Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method.

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain  excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Monosynaptic convergence of chorda tympani and glossopharyngeal afferents onto ascending relay neurons in the nucleus of the solitary tract: a high-resolution confocal and correlative electron microscopy approach.

Physiological studies suggest convergence of chorda tympani and glossopharyngeal afferent axons onto single neurons of the rostral nucleus of the solitary tract (rNTS), but anatomical evidence has been elusive. The current study uses high-magnification confocal microscopy to identify putative synaptic contacts from afferent fibers of the two nerves onto individual projection neurons. Imaged tissue is revisualized with electron microscopy, confirming that overlapping fluorescent signals in confocal z-stacks accurately identify appositions between labeled terminal and dendrite pairs. Monte Carlo modeling reveals that the probability of overlapping fluorophores is stochastically unrelated to the density of afferent label, suggesting that convergent innervation in the rNTS is selective rather than opportunistic. Putative synaptic contacts from each nerve are often compartmentalized onto dendrite segments of convergently innervated neurons. These results have important implications for orosensory processing in the rNTS, and the techniques presented here have applications in investigations of neural microcircuitry with an emphasis on innervation patterning.

A cost-benefit analysis of neuronal morphology.

Over hundreds of millions of years, evolution has optimized brain design to maximize its functionality while minimizing costs associated with building and maintenance. This observation suggests that one can use optimization theory to rationalize various features of brain design. Here, we attempt to explain the dimensions and branching structure of dendritic arbors by minimizing dendritic cost for given potential synaptic connectivity. Assuming only that dendritic cost increases with total dendritic length and path length from synapses to soma, we find that branching, planar, and compact dendritic arbors, such as those belonging to Purkinje cells in the cerebellum, are optimal. The theory predicts that adjacent Purkinje dendritic arbors should spatially segregate. In addition, we propose two explicit cost function expressions, falsifiable by measuring dendritic caliber near bifurcations.

Correlative electron and confocal microscopy assessment of synapse localization in the central nervous system of an insect.

Excellent methods exist to analyze sub-neuronal structures, such as synapses, at nanometer resolution with electron microscopy. However, due to methodological constraints, electron microscopy is feasible only for small volumes of fixed tissue. By contrast, confocal or two-photon laser scanning microscopy is well suited to obtain neuronal structures from large volumes of living or fixed tissue at sub-micrometer resolution. Therefore, a gap exists when analyzing synaptic organization of neuropils, or the distribution of synapses throughout the dendritic trees of individual neurons, and it would be advantageous to use confocal microscopy to investigate the synaptic organization of central neuropils. This study uses correlative electron and confocal microscopy from the same tissue sections to test whether synapsin I-immunopositive puncta can be analyzed at the light microscopy level to estimate the distributions of synaptic sites within central motor neuropils and along reconstructed dendritic surfaces in an insect ventral nerve cord. It demonstrates that every type 1 synaptic terminal can be detected as a distinct punctum by synapsin I-immunolabeling and confocal microscopy. Furthermore, it provides data indicating that co-localization analysis from confocal image stacks as recently published provides a good estimate for quantifying the distribution patterns of input synapses through dendritic trees.

A cross-platform freeware tool for digital reconstruction of neuronal arborizations from image stacks.

Digital reconstruction of neuronal arborizations is an important step in the quantitative investigation of cellular neuroanatomy. In this process, neurites imaged by microscopy are semi-manually traced through the use of specialized computer software and represented as binary trees of branching cylinders (or truncated cones). Such form of the reconstruction files is efficient and parsimonious, and allows extensive morphometric analysis as well as the implementation of biophysical models of electrophysiology. Here, we describe Neuron_ Morpho, a plugin for the popular Java application ImageJ that mediates the digital reconstruction of neurons from image stacks. Both the executable and code of Neuron_ Morpho are freely distributed (www.maths. soton.ac.uk/staff/D'Alessandro/morpho or www.krasnow.gmu.edu/L-Neuron), and are compatible with all major computer platforms (including Windows, Mac, and Linux). We tested Neuron_Morpho by reconstructing two neurons from each of the two preparations representing different brain areas (hippocampus and cerebellum), neuritic type (pyramidal cell dendrites and olivar axonal projection terminals), and labeling method (rapid Golgi impregnation and anterograde dextran amine), and quantitatively comparing the resulting morphologies to those of the same cells reconstructed with the standard commercial system, Neurolucida. None of the numerous morphometric measures that were analyzed displayed any significant or systematic difference between the two reconstructing systems.

Glutamate escape from a tortuous synaptic cleft of the hippocampal mossy fibre synapse.

The time course of neurotransmitter in the synaptic cleft contributes substantially to the fast kinetics of synaptic signalling. Hippocampal mossy fibres (MFs), a well-characterised excitatory pathway from dentate granule cells to the hippocampus proper, form large glutamatergic synapses at branched spiny structures in CA3 pyramidal cell dendrites. To what extent transmission at these synapses is affected by retarded glutamate clearance from the large tortuous synaptic cleft is not known. Here, we propose a simple geometrical approximation representing the 'typical' geometry of thorny excrescences that form the tortuous cleft interface at a MF synapse. We then employ Monte Carlo simulations to monitor movements of 3000 individual glutamate molecules released within the cleft. The results predict that, in the absence of neuronal glutamate transporters, it should take approximately 10 ms for 50% and 60-70 ms for 90% of glutamate molecules to escape the MF synapse.

Quantified assessment of terminal density and innervation.

Stereological methods allow for the determination of cell numbers, terminal densities, and, subsequently, the estimation of terminal arbor size within a given brain nucleus. This unit provides an explanation for determining the terminal arbor size of dopaminergic neurons of the nigrostriatal pathway in rodents. In contrast to previously used single-axon reconstructions, these stereological methods allow for quick and easy determination of terminal arbor size.

Subthreshold voltage noise due to channel fluctuations in active neuronal membranes.

Voltage-gated ion channels in neuronal membranes fluctuate randomly between different conformational states due to thermal agitation. Fluctuations between conducting and nonconducting states give rise to noisy membrane currents and subthreshold voltage fluctuations and may contribute to variability in spike timing. Here we study subthreshold voltage fluctuations due to active voltage-gated Na+ and K+ channels as predicted by two commonly used kinetic schemes: the Mainen et al. (1995) (MJHS) kinetic scheme, which has been used to model dendritic channels in cortical neurons, and the classical Hodgkin-Huxley (1952) (HH) kinetic scheme for the squid giant axon. We compute the magnitudes, amplitude distributions, and power spectral densities of the voltage noise in isopotential membrane patches predicted by these kinetic schemes. For both schemes, noise magnitudes increase rapidly with depolarization from rest. Noise is larger for smaller patch areas but is smaller for increased model temperatures. We contrast the results from Monte Carlo simulations of the stochastic nonlinear kinetic schemes with analytical, closed-form expressions derived using passive and quasi-active linear approximations to the kinetic schemes. For all subthreshold voltage ranges, the quasi-active linearized approximation is accurate within 8% and may thus be used in large-scale simulations of realistic neuronal geometries.

Critical assessment of the involvement of perforations, spinules, and spine branching in hippocampal synapse formation.

Several studies propose that long-term enhancement of synaptic transmission between neurons results from the enlargement, perforation, and splitting of synapses and dendritic spines. Unbiased analyses through serial electron microscopy were used to assess the morphological basis for synapse spilitting in hippocampal area CA1. Few perforated synapses and almost no split (i.e., branched) spines occurred at postnatal day 15, an age of high synaptogenesis; thus, synapse splitting is unlikely to be important during development. The synapse splitting hypothesis predicts an intermediate stage of branched spines with both heads sharing the same presynaptic bouton. Ninety-one branched dendritic spines were traced through serial sections, and the different branches never synapsed with the same presynaptic bouton. Projections from spines, called "spinules," have been thought to extend from perforations in the postsynaptic density (PSD), thereby dividing the presynaptic bouton. Forty-six spinules were traced, and only 13% emerged from perforations in the PSD. Most spinules emerged from the edges of nonperforated PSDs, or from spine necks, where they extended into boutons that were not presynaptic to the spine. In summary, these morphological characteristics are inconsistent with synapse and spine splitting. An alternative is discussed whereby perforated synapses and spinules are transient components of synaptic activation, and branched spines appear from synapses forming in close proximity to one another.

Comparison of the topology and growth rules of motoneuronal dendrites.

The complexity, shape, and branching modes of the dendrites of spinal motoneurons were compared in cat, rat, and frog using topological analysis and growth models. The complexity of motoneuronal dendrites, measured as the mean number of terminal segments, varied significantly among samples and was related to contractile properties of innervated motor units. Despite this variation, all mature motoneurons having a mean number of terminal segments per dendrite greater than ten (up to 24.3) exhibited a narrow range of values of coefficients describing the symmetry of tree shapes (0.42-0.47). This implies low variability in the topological shape of motoneuronal dendrites of different animals. This similarity of tree shapes proved to be a result of the similarity of growth rules. The growth of the dendrites could be described to a first approximation by a two-parameter (Q and S) model called the QS model and by a multitype Markovian model. The estimation of parameters of the QS model, in which parameter Q is related to the probability of branching of intermediate segments, revealed that Q was equal or close to 0, implying that branching of dendrites is restricted to terminal segments. The estimates of the parameter S, which describes whether the probability of branching increases (S < 0) or decreases (S > 0) exponentially with segment order, were positive. This was in agreement with the results of estimation of probabilities of branching provided by the Markovian model, which showed that the branching probabilities decreased with segment order in an exponential manner in most of the neurons studied. The QS and Markovian models involve different assumptions about the sequence and timing of branching events, and selection of the best model can provide insight into details of dendritic outgrowth. Extensive simulation of tree outgrowth using a Markovian model revealed significant differences between stimulated trees and real dendrites, particularly with regard to variability of the number of terminals and to symmetry. In contrast, the QS model provided a good fit to the mean values and standard deviations of basic topological parameters. This model is adequate to describe the shape of mature motoneuronal dendrites. It implies that dendritic branches have many opportunities to bifurcate during the whole time of development and that bifurcating potency of a branch is a function of the number and position of other branches of that dendrite. Combined with analysis of metrical properties such as lengths of segments, the QS model can assist in a quantitative analysis of development and plasticity.

Repeated confocal imaging of individual dendritic spines in the living hippocampal slice: evidence for changes in length and orientation associated with chemically induced LTP.

Using confocal microscopy in conjunction with microdrop application of Dil, we have imaged and measured individual dendritic spines of living hippocampal CA1 pyramidal neurons in acute brain slices, before and approximately 3 hr after induction of long-term potentiation by chemical means. Statistical analysis of changes in the length of individual spines, and comparison with results of Monte Carlo simulations, suggests that two forms of structural change occur in chemically induced long-term potentiation: growth of a subpopulation of small spines, and angular displacement of spines. These changes could provide a structural basis for the expression of long-term potentiation.

A note on the distribution of dendritic spines.

Some previously published data concerning the spatial distributions of dendritic spines are re-examined. It appears that such distributions are not completely random, but show a tendency towards repulsion, that is to say a deficiency of short inter-spine distances and an excess of long distances when compared to a totally random model. While this lack of total randomness contradicts an assumption underlying a previously published technique for estimating total spine number, it does not appear to seriously interfere with this technique.

Morphological details of primate axons and dendrites revealed by extracellular injection of biocytin: an economic and reliable alternative to PHA-L.

The objective of this study was to determine if biocytin would reliably label details of distant axons and dendrites when injected extracellularly in primates. Biocytin (2.5-5%) was injected iontophoretically or by pressure into several areas of the visual and somatosensory systems of macaque monkeys, squirrel monkeys, tree shrews and galagos. After survival times that ranged from 9 h to 2 weeks, fine details of anterogradely filled axons and/or retrogradely filled dendrites were reliably revealed with an avidin-biotin-HRP complex (ABC solution) that was enhanced with heavy metals. Biocytin labeling was successfully combined with choline acetyltransferase (ChAT) or cytochrome oxidase (CO) histochemistry to reveal double-labeled cells. Our results show that biocytin is a versatile, easy-to-use label that completely fills cell processes both anterogradely and retrogradely in several primate species.

Quantitative assessment of dendritic branching and spine densities of neurons of hippocampal embryonic tissue transplanted into juvenile neocortex.

Embryonic 18-day-old hippocampal tissue was transplanted into a fresh cavity in frontal neocortex in 18-day-old (juvenile) Wistar rats. The transplant was examined after 2-5 months. The rapid Golgi procedure was used to assess quantitatively the dendritic branching (intersections and branching orders) and spine numbers per neuronal region, and spine densities (per 20 microns segment) on the pyramidal neurons, granule neurons and other types. The results revealed that the graft neurons generally have significant deviations from those of normal native hippocampal neurons. The aberrations were present in both pyramidal and granule cells. There were also many neurons which could not be classified into the known types. The pyramidal neurons showed a significant deficiency in dendritic branching, more often in basal dendrites than in apical dendrites. Apical dendrites of the graft pyramidal neurons also developed spine densities relatively better than basal dendrites, and sometimes significantly higher than even normal neurons. The significant deficiency in spine numbers of basal dendrites was primarily due to deficiency in dendritic branching rather than to spine density. On the contrary, the granule cells developed significantly less of both dendritic branching and spine densities (hence spines per neuron) than normal hippocampal granule neurons. The unclassifiable neuronal types had generally high dendritic branching and spine densities relative to the other neurons of the graft. The study discusses the importance and necessity of making quantitative assessments of neurons to understand whether the apparently normal neurons of transplants are really normal, and how far they are deviant. So far, quantitative assessments have been seldom reported in this important area of research, hence, this study is the first of its kind to highlight it.

Characterization of muscarinic acetylcholine receptors in rat cerebral cortex slices with concomitant morphological and physiological assessment of tissue viability.

We have begun studies on regulatory mechanisms of muscarinic acetylcholine receptors (mAChRs) in slices of rat cerebral cortex. This paper, the first of two, deals with the viability of the cells in the slices (a prerequisite for studying receptor regulation) and provides a characterization of binding sites for [3H]N-methyl scopolamine ([3H]NMS) and [3H]quinuclidinyl benzylate ([3H]QNB) in this preparation. Trypan blue exclusion tests in 400-microns-thick cortical slices showed a number of dead cells in a 100 microns zone from each cut edge, for a total of about 15-30% of all cells in the slice. In agreement with previous reports, electron microscopy revealed healthy tissue in the middle of the slice, but after incubation for several hours, swollen cells and dendrites were seen without cytoplasmic organelles. Axon terminals, however, were still seen to synapse upon these processes. Electrophysiological single unit recordings showed spontaneous action potentials in the slices. For receptor binding experiments, slices were incubated with either [3H]NMS, a hydrophilic mAChR ligand which does not penetrate the cell membrane, or the lipophilic ligand [3H]QNB which readily enters cells. For both ligands, equilibrium binding was reached after 8 h at 4 degrees C, and after 3 h at 30 degrees C. Binding of both ligands could be displaced by unlabelled atropine sulphate, NMS or QNB. Saturation binding curves yielded a Bmax of 2187 fmol/mg protein for [3H]QNB (reflecting all mAChRs) and 1335 fmol/mg protein for [3H]NMS (only mAChRs on the cell surface) at 30 degrees C. Kd values were 8.2 and 5.2 nM for [3H]QNB and [3H]NMS, respectively. These values are high compared with values obtained from homogenates, frozen sections or dissociated cells, and presumably reflect the use of intact, living tissue. These data are probably a better reflection of the actual, in vivo mAChR number and affinity than those obtained from dead tissue. This slice preparation suggests itself as a simple but effective method with which to study the regulation of mAChRs in living brain tissue.

Ultrastructural investigation into the influence of ethanol on synaptic maturation in rat neocortex. I. Qualitative assessment.

Male rats were exposed daily to ethanol vapour from 3 days of age, and their brains were examined at 7, 14, 21, 56 and 76 days postnatally. Control animals were examined at each age, and ethanol-rehabilitated animals were examined at 56 and 76 days postnatally. Tissue from the parietal cortex of each animal stained with osmium tetroxide and with ethanolic-phosphotungstic acid (E-PTA) was analyzed by qualitative ultrastructural techniques. The ethanol flow rate in the incubation chamber was adjusted to maintain the blood ethanol level as close as possible to 0.1 g/100 ml. Ethanol-treated rats weighed less than ethanol-rehabilitated animals at days 56 and 76. At day 7 synapses were formed between axons and dendritic growth cones, dendritic shafts and filopodia in control and ethanol-treated tissue. At days 14 and 21 well-developed axodendritic and axospinous synapses were evident in both groups. The neuropil of 56- and 76-day-old material was similar in the control and treatment groups, except that there was an enlargement of dendritic profiles in the 56-day-old ethanol-treated material. Perforated synapses were most common in 56-day-old ethanol-treated material, with degenerating synapses most common in 56-day-old (and to a lesser extent 76-day-old) ethanol-rehabilitated material. No obvious differences were detected between any of the groups of the E-PTA-stained material. The presence of degenerating and perforated synapses suggests that synaptic remodelling is occurring, and this may be a means of adapting synaptic mechanisms to the functional demands imposed by ethanol.

Postnatal dendritic development in motoneurons: evaluation by a Monte Carlo technique.

A Monte Carlo method was used to quantitate the dendritic characteristics of horseradish peroxidase-injected lumbar motoneurons (MNs) from kittens 44 to 73 days old (during the postnatal interval when motor cortical input/output linkages mature) and compared with those from adults. All of the 6 MNs analyzed were within the same range of somal sizes (40-61 micron) and dendritic domain volumes. However, two-dimensional distributions of dendritic diameter versus distance from the soma revealed that in the youngest MNs' (44 and 51 days) most dendritic processes (82 and 84%) were less than 2 micron in diameter, and had no dendrites greater than 5 micron. The adult MNs had large dendrites (20 and 24% were greater than 5 mu), while the kittens of intermediate age (66 and 73 days) exhibited both adult-like (6 and 13% greater than 5 micron) and immature (48 and 73% less than 2 micron) characteristics. "Hit probabilities" calculated from the Monte Carlo data suggest that immature MNs' dendrites may present denser targets per unit volume for ingrowing growth cones than adult MNs, and may facilitate the probability of contact with appropriate afferent axons.

Development of the brain stem reticular core: an assessment of dendritic state and configuration in the perinatal rat.

The brain stems of normal newborn, 11- and 20-day-old rats were examined using Golgi techniques in the light and electron microscope. Patterns of dendritic branching and growth of brain stem reticular core neurons were analyzed by projection drawing and quantitative methods. The number of protospines and dendritic varicosities was counted on proximal and distal dendritic segments. The amount of DNA and protein in the brain stem was determined using colorimetric methods. During the course of early development, the sparsely branched reticular core dendrites extend rapidly into the neuropil. The dendritic branching of these neurons increases slightly but the number of primary dendrites remains constant. Protospine development follows a regular progression increasing in number to 11 days, then declining to 20 days as protospines are resorbed onto the dendritic surface. Dendritic varicosities often contain additional vesicles as a source of dendritic membrane and subsequent preterminal dendritic growth. The gradual decline of dendritic varicosities, first proximally then distally, indicates a shift of growth toward the distal extreme of the dendrite.