A study in developing visual systems with a new method of staining neurones and their processes in fixed tissue

Carbocyanine dyes, fluorescent lipophilic substances used for optical recordings of membrane voltage and for studies of membrane fluidity, have recently been shown to provide intense and long-lasting staining of neurones in vivo and in vitro (Schwartz & Agranoff, 1981; Honig & Hume, 1985, 1986; Catsicas, Thanos & Clarke, 1986; Landmesser & Honig, 1986; Thanos & Bonhoeffer, 1987). We report here that two of these dyes, diI (1,1',dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate) and diO (3,3'-dioctadecyloxacarbocyanine perchlorate), can also label neurones in embryonic mouse and chicken brain tissue that has been previously fixed in aldehyde fixatives. Neuronal processes and perikarya can be labelled along considerable distances in both anterograde and retrograde directions. The staining of processes and cells, including their finest extensions is smooth and clear, rivalling intracellular injections of HRP or Lucifer Yellow. The appearance and time course of progression of the staining along axons suggest that the staining in fixed tissue occurs due to a process of diffusion of dyes along the plasma membranes of cells. This technique has allowed us to study the first stages in the development of optic fibres in mouse embryos, especially at the optic chiasm. The early retinal projection (E13-E13 1/2) is mainly crossed, but some optic fibres grow to the ipsilateral side of the brain at the outset. Retrogradely labelled ganglion cells from the dorsocentral area of the retina participate in the formation of both the ipsilateral and the contralateral projection. Thus, at early stages, crossed and uncrossed projections arise from identical subregions of the retina and the partition of the retina with respect to the laterality of its projection arises later.     

Use of the procedure of photo-oxidation of fluorescent preparations of autopsy material of the human brain for light and electron microscopy]

Neurons and fibers from human autopsy material were labeled by application of Lucifer Yellow or diI. Dye-filled neurons and fibers were immersed in a diaminobenzidine and potassium cyanide solutions and irradiated with epifluorescent illumination until all visible fluorescence had faded. This simple photo-oxidation procedure resulted in the autopsy human tissue of a stable, nonfading, brown reaction product, visible with the light microscope providing a Golgi-like image, and EM level. This methodological procedure was successfully used for revealing detailed morphology of neurons with dendritic spines, axons with collaterals and varicosities in the human cortical areas and hippocampus.     

The response of cultured trigeminal and spinal cord motoneurons to nerve growth factor

Dissociated neurons from the trigeminal (V) region of the metencephalic basal plate or the ventral spinal cord from chick embryos of Day 4 (V basal plate) or Day 5 (spinal cord) were cultured on a laminin substratum either in the presence of nerve growth factor (NGF) or in control medium. Assessment was made of neuronal survival, the amount of neurite elaborated, and the percentage of neurons initiating neurites. The presence of motoneurons was verified by retrograde labeling with the fluorescent dye diI. NGF was found to significantly increase the quantity of neuritic processes produced by the spinal cord dissociates at both 24 and 48 hr in vitro. The percentage of neurons initiating neuritic processes was significantly increased by NGF in the trigeminal population at 48 hr in vitro. Neuronal survival was not enhanced by NGF in either group. Both trigeminal and spinal cord neurons were also found to specifically bind 125I-NGF in culture. These results provide direct evidence for an influence of NGF on process formation of early embryonic motoneurons in culture.     

Resolving the Detailed Structure of Cortical and Thalamic Neurons in the Adult Rat Brain with Refined Biotinylated Dextran Amine Labeling

Biotinylated dextran amine (BDA) has been used frequently for both anterograde and retrograde pathway tracing in the central nervous system. Typically, BDA labels axons and cell somas in sufficient detail to identify their topographical location accurately. However, BDA labeling often has proved to be inadequate to resolve the fine structural details of axon arbors or the dendrites of neurons at a distance from the site of BDA injection. To overcome this limitation, we varied several experimental parameters associated with the BDA labeling of neurons in the adult rat brain in order to improve the sensitivity of the method. Specifically, we compared the effect on labeling sensitivity of: (a) using 3,000 or 10,000 MW BDA; (b) injecting different volumes of BDA; (c) co-injecting BDA with NMDA; and (d) employing various post-injection survival times. Following the extracellular injection of BDA into the visual cortex, labeled cells and axons were observed in both cortical and thalamic areas of all animals studied. However, the detailed morphology of axon arbors and distal dendrites was evident only under optimal conditions for BDA labeling that take into account the: molecular weight of the BDA used, concentration and volume of BDA injected, post-injection survival time, and toning of the resolved BDA with gold and silver. In these instances, anterogradely labeled axons and retrogradely labeled dendrites were resolved in fine detail, approximating that which can be achieved with intracellularly injected compounds such as biocytin or fluorescent dyes.     

Fluorescent carbocyanine dyes allow living neurons of identified origin to be studied in long-term cultures

A prerequisite for many studies of neurons in culture is a means of determining their original identity. We needed such a technique to study the interactions in vitro between a class of spinal cord neurons, sympathetic preganglionic neurons, and their normal target, neurons from the sympathetic chain. Here, we describe how we use two highly fluorescent carbocyanine dyes, which differ in color but are otherwise similar, to identify neurons in culture. The long carbon chain carbocyanine dyes we use are lipid-soluble and so become incorporated into the plasma membrane. Neurons can be labeled either retrogradely or during dissociation. Some of the labeled membrane gradually becomes internalized and retains its fluorescence, allowing identification of cells for several weeks in culture. These dyes do not affect the survival, development, or basic physiological properties of neurons and do not spread detectably from labeled to unlabeled neurons. It seems likely that cells become retrogradely labeled mainly by lateral diffusion of dye in the plane of the membrane. If so, carbocyanine dyes may be most useful for retrograde labeling over relatively short distances. An additional feature of carbocyanine labeling is that neuronal processes are brightly fluorescent for the first few days in culture, presumably because dye rapidly diffuses into newly inserted membrane. We have used carbocyanine dyes to identify sympathetic preganglionic neurons in culture. Our results indicate that preganglionic neurons can survive in the absence of their target cells and that several aspects of their differentiation in the absence of target appear normal.     

Innervation of the superficial pineal of the rat using retrograde tracing methods

Fast blue (FB), rhodamine microspheres (RH), horseradish peroxidase (HRP), and wheat germ agglutinin-horseradish peroxidase conjugate (WGA-HRP) were used as retrograde tracers to study the innervation of the rat superficial pineal gland (SP). One of the tracers was injected into the gland of each animal. All four retrograde tracers injected into the gland always labeled neurons in the superior cervical ganglia (SCG). No retrograde labeling was ever seen in the suprachiasmatic nuclei, paraventricular hypothalamic nuclei, lateral hypothalamus, habenular nuclei, amygdalar nuclei, or superior salivatory nuclei. Retrograde labeling was seen in the anterior hypothalamic nuclei, anterior thalamic nuclei, lateral geniculate bodies, and midbrain tectal structures when a tracer spread from the injection site to the overlying cortex, tectum, or commissures. Control studies included injection of tracer into the subarachnoid space around the SP or into structures adjacent to the SP. Only the injection of FB or WGA-HRP into the subarachnoid space labeled neurons in the SCG. This labeling was probably due to the spread of tracer to the choroid plexus. These results agree with recent work confirming the existence of a direct projection of the SCG into the interstitium around pinealocytes. The evidence does not substantiate an innervation originating in the habenular nuclei; the superior salivatory nuclei; or any other diencephalic, midbrain, pontine, or medullary structure.     

Neurosecretory cells in the circadian-clock system of the scorpion,Androctonus australis

Summary The somata of the efferent neurosecretory fibers that control the circadian sensitivity rhythm in the median eyes of the scorpion, Androctonus australis, were detected in the brain by retrograde labeling with Lucifer Yellow CH. A total of 20–40 neurons are arranged in two groups displaying a bilaterally symmetrical, marginal position near the circumesophageal connectives. Half the cells in each group send fibers into the ipsilateral optic nerve; the fibers from the other half enter the contralateral optic nerve.     

Characterization of the autonomic innervation of mammary gland in lactating rats studied by retrograde transynaptic virus labeling and immunohistochemistry

The aim of experiments was to characterize the neurons of the autonomic chain that innervates the nipple and the mammary gland of lactating rats using retrograde transynaptic virus labeling and neurotransmitter and neuropeptide immunohistochemistry. Two days after injection of green fluorescence protein labeled virus in two nipples and underlying mammary glands, labeling was observed in the ipsilateral paravertebral sympathetic trunk and the lateral horn. Three days after inoculation the labeling appeared in the brain stem and the hypothalamic paraventricular nucleus. Above the spinal cord the labeling was bilateral. A subpopulation of virus labeled cells in the paraventricular nuclei synthesized oxytocin. Labeled neurons in the lateral horn showed cholinergic immunoreactivity. These cholinergic neurons innervated the paravertebral ganglia where the virus labeled neurons were partially noradrenergic. The noradrenergic fibers in the mammary gland innervate the smooth muscle wall of vessels, but not the mammary gland in rats. The neurons in the lateral horn receive afferents from the brain stem, and paraventricular nucleus and these afferents are noradrenergic and oxytocinergic. New findings in our work: Some oxytocinergic fibers may descend to the neurons of the lateral horn which innervate noradrenergic neurons in the paravertebral sympathetic trunk, and in turn these noradrenergic neurons reach the vessels of the mammary gland.     

Successful retrograde transport of fluorescent latex nanospheres in the cerebral cortex of the macaque monkey.

Retrograde axonal transport of latex nanospheres offers a means of delivering chemical agents to a targeted region of the central nervous system (CNS). In this study we performed microinjections of latex nanospheres into the cerebral cortex of cynomolgus monkeys and observed successful retrograde labeling of neurons in the contralateral region. Our data indicate the successful use of this delivery system, reported in studies using other animals, may also be achievable with primates as well.     

Long-range neuronal circuits underlying the interaction between sensory and motor cortex

In the rodent vibrissal system, active sensation and sensorimotor integration are mediated in part by connections between barrel cortex and vibrissal motor cortex. Little is known about how these structures interact at the level of neurons. We used Channelrhodopsin-2 (ChR2) expression, combined with anterograde and retrograde labeling, to map connections between barrel cortex and pyramidal neurons in mouse motor cortex. Barrel cortex axons preferentially targeted upper layer (L2/3, L5A) neurons in motor cortex; input to neurons projecting back to barrel cortex was particularly strong. Barrel cortex input to deeper layers (L5B, L6) of motor cortex, including neurons projecting to the brainstem, was weak, despite pronounced geometric overlap of dendrites with axons from barrel cortex. Neurons in different layers received barrel cortex input within stereotyped dendritic domains. The cortico-cortical neurons in superficial layers of motor cortex thus couple motor and sensory signals and might mediate sensorimotor integration and motor learning.     

Rapid labeling of neuronal populations by ballistic delivery of fluorescent dyes

Particle-mediated ballistic delivery of fluorescent dyes has been recently used to label neuronal populations in a rapid and efficient fashion. Here we describe detailed protocols for this technique as well as recent improvements in its implementation. This technique allows rapid labeling of entire neurons in a Golgi-like manner after membranes of individual neurons are contacted by particles coated with lipophilic dyes. Neurons can be labeled by dyes of different colors at controlled densities to facilitate the study of structural interactions between cells. Furthermore, in conjunction with other histochemical labeling methods, the technique can be used to study changes in neuronal structures associated with pathologic processes in animal models or postmortem human brain. In addition to lipophilic dyes, water-soluble molecules such as calcium indicators can also be delivered efficiently with this technique. The method of ballistic delivery of indicators thus provides new avenues to probe the structure and function of the nervous system.     

3H]-GABA selective retrograde labeling of neurons in the pigeon thalamo-Wulst pathway

Evidence is presented which is consistent with a specific retrograde labeling of GABAergic neurons following [3H]-GABA application in their zone of termination. [3H]-GABA injection in the pigeon Wulst leads to perikaryal retrograde labeling in the ipsilateral thalamic visual relay, n. dorsolateralis anterior thalami, pars lateralis (DLLv). This result gives further support to the biochemical evidence of the existence, in the pigeon, of a GABAergic projection from DLLv to the ipsilateral visual Wulst.     

Combined immunohistochemical and retrograde tracing reveals little evidence of innervation of the rat dentate gyrus by midbrain dopamine neurons

Although the functional neuroanatomy of the midbrain dopamine (mDA) system has been well characterized, the literature regarding its capacity to innervate the hippocampal formation has been inconsistent. The lack of expression of definitive markers for dopaminergic fibers, such as the dopamine transporter, in the hippocampus has complicated studies in this area. Here we have used immunohistochemical techniques to characterize the tyrosine hydroxylase expressing fiber network in the rat hippocampus, combined with retrograde tracing from the dentate gyrus to assess the capacity for afferent innervation by mDA neurons. The results indicate that virtually all tyrosine hydroxylase fibers throughout the hippocampus are of a noradrenergic phenotype, while the overlying cortex contains both dopaminergic and noradrenergic fiber networks. Furthermore, retrograde tracing from the dentate gyrus robustly labels tyrosine hydroxylase-immunoreactive noradrenergic neurons in the locus coeruleus but not mDA neurons.     

A novel method for simultaneous anterograde and retrograde labeling of spinal cord motor tracts in the same animal.

Examination of repaired spinal cord tracts has usually required separate groups of animals for anterograde and retrograde tracing owing to the incompatibility of techniques such as tissue fixation. However, anterograde and retrograde labeling of different animals subjected to the same repair may not allow accurate examination of that repair strategy because widely variable results can occur in animals subjected to the same strategy. We have developed a reliable method of labeling spinal cord motor tracts bidirectionally in the same animal using DiI, a lipophilic dye, to anterogradely label the corticospinal tract and Fluoro-Gold (FG) to retrogradely label cortical and brainstem neurons of several spinal cord motor tracts in normal and injured adult rats. Other tracer combinations (lipophilic dyes or fluorescent dextrans) were also investigated but were less effective. We also developed methods to minimize autofluorescence with the DiI/FG technique, and found that the DiI/FG technique is compatible with decalcification and immunohistochemistry for several markers relevant for studies of spinal cord regeneration. Thus, the use of anterograde DiI and retrograde FG is a novel technique for bidirectional labeling of the motor tracts of the adult spinal cord with fluorescent tracers and should be useful for demonstrating neurite regeneration in studies of spinal cord repair.(J Histochem Cytochem 49:1111-1122, 2001)     

The connectivity of the brain: multi-level quantitative analysis

 We develop a mathematical formalism or calculating connectivity volumes generated by specific topologies with various physical packing strategies. We consider four topologies (full, random, nearest-neighbor, and modular connectivity) and three physical models: (i) interior packing, where neurons and connection fibers are intermixed, (ii) sheeted packing where neurons are located on a sheet with fibers running underneath, and (iii) exterior packing where the neurons are located at the surfaces of a cube or sphere with fibers taking up the internal volume. By extensive cross-referencing of available human neuroanatomical data we produce a consistent set of parameters for the whole brain, the cerebral cortex, and the cerebellar cortex. By comparing these inferred values with those predicted by the expressions, we draw the following general conclusions for the human brain, cortex, and cerebellum: (i) Interior packing is less efficient than exterior packing (in a sphere). (ii) Fully and randomly connected topologies are extremely inefficient. More specifically we find evidence that different topologies and physical packing strategies might be used at different scales. (iii) For the human brain at a macrostructural level, modular topologies on an exterior sphere approach the data most closely. (iv) On a mesostructural level, laminarization and columnarization are evidence of the superior efficiency of organizing the wiring as sheets. (v) Within sheets, microstructures emerge in which interior models are shown to be the most efficient. With regard to interspecies similarities and differences we conjecture (vi) that the remarkable constancy of number of neurons per underlying square millimeter of cortex may be the result of evolution minimizing interneuron distance in grey matter, and (vii) that the topologies that best fit the human brain data should not be assumed to apply to other mammals, such as the mouse for which we show that a random topology may be feasible for the cortex. Received: 14 December 1994/Accepted in revised form: 23 May 1995     

Adenovirus-mediated WGA gene delivery for transsynaptic labeling of mouse olfactory pathways

Detailed knowledge of neuronal connectivity patterns is indispensable for studies of various aspects of brain functions. We previously established a genetic strategy for visualization of multisynaptic neural pathways by expressing wheat germ agglutinin (WGA) transgene under the control of neuron type-specific promoter elements in transgenic mice and Drosophila. In this paper, we have developed a WGA-expressing recombinant adenoviral vector system and applied it for analysis of the olfactory system. When the WGA-expressing adenovirus was infused into a mouse nostril, various types of cells throughout the olfactory epithelium were infected and expressed WGA protein robustly. WGA transgene products in the olfactory sensory neurons were anterogradely transported along their axons to the olfactory bulb and transsynaptically transferred in glomeruli to dendrites of the second-order neurons, mitral and tufted cells. WGA protein was further conveyed via the lateral olfactory tract to the olfactory cortical areas including the anterior olfactory nucleus, olfactory tubercle, piriform cortex and lateral entorhinal cortex. In addition, transsynaptic retrograde labeling was observed in cholinergic neurons in the horizontal limb of diagonal band, serotonergic neurons in the median raphe nucleus, and noradrenergic neurons in the locus coeruleus, all of which project centrifugal fibers to the olfactory bulb. Thus, the WGA-expressing adenovirus is a useful and powerful tool for tracing neural pathways and could be used in animals that are not amenable to the transgenic technology.     

Divergence between axonal collaterals of thalamic neurons running to the cat visual and association cortex

Projections from thalamic neurons to the visual (area 17) and parietal association cortex (area 7) were investigated in cats by means of retrograde axonal transport of fluorescent dyes. Pulvinar neurons may be divided into three groups on the basis of their connections with these areas: those projecting to area 7 (the largest (the largest group of cells), those projecting to area 17 (the smaller group), and others sending out axons to two cortical areas at the same time (a few isolated units). The two first groups only were found in the posterolateral thalamus. Divergence between axonal collaterals of pulvinar neurons may be responsible for parallel routes of information transmission to the visual and association cortex.Institute of Higher Nervous Activity and Neurophysiology, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 22, No. 4, pp. 513–520, July–August, 1990.     

GABA-immunohistochemistry as a label for identifying types of local interneurons and their synaptic contacts in the antennal lobes of the American cockroach

Summary Synaptic contacts between GABA-immunoreactive neurons, antennal receptor fibers and non-GABA-immunoreactive neurons in the glomerular neuropil of the antennal lobes have been identified by means of a combination of (i) immunohistochemical labeling and (ii) labeling of afferent fibers of the antenna by experimentally induced degeneration. Characteristic contacts of these neurons are: a) Serially arranged polysynaptic contacts between degenerated antennal fibers, GABA-immunoreactive neurons and non-GABA-immunoreactive neurons. b) Monosynaptic contacts between degenerated antennal fibers and non-GABA-immunoreactive neurons. c) Reciprocal synaptic contacts between immunostained and non-stained neurons and synaptic contacts between individual GABA-immunoreactive neurons. d) Synaptic output contacts of GABA-immunoreactive neurons with degenerated antennal fibers.GABA-immunoreactive neuron profiles in the glomeruli are assigned to multiglomerular local interneurons (Distler 1989a); non-immunolabeled profiles may be assigned to projection neurons and other not yet identified interneurons.     

Localization of sources of thalamic inputs into the sensorimotor cortex in the rabbit using retrograde axonal transport technique

It was shown that the rabbit sensorimotor cortex received afferent fibers from neurons located in the specific, nonspecific, and association thalamic nuclei using the retrograde axonal transport technique. The distribution, dimensions, and shape of the somata of relay neurons spread through the thalamic nuclei were analyzed. The total number of neurons sending out thalamo-sensorimotor-cortical fibers was calculated and the coordinates of loci with the highest density of these cells in each thalamic nucleus were identified. Multipolar and stellate cells with somata measuring 12–20 µm and 10–15 µm in diameter, respectively, prevailed amongst relay neurons. Amongst the specific nuclei, the majority of afferent fibers are sent out by the ventrolateral, ventral anterior, and anterior ventral nuclei. A comparable number of afferent fibers are sent out by the mediodorsal and paracentral nuclei; these split up among the association nuclei and paracentral nuclei, respectively. It is suggested that afferents from many different groups of thalamic nuclei are essential for the sensorimotor cortex to participate in thalamocortical interaction.Institute of Higher Nervous Activity and Neurophysiology, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 19, No. 1, pp. 87–94, January–February, 1987.     

GABA-immunohistochemistry as a label for identifying types of local interneurons and their synaptic contacts in the antennal lobes of the American cockroach

Synaptic contacts between GABA-immunoreactive neurons, antennal receptor fibers and non-GABA-immunoreactive neurons in the glomerular neuropil of the antennal lobes have been identified by means of a combination of (i) immunohistochemical labeling and (ii) labeling of afferent fibers of the antenna by experimentally induced degeneration. Characteristic contacts of these neurons are: a) Serially arranged polysynaptic contacts between degenerated antennal fibers, GABA-immunoreactive neurons and non-GABA-immunoreactive neurons. b) Monosynaptic contacts between degenerated antennal fibers and non-GABA-immunoreactive neurons. c) Reciprocal synaptic contacts between immunostained and non-stained neurons and synaptic contacts between individual GABA-immunoreactive neurons. d) Synaptic output contacts of GABA-immunoreactive neurons with degenerated antennal fibers. GABA-immunoreactive neuron profiles in the glomeruli are assigned to multiglomerular local interneurons (Distler 1989a); non-immunolabeled profiles may be assigned to projection neurons and other not yet identified interneurons.