Neuronal composition of processing modules in human V1: laminar density for neuronal and non-neuronal populations and a comparison with macaque.

The neuronal composition of homologous brain regions in different primates is important for understanding their processing capacities. Primary visual cortex (V1) has been widely studied in different members of the catarrhines. Neuronal density is considered to be central in defining the structure-function relationship. In human, there are large variations in the reported neuronal density from prior studies. We found the neuronal density in human V1 was 79,000 neurons/mm3, which is 35% of the neuronal density previously determined in macaque V1. Laminar density was proportionally similar between human and macaque. In V1, the ocular dominance column (ODC) contains the circuits for the emergence of orientation preference and spatial processing of a point image in many mammalian species. Analysis of the total neurons in an ODC and of the full number of neurons in macular vision (the central 15°) indicates that humans have 1.3× more neurons than macaques even though the density of neurons in macaque is 3× the density in human V1. We propose that the number of neurons in a functional processing unit rather than the number of neurons under a mm2 of cortex is more appropriate for cortical comparisons across species.

Functional Clusters of Neurons in Layer 6 of Macaque V1.

Layer 6 appears to perform a very important role in the function of macaque primary visual cortex, V1, but not enough is understood about the functional characteristics of neurons in the layer 6 population. It is unclear to what extent the population is homogeneous with respect to their visual properties or if one can identify distinct subpopulations. Here we performed a cluster analysis based on measurements of the responses of single neurons in layer 6 of primary visual cortex in male macaque monkeys (Macaca fascicularis) to achromatic grating stimuli that varied in orientation, direction of motion, spatial and temporal frequency, and contrast. The visual stimuli were presented in a stimulus window that was also varied in size. Using the responses to parametric variation in these stimulus variables, we extracted a number of tuning response measures and used them in the cluster analysis. Six main clusters emerged along with some smaller clusters. Additionally, we asked whether parameter distributions from each of the clusters were statistically different. There were clear separations of parameters between some of the clusters, particularly for f1/f0 ratio, direction selectivity, and temporal frequency bandwidth, but other dimensions also showed differences between clusters. Our data suggest that in layer 6 there are multiple parallel circuits that provide information about different aspects of the visual stimulus.SIGNIFICANCE STATEMENT The cortex is multilayered and is involved in many high-level computations. In the current study, we have asked whether there are subpopulations of neurons, clusters, in layer 6 of cortex with different functional tuning properties that provide information about different aspects of the visual image. We identified six major functional clusters within layer 6. These findings show that there is much more complexity to the circuits in cortex than previously demonstrated and open up a new avenue for experimental investigation within layers of other cortical areas and for the elaboration of models of circuit function that incorporate many parallel pathways with different functional roles.

Major Feedforward Thalamic Input Into Layer 4C of Primary Visual Cortex in Primate.

One of the underlying principles of how mammalian circuits are constructed is the relative influence of feedforward to recurrent synaptic drive. It has been dogma in sensory systems that the thalamic feedforward input is relatively weak and that there is a large amplification of the input signal by recurrent feedback. Here we show that in trichromatic primates there is a major feedforward input to layer 4C of primary visual cortex. Using a combination of 3D-electron-microscopy and 3D-confocal imaging of thalamic boutons we found that the average feedforward contribution was about 20% of the total excitatory input in the parvocellular (P) pathway, about 3 times the currently accepted values for primates. In the magnocellular (M) pathway it was around 15%, nearly twice the currently accepted values. New methods showed the total synaptic and cell densities were as much as 150% of currently accepted values. The new estimates of contributions of feedforward synaptic inputs into visual cortex call for a major revision of the design of the canonical cortical circuit.

Densities and Laminar Distributions of Kv3.1b-, PV-, GABA-, and SMI-32-Immunoreactive Neurons in Macaque Area V1.

The Kv3.1b potassium channel subunit is associated with narrow spike widths and fast-spiking properties. In macaque primary visual cortex (V1), subsets of neurons have previously been found to be Kv3.1b-immunoreactive (ir) but not parvalbumin (PV)-ir or not GABA-ir, suggesting that they may be both fast-spiking and excitatory. This population includes Meynert cells, the large layer 5/6 pyramidal neurons that are also labeled by the neurofilament antibody SMI-32. In the present study, triple immunofluorescence labeling and confocal microscopy were used to measure the distribution of Kv3.1b-ir, non-PV-ir, non-GABA-ir neurons across cortical depth in V1, and to determine whether, like the Meynert cells, other Kv3.1b-ir excitatory neurons were also SMI-32-ir pyramidal neurons. We found that Kv3.1b-ir, non-PV-ir, non-GABA-ir neurons were most prevalent in the M pathway-associated layers 4 Cα and 4B. GABAergic neurons accounted for a smaller fraction (11%) of the total neuronal population across layers 1-6 than has previously been reported. Of Kv3.1b-ir neurons, PV expression reliably indicated GABA expression. Kv3.1b-ir, non-PV-ir neurons varied in SMI-32 coimmunoreactivity. The results suggest the existence of a heterogeneous population of excitatory neurons in macaque V1 with the potential for sustained high firing rates, and these neurons were particularly abundant in layers 4B and 4 Cα.

Asymmetric Dichoptic Masking in Visual Cortex of Amblyopic Macaque Monkeys.

In amblyopia, abnormal visual experience leads to an extreme form of eye dominance, in which vision through the nondominant eye is degraded. A key aspect of this disorder is perceptual suppression: the image seen by the stronger eye often dominates during binocular viewing, blocking the image of the weaker eye from reaching awareness. Interocular suppression is the focus of ongoing work aimed at understanding and treating amblyopia, yet its physiological basis remains unknown. We measured binocular interactions in visual cortex of anesthetized amblyopic monkeys (female Macaca nemestrina), using 96-channel "Utah" arrays to record from populations of neurons in V1 and V2. In an experiment reported recently (Hallum et al., 2017), we found that reduced excitatory input from the amblyopic eye (AE) revealed a form of balanced binocular suppression that is unaltered in amblyopia. Here, we report on the modulation of the gain of excitatory signals from the AE by signals from its dominant fellow eye (FE). Using a dichoptic masking technique, we found that AE responses to grating stimuli were attenuated by the presentation of a noise mask to the FE, as in a normal control animal. Responses to FE stimuli, by contrast, could not be masked from the AE. We conclude that a weakened ability of the amblyopic eye to modulate cortical response gain creates an imbalance of suppression that favors the dominant eye.SIGNIFICANCE STATEMENT In amblyopia, vision in one eye is impaired as a result of abnormal early visual experience. Behavioral observations in humans with amblyopia suggest that much of their visual loss is due to active suppression of their amblyopic eye. Here we describe experiments in which we studied binocular interactions in macaques with experimentally induced amblyopia. In normal monkeys, the gain of neuronal response to stimulation of one eye is modulated by contrast in the other eye, but in monkeys with amblyopia the balance of gain modulation is altered so that the weaker, amblyopic eye has little effect while the stronger fellow eye has a strong effect. This asymmetric suppression may be a key component of the perceptual losses in amblyopia.

Altered Balance of Receptive Field Excitation and Suppression in Visual Cortex of Amblyopic Macaque Monkeys.

In amblyopia, a visual disorder caused by abnormal visual experience during development, the amblyopic eye (AE) loses visual sensitivity whereas the fellow eye (FE) is largely unaffected. Binocular vision in amblyopes is often disrupted by interocular suppression. We used 96-electrode arrays to record neurons and neuronal groups in areas V1 and V2 of six female macaque monkeys (Macaca nemestrina) made amblyopic by artificial strabismus or anisometropia in early life, as well as two visually normal female controls. To measure suppressive binocular interactions directly, we recorded neuronal responses to dichoptic stimulation. We stimulated both eyes simultaneously with large sinusoidal gratings, controlling their contrast independently with raised-cosine modulators of different orientations and spatial frequencies. We modeled each eye's receptive field at each cortical site using a difference of Gaussian envelopes and derived estimates of the strength of central excitation and surround suppression. We used these estimates to calculate ocular dominance separately for excitation and suppression. Excitatory drive from the FE dominated amblyopic visual cortex, especially in more severe amblyopes, but suppression from both the FE and AEs was prevalent in all animals. This imbalance created strong interocular suppression in deep amblyopes: increasing contrast in the AE decreased responses at binocular cortical sites. These response patterns reveal mechanisms that likely contribute to the interocular suppression that disrupts vision in amblyopes.SIGNIFICANCE STATEMENT Amblyopia is a developmental visual disorder that alters both monocular vision and binocular interaction. Using microelectrode arrays, we examined binocular interaction in primary visual cortex and V2 of six amblyopic macaque monkeys (Macaca nemestrina) and two visually normal controls. By stimulating the eyes dichoptically, we showed that, in amblyopic cortex, the binocular combination of signals is altered. The excitatory influence of the two eyes is imbalanced to a degree that can be predicted from the severity of amblyopia, whereas suppression from both eyes is prevalent in all animals. This altered balance of excitation and suppression reflects mechanisms that may contribute to the interocular perceptual suppression that disrupts vision in amblyopes.

Population representation of visual information in areas V1 and V2 of amblyopic macaques.

Amblyopia is a developmental disorder resulting in poor vision in one eye. The mechanism by which input to the affected eye is prevented from reaching the level of awareness remains poorly understood. We recorded simultaneously from large populations of neurons in the supragranular layers of areas V1 and V2 in 6 macaques that were made amblyopic by rearing with artificial strabismus or anisometropia, and 1 normally reared control. In agreement with previous reports, we found that cortical neuronal signals driven through the amblyopic eyes were reduced, and that cortical neurons were on average more strongly driven by the non-amblyopic than by the amblyopic eyes. We analyzed multiunit recordings using standard population decoding methods, and found that visual signals from the amblyopic eye, while weakened, were not degraded enough to explain the behavioral deficits. Thus additional losses must arise in downstream processing. We tested the idea that under monocular viewing conditions, only signals from neurons dominated by - rather than driven by - the open eye might be used. This reduces the proportion of neuronal signals available from the amblyopic eye, and amplifies the interocular difference observed at the level of single neurons. We conclude that amblyopia might arise in part from degradation in the neuronal signals from the amblyopic eye, and in part from a reduction in the number of signals processed by downstream areas.

Reduced density of geniculocortical terminals in foveal layer 4A in the macaque primary visual cortex: relationship to S-cone density.

The S-cone system is closely linked to the perception of blue/yellow. The trichromatic system of Old-World monkeys and humans has relatively few S-cones in the fovea. In this study, we investigated the distribution of putative S-cone afferents in macaques primary visual cortex (V1) which form a characteristic honeycomb arrangement in layer 4A. It was hypothesized that if there were a low number of S-cone opponent projecting neurons in central vision then this would be seen as a reduction in afferents in foveal layer 4A. Recent studies have shown that the vesicular glutamate transporter 2 (VGlut2) is a marker for thalamic afferent terminals in cortex. The distribution of VGlut2-immunoreactive (-ir) terminals was studied in the foveal and perifoveal representation of V1. It was found that there was a substantial reduction in the terminal density in the foveal representation: the density was 5-6 times lower in the foveal V1 than in regions representing perifoveal eccentricities of 1°-2° and beyond. These findings may provide the cortical substrate of foveal tritanopia, the reduced blue perceptual ability for small fields in the center of gaze.

Distribution of vesicular glutamate transporter 2 (VGluT2) in the primary visual cortex of the macaque and human.

The majority of thalamic terminals in V1 arise from lateral geniculate nucleus (LGN) afferents. Thalamic afferent terminals are preferentially labeled by an isoform of the vesicular glutamate transporter, VGluT2. The goal of our study was to determine the distribution of VGluT2-ir puncta in macaque and human visual cortex. First, we investigated the distribution of VGluT2-ir puncta in all layers of macaque monkey primary visual cortex (V1), and found a very close correspondence between the known distribution of LGN afferents from previous studies and the distribution of VGluT2-immunoreactive (-ir) puncta. There was also a close correspondence between cytochrome oxidase density and VGluT2-ir puncta distribution. After validating the correspondence in macaque, we made a comparative study in human V1. In many aspects, the distribution of VGluT2-ir puncta in human was qualitatively similar to that of the macaque: high densities in layer 4C, patches of VGluT2-ir puncta in the supragranular layer (2/3), lower but clear distribution in layers 1 and 6, and very few puncta in layers 5 and 4B. However, there were also important differences between macaques and humans. In layer 4A of human, there was a sparse distribution of VGluT2-ir puncta, whereas in macaque, there was a dense distribution with the characteristic honeycomb organization. The results suggest important changes in the pattern of cortical VGluT2 immunostaining that may be related to evolutionary differences in the cortical organization of LGN afferents between Old World monkeys and humans.

Abnormal tau phosphorylation in the thorny excrescences of CA3 hippocampal neurons in patients with Alzheimer's disease.

A key symptom in the early stages of Alzheimer's disease (AD) is the loss of declarative memory. The anatomical substrate that supports this kind of memory involves the neural circuits of the medial temporal lobe, and in particular, of the hippocampal formation and adjacent cortex. A main feature of AD is the abnormal phosphorylation of the tau protein and the presence of tangles. The sequence of cellular changes related to tau phosphorylation and tangle formation has been studied with an antibody that binds to diffuse phosphotau (AT8). Moreover, another tau antibody (PHF-1) has been used to follow the pathway of neurofibrillary (tau aggregation) degeneration in AD. We have used a variety of quantitative immunocytochemical techniques and confocal microscopy to visualize and characterize neurons labeled with AT8 and PHF-1 antibodies. We present here the rather unexpected discovery that in AD, there is conspicuous abnormal phosphorylation of the tau protein in a selective subset of dendritic spines. We identified these spines as the typical thorny excrescences of hippocampal CA3 neurons in a pre-tangle state. Since thorny excrescences represent a major synaptic target of granule cell axons (mossy fibers), such aberrant phosphorylation may play an essential role in the memory impairment typical of AD patients.

GABAergic complex basket formations in the human neocortex.

Certain GABAergic interneurons in the cerebral cortex, basket cells, establish multiple connections with cell bodies that typically outline the somata and proximal dendrites of pyramidal cells. During studies into the distribution of the vesicular GABA transporter (VGAT) in the human cerebral cortex, we were struck by the presence of a very dense, pericellular arrangement of multiple VGAT-immunoreactive (-ir) terminals in certain cortical areas. We called these terminals "Complex basket formations" (Cbk-formations) to distinguish them from the simpler and more typical pericellular GABAergic innervations of most cortical neurons. Here we examined the distribution of these VGAT-ir Cbk-formations in various cortical areas, including the somatosensory (area 3b), visual (areas 17 and 18), motor (area 4), associative frontal (dorsolateral areas 9, 10, 45, 46, and orbital areas 11, 12, 13, 14, 47), associative temporal (areas 20, 21, 22, and 38), and limbic cingulate areas (areas 24, 32). Furthermore, we used dual or triple staining techniques to study the chemical nature of the innervated cells. We found that VGAT-ir Cbk-formations were most frequently found in area 4 followed by areas 3b, 13, and 18. In addition, they were mostly observed in layer III, except in area 17, where they were most dense in layer IV. We also found that 70% of the innervated neurons were pyramidal cells, while the remaining 30% were multipolar cells. Most of these multipolar cells expressed the calcium-binding protein parvalbumin and the lectin Vicia villosa agglutinin.

Pericellular innervation of neurons expressing abnormally hyperphosphorylated tau in the hippocampal formation of Alzheimer's disease patients.

Neurofibrillary tangles (NFT) represent one of the main neuropathological features in the cerebral cortex associated with Alzheimer's disease (AD). This neurofibrillary lesion involves the accumulation of abnormally hyperphosphorylated or abnormally phosphorylated microtubule-associated protein tau into paired helical filaments (PHF-tau) within neurons. We have used immunocytochemical techniques and confocal microscopy reconstructions to examine the distribution of PHF-tau-immunoreactive (ir) cells, and their perisomatic GABAergic and glutamatergic innervations in the hippocampal formation and adjacent cortex of AD patients. Furthermore, correlative light and electron microscopy was employed to examine these neurons and the perisomatic synapses. We observed two patterns of staining in PHF-tau-ir neurons, pattern I (without NFT) and pattern II (with NFT), the distribution of which varies according to the cortical layer and area. Furthermore, the distribution of both GABAergic and glutamatergic terminals around the soma and proximal processes of PHF-tau-ir neurons does not seem to be altered as it is indistinguishable from both control cases and from adjacent neurons that did not contain PHF-tau. At the electron microscope level, a normal looking neuropil with typical symmetric and asymmetric synapses was observed around PHF-tau-ir neurons. These observations suggest that the synaptic connectivity around the perisomatic region of these PHF-tau-ir neurons was apparently unaltered.

The histological slides and drawings of cajal.

Ramón y Cajal's studies in the field of neuroscience provoked a radical change in the course of its history. For this reason he is considered as the father of modern neuroscience. Some of his original preparations are housed at the Cajal Museum (Cajal Institute, CSIC, Madrid, Spain). In this article, we catalogue and analyse more than 4,500 of Cajal's histological preparations, the same preparations he used during his scientific career. Furthermore, we catalogued Cajal's original correspondence, both manuscripts and personal letters, drawings and plates. This is the first time anyone has compiled an account of Cajal's enormous scientific production, offering some curious insights into his work and his legacy.

Cajal's achievements in the field of the development of dendritic arbors.

In 1909, Cajal published an up-dated version in French (Cajal, 1909-1911) of his main work Texture of the Nervous System of Man and Vertebrates (Cajal, 1899-1904), considered the most important book devoted to the nervous system. Owing that last year was the centenary of this publication, we decided to produce an article focused on Cajals description of the morphological changes that dendritic trees undergo during development. We will emphasize his brilliant hypotheses explaining the modelling of dendritic trees (the neurotropic hypothesis and the role of neuronal activity in the patterning of the dendritic trees), and the status of this topic in present day Neuroscience. Here, we will show original photographs taken from a selected collection of Cajals slides housed in the Cajal Museum (Instituto Cajal, CSIC, Madrid, Spain) illustrating the principal changes in neuronal morphology at different stages of development of the spinal cord, cerebellum and cerebral cortex. We will also discuss Cajals initial proposals regarding the influence of neurotropic substances (chemotactic hypothesis) and neural activity in the modelling of the dendritic tree, as well as the evidence that later confirmed these theories.

Dendritic spines and development: towards a unifying model of spinogenesis--a present day review of Cajal's histological slides and drawings.

Dendritic spines receive the majority of excitatory connections in the central nervous system, and, thus, they are key structures in the regulation of neural activity. Hence, the cellular and molecular mechanisms underlying their generation and plasticity, both during development and in adulthood, are a matter of fundamental and practical interest. Indeed, a better understanding of these mechanisms should provide clues to the development of novel clinical therapies. Here, we present original results obtained from high-quality images of Cajal's histological preparations, stored at the Cajal Museum (Instituto Cajal, CSIC), obtained using extended focus imaging, three-dimensional reconstruction, and rendering. Based on the data available in the literature regarding the formation of dendritic spines during development and our results, we propose a unifying model for dendritic spine development.

Diminished perisomatic GABAergic terminals on cortical neurons adjacent to amyloid plaques.

One of the main pathological hallmarks of Alzheimer's disease (AD) is the accumulation of plaques in the cerebral cortex, which may appear either in the neuropil or in direct association with neuronal somata. Since different axonal systems innervate the dendritic (mostly glutamatergic) and perisomatic (mostly GABAergic) regions of neurons, the accumulation of plaques in the neuropil or associated with the soma might produce different alterations to synaptic circuits. We have used a variety of conventional light, confocal and electron microscopy techniques to study their relationship with neuronal somata in the cerebral cortex from AD patients and APP/PS1 transgenic mice. The main finding was that the membrane surfaces of neurons (mainly pyramidal cells) in contact with plaques lack GABAergic perisomatic synapses. Since these perisomatic synapses are thought to exert a strong influence on the output of pyramidal cells, their loss may lead to the hyperactivity of the neurons in contact with plaques. These results suggest that plaques modify circuits in a more selective manner than previously thought.

The growth cone as seen through Cajal's original histological preparations and publications.

During the development of the nervous system, each neuron must contact its appropriate target cell in order to establish its specific connections. More than a century ago, Ramon y Cajal discovered an amoeboid-like structure at the end of the axon of developing nerve cells. He called this structure the growth cone [cono de crecimiento] and he proposed that this structure was guided towards its target tissue by chemical substances secreted by the different cells that line its course. We have reviewed the discovery of the growth cone by Cajal using his original publications, his original scientific drawings, and by studying his histological preparations conserved at the "Instituto Cajal" (Madrid, Spain).(1) We found a very good correlation between the structure of the growth cone in the Golgi-impregnated and reduced silver-nitrate-stained material used by Cajal, and that which is revealed with present-day methods. Finally, Cajal's view of the function of the growth cone and his chemotactic hypothesis will also be considered in the light of present-day knowledge.

Cajal's contributions to the study of Alzheimer's disease.

Last year 2006, we commemorated two important events in the history of Neuroscience. One hundred years ago, on November 3, Alois Alzheimer (1864-1915) presented the first case of a patient with symptoms of a disease that later would be called Alzheimer's disease. One month later, on December 10, Santiago Ramón y Cajal (1852-1934) and Camilo Golgi (1843-1926) received the Nobel Prize "in recognition of their work on the structure of the Nervous System". These facts seem not to be related, but working in the Museum Cajal we found 37 histological preparations of material from patients suffering from Alzheimer's disease, revealing that Cajal also studied this disease. This paper deals with Cajal's contribution to the study of Alzheimer's disease and it is fully illustrated by original pictures of Cajal's slides preserved in the Cajal Museum, Madrid.

Cajal's contributions to glia research.

In 1906, Santiago Ramón y Cajal was awarded the Nobel Prize in Physiology or Medicine in recognition of his work on the structure of neurons and their connections. What is less well known is that he also had a keen interest in glia and developed specific staining methods for their study. In addition to describing their morphology, he speculated on a role for glia in sleep and wakefulness and even in executive brain functions such as attention. In this article, we focus on Cajal's histological research into glial cells; this research includes original drawings of astrocytes, oligodendrocytes, microglia and radial glia, as well as his scientific writings. We aim to show that, concerning glia as well as neurons, Cajal was far ahead of his time.

The discovery of dendritic spines by Cajal in 1888 and its relevance in the present neuroscience.

The year 2006 marks the centenary of the Nobel Prize for Physiology or Medicine awarded to Santiago Ramón y Cajal and Camilo Golgi, "in recognition of their work on the structure of the nervous system". Their discoveries are keys to understanding the present neuroscience, for instance, the discovery of dendritic spines. Cajal discovered dendritic spines in 1888 with the Golgi method, although other contemporary scientists thought that they were silver precipitates. Dendritic spines were demonstrated definitively as real structures by Cajal with the Methylene Blue in 1896. Many of the observations of Cajal and other contemporary scientists about dendritic spines are active fields of research of present neuroscience, for instance, their morphology, distribution, density, development and function. This article will deal with the main contributions of Cajal and other contemporary scientists about dendritic spines. We will analyse their contributions from the historical and present point of view. In addition, we will show high quality images of Cajal's original preparations and drawings related with this discovery.