Golgi Preparations Research Articles

Glutamate released by Cajal-Retzius cells impacts specific hippocampal circuits and behaviors.

SUMMARYThe impact of Cajal-Retzius cells on the regulation of hippocampal circuits and related behaviors is unresolved. Here, we directly address this issue by impairing the glutamatergic output of Cajal-Retzius cells with the conditional ablation of vGluT2, which is their main vesicular glutamate transporter. Although two distinct conditional knockout lines do not reveal major alterations in hippocampal-layer organization and dendritic length of principal neurons or GABAergic cells, we find parallel deficits in specific hippocampal-dependent behaviors and in their putative underlying microcircuits. First, conditional knockout animals show increased innate anxiety and decreased feedforward GABAergic inhibition on dentate gyrus granule cells. Second, we observe impaired spatial memory processing, which is associated with decreased spine density and reduced AMPA/NMDA ratio of postsynaptic responses at the perforant- and entorhino-hippocampal pathways. We conclude that glutamate synaptically released by Cajal-Retzius cells is critical for the regulation of hippocampal microcircuits and specific types of behaviors.

CD40L Reverse Signaling Influences Dendrite Spine Morphology and Expression of PSD-95 and Rho Small GTPases.

CD40-activated CD40L reverse signaling is a major physiological regulator of neural process growth from many kinds of developing neurons. Here we have investigated whether CD40L-reverse signaling also influences dendrite spine number and morphology in striatal medium spiny neurons (MSNs). Golgi preparations revealed no differences in the spine density, but because the dendrite arbors of MSNs were larger and branched in Cd40–/– mice, the total number of spines was greater in Cd40–/– mice. We also detected more mature spines compared with wild-type littermates. Western blot revealed that MSN cultures from Cd40–/– mice had significantly less PSD-95 and there were changes in RhoA/B/C and Cdc42. Immunocytochemistry revealed that PSD-95 was clustered in spines in Cd40–/– neurons compared with more diffuse labeling in Cd40+/+ neurons. Activation of CD40L-reverse signaling with CD40-Fc prevented the changes observed in Cd40–/– cultures. Our findings suggest that CD40L-reverse signaling influences dendrite spine morphology and related protein expression and distribution.

Synaptic organization of the dorsal lateral geniculate nucleus.

A half century after Ray Guillery's classic descriptions of cell types, axon types, and synaptic architecture of the dorsal lateral geniculate nucleus, the functional organization of this nucleus, as well as all other thalamic nuclei, is still of enormous interest. This review will focus on two classic papers written by Ray Guillery: 'A study of Golgi preparations from the dorsal lateral geniculate nucleus of the adult cat', and 'The organization of synaptic interconnections in the laminae of the dorsal lateral geniculate nucleus of the cat', as well as the studies that most directly followed from the insights these landmark manuscripts provided. It is hoped that this review will honor Ray Guillery by encouraging further investigations of the synaptic organization of the dorsal thalamus.

Neurons of human nucleus accumbens

Nucleus accumbens is a part of the ventral striatum also known as a drug active brain region, especially related with drug addiction. The aim of the study was to investigate the Golgi morphology of the nucleus accumbens neurons. The study was performed on the frontal and sagittal sections of 15 human brains by the Golgi Kopsch method. We classified neurons in the human nucleus accumbens according to their morphology and size into four types: type I--fusiform neurons; type II--fusiform neurons with lateral dendrite, arising from a part of the cell body; type III--pyramidal-like neuron; type IV--multipolar neuron. The medium spiny neurons, which are mostly noted regarding to the drug addictive conditions of the brain, correspond to the type IV--multipolar neurons. Two regions of human nucleus accumbens could be clearly recognized on Nissl and Golgi preparations each containing different predominant neuronal types. Central part of nucleus accumbens, core region, has a low density of impregnated neurons with predominant type III, pyramidal-like neurons, with spines on secondary branches and rare type IV, multipolar neurons. Contrary to the core, peripheral region, shell of nucleus, has a high density of impregnated neurons predominantly contained of type I and type IV--multipolar neurons, which all are rich in spines on secondary and tertiary dendritic branches. Our results indicate great morphological variability of human nucleus accumbens neurons. This requires further investigations and clarifying clinical significance of this important brain region.

Morphometric Analysis in Neurodegenerative Disorders

AbstractGolgi impregnation has been commonly used for neuronal morphometry in a number of neurological diseases and brain‐disorder studies. Introduced more than a century ago, it is still the standard and state‐of‐the‐art technique for visualization of neuronal architecture. We successfully applied the Golgi method to mouse, rat, monkey, and human brain tissues for studying both the normal and abnormal morphology of neurons. We were able to discover subtle morphological alterations in neuronal dendrites and dendritic spines in different brain areas. Although Golgi preparations can be examined by electronic microscopy, we used light microscopy and reconstruction using Neurolucida software to quantitatively explore the relationship between total dendritic length and spine density in different types of neurons. This unit summarizes the methodology used to quantify neuronal abnormalities and discusses the utility of these techniques in different models of neurodegeneration. Curr. Protoc. Toxicol. 46:12.16.1‐12.16.14. © 2010 by John Wiley & Sons, Inc.

Morphometric analysis in neurodegenerative disorders.

The study of dendritic length and spine density has become a standard in the analysis of neuronal abnormalities since a considerable number of neurological diseases have their foundation in alterations in these structures. One of the best ways to study possible alterations in neuronal morphometry is the use of Golgi impregnation. Introduced more than a century ago, it is still the standard and state-of-the-art technique for visualization of neuronal architecture. We successfully applied the Golgi method to mouse, rat, monkey and human brain tissues for studying both the normal and abnormal morphology of neurons. We were able to discover subtle morphological alterations in neuronal dendrites and dendritic spines in different brain areas. Although Golgi preparations can be examined by electronic microscopy, we used light microscopy and Neurolucida reconstruction to quantitatively explore the relationship between total dendritic length and spine density in different types of neurons. This review summarizes the methodology used to quantify neuronal abnormalities and discusses the utility of these techniques in different models of neurodegeneration.

Mapping synaptic pathology within cerebral cortical circuits in subjects with schizophrenia

Converging lines of evidence indicate that schizophrenia is characterized by impairments of synaptic machinery within cerebral cortical circuits. Efforts to localize these alterations in brain tissue from subjects with schizophrenia have frequently been limited to the quantification of structures that are non-selectively identified (e.g., dendritic spines labeled in Golgi preparations, axon boutons labeled with synaptophysin), or to quantification of proteins using methods unable to resolve relevant cellular compartments. Multiple label fluorescence confocal microscopy represents a means to circumvent many of these limitations, by concurrently extracting information regarding the number, morphology, and relative protein content of synaptic structures. An important adaptation required for studies of human disease is coupling this approach to stereologic methods for systematic random sampling of relevant brain regions. In this review article we consider the application of multiple label fluorescence confocal microscopy to the mapping of synaptic alterations in subjects with schizophrenia and describe the application of a novel, readily automated, iterative intensity/morphological segmentation algorithm for the extraction of information regarding synaptic structure number, size, and relative protein level from tissue sections obtained using unbiased stereological principles of sampling. In this context, we provide examples of the examination of pre- and post-synaptic structures within excitatory and inhibitory circuits of the cerebral cortex.

The Development of Developmental Neuroscience

### Introduction Forty years ago in the emerging field of developmental neurobiology, neuroanatomists studied cellular development in their favorite brain region by annotating static Golgi preparations at different time points. Cell biologists examined how growth cones grow in tissue culture,

Chronic stress induces dendritic atrophy in the rat medial geniculate nucleus: Effects on auditory conditioning

Chronic stress induces dendritic atrophy in the inferior colliculus (IC, auditory mesencephalon) and impairs auditory avoidance conditioning. The aim of this study was to determine in Golgi preparations and in cued fear conditioning whether stress affects other auditory components, like the thalamic medial geniculate nucleus (MG) or the posterior thalamic nucleus (PO), in Sprague–Dawley rats. Chronic restraint stress produced a significant dendritic atrophy in the MG (stress: 407±55μm; control: 808±120μm; p<0.01) but did not affect auditory fear conditioning. The last result was in apparent contrast with the fact that stress impairs both the acquisition of auditory avoidance conditioned responses and the dendritic structure in two major nuclei of the auditory system. In order to analyze this disagreement, we investigated whether the stress-related freezing to tone occurring in the fear conditioning protocol corresponded to a conditioned or an unconditioned fear response, using changes in tone instead of light throughout conditioning trials. Chronic stress significantly enhanced visual fear conditioning in stressed animals compared to controls (stress: 58.9±8.42%, control: 23.31±8.01%; p<0.05), but this fear enhancement was related to unconditioned fear. Conversely, chronic stress did not affect the morphology of the PO (subserving both auditory and somatosensory information) or the corresponding auditory and somatosensory unconditioned responses (acoustic startle response and escape behavior). Our results suggest that the auditory conditioned stimulus can be processed in part independently of the IC and MG in the stressed animals, and sent to the amygdala via the PO inducing unconditioned fear. Comparable alterations could be produced in major depression.

Reflections

CNS white matter from higher vertebrates and cultured differentiated oligodendrocytes are nonpermissive substrates for neurite growth and fibroblast spreading. Membrane proteins of 35 kd and 250 kd with highly nonpermissive substrate properties could be extracted from CNS myelin fractions. Monoclonal antibodies were raised against these proteins: IN-1 and IN-2 bound both to the 35 kd and 250 kd inhibitors and to the surface to differentiated cultured oligodendrocytes. Adsorption of nonpermissive CNS myelin or nonpermissive oligodendrocytes with either antibody markedly improved their substrate properties. Optic nerve explants injected with IN-1 or IN-2 allowed axon ingrowth of cocultured sensory and sympathetic neurons. We conclude that the nonpermissive substrate properties of CNS white matter are due to these membrane proteins on the surface of differentiated oligodendrocytes and to their in vivo product, myelin.

Golgi-type I and Golgi-type II neurons in the ventral anterior thalamic nucleus of the adult human: morphological features and quantitative analysis.

The morphological and quantitative features of neurons in the adult human ventral anterior thalamic nucleus were studied in Golgi preparations. Two neuronal types were found and their quantitative features were studied. Golgi-type I neurons were medium to large cells with dense dendritic trees and dendritic protrusions and short hair-like appendages. They have somatic mean diameter of 30.8 microm (+/-9.4, n = 85). They have an average 100.3 dendritic branches, 48.97 dendritic branching points, and 58.85 dendritic tips. The mean diameters of their primary, secondary, and tertiary dendrites were 3.1 microm (+/-1, n = 80), 1.85 microm (+/-0.8, n = 145), and 1.5 microm (+/-0.4, n = 160), respectively. Golgi-type II neurons were small to medium cells with few sparsely branching dendrites and dendritic stalked appendages with or without terminal swellings. They have somatic mean diameters of 22.2 microm (+/-5.8, n = 120). They have an average 33.76 dendritic branches, 16.49 dendritic branching points, and 21.97 dendritic tips. The mean diameters of their primary, secondary, and tertiary dendrites were 1.6 microm (+/-0.86, n = 70), 1.15 microm (+/-0.55, n = 118), and 1 microm (+/-0.70, n = 95), respectively. These quantitative data may form the basis for further quantitative studies involving aging or some degenerative diseases that may affect cell bodies and/or dendritic trees of the Golgi-type I and/or Golgi-type II thalamic neurons.

“Small-tufted” ganglion cells and two visual systems for the detection of object motion in rabbit retina

Small-tufted (ST) ganglion cells of rabbit retina are divided into eight types based upon morphology, branching pattern, level of dendritic stratification, and quantitative dimensional analysis. Only one of these types has been previously characterized in Golgi preparations, and four may be discerned in the work of others. Given their small dendritic-field size, and assuming uniform mosaics of each across the retina, ST cells comprise about 45% of all rabbit ganglion cells, and are therefore of major functional significance. Four ST cells occur as two paramorphic (a/b) pairs, and thus belong to class III, as previously defined. Four branch in sublaminae a and b of the inner plexiform layer (IPL) and therefore belong to class IV. ST cells have small cell bodies 10-15 microm in diameter, small axons 0.7-1.3 microm in diameter, and small dendritic-field diameters, 40-110 microm in mid-visual streak. The dendrites of ST cells are highly branched, and bear spines and appendages of varying length, but vary from type to type. Class III.2 cells and class III.3 cells are partly bistratified. Class IV small-tufted cells differ characteristically in multiple features of dendritic branching and stratification. Class III small-tufted cells apparently have concentric (ON-center and OFF-center) receptive fields and may have "sluggish-transient" (class III.2) and "sluggish-sustained" (class III.3) physiology. Class IV cells include the "local-edge-detector" (LED) (class IVst1), and are all expected to give ON-OFF responses to small, centered, slowly moving visual stimuli. Based upon systematic variation in dendritic-field size across the retina, ST cells may be divided into two groups. In this "universal prey" species, they may belong to two systems of motion detection, typified by ON-OFF directionally selective and LED ganglion cells, respectively, specialized for detection of rapid motion at the horizon for land-based predators, and slow motion for airborne predators.

The axon arbourisation of nuclei isthmi neurons in the optic tectum of the chick and pigeon. A Golgi and anterograde tracer-study

The optic tectum is reciprocally connected to the nuclei isthmi pars magnocellularis (Imc) and pars parvocellularis (Ipc), which have different modulatory effects on optic transmission. We studied the axon arbourisation of these isthmic nuclei in the optic tectum in order to differentiate between them using Golgi-impregnated preparations both in chickens and pigeons. In addition, sections from animals injected with the anterograde tracer biotinylated dextran-amine (BDA) into the Imc were examined in the bright-field and electron microscope to identify the axon arbourisations and terminals. Also, GABA immunogold stained sections were examined in the electron microscope. In Golgi preparations, slab-like (or poplar tree-like) axon terminal arbourisations of both magnocellular and parvocellular isthmic nuclei neurons were found extending to the tectal surface, with similar branching patterns, but different lengths. The axon arbourisations extending from layer 5 of the optic tectum to the surface were termed type 1, whereas those extending from the internal (12-11) layers to the tectal surface were termed type 2. Type 2 arbourisations very closely matched arbourisations observed in BDA injected material, indicating that Imc neurons gave rise to type 2 arbourisations. The two kinds of axon arbourisation in the external tectal layers were alike in both types of bird, except for the width, which was about 10 mum larger in the type 2 axon arbour. Controlling for size, there was no significant difference between chicks and pigeons. The significance of these afferents in the optic tectum is discussed.

Chronic stress impairs acoustic conditioning more than visual conditioning in rats: Morphological and behavioural evidence

Chronic stress affects brain areas involved in learning and emotional responses. These alterations have been related with the development of cognitive deficits in major depression. The aim of this study was to determine the effect of chronic immobilization stress on the auditory and visual mesencephalic regions in the rat brain. We analyzed in Golgi preparations whether stress impairs the neuronal morphology of the inferior (auditory processing) and superior colliculi (visual processing). Afterward, we examined the effect of stress on acoustic and visual conditioning using an avoidance conditioning test. We found that stress induced dendritic atrophy in inferior colliculus neurons and did not affect neuronal morphology in the superior colliculus. Furthermore, stressed rats showed a stronger impairment in acoustic conditioning than in visual conditioning. Fifteen days post-stress the inferior colliculus neurons completely restored their dendritic structure, showing a high level of neural plasticity that is correlated with an improvement in acoustic learning. These results suggest that chronic stress has more deleterious effects in the subcortical auditory system than in the visual system and may affect the aversive system and fear-like behaviors. Our study opens a new approach to understand the pathophysiology of stress and stress-related disorders such as major depression.

Pioneering a golden age of cerebral microcircuits: The births of the combined Golgi–electron microscope methods

Theodor W. Blackstad devised methods by which the synaptic connectivity of neuron somata and their dendritic and axonal processes in the CNS could be analyzed by the combined use of light and electron microscope techniques. His first publication on that subject dates from 1965 and was contemporary to the independent research by William K. Stell. The Golgi method was an obvious neuronal marker at those times, and Blackstad and Stell showed that the Golgi precipitate is electron-dense and intracellular and, therefore, it could help identify in the electron microscope, with great accuracy, profiles of neurons initially visualized in light microscopy. Besides this convergent research, Blackstad demonstrated for the first time that anterograde axonal degeneration could be combined with the Golgi–electron microscope method, allowing the identification of the neurons whose dendritic or somatic profiles were postsynaptic to the severed axonal afferent projections. Last, but not least, Blackstad pioneered de-impregnation techniques for electron microscopy of Golgi preparations. This had a great impact in the study of synaptic circuitry. The present account is a remembrance of the events that linked these early attempts with the development of a de-impregnation method based on gold toning by Alan Peters and the present author.

Tri-dimensional morphometric analysis of astrocytic processes with high voltage electron microscopy of thick Golgi preparations.

A characteristic feature of the astrocytic processes is to assume the form of shin sheets or lamellate coverings of other brain constituents. We analyzed the extensive and finely divided processes of the protoplasmic astrocyte in the molecular layer of the rat dentate gyrus by means of computer electron tomography and stereo-photogrammetry using tilted high voltage electron microscope images of thick Golgi preparations. The surface area and volume of the astrocytic processes were measured and the surface/volume ratios were estimated. The surface/volume ratios of astrocytic processes in the neuropile ranged from 18.9 to 33.0 per microm, and the mean value was 26.2+/-5.0 per microm. The values were roughly comparable to those previously reported for the microdomain of Bergmann glia cell terminal processes in the rat cerebellum, which were estimated from reconstructions using thin serial section electron microscope images. The large surface to volume ratio of the astrocytic processes in the neuropile resulted from the lamellar nature of the processes interposed between other cellular elements, and may reflect the functional activities of the astrocyte. The results suggest the usefulness of the electron tomography and stereo-photogrammetry for three-dimensional morphometrical analysis of the astrocytic processes, although both techniques can be expected to be refined further in order to provide more precise measurements of these complicated processes.

The Ramifications and Terminals of Optic Fibres in Layers 2 and 3 of the Avian Optic Tectum: A Golgi and Light and Electron Microscopic Anterograde Tracer Study

The ramification patterns and terminals of optic fibres in layers 2 and 3 of the optic tectum were studied in Golgi-stained and immunolabelled preparations made from the brains of chicks and pigeons. The different neuronal structures of layers 2 and 3 were also examined. In Golgi preparations, two types of optic fibre were found both in chick and pigeon tectum according to their thickness and terminal branching patterns. The same types of optic fibres were also found to be present in the anterograde tracer experiments after iontophoresis of biotinylated dextran amine into the optic nerve. The varicose terminals of thin fibres mostly terminated on terminal dendritic sections of radiate and pyramidal-like neurons, contacting them on their apical dendrites. The medium-thick fibre terminals in layer 2 mainly established synapses with horizontally extending dendrites, which may therefore be contacts with inhibitory local circuit neurons. The medium-thick optic fibre bushy-like arborisation in layer 3 established synapses with larger dendrites and also stem dendrites. Their terminals formed groups with different dendritic profiles, some of which were partly covered by glial processes, and/or were located among converging dendrites. The presence of these glomerular-like synapses in layer 3 proves that the optic terminals in layer 3 also take part in the transmission of optic impulses to the nucleus rotundus.

Neuropathology of Rett syndrome.

Rett Syndrome is unlike any other pediatric neurologic disease, and its clinical-pathologic correlation can not be defined with standard histology techniques. Based on hypotheses suggested by careful clinical observations, the nervous system of the Rett child has been explored utilizing morphometry, golgi preparations, computerized tomography, magnetic resonance imaging, chemistry, immunocytochemistry, autoradiography, and molecular biologic techniques. From these many perspectives we conclude that Rett syndrome is not a typical degenerative disorder, storage disorder, nor the result of gross malformation, infectious or neoplastic processes. There remain regions of the brain that have not been studied in detail but the available data suggest that the neuropathology of Rett syndrome can be summarized as follows: the Rett brain is small for the age and the height of the patient; it does not become progressively smaller over three to four decades; it has small dendritic trees in pyramidal neurons of layers III and V in selected lobes (frontal, motor, and temporal); it has small neurons with an increased neuronal packing density; it has an immature expression of microtubular protein-2 and cyclooxygenase; it exhibits a changing pattern of neurotransmitter receptors with an apparent reduction in many neurotransmitters, possibly contributing to some symptomatology. A mutation in Mecp2 causes this unique disorder of brain development. Neuronal mosaicism for normal and mutated Mecp2 produces a consistent phenotype in the classic female patient and a small brain with some preserved islands of function, but with an inability to support hand use and speech. This paper summarizes our current observations about neuropathology of Rett syndrome. MRDD Research Reviews 2002;8:72-76.

Chapter 25 Neuroplasticity in the damaged dentate gyrus of the epileptic brain

Using Golgi preparations, Cajal described many cell types and connections of the dentate gyrus. He described granule cells as having a round or elliptical cell body with their long axis perpendicular to the granule cell layer, dendrites arising from one pole and an axon arising from the other. Cajal apparently never studied the brains from epileptic animals or humans, and thus did not report on changes in granule cell morphology after epilepsy. Several neuroplastic changes have been described in the dentate gyrus of epileptic mammals in the past decade or so using modern methods. Two changes involving their processes include mossy fiber sprouting of granule cell axons into the inner molecular layer of the dentate gyrus and the formation of hilar basal dendrites. Two changes associated with increased neurogenesis of granule cells in the epileptic brain include hilar ectopic granule cells and the dispersion of the granule cell layer. The significance of the first two changes is that granule cell axon collaterals establish additional synapses with apical and basal dendrites of granule cells, and these connections contribute to new recurrent excitatory circuitry. The significance of increased neurogenesis is that granule cells are migrating into inappropriate areas (deep hilus) or excessive numbers of granule cells accumulate in the layer (dispersion). These data on the epileptic dentate gyrus show that granule cells may change their axonal and dendritic arbors as well as their numbers and position to respond to altered activity possibly caused by decreased inhibition. These findings indicate that the dentate gyrus shows several neuroplastic changes following temporal lobe epilepsy.

Luis Simarro Lacabra [1851–1921]: From Golgi to Cajal Through Simarro, via Ranvier

Knowledge of cerebral structure and function in its modern form can be traced to the neurone doctrine based largely on the work of Santiago Ramón y Cajal [1852–1934] and his lifelong exploitation of the Golgi method. Cajal openly acknowledged his debt to the neuropsychiatrist Luis Simarro Lacabra [1851–1921] who introduced him to the method in 1887, and recalled that the sight of the silver-impregnated nerve cells was the turning point which led him to abandon general anatomy and concentrate on neurohistology. Simarro, who dissipated his free time in trying to improve not only the scientific but also the political world around him, was able to produce exciting Golgi preparations of the cerebral cortex after he returned from voluntary exile in Paris from 1880 to 1885. Certainly it was there that he learned the methods of experimental histology from Louis-Antoine Ranvier [1835–1922] whose laboratory exercises, in the guise of lectures, he attended assiduously.