolateral striatal territory. Responding nigrostriatal neurons were located in the medial substantia nigra and adjacent VTA. Our results strongly suggest a functional link between ventromedial, emotional‐motivational striatum, and the sensorimotor dorsal striatum via dopaminergic nigrostriatal neurons.

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Analysis of Brain Projection Systems Using Third-Generation Neuroanatomical Tracers and Multiple Fluorescence Laser Scanning Microscopy

Wouterlood

2014

Neuromethods

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Confocal Laser Scanning: of Instrument, Computer Processing, and Men

Cited by 4 publications

References 25 publications

A Quantitative Comparison of Inhibitory Interneuron Size and Distribution between Mouse and Macaque V1, Using Calcium-Binding Proteins

A Quantitative Comparison of Inhibitory Interneuron Size and Distribution between Mouse and Macaque V1, Using Calcium-Binding Proteins

Mesencephalic dopamine neurons interfacing the shell of nucleus accumbens and the dorsolateral striatum in the rat

Analysis of Brain Projection Systems Using Third-Generation Neuroanatomical Tracers and Multiple Fluorescence Laser Scanning Microscopy

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