Carbocyanine Dye DiI Research Articles

Over 30 Years of DiI Use for Human Neuroanatomical Tract Tracing: A Scoping Review.

In the present study, we conducted a scoping review to provide an overview of the existing literature on the carbocyanine dye DiI, in human neuroanatomical tract tracing. The PubMed, Scopus, and Web of Science databases were systematically searched. We identified 61 studies published during the last three decades. While studies incorporated specimens across human life from the embryonic stage onwards, the majority of studies focused on adult human tissue. Studies that utilized peripheral nervous system (PNS) tissue were a minority, with the majority of studies focusing on the central nervous system (CNS). The most common topic of interest in previous tract tracing investigations was the connectivity of the visual pathway. DiI crystals were more commonly applied. Nevertheless, several studies utilized DiI in a paste or dissolved form. The maximum tracing distance and tracing speed achieved was, respectively, 70 mm and 1 mm/h. We identified studies that focused on optimizing tracing efficacy by varying parameters such as fixation, incubation temperature, dye re-application, or the application of electric fields. Additional studies aimed at broadening the scope of DiI use by assessing the utility of archival tissue and compatibility of tissue clearing in DiI applications. A combination of DiI tracing and immunohistochemistry in double-labeling studies have been shown to provide the means for assessing connectivity of phenotypically defined human CNS and PNS neuronal populations.

Afferent and efferent connections of the nucleus posterior tuberis in the firemouth cichlid, Thorichthys meeki

The nucleus posterior tuberis (NPT) in teleost fishes, also called posterior tuberal nucleus, is situated in the posterior tuberculum of the diencephalon. It is fused across the midline and densely packed with small cells, but little is known about its connections. In this study, the afferent and efferent connections of the NPT were examined by means of tracer applications of the carbocyanine dye DiI in the firemouth cichlid, Thorichthys meeki. Retrogradely labeled cell bodies were found in the corpus mamillare and nucleus periventricularis of the inferior lobe; and anterogradely labeled terminal fibers were detected in the medial zone of the dorsal telencephalon, medial part of the nucleus lateralis tuberis, dorsal posterior thalamic nucleus, torus lateralis, medial part of the nucleus diffusus of the inferior lobe, and tectum opticum. All these connections show an ipsilateral tendency. The NPT is apparently a significant relay nucleus in the diencephalon of T. meeki, and possibly involved in a variety of feedback circuits. It seems also to be part of a tecto-hypothalamo-telencephalic pathway in cichlids.

Acute slice preparation for electrophysiology increases spine numbers equivalently in the male and female juvenile hippocampus: a DiI labeling study.

Hippocampal slices are widely used for in vitro electrophysiological experiments to study underlying mechanisms for synaptic transmission and plasticity, and there is a growing appreciation for sex differences in synaptic plasticity. To date, several studies have shown that the process of making slices from male animals can induce synaptogenesis in cornu ammonis area 1 (CA1) pyramidal cells, but there is a paucity of data for females and other brain regions. In the current study we use microcrystals of the lipophilic carbocyanine dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) to stain individual neurons in the CA1 and dentate gyrus (DG) hippocampal subfields of postnatal day 21 male and female rats. We show that the preparation of sections for electrophysiology produces significant increases in spines in sections obtained from females, similar to that observed in males. We also show that the procedures used for in vitro electrophysiology also result in significant spine increases in the DG and CA1 subfields. These results demonstrate the utility of this refined DiI procedure for staining neuronal dendrites and spines. They also show, for the first time, that in vitro electrophysiology slice preparations enhance spine numbers on hippocampal cells equivalently in both juvenile females and males.NEW & NOTEWORTHY This study introduces a new DiI technique that elucidates differences in spine numbers in juvenile female and male hippocampus, and shows that slice preparations for hippocampal electrophysiology in vitro may mask these differences.

Multispectral labeling of embryonic cells with lipophilic carbocyanine dyes.

Incubation of hatched Strongylocentrotus purpuratus sea urchin embryos or larvae with suspensions of the carbocyanine dyes DiI, DiO, and DiD resulted in the random labeling of membranes of some ectodermal epithelial cells and blastocoelar cells, producing a range of differentially colored cells that can be tracked during development. Simultaneous application of soluble Vybrant® preparations of the three dyes resulted in similar labeling of each cell. Dye labeling of the ectoderm was nearly eliminated by deciliation and some ciliated squamous epithelial cells adjacent to labelled cells were refractory to Vybrant® dye uptake irrespective of concentration or duration of treatment, together suggesting local variation in the properties of cell membranes or cilia. Furthermore, single cells possessing distinctive morphological features were detected.

Neural basis of the stress response in a pufferfish, Takifugu obscurus.

When faced with danger, pufferfish react with both a fast-start escape response and inflation behavior. The neural basis of these stress responses in the pufferfish has not been described. The present study reveals that during inflation behavior, the buccal cavity expands and compresses as a pump to control the direction of water flow and draws water in and out. The inflation involves a series of major anatomical modifications of the head. The greatly enlarged first branchiostegal ray and its associated hyohyoideus abductor muscle are the key mechanisms responsible for this behavior. The nerve branch innervating the hyohyoideus abductor muscle is highly developed, and its central origin at the motor nucleus of the VIIth cranial nerve was revealed by tract-tracing using the carbocyanine dye DiI. The central connections from its origin were found to be several motor nuclei in the medulla and the spinal cord, the nuclei of cranial nerve III and IV in the mesencephalon, and the pretectalis superficialis and periventricular preoptic nuclei in the diencephalon. Both the sympathetic cells and the parvocellular part of the periventricular preoptic nucleus might play a neuro-endocrine role in the rapid movement of the buccal cavity during puffing behavior in this species of pufferfish. The central circuit revealed by this study is hypothesized to mediate the C-start escape behavior and puffing behavior.

Primary Acute Lymphoblastic Leukemia Cells Create a Drug-Resistant Niche By Tunneling Nanotube Signaling with Mesenchymal Stromal Cells

Background: Acute lymphoblastic leukemia (ALL) cells reside in the bone marrow microenvironment, which nurtures and protects cells from chemotherapeutic drugs. Bidirectional signaling between leukemic cells and their niche is shown to be essential for these processes. A major gap in our knowledge is the lack of insight into the functional mechanism regulating and controlling this crosstalk. Tunneling nanotubes (TNTs) have recently been reported as a novel mode of communication between eukaryotic cells, which can facilitate the transport of several types of cellular cargo.Objectives: This study aims to investigate the presence and functional importance of TNTs in the leukemic niche.Results: Here, we show for the first time that TNT signaling occurs between primary patient-derived leukemic cells and primary mesenchymal stromal cells using flow cytometry and time-lapse confocal microscopy. TNTs form within minutes after the start of co-culture and efficiently transfer lipophilic carbocyanine dye DiI from ALL cells towards MSCs. Dye transfer was significantly reduced when TNT signaling was inhibited using three independent experimental setups: actin inhibition, mechanical disruption through gentle shaking of cell cultures, and physical separation using a 3.0 µm pore-sized transwell system (4-fold, 5-fold and 35-fold reduction, respectively). In reciprocal experiments we also observed dye transfer via TNTs from MSCs to BCP-ALL cells, but the magnitude was strikingly less (175-fold, p ≤ 0.001). In addition, we show that leukemic cells use TNTs to modulate their microenvironment by directing non-malignant stromal cells to produce pro-survival cytokines. We observed patient-specific cytokine signatures after co-culture of primary ALL cells with different primary MSCs. For example, IP10/CXCL10 levels increased more than 1000-fold when patient ALL#1 cells were co-cultured with primary MSCs, but were undetectable in co-cultures from patient ALL#2 cells. Induction of all cytokines was dependent on TNT signaling, as TNT inhibition significantly lowered cytokine production (p ≤ 0.001). Importantly, we show that TNT signaling is functionally important for leukemic cell survival and stroma-driven drug resistance. Leukemic cell viability was assessed by flow cytometry after staining with Annexin V, Propidium Iodide and CD19. Primary leukemic cell survival significantly increased in 5-day co-cultures with primary MSCs compared to mono-culture (p ≤ 0.001). When TNT formation was prevented by shaking of these co-cultures or by transwell conditions, the cell viability reduced 3.5- and 3.6-fold, respectively (p ≤ 0.001). TNT inhibition similarly reduced the survival of primary ALL cells in co-culture with MSCs during prednisolone exposure (p ≤ 0.01). Co-culture with primary MSCs also induced resistance to prednisolone 2.5-fold compared to mono-culture in a proliferative setting, using the BCP-ALL cell line NALM6. Inhibition of TNT formation by shaking or transwell conditions significantly reduced this effect: cells in co-culture were only 1.4- and 1.1-fold more resistant compared to mono-culture, respectively (p ≤ 0.01).Conclusion: The presented study identifies TNT formation as a major regulator of interaction between ALL cells and their bone marrow niche, which facilitates signaling mainly from leukemic cells towards MSCs. This signaling drives the release of cytokines within the microenvironment. Disruption of tunneling nanotubes inhibits this release, diminishes the survival benefit that MSCs provide to primary ALL cells, and sensitizes ALL cells to the important anti-leukemic drug prednisolone. This observation gives insight into the pathogenesis of BCP-ALL and opens new avenues to develop more effective therapies that interfere with the leukemic niche. DisclosuresNo relevant conflicts of interest to declare.

Развитие хабенуло-интерпедункулярного тракта у крыс

Development of the habenulointerpeduncular tract has been carried out on fixed brain preparations obtained from 21 day rat embryos and from neonatal animals on the 0 and 9 days of postnatal development by diffusion oflipophilic fluorescent carbocyanine dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) through neuron membranes. The marker was introduced into the nuclei of the habenula, the interpeduncular nucleus, and into the area of raphe nuclei. Neurons and fibers that contained Dil were identified on vibratome sections by fluorescent and confocal microscopy. we have found that reciprocal links between the lateral habenula nucleus and raphe nuclei are formed in the prenatal period by stage E21. Raphe nuclei innervating neurons were located in dorso- and ventrocaudal parts of the lateral habenula nucleus. Projections of the medial habenula nucleus onto interpeduncular nucleus were found only in the postnatal P2 period. Neurons that provide a source of these projections form characteristic assemblies inside the medial habenula nucleus. Therefore, the present study for the first time describes heterogenic formation of different projection systems that are involved in the habenulointerpeduncular tract of rats at perinatal ontogenesis.

Whole-Mount Imaging of the Mouse Hindlimb Vasculature Using the Lipophilic Carbocyanine Dye DiI.

The availability of transgenic disease backgrounds and the accessibility of molecular research reagents have contributed to make the mouse ischemic hindlimb the model of choice for many studies of angiogenesis, and to investigate new treatments for peripheral artery disease. A limitation of these models involves our inability to easily visualize the regenerated vascular architecture. Approaches such as micro-computed tomography and micro-angiography are expensive, technically demanding and not available to many laboratories. Here we describe a rapid and inexpensive adaptation of a vascular staining procedure for precise imaging of the mouse hindlimb vasculature. We introduced two technical modifications and an analytical extension to the original method including (i) pre-skinning of the muscle prior to fixation that preserves tissue integrity, (ii) mild pressure-desiccation subsequent to fixing that enhances resolution and image penetration, and (iii) reconstruction of confocal data into 3D images. The procedure provides resolution that is equivalent or superior to other approaches at a fraction of the cost, time and technology required.

Neurochemical Organization and Connections of the Cerebral Preglomerular Complex of the Masu Salmon

Using immunohistochemical labeling of the cells containing neuronal NO synthase (nNOS), tyrosine hydroxylase (TH), GABA, and parvalbumin (PA), as well as histochemical marking of choline acetyltransferase-containing neurons, we examined the neurochemical organization of the glomerular nuclei and preglomerular complex in the brain of the masu salmon (Oncorhynchus masou). Injections of the carbocyanine dye DiI allowed us to examine projections of neurons of the preglomerular and mammillary nuclei in the salmon brain. We showed that cholinergic, GABA-, PA-, TH-, and nNOS-immunopositive neurons belonging to different morphological types are present in the glomerular and medial preglomerular nuclei. The analysis of correlations between morphometric characteristics of the cells belonging to different neurochemical types and densitometric estimates of amounts of neurochemical agents present in these cells allowed us to hypothesize that there are close morphofunctional interrelations in cell populations possessing different neurochemical and morphometric characteristics. These interrelations of the cells belonging to different chemotypes are, probably, realized as mediatory/modulatory ones. The presence of a great number of small slightly differentiated cells in the preglomerular and glomerular nuclei allows us to suppose that the growth of the greatest sensory center of the salmon brain is provided by neuroblasts that migrate from the proliferative zones in the course of postembryonal neurogenesis. It is also hypothesized that NO, TH, and GABA are involved in paracrine control of the postnatal morphogenesis of the salmon preglomerular complex. The data obtained by hodological analysis indicate that the nuclei of the preglomerular complex obtain afferent projections from the dorsomedial and ventroventral telencephalic regions, preoptic nucleus, periventricular layer of the tectum, and posterior central thalamic nucleus. Our study demonstrated the existence of reciprocal functional connections between the preglomerular complex (most important diencephalic center for transmission of sensory information) and dorsomedial and ventral regions of the telencephalon in the masu salmon.

J-type aggregation of squaraine dye Sq-2Me in surfactant solutions

The influence of anionic sodium dodecyl sulphate (SDS) on aggregation behavior of squaraine dye Sq-2Me has been studied in dimethylformamide–water (95%) (DMF–W) binary solutions by optical spectroscopy. The addition of surfactants at concentrations above the critical micelle concentration into a DMF–W (95%) solution is found to promote the dye ordered aggregation. The structure of Sq-2Me aggregates formed is analyzed using the exciton theory. In solutions with SDS micelles, formation of Sq-2Me J-type dimers of co-planar inclined geometry with an angle between the chromophore long axes and chromophore center-to-center line ( θ) of 47 ° and center-to-center distance ( R) of 5 Å has been revealed. The interaction of Sq-2Me with other hydrophobic molecules (carbocyanine dye DiI) solubilized at equal concentrations in SDS micelles is revealed to prevent the Sq-2Me dimer formation. In such a solution, fluorescent resonance energy transfer between donor dye DiI and acceptor Sq-2Me is observed.

Differential bulbar and extrabulbar projections of diverse olfactory receptor neuron populations in the adult zebrafish (Danio rerio)

Immunohistochemical methods were used to characterize the expression of two calcium-binding proteins, calretinin (CR) and S100, in the olfactory rosette of the adult zebrafish. These proteins are expressed in different sets of sensory neurons, and together represent a large proportion of these cells. Double immunofluorescence for CR and Gα(olf) protein, and CR immunoelectron microscopy, indicated that most CR-immunoreactive (ir) cells were ciliary neurons. Differential S100- and CR-ir projections to glomerular fields of the olfactory bulb were also observed, although these projections overlap in some glomeruli. Application of the carbocyanine dye DiI to either S100-ir or CR-ir glomerular regions led to labeling of cells mostly similar to S100-ir and CR-ir neurons, respectively. Instead, these bulbar regions project to similar telencephalic targets. On the other hand, antibodies against keyhole limpet hemocyanin (KLH)-stained numerous sensory cells in the olfactory rosette, including cells that were CR- and S100-negative. This antiserum also stained most primary bulbar projections and revealed extrabulbar olfactory primary projections coursing to the ventral area of the telencephalon through the medial olfactory tract. This extrabulbar projection was confirmed by tract-tracing with DiI. A loose association of this extrabulbar primary olfactory projection and the catecholaminergic populations of the ventral area was also observed with double tyrosine hydroxylase/KLH-like immunohistochemistry. Comparison between KLH-like-ir pathways and the structures revealed by FMRFamide immunohistochemistry (a marker of terminal ganglion cells and fibers) indicated that the KLH-like-ir extrabulbar projection was different from the terminal nerve system. The significance of the extrabulbar olfactory projection of zebrafish is discussed.

Central Projections of Primary Sensory Afferents to the Spinal Dorsal Horn in the Long-Tailed Stingray, Himantura fai

The central projections of primary sensory afferents innervating the caudal region of the pectoral fin of the long-tailed stingray (Himantura fai) were labeled by applying the lipophilic carbocyanine dye DiI to the dorsal roots in fixed tissue. These observations were complemented by examination of hemotoxylin and eosin-stained paraffin sections of the dorsal root entry zone, and transmission electron microscopy of the dorsal horn. Transverse sections of the sensory nerve and dorsal root revealed two distinct myelinated axon sizes in the sensory nerve. Although the thick and thin axons do not appear to group together in the sensory nerves and dorsal root, they segregate into a dorsally directed bundle of thin fibers and a more horizontally directed bundle of thick fibers soon after entering the spinal cord. In DiI-labeled horizontal sections, fibers were observed to enter the spinal cord and diverge into rostrally and caudally directed trajectories. Branching varicose axons could be traced in the dorsal horn gray matter in the segment of entry and about half of the adjacent rostral and caudal segments. In transverse and sagittal sections, DiI-labeled afferents were seen to innervate the superficial and, to a lesser extent, deeper laminae of the dorsal horn, but not the ventral horn. Electron microscopy of unlabeled dorsal horn sections revealed a variety of synaptic morphologies including large presynaptic elements (some containing dense-core vesicles) making synaptic contacts with multiple processes in a glomerular arrangement; in this respect, the synaptic ultrastructure is broadly similar to that seen in the dorsal horn of rodents and other mammals.

Descriptive findings on the morphology of dendritic spines in the rat medial amygdala

The rat posterodorsal medial amygdala (MePD) is a brain area in which gonadal hormones induce notable plastic effects in the density of dendritic spines. Dendritic spines are post-synaptic specializations whose shape and spacing change neuronal excitability. Our aim was to obtain new data on the dendritic spines morphology and density from MePD neurons using the carbocyanine dye DiI under confocal microscopy. In adult male rats, the dendritic spine density of the medial branches of the left MePD (mean ± SD) was 1.15 ± 0.67 spines/dendritic μm. From the total sampled, approximately 53% of the spines were classified as thin, 22.5% as “mushroom-like”, and 21.5% as stubby/wide. Other spine shapes (3%) included those ramified, with a filopodium-like or a gemule appearance, and others with a protruding spinule. Additional experiment joining DiI and synaptophysin (a pre-synaptic protein) labeling suggested synaptic sites on dendritic shafts and spines. Dendritic spines showed synaptophysin puncta close to their head and neck, although some spines had no evident labeled puncta on them or, conversely, multiple puncta appeared upon one spine. These results advance previous light microscopy results by revealing features and complexities of the dendritic spines at the same time that give new insight on the possible synaptic organization of the adult rat MePD.

Microglial activation and ganglion cells damages in the rat retina with early stage streptozotocin-induced diabetes

Objective To observe the relationship between retinal microglial activations and ganglion cell (RGC) damages in early-stage diabetic rats. Methods A total of 20 Sprague-Dawley(SD)rats were randomly divided into 4 groups (each with 5 rats): 1 month control group, 1 month diabetes group, 3 month control group, 3 month diabetes group. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ). The RGCs of all rats were retrograde labeled by carbocyanine dye DiI injected at the superior colliculi. Microglial cells and RGCs in retinal flat-mounts and sections were stained immunohistochemically and recorded under confocal microscope. Results The diabetic microglial cells were amoeboid and ovoid with fewer processes on retinal flat-mounts. The density of microglial cells which phagocytosed Dil particles in the RGC layer significantly increased in the 3-month diabetes group(P<0.01). The density of microglial cells in the RGC layer significantly increased in the 1- and 3- month diabetes group (P<0. 05). However there were more microglial cells in the RGC layer in the 3-month diabetes group than the 1-month diabetes group (P < 0.0001 ). Significant correlation was found between the amount of microglial cells and that of RGCs in the early-stage of diabetes. Conclusions Microglial cell activation has close relationship with the RGC damages in early-stage diabetic rats. Key words: Diabetic retinopathy/Phisiopathology; Microglia/physiology; Retinal ganglion cells/physiology; Diabetes mellitus, experimental

Fewer Latent Herpes Simplex Virus Type 1 and Cytotoxic T Cells Occur in the Ophthalmic Division than in the Maxillary and Mandibular Divisions of the Human Trigeminal Ganglion and Nerve

Following primary infection of the mouth, herpes simplex virus type 1 (HSV-1) travels retrogradely along the maxillary (V2) or mandibular (V3) nerve to the trigeminal ganglion (TG), where it establishes lifelong latency. Symptomatic HSV-1 reactivations frequently manifest as herpes labialis, while ocular HSV-1 disease is rare. We investigated whether these clinical observations are mirrored by the distribution of latent HSV-1 as well as cytotoxic T-cell infiltration around the nerve cell bodies and in the nerve fibers. The three divisions of the TG were separated by using neurofilament staining and carbocyanine dye Di-I tracing and then screened by in situ hybridization for the presence of HSV-1 latency-associated transcript (LAT). The T-cell distribution and the pattern of cytolytic molecule expression were evaluated by immunohistochemistry. The Di-I-labeled neurons were largely confined to the nerve entry zone of the traced nerve branches. Very few Di-I-labeled neurons were found in adjacent divisions due to traversing fiber bundles. LAT was abundant in the V2 and V3 divisions of all TG but was scarce or totally absent in the ophthalmic (V1) division. CD8(+) T cells were found in all three divisions of the TG and in the respective nerves, clearly clustering in V2 and V3, which is indicative of a chronic inflammation. Only T cells surrounding neurons in the V2 and V3 ganglionic divisions expressed granzyme B. In conclusion, the large accumulation of LAT and cytotoxic T cells in the V2 and V3 but not in the V1 division of the TG reflects the sites supplied by the sensory fibers and the clinical reactivation patterns.

Descending brain-spinal cord projections in a primitive vertebrate, the lamprey: Cerebrospinal fluid-contacting and dopaminergic neurons

We used Neurobiotin as a retrograde tract tracer in both larval and adult sea lampreys and observed a number of neuronal brainstem populations (mainly reticular and octaval populations and some diencephalic nuclei) that project to the spinal cord, in agreement with the results of previous tracer studies. We also observed small labeled neurons in the ventral hypothalamus, the mammillary region, and the paratubercular nucleus, nuclei that were not reported as spinal projecting. Notably, most of the labeled cells of the mammillary region and some of the ventral hypothalamus were cerebrospinal fluid-contacting (CSF-c) neurons. Combined tract tracing and immunocytochemistry showed that some of the labeled neurons of the mammillary and paratubercular nuclei were dopamine immunoreactive. In addition, some CSF-c cells were labeled in the caudal rhombencephalon and rostral spinal cord, and many were also dopamine immunoreactive. Results with other tracers (biotinylated dextran amines, horseradish peroxidase, and the carbocyanine dye DiI) also demonstrated that the molecular weight or the molecular nature of the tracer was determinant in revealing diencephalic cells with very thin axons. The results show that descending systems afferent to the spinal cord in lampreys are more varied than previously reported, and reveal a descending projection from CSF-c cells, which is unknown in vertebrates. The present results also reveal the existence of large differences between agnathans and gnathostomes in the organization of the dopaminergic cells that project to the spinal cord.

Direct labeling and visualization of blood vessels with lipophilic carbocyanine dye DiI

We describe a protocol to rapidly and reliably visualize blood vessels in experimental animals. Blood vessels are directly labeled by cardiac perfusion using a specially formulated aqueous solution containing 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), a lipophilic carbocyanine dye, which incorporates into endothelial cell membranes upon contact. By lateral diffusion, DiI also stains membrane structures, including angiogenic sprouts and pseudopodial processes that are not in direct contact. Tissues can be immediately examined by conventional and confocal fluorescence microscopy. High-quality serial optical sections using confocal microscopy are obtainable from thick tissue sections, especially at low magnification, for three-dimensional reconstruction. It takes less than 1 h to stain the vasculature in a whole animal. Compared with alternative techniques to visualize blood vessels, including space-occupying materials such as India ink or fluorescent dye-conjugated dextran, the corrosion casting technique, endothelial cell-specific markers and lectins, the present method simplifies the visualization of blood vessels and data analysis.

Fluorescent double-labeling with carbocyanine neuronal tracing and immunohistochemistry using a cholesterol-specific detergent digitonin

The fluorescent carbocyanine dye DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate) has been widely used for tracing of neuronal pathways. To examine identities of the DiI-labeled neuronal pathways, it is desirable to combine DiI labeling with immunofluorescent staining. However, DiI labeling and immunofluorescent staining are not well compatible, mainly because treatment of DiI-labeled neurons with detergents, which are commonly used for immunohistochemistry, results in high levels of diffusion of the DiI label. In this study, we searched for detergents that are compatible with DiI labeling, and found that a cholesterol-specific detergent digitonin is useful for fluorescent double-labeling with DiI tracing and immunohistochemistry. We show that digitonin treatment, in contrast to Triton X-100, methanol and Nonidet P-40 treatment, preserves DiI labeling, even at higher concentrations. We also show that digitonin also preserves the signal of a DiI derivative CM-DiI. Moreover, we demonstrate that digitonin efficiently increases antibody penetration into brain sections. As a result, immunohistochemical images obtained with digitonin treatment are as good as those obtained with Triton X-100 treatment. In addition, we also try another cholesterol-specific detergent quillaja saponin, but find that it degrades the DiI label. Our simple double-labeling protocol using digitonin should prove useful in enabling detailed examination of the neuronal circuitry of the nervous system.

Early segregation of layered projections from the lateral superior olivary nucleus to the central nucleus of the inferior colliculus in the neonatal cat

The central nucleus of the inferior colliculus (IC) is a laminated structure that receives multiple converging afferent projections. These projections terminate in a layered arrangement and are aligned with dendritic arbors of the predominant disc-shaped neurons, forming fibrodendritic laminae. Within this structural framework, inputs terminate in a precise manner, establishing a mosaic of partially overlapping domains that likely define functional compartments. Although several of these patterned inputs have been described in the adult, relatively little is known about their organization prior to hearing onset. The present study used the lipophilic carbocyanine dyes DiI and DiD to examine the ipsilateral and contralateral projections from the lateral superior olivary (LSO) nucleus to the IC in a developmental series of paraformaldehyde-fixed kitten tissue. By birth, the crossed and uncrossed projections had reached the IC and were distributed across the frequency axis of the central nucleus. At this earliest postnatal stage, projections already exhibited a characteristic banded arrangement similar to that described in the adult. The heaviest terminal fields of the two inputs were always complementary in nature, with the ipsilateral input appearing slightly denser. This early arrangement of interdigitating ipsilateral and contralateral LSO axonal bands that occupy adjacent sublayers supports the idea that the initial establishment of this highly organized mosaic of inputs that defines distinct synaptic domains within the IC occurs largely in the absence of auditory experience. Potential developmental mechanisms that may shape these highly ordered inputs prior to hearing onset are discussed.

Labeling of dendritic spines with the carbocyanine dye DiI for confocal microscopic imaging in lightly fixed cortical slices

Visualization of dendritic spines is an important tool for researches on structural synaptic plasticity. Fluorescent labeling of the dendrites and spines followed by confocal microscopy permits imaging a large population of dendritic spines with a higher resolution. We sought to establish an optimal protocol to label neurons in cortical slices with the carbocyanine dye DiI for confocal microscopic imaging of dendritic spines. DiI finely labeled dendrites and spines in slices prefixed (by cardiac perfusion) with 1.5% paraformaldehyde to the similar extent that could be achieved in live preparation. In contrast, fixation with 4% paraformaldehyde severely compromised dye diffusion. Confocal microscopy showed that structural integrity of dendrites and spines was preserved much better in lightly (1.5%) fixed slices than those prepared without fixation. Quantitative measurement revealed that spine density was lower in live slices than that counted in lightly fixed slices, suggesting that fixation is necessary for an adequate evaluation of spine density. The quality of confocal microscopic images obtained from lightly fixed slices allowed us to observe distinctive morphologies such as branched spines and dendritic filopodium, which may be indicative of structural changes at synapses. This method will thus be useful for studying structural synaptic plasticity.