Biocytin-Labeling in Whole-Cell Recording: Electrophysiological and Morphological Properties of Pyramidal Neurons in CYLD-Deficient Mice.

Biocytin, a chemical compound that is an amide formed from the vitamin biotin and the amino acid L-lysine, has been used as a histological dye to stain nerve cells. Electrophysiological activity and morphology are two key characteristics of neurons, but revealing both the electrophysiological and morphological properties of the same neuron is challenging. This article introduces a detailed and easy-to-operate procedure for single-cell labeling in combination with whole-cell patch-clamp recording. Using a recording electrode filled with a biocytin-containing internal solution, we demonstrate the electrophysiological and morphological characteristics of pyramidal (PNs), medial spiny (MSNs) and parvalbumin neurons (PVs) in brain slices, where the electrophysiological and morphological properties of the same individual cell are elucidated. We first introduce a protocol for whole-cell patch-clamp recording in various neurons, coupled with the intracellular diffusion of biocytin delivered by the glass capillary of the recording electrode, followed by a post hoc procedure to reveal the architecture and morphology of biocytin-labeled neurons. An analysis of action potentials (APs) and neuronal morphology, including the dendritic length, number of intersections, and spine density of biocytin-labeled neurons, were performed using ClampFit and Fiji Image (ImageJ), respectively. Next, to take advantage of the techniques introduced above, we uncovered defects in the APs and the dendritic spines of PNs in the primary motor cortex (M1) of deubiquitinase cylindromatosis (CYLD) knock-out (Cyld-/-) mice. In summary, this article provides a detailed methodology for revealing the morphology as well as the electrophysiological activity of a single neuron that will have many applications in neurobiology.

Repetitively burst-spiking neurons in reeler mice show conserved but also highly variable morphological features of layer Vb-fated "thick-tufted" pyramidal cells.

Reelin is a large extracellular glycoprotein that is secreted by Cajal-Retzius cells during embryonic development to regulate neuronal migration and cell proliferation but it also seems to regulate ion channel distribution and synaptic vesicle release properties of excitatory neurons well into adulthood. Mouse mutants with a compromised reelin signaling cascade show a highly disorganized neocortex but the basic connectional features of the displaced excitatory principal cells seem to be relatively intact. Very little is known, however, about the intrinsic electrophysiological and morphological properties of individual cells in the reeler cortex. Repetitive burst-spiking (RB) is a unique property of large, thick-tufted pyramidal cells of wild-type layer Vb exclusively, which project to several subcortical targets. In addition, they are known to possess sparse but far-reaching intracortical recurrent collaterals. Here, we compared the electrophysiological properties and morphological features of neurons in the reeler primary somatosensory cortex with those of wild-type controls. Whereas in wild-type mice, RB pyramidal cells were only detected in layer Vb, and the vast majority of reeler RB pyramidal cells were found in the superficial third of the cortical depth. There were no obvious differences in the intrinsic electrophysiological properties and basic morphological features (such as soma size or the number of dendrites) were also well preserved. However, the spatial orientation of the entire dendritic tree was highly variable in the reeler neocortex, whereas it was completely stereotyped in wild-type mice. It seems that basic quantitative features of layer Vb-fated RB pyramidal cells are well conserved in the highly disorganized mutant neocortex, whereas qualitative morphological features vary, possibly to properly orient toward the appropriate input pathways, which are known to show an atypical oblique path through the reeler cortex. The oblique dendritic orientation thus presumably reflects a re-orientation of dendritic input domains toward spatially highly disorganized afferent projections.

Patch-clamp Recordings and Single Fiber Labeling from Spiral Ganglion Somata in Acutely Prepared Semi-intact Cochleae from Neonatal Rats.

Spiral ganglion neurons (SGN) are the primary neuronal pathway for transmitting sensory information from the inner ear to the brainstem. Recent studies have revealed significant biophysical and molecular diversity indicating that auditory neurons are comprised of sub-groups whose intrinsic properties contribute to their diverse functions. Previous approaches for studying the intrinsic biophysical properties of spiral ganglion neurons relied on patch-clamp and molecular analysis of cultured somata that were disconnected from their pre-synaptic hair cell partners. In the absence of the information provided by cell-to-cell connectivity, such studies could not associate biophysical diversity with functional sub-groups. Here we describe a protocol for preparing, recording, and labeling spiral ganglion neurons in a semi-intact ex-vivo preparation. In these preparations, the cell bodies of spiral ganglion neurons remain connected to their hair cell partners. The recordings are completed within 4 hours of euthanasia, alleviating concerns about whether long culture times and culture conditions change the intrinsic properties of neurons.

Minimizing shrinkage of acute brain slices using metal spacers during histological embedding.

The morphological structure of neurons provides the basis for their functions and is a major focus of contemporary neuroscience studies. Intracellular staining of single cells in acute slices is a well-established approach, offering high-resolution information on neuronal morphology, complementing their physiology. Despite major technical advances, however, a common histological artifact often precludes precise morphological analysis: shrinkage of the sampled tissue after embedding for microscopy. Here, we describe a new approach using a metal spacer, sandwiched between two coverslips to reduce shrinkage of whole-mount slice preparations during embedding with aqueous mounting medium under a coverslip. This approach additionally allows imaging the slices from both sides to obtain better quality images of deeper structures. We demonstrate that the use of this spacer system can efficiently and stably reduce the shrinkage of slices, whereas conventional embedding methods without spacer or with agar spacer cause severe, progressive shrinkage after embedding. We further show that the shrinkage of slices is not uniform and artifacts in morphology and anatomical parameters produced cannot be compensated using linear correction algorithms. Our study, thus, emphasizes the importance of preventing the deformation of slice preparations and offers an effective means for reducing shrinkage and associated artifacts during embedding.

Competitive binding assay for biotin and biotin derivatives, based on avidin and biotin-4-fluorescein.

Biotinylated molecules are extensively employed in bioanalytics and biotechnology. The currently available assays for quantification of biotin groups suffer from low sensitivity, low accuracy, or provide highly variable responses for different biotin derivatives. We developed a competitive binding assay in which avidin was pre-blocked to different extents by the biotinylated analyte and a constant amount of biotin-4-fluorescein (B4F) was added, resulting in strong quenching of the B4F. The assay was robust and the shape of the titration curve immediately revealed whether the data were reliable or perturbed by steric hindrance in case of large biotin derivatives. These advantages justified well the 10× higher sample consumption (~0.6nmol) compared to single point assays. The assay was applied to a representative set of small biotin derivatives and validated by cross-control with the well-established 2-anilinonaphthalene-6-sulfonic acid (2,6-ANS) binding assay. In comparison to the 2,6-ANS binding assay, the lower precision (±10%) was compensated by the 100-fold higher sensitivity and the deviations from the ANS assay were ≤5%. In comparison to the more sensitive biotin group assays, the new assay has the advantage of minimal bias for different biotin derivatives. In case of biotinylated DNA with 30 nucleotides, steric hindrance was found to reduce the accuracy of biotin group determination; this problem was overcome by partial digestion to n≤5 nucleotide residues with a 3'-exonuclease. The newly proposed biotin group assay offers a useful compromise in terms of sensitivity, precision, trueness, and robustness.

A subset of taste receptor cells express biocytin-permeable channels activated by reducing extracellular Ca2+ concentration.

Taste receptor cells (type II cells) transmit taste information to taste nerve fibres via ATP-permeable channels, including calcium homeostasis modulator (CALHM), connexin and/or pannexin1 channels, via the paracrine release of adenosine triphosphate (ATP) as a predominant transmitter. In the present study, we demonstrate that extracellular Ca2+ -dependent biocytin-permeable channels are present in a subset of type II cells in mouse fungiform taste buds using biocytin uptake, immunohistochemistry and in situ whole-cell recordings. Type II cells were labelled with biocytin in an extracellular Ca2+ concentration ([Ca2+ ]out )-sensitive manner. We found that the ratio of biocytin-labelled type II cells to type II cells per taste bud was approximately 20% in 2 mM Ca2+ saline, and this ratio increased to approximately 50% in nominally Ca2+ -free saline. The addition of 300 µM GdCl3 , which inhibits various channels including CALHM1 channels, significantly inhibited biocytin labelling in nominally Ca2+ -free saline, whereas the addition of 20 µM ruthenium red did not. Moreover, Cs+ -insensitive currents increased in nominally Ca2+ -free saline in approximately 40% of type II cells. These increased currents appeared at a potential of above -35 mV, reversed at approximately +10 mV and increased with depolarization. These results suggest that biocytin labels type II cells via ion channels activated by [Ca2+ ]out reduction, probably "CALHM-like" channels, on the basolateral membrane and that taste receptor cells can be categorized into two groups based on differences in the expression levels of [Ca2+ ]out -dependent biocytin-permeable channels. These data indicate electrophysiological and pharmacologically relevant properties of biocytin-permeable channels and suggest their contributions to taste signal transduction.

Morphology of the utricular otolith organ in the toadfish, Opsanus tau.

The utricle provides the vestibular reflex pathways with the sensory codes of inertial acceleration of self-motion and head orientation with respect to gravity to control balance and equilibrium. Here we present an anatomical description of this structure in the adult oyster toadfish and establish a morphological basis for interpretation of subsequent functional studies. Light, scanning, and transmission electron microscopy techniques were applied to visualize the sensory epithelium at varying levels of detail, its neural innervation and its synaptic organization. Scanning electron microscopy was used to visualize otolith mass and morphological polarization patterns of hair cells. Afferent nerve fibers were visualized following labeling with biocytin, and light microscope images were used to make three-dimensional (3-D) reconstructions of individual labeled afferents to identify dendritic morphology with respect to epithelial location. Transmission electron micrographs were compiled to create a serial 3-D reconstruction of a labeled afferent over a segment of its dendritic field and to examine the cell-afferent synaptic contacts. Major observations are: a well-defined striola, medial and lateral extra-striolar regions with a zonal organization of hair bundles; prominent lacinia projecting laterally; dependence of hair cell density on macular location; narrow afferent dendritic fields that follow the hair bundle polarization; synaptic specializations issued by afferents are typically directed towards a limited number of 7-13 hair cells, but larger dendritic fields in the medial extra-striola can be associated with > 20 hair cells also; and hair cell synaptic bodies can be confined to only an individual afferent or can synapse upon several afferents.

Biocytin-Based pH-Stimuli Responsive Supramolecular Multivariant Hydrogelator for Potential Applications.

Self-assembly is one of the important and fruitful methods to develop various biomaterials, especially hydrogels, from biologically important small molecules. Two such molecules are d-biotin (B-vitamin), which plays several vital roles in biology and is used in the biomedical field, and biocytin, which is used as an intracellular marker and also to identify neurons. Despite the significance of these biomolecules, their usage in the form of hydrogel is very limited. Herein, for the first time, a pH-stimuli responsive supramolecular hydrogel based on biocytin, an amidation product of d-biotin and l-lysine, has been developed to extend the use of d-biotin, biocytin, and l-lysine in the biomedical field. The newly developed hydrogelator such as Nα-(fluorenylmethoxycarbonyl)-d-biocytin) [FmB] exhibited pH-dependent hydrogelation and exhibited a thixotropic and thermally reversible nature at a physiological pH, while it displayed a supergelator nature at an acidic pH. Electron microscopic analysis of FmB hydrogel indicated the formation of a fibrous network structure. Various biophysical measurements including CD, FT-IR, fluorescence, and powder XRD demonstrated that intermolecular interactions such as aromatic and hydrogen bonding stabilize the fibrous structure of FmB. Importantly, the FmB hydrogel was found to be viable for the cells and displayed specific bactericidal activity against Escherichia coli. Since making hydrogels directly with drugs or vitamins rather than inclusion in other hydrogels (matrix) may provide new biomaterials that can act as self-delivery systems, we believe that the newly developed multivariant biocytin-based supramolecular hydrogel can significantly advance the application of biotin and biocytin in various fields.

Efferent Axonal Projections of the Habenular Complex in the Fire-Bellied Toad Bombina orientalis.

The habenular complex and its associated axonal pathways are often thought of as phylogenetically conserved features of the brain among vertebrates despite the fact that detailed studies of this brain region are limited to a few species. Here, the gross morphology and axonal projection pattern of the habenular complex of an anuran amphibian, the fire-bellied toad Bombina orientalis, was studied to allow comparison with the situation in other vertebrates. Axonal pathways were traced using biocytin applications in dissected brain preparations. The results show that the rostral part of the left dorsal nucleus is enlarged in this species, while the rostral ventral nucleus and caudal parts do not show left-right size differences. Biocytin applications revealed widespread axonal projections of the habenular complex to the posterior tuberculum/dorsal hypothalamic region, ventral tegmentum, interpeduncular nucleus (IPN), and raphe median. Additionally, axons targeting the lateral hypothalamus originated from the ventral habenular nuclei. The results also suggest an asymmetrical pattern of projection to the IPN in the rostral part of the habenular complex, where the left habenula preferentially targeted the dorsal IPN while the right habenula preferentially targeted the ventral IPN. The caudal habenular nuclei showed no asymmetry of projections as both sides targeted the ventral IPN. Comparison of the habenular complex axonal connectivity across vertebrates argues against strong phylogenetic conservation of the axonal projection patterns of different habenular nuclei.

Advances and perspectives in tissue clearing using CLARITY.

CLARITY is a tissue clearing method, which enables immunostaining and imaging of large volumes for 3D-reconstruction. The method was initially time-consuming, expensive and relied on electrophoresis to remove lipids to make the tissue transparent. Since then several improvements and simplifications have emerged, such as passive clearing (PACT) and methods to improve tissue staining. Here, we review advances and compare current applications with the aim of highlighting needed improvements as well as aiding selection of the specific protocol for use in future investigations.

A novel relay nucleus between the inferior colliculus and the optic tectum in the chicken (Gallus gallus).

Processing multimodal sensory information is vital for behaving animals in many contexts. The barn owl, an auditory specialist, is a classic model for studying multisensory integration. In the barn owl, spatial auditory information is conveyed to the optic tectum (TeO) by a direct projection from the external nucleus of the inferior colliculus (ICX). In contrast, evidence of an integration of visual and auditory information in auditory generalist avian species is completely lacking. In particular, it is not known whether in auditory generalist species the ICX projects to the TeO at all. Here we use various retrograde and anterograde tracing techniques both in vivo and in vitro, intracellular fillings of neurons in vitro, and whole-cell patch recordings to characterize the connectivity between ICX and TeO in the chicken. We found that there is a direct projection from ICX to the TeO in the chicken, although this is small and only to the deeper layers (layers 13-15) of the TeO. However, we found a relay area interposed among the IC, the TeO, and the isthmic complex that receives strong synaptic input from the ICX and projects broadly upon the intermediate and deep layers of the TeO. This area is an external portion of the formatio reticularis lateralis (FRLx). In addition to the projection to the TeO, cells in FRLx send, via collaterals, descending projections through tectopontine-tectoreticular pathways. This newly described connection from the inferior colliculus to the TeO provides a solid basis for visual-auditory integration in an auditory generalist bird. J. Comp. Neurol. 525:513-534, 2017. © 2016 Wiley Periodicals, Inc.

Microconnectomics of the pretectum and ventral thalamus in the chicken (Gallus gallus).

The avian pretectal and ventrothalamic nuclei, encompassing the griseum tectale (GT), n. lentiformis mesencephali (LM), and n. geniculatus lateralis pars ventralis (GLv), are prominent retinorecipient structures related to optic flow operations and visuomotor control. Hence, a close coordination of these neural circuits is to be expected. Yet the connectivity among these nuclei is poorly known. Here, using intracellular labeling and in situ hybridization, we investigated the detailed morphology, connectivity, and neurochemical identity of neurons in these nuclei. Two different cell types exist in the GT: one that generates an axonal projection to the optic tectum (TeO), LM, GLv, and n. intercalatus thalami (ICT), and a second population that only projects to the LM and GLv. In situ hybridization revealed that most neurons in the GT express the vesicular glutamate transporter (VGluT2) mRNA, indicating a glutamatergic identity. In the LM, three morphological cell types were defined, two of which project axons towards dorsal targets. The LM neurons showed strong VGluT2 expression. Finally, the cells located in the GLv project to the TeO, LM, GT, n. principalis precommisuralis (PPC), and ICT. All neurons in the GLv showed strong expression of the vesicular inhibitory amino acid transporter (VIAAT) mRNA, suggesting a GABAergic identity. Our results show that the pretectal and ventrothalamic nuclei are highly interconnected, especially by glutamatergic and GABAergic neurons from the GT and GLv, respectively. This complex morphology and connectivity might be required to organize orienting visuomotor behaviors and coordinate the specific optic flow patterns that they induce. J. Comp. Neurol. 524:2208-2229, 2016. © 2015 Wiley Periodicals, Inc.

Fast neuronal labeling in live tissue using a biocytin conjugated fluorescent probe.

BACKGROUND: Biocytin has found numerous uses as a neuronal tracer, since it shows both antero- and retrograde transport in neuronal tracts. The main advantage of biocytin lies in the comprehensive intracellular distribution of the molecule, and in effective detection using avidin-based reactions. The main drawback is that biocytin cannot be visualized in live tissue. NEW METHOD: We demonstrate that TMR biocytin, a conjugate of biocytin and a rhodamine fluorophore, is an effective neuronal tracer in live tissue when applied by electroporation. RESULTS: The initial fiber transport velocity of TMR biocytin is high-5.4mm/h. TMR biocytin can be used in conjunction with AM calcium dyes to label neuronal somas from distances of several millimetres, and record calcium transients during the course of a few hours. Juxtacellular application of TMR biocytin leads to fast anterograde transport with labeling of local synapses within 10min. TMR biocytin is fixable, stable during methyl salicylate clearing, and can be visualized deep in nervous tissue. COMPARISON WITH EXISTING METHODS: Retrograde labeling with TMR biocytin enables long-range neuronal visualization and concurrent calcium imaging after only a few hours, which is substantially faster than other fluorescence-based tracers. The green emitting Atto 488 biotin is also taken up and transported retrogradely, but it is not compatible with standard green emitting calcium dyes. CONCLUSIONS: TMR biocytin is an attractive neuronal tracer. It labels neurons fast over long distances, and it can be used in conjunction with calcium dyes to report on neuronal activity in retrogradely labeled live neurons.

Relationships between dendritic morphology, spatial distribution and firing patterns in rat layer 1 neurons

The cortical layer 1 contains mainly small interneurons, which have traditionally been classified according to their axonal morphology. The dendritic morphology of these cells, however, has received little attention and remains ill defined. Very little is known about how the dendritic morphology and spatial distribution of these cells may relate to functional neuronal properties. We used biocytin labeling and whole cell patch clamp recordings, associated with digital reconstruction and quantitative morphological analysis, to assess correlations between dendritic morphology, spatial distribution and membrane properties of rat layer 1 neurons. A total of 106 cells were recorded, labeled and subjected to morphological analysis. Based on the quantitative patterns of their dendritic arbor, cells were divided into four major morphotypes: horizontal, radial, ascendant, and descendant cells. Descendant cells exhibited a highly distinct spatial distribution in relation to other morphotypes, suggesting that they may have a distinct function in these cortical circuits. A significant difference was also found in the distribution of firing patterns between each morphotype and between the neuronal populations of each sublayer. Passive membrane properties were, however, statistically homogeneous among all subgroups. We speculate that the differences observed in active membrane properties might be related to differences in the synaptic input of specific types of afferent fibers and to differences in the computational roles of each morphotype in layer 1 circuits. Our findings provide new insights into dendritic morphology and neuronal spatial distribution in layer 1 circuits, indicating that variations in these properties may be correlated with distinct physiological functions.

Biocytin-derived MRI contrast agent for longitudinal brain connectivity studies.

To investigate the connectivity of brain networks noninvasively and dynamically, we have developed a new strategy to functionalize neuronal tracers and designed a biocompatible probe that can be visualized in vivo using magnetic resonance imaging (MRI). Furthermore, the multimodal design used allows combined ex vivo studies with microscopic spatial resolution by conventional histochemical techniques. We present data on the functionalization of biocytin, a well-known neuronal tract tracer, and demonstrate the validity of the approach by showing brain networks of cortical connectivity in live rats under MRI, together with the corresponding microscopic details, such as fibers and neuronal morphology under light microscopy. We further demonstrate that the developed molecule is the first MRI-visible probe to preferentially trace retrograde connections. Our study offers a new platform for the development of multimodal molecular imaging tools of broad interest in neuroscience, that capture in vivo the dynamics of large scale neural networks together with their microscopic characteristics, thereby spanning several organizational levels.

Improved neuronal tract tracing with stable biocytin-derived neuroimaging agents.

One of the main characteristics of brains is their profuse connectivity at different spatial scales. Understanding brain function evidently first requires a comprehensive description of neuronal anatomical connections. Not surprisingly a large number of histological markers were developed over the years that can be used for tracing mono- or polysynaptic connections. Biocytin is a classical neuroanatomical tracer commonly used to map brain connectivity. However, the endogenous degradation of the molecule by the action of biotinidase enzymes precludes its applicability in long-term experiments and limits the quality and completeness of the rendered connections. With the aim to improve the stability of this classical tracer, two novel biocytin-derived compounds were designed and synthesized. Here we present their greatly improved stability in biological tissue along with retained capacity to function as neuronal tracers. The experiments, 24 and 96 h postinjection, demonstrated that the newly synthesized molecules yielded more detailed and complete information about brain networks than that obtained with conventional biocytin. Preliminary results suggest that the reported molecular designs can be further diversified for use as multimodal tracers in combined MRI and optical or electron microscopy experiments.

NADPH-Diaphorase Containing Neurons and Biocytin-labelled Axon Terminals in the Visual Cortex of Adult Rats Malnourished During Development.

This is the first report of the effects of malnutrition during brain development on biocytin-labelled axon terminals and histochemical pattern of NADPH-diaphorase (NADPH-d)-containing neurons in area 17 of the adult rat. Wistar rats (n = 6) were submitted early in life (from gestation up to 42 days of age) to a multideficient diet (the 'regional basic diet' (RBD) of low-income human populations of north-east Brazil, containing only 8% protein). From day 43 up to adulthood (135-212 days), they were switched to a commercial laboratory chow diet (Purina do Brasil Ltda), with 22% protein. These animals were compared to control rats (n = 11), fed the laboratory chow diet until adulthood. The brains of four adult malnourished and five controls were processed according to the indirect method of the malic enzyme to reveal NADPH-d-containing neurons. Five other adult subjects (three controls and two malnourished) received iontophoretic injections of the tracer biocytin in area 17and were processed according to the glucose oxidase-DAB-nickel protocol in order to visualize axon terminals filled with biocytin. Three other control rats were processed for both techniques. In these last brains, no double-labelled cells could be found, suggesting that the NADPH-d-containing-neurons and the biocytin-labelled ones belong to different groups of cells, in area 17. The appendages of the NADPH-d-positive cells showed minor qualitative and quantitative differences between undernourished and control rats. The soma area of these cells was reduced in the white matter of malnourished rats, as compared to the controls (468.6 ± 54.3 µm(2); n = 4 and 515.4 ± 30.5 µm(2);n = 8, respectively; p < 0.05). The cell density (cells/mm(2)) was greater in the malnourished group than in the control, both at the grey matter (16.6 ± 4.4; n = 4 and 11.3 ± 4.3; n = 8, respectively; p < 0.05) and at the white matter (55.9 ± 15.7; n = 4 and 24.4 ± 8.5; n = 8; p < 0.005). The number of potential synaptic sites in the biocytin-labelled axon terminals was reduced as compared to the control (126 ± 33 boutons/mm, n = 32 and 160 ± 37; n = 30, respectively; p < 0.01). The results indicate that the rat area 17 is affected differently by early malnutrition, regarding biocytin-labelled axon terminals, on the one hand, and NADPH-d-containing neurons, on the other. Concerning these last cells, the data also suggest that they are less sensitive to the injury represented by early malnutrition.