The cytoarchitecture and cortical connections of the ventral motor region are investigated using Nissl, and NeuN staining methods and the fluorescent retrograde tract tracing technique in the rhesus monkey. On the basis of gradual laminar differentiation, it is shown that the ventral motor region stems from the ventral proisocortical area (anterior insula and dorsal Sylvian opercular region). The cytoarchitecture of the ventral motor region is shown to progress in three lines, as we have recently shown for the dorsal motor region. Namely, root (anterior insular and dorsal Sylvian opercular area ProM), belt (ventral premotor cortex) and core (precentral motor cortex) lines. This stepwise architectonic organization is supported by the overall patterns of corticocortical connections. Areas in each line are sequentially interconnected (intralineal connections) and all lines are interconnected (interlinear connections). Moreover, root areas, as well as some of the belt areas of the ventral and dorsal trend are interconnected. The ventral motor region is also connected with the ventral somatosensory areas in a topographic manner. The root and belt areas of ventral motor region are connected with paralimbic, multimodal and prefrontal (outer belt) areas. In contrast, the core area has a comparatively more restricted pattern of corticocortical connections. This architectonic and connectional organization is consistent in part, with the functional organization of the ventral motor region as reported in behavioral and neuroimaging studies which include the mediation of facial expression and emotion, communication, phonic articulation, and language in human.

The cytoarchitecture and cortical connections of the anterior cingulate, medial and dorsal premotor, and precentral region are investigated using the Nissl and NeuN staining methods and the fluorescent retrograde tract tracing technique. There is a gradual stepwise laminar change in the cytoarchitectonic organization from the proisocortical anterior cingulate region, through the lower and upper banks of the cingulate sulcus, to the dorsolateral isocortical premotor and precentral motor regions of the frontal lobe. These changes are characterized by a gradational emphasis on the lower stratum layers (V and VI) in the proisocortical cingulate region to the upper stratum layers (II and III) in the premotor and precentral motor region. This is accompanied by a progressive widening of layers III and VI, a poorly delineated border between layers III and V and a sequential increase in the size of layer V neurons culminating in the presence of giant Betz cells in the precentral motor region. The overall patterns of corticocortical connections paralleled the sequential changes in cytoarchitectonic organization. The proisocortical areas have connections with cingulate motor, supplementary motor, premotor and precentral motor areas on the one hand and have widespread connections with the frontal, parietal, temporal and multimodal association cortex and limbic regions on the other. The dorsal premotor areas have connections with the proisocortical areas including cingulate motor areas and supplementary motor area on the one hand, and premotor and precentral motor cortex on the other. Additionally, this region has significant connections with posterior parietal cortex and limited connections with prefrontal, limbic and multimodal regions. The precentral motor cortex also has connections with the proisocortical areas and premotor areas. Its other connections are limited to the somatosensory regions of the parietal lobe. Since the isocortical motor areas on the dorsal convexity mediate voluntary motor function, their close connectional relationship with the cingulate areas form a pivotal limbic-motor interface that could provide critical sources of cognitive, emotional and motivational influence on complex motor function. (C) 2011 Elsevier Inc. All rights reserved.

This article reports the development of an optical imaging technique, confocal light absorption and scattering spectroscopic (CLASS) microscopy, capable of noninvasively determining the dimensions and other physical properties of single subcellular organelles. CLASS microscopy combines the principles of light-scattering spectroscopy (LSS) with confocal microscopy. LSS is an optical technique that relates the spectroscopic properties of light elastically scattered by small particles to their size, refractive index, and shape. The multispectral nature of LSS enables it to measure internal cell structures much smaller than the diffraction limit without damaging the cell or requiring exogenous markers, which could affect cell function. Scanning the confocal volume across the sample creates an image. CLASS microscopy approaches the accuracy of electron microscopy but is nondestructive and does not require the contrast agents common to optical microscopy. It provides unique capabilities to study functions of viable cells, which are beyond the capabilities of other techniques.

We recently developed a novel optical method for observing submicron intracellular structures in living cells which is called confocal light absorption and scattering spectroscopic (CLASS) microscopy. It combines confocal microscopy, a well-established high-resolution microscopic technique, with light scattering spectroscopy (LSS). CLASS microscopy requires no exogenous labels and is capable of imaging and continuously monitoring individual viable cells, enabling the observation of cell and organelle functioning at scales on the order of 100 nm with 10 nm accuracy. To demonstrate the ability of the CLASS microscope to monitor unstained living cells on submicrometer scale we studied human bronchial epithelial cells undergoing apoptosis. Fluorescence microscopy of living cells requires application of molecular markers which can affect normal cell functioning. CLASS microscopy is not affected by this avoiding potential interference of fluorescence molecular markers with cell processes. In addition, it provides not only size information but also information about the biochemical and physical properties of the cell. CLASS microscopy can provide unique capabilities for the study of cell interactions with the environment, cell reproduction and growth and other functions of viable cells, which are inaccessible by other techniques.

We report the development of a novel optical imaging technique, confocal light scattering spectroscopic microscopy, capable of non-invasively determining the dimensions and other physical properties of single subcellular organelles with 5 nm accuracy.

The cytoarchitecture and connections of the caudal cingulate and medial somatosensory areas were investigated in the rhesus monkey. There is a stepwise laminar differentiation starting from retrosplenial area 30 towards the isocortical regions of the medial parietal cortex. This includes a gradational emphasis on supragranular laminar organization and general reduction of the infragranular neurons as one proceeds from area 30 toward the medial parietal regions, including areas 3, 1, 2, 5, 31, and the supplementary sensory area (SSA). This trend includes a progressive increase in layer IV neurons. Area 23c in the lower bank and transitional somatosensory area (TSA) in the upper bank of the cingulate sulcus appear as nodal points. From area 23c and TSA the architectonic progression can be traced in three directions: one culminates in areas 3a and 3b (core line), the second in areas 1, 2, and 5 (belt line), and the third in areas 31 and SSA (root line). These architectonic gradients are reflected in the connections of these regions. Thus, cingulate areas (30, 23a, and 23b) are connected with area 23c and TSA on the one hand and have widespread connections with parieto-temporal, frontal, and parahippocampal (limbic) regions on the other. Area 23c has connections with areas 30, 23a and b, and TSA as well as with medial somatosensory areas 3, 1, 2, 5, and SSA. Area 23c also has connections with parietotemporal, frontal, and limbic areas similar to areas 30, 23a, and 23b. Area TSA, like area 23c, has connections with areas 3, 1, 2, 5, and SSA. However, it has only limited connections with the parietotemporal and frontal regions and none with the parahippocampal gyrus. Medial area 3 is mainly connected to medial and dorsal sensory areas 3, 1, 2, 5, and SSA and to areas 4 and 6 as well as to supplementary (M2 or area 6m), rostral cingulate (M3 or areas 24c and d), and caudal cingulate (M4 or areas 23c and d) motor cortices. Thus, in parallel with the architectonic gradient of laminar differentiation, there is also a progressive shift in the pattern of corticocortical connections. Cingulate areas have widespread connections with limbic, parietotemporal, and frontal association areas, whereas parietal area 3 has more restricted connections with adjacent somatosensory and motor cortices. TSA is primarily related to the somatosensory-motor areas and has limited connections with the parietotemporal and frontal association cortices.

A long-standing impediment for applications of optical techniques in cellular biology is the inability to characterize subcellular structures whose dimensions are much less than about 1 mum. In this paper, we describe a method based on light scattering spectroscopy that can find the size distribution of subcellular organelles as small as 100 nm with an accuracy of 20 nm. We report experiments using aqueous suspensions of subcellular organelles enriched in mitochondria, zymogen granules, and microsomes. From the observed light scattering spectra, we extract size distributions that are in excellent agreement with the results of electron microscopy. Further studies are underway to extract the shapes of organelles in addition to their sizes.

The connections of the frontoparietal opercular areas were studied in rhesus monkeys by using antero- and retrograde tracer techniques. The rostral opercular cortex including the gustatory and proisocortical motor (ProM) areas is connected with precentral areas 3, 1, and 2 as well as with the rostral portion of the opercular area which resembles the second somatosensory type of cortex (area SII) and the ventral portion of area 6. Its distant connections are with the ventral portion of prefrontal areas 46, 11, 12, and 13 as well as with the rostral insula and cingulate motor area (CMAr). The mid opercular region (areas 1 and 2) is connected with pre- and postcentral areas 3, 1, and 2 as well as SII. Additionally, it is connected with the ventral portion of area 6, area 44, area ProM, the gustatory area, and the rostral insula. Its distant connections are with area 4, the ventral portion of area 46, area 7b, and area POa in the intraparietal sulcus (IPS). The rostral parietal opercular region is connected with the postcentral portions of areas 3, 1, and 2; areas 5, 7, and SII; the gustatory area; and the vestibular area. Its other connections are with area 4, area 44, the ventral portion of area 46, area ProM, CMAr, and the supplementary motor area (SMA). The caudal opercular region is connected with the dorsal portion of area 3; areas 2, 5, and 7a; and areas PEa as well as IPd of IPS. It is also connected with area SII, insula, and the superior temporal sulcus. Its distant connections are with area 44; the dorsal portion of area 8 and the ventral portion of area 46; as well as CMAr, SMA, and the supplementary sensory area. This connectivity suggests that the ventral somatosensory areas are involved in sensorimotor activities mainly related to head, neck, and face structures as well as to taste. Additionally, these areas may have a role in frontal (working) and temporal (tactile) memory systems.

The mechanisms by which cortical neurons perform spatial and temporal integration of synaptic inputs are dependent, in large part, on the numbers, types, and distributions of their synapses. To further our understanding of these integrative mechanisms, we examined the distribution of synapses on identified classes of cortical neurons. Pyramidal cells in the cat motor cortex projecting either to the ipsilateral somatosensory cortex or to the spinal cord were labeled by the retrograde transport of horseradish peroxidase. Entire soma of selected corticocortical and corticospinal cells were examined using serial-section electron microscopy. The profiles of these somata and the synapses formed with each of these profiles were reconstructed from each thin section with a computer-aided morphometry system. All somatic synapses were of the symmetrical, presumably inhibitory type. For both cell types, these synapses were not homogeneously distributed over the somatic membrane, but were clustered at several discrete zones. The number and density of synapses on the somata of different corticocortical and corticospinal neurons were not significantly different. However, the density of these synapses was inversely correlated with the size of their postsynaptic somata. We discuss the significance of these findings to the integrative properties of cortical neurons.

Nitric oxide may be a key mediator of excitotoxic neuronal injury in the central nervous system. We examined the effects of the neuronal nitric oxide synthase inhibitor 7-nitroindazole (7-NI) on excitotoxic striatal lesions. 7-NI significantly attenuated lesions produced by intrastriatal injections of NMDA, but not kainic acid or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) 7-NI attenuated secondary striatal excitotoxic lesions produced by the succinate dehydrogenase inhibitor malonate, and the protection was reversed by L-arginine but not by D-arginine, 7-NI produced nearly complete protection against striatal lesions produced by systemic administration of 3-nitropropionic acid (3-NP), another succinate dehydrogenase inhibitor, 7-NI protected against malonate induced decreases in ATP, and increases in lactate, as assessed by 1H magnetic resonance spectroscopy. 7-NI had no effects on spontaneous electrophysiologic activity in the striatum in vivo, suggesting that its effects were not mediated by an interaction with excitatory amino acid receptors. 7-NI attenuated increases in hydroxyl radical, 8-hydroxy-2-deoxyguanosine and 3-nitrotyrosine generation in vivo, which may be a consequence of peroxynitrite formation. The present results implicate neuronal nitric oxide generation in the pathogenesis of both direct and secondary excitotoxic neuronal injury in vivo. As such they suggest that neuronal nitric oxide synthase inhibitors may be useful in the treatment of neurologic diseases in which excitotoxic mechanisms play a role.

We previously reported on the histologic and neurochemical features of quinolinic acid lesions in primates which produce many of the characteristic features of Huntington's disease (HD). We now report on the effects of apomorphine in generating a movement disorder in four of these animals. Animals were tested with saline or apomorphine both before and after the lesions. All animals showed few spontaneous abnormal movements after lesioning, but showed marked dyskinetic movements following apomorphine administration. These results show that excitotoxin lesions in primates can produce an apomorphine-inducible movement disorder which closely resembles that of HD.

Excitotoxin lesions induced by quinolinic acid (QA) were made unilaterally in the caudate nucleus and putamen of 12 rhesus monkeys. Both acute (2-3 weeks) and chronic (4-6 months) effects were evaluated. Excitotoxin striatal lesions were characterized by a central zone of intense astrogliosis and marked neuronal depletion, which was surrounded by a transition zone in which there was partial neuronal sparing throughout the entire lesioned side. Immunocytochemical and enzyme histochemical markers for both large and medium-sized aspiny- and spiny-striatal neurons clearly demonstrated a selective pattern of neuronal vulnerability to the excitotoxic effects of QA within lesioned striata. Medium-sized spiny neurons containing calbindin Dk28, enkephalin, and substance P were disproportionately lost, while aspiny neuronal subpopulations containing NADPH diaphorase (NADPH-d) and choline acetyltransferase activity (ChAT) were relatively spared. Combined labeling by NADPH-d enzyme histochemistry and Nissl staining, as well as NADPH-d histochemistry and calbindin Dk28 immunocytochemistry, demonstrated significant increases in the ratio of aspiny to spiny neurons within the lesioned striata. Neurochemical measurements confirmed a loss of GABA and substance P-like immunoreactivity yet no significant depletion of somatostatin-like immunoreactivity, neuropeptide Y-like immunoreactivity, or ChAT were seen. The striatal patch-matrix pattern persisted, as demonstrated by acetylcholinesterase activity. The pattern was altered, however, in the chronic animals, such that the matrix zone was significantly reduced, while the total area of patches remained within normal limits. Ultrastructural analysis confirmed axon sparing lesions with neuronal loss and astrogliosis. Pretreatment of 3 monkeys with MK-801, a noncompetitive N-methyl-d-aspartate (NMDA) antagonist, blocked striatal QA neurotoxicity. The present results provide an experimental primate model which closely resembles the neuropathologic and neurochemical features of Huntington's disease. These findings further strengthen the possibility that an NMDA receptor-mediated excitotoxic process plays a role in the pathogenesis of this disorder.

Morphological characteristics of the neurons of the auditory cortical areas of the rhesus monkey were investigated using Golgi and horseradish peroxidase methods. Neurons of the auditory cortices can be segregated into two categories, spinous and nonspinous, which can be further subclassified according to their dendritic arrays. The spinous neurons include pyramidal, “star pyramid,” multipolar, and bipolar cells. As in other cortices, pyramidal cells are found in layers II–VI and appear to be the most numerous of all cortical neurons. The “star pyramids” have radially oriented dendrites with a less prominent apical shaft and are found mainly in the middle cortical layers. The spinous multipolar neurons are also found in the middle cortical layers and have their dendrites radially arrayed but have no apical dendrite. The spinous bipolar cells, found in the infragranular layers, occur most frequently in the lateral auditory association cortex. The nonspinous neurons include neurogliaform, multipolar, bitufted, and bipolar cells and are found in all cortical layers. The neurogliaform cells are the smallest of all neurons and have radially arrayed, recurving dendrites. The nonspinous multipolar cells also have radially arrayed dendrites but vary in size from being confined to one cortical layer to extending across four laminae. The bitufted neurons are subclassified into three groups: neurons whose primary dendrites arise radially from their somata, those whose dendrites arise from two poles of their somata, and those that have a single primary dendrite arising from one pole and multiple dendrites from another pole of their somata. The nonspinous bipolar cells also have several variants but usually have dendrites arising from two poles of the somata. The chemical characteristics of the auditory neurons were investigated using histochemical and immunocytochemical methods. Peptidergic neurons, i.e., cholecytokinin-, vasoactive intestinal polypeptide-, somatostatin-, and substance P-reactive neurons are found in the various subregions of the auditory cortices and are distributed differentially in the cortical laminae. These neurons are of the nonpyramidal type. Gamma aminobutyric acid-reactive neurons are also nonpyramidal cells and they are found in all cortical layers. Their numbers varied among the cortical laminae in the different auditory regions. The nicotinamide adenine dinucleotide phosphate-diaphorase labeled neurons are quite numerous and are strictly of the nonpyramidal type. They are also found in all cortical laminae. Cytochrome oxidase-labeled cells are of both pyramidal and nonpyramidal types. They are distributed in the central cortical laminae and are found mainly in the auditory association areas.

This study identifies the neuronal types of the rhesus monkey lateral entorhinal cortex (LEC) and discusses the importance of these data in the context of the connectional patterns of the LEC and the possible role of these cells in neurodegenerative diseases. These neuronal types were characterized with the aid of Golgi impregnation techniques. These characterizations were based upon their spine densities, dendritic arrays, and, where possible, axonal arborizations. The cells could be segregated into only spinous and sparsely spinous types. The most numerous spinous types were pyramidal neurons. Other spinous types included multipolar, vertical bipolar and bitufted, and vertical tripolar neurons. The sparsely spinous neuronal types consisted of multipolar, horizontal bipolar and bitufted, and neurogliaform cells. These cells were further classified with the aid of histochemical stains and immunocytochemical markers. Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry stained multipolar, bipolar, and bitufted neurons. Stain for cytochrome oxidase (CO) was found in pyramidal and nonpyramidal cell types. Immunocytochemical techniques revealed several nonpyramidal neurons that contain somatostatin (Som) or substance P (SP). This study complements previous analyses of the neuronal components described in the LEC and adds further information about the distribution of selected neurochemicals within this cortex.

The interhemispheric and ipsilateral afferents of the superior temporal region (STR) were investigated with the aid of fluorescent retrograde tracers (Diamidino Yellow and Fast Blue). Different tracers were injected in selected cortical areas of the STR of each hemisphere of four rhesus monkeys. The results show that the interhemispheric afferents originate not only from the homotopic but also from heterotopic areas. The heterotopic areas giving rise to interhemispheric projections correspond to cortical areas of the origin of the ipsilateral projections. Although there is considerable overlap of labeled neurons of both afferent systems, only occasional double-labeled neurons are found. Whereas the laminar patterns of ipsilateral neurons of origin vary considerably, the interhemispheric projection neurons are located mainly in cortical layer III. This study provides additional information about the ipsilateral connectional organization of the superior temporal region. That is, the primary auditory area receives projections not only from adjacent lateral and medial cortical regions but also from adjoining rostral and caudal cortical regions. Thus, the highly differentiated primary auditory cortical area receives strong projections from the surrounding less-differentiated cortical regions. This connectional pattern is discussed from the perspective of the growth ring concept of cortical development.

The objective of this study was to identify neurons in layer IV of the monkey primary auditory cortex (area KA) that are postsynaptic to thalamocortical axon terminals. Thalamocortical axon terminals were labeled by lesion-induced degeneration; neurons postsynaptic to these afferents were labeled by the Golgi/EM method followed by postembedding immunocytochemistry. Five of the six non-pyramidal neurons examined received synapses from thalamocortical axon terminals. All of these cells were immunoreactive for γ-aminobutyric acid (GABA). One of the cells stained also with an antiserum to somatostatin, and another for cholecystokinin. None of the cells examined were immunoreactive to substance P, and in no instance were two different peptides co-localized within the same GABA-positive neuron.

The anterograde transport and immunohistochemical demonstration of the lectin, Phaseolus vulgaris-leucoagglutinin (PHA-L) has been used to label thalamocortical axon terminals in barrels of mouse SmI cortex. The reaction product is visible with both the light and electron microscopes so that the distribution of axons and the types of synapses they form can be determined.

Lesion-induced axonal degeneration and autoradiography-electron microscopy have been the only reliable anterograde axonal markers available for electron microscopic examination of neuronal circuitry. However, these methods have their limitations. Recently, Phaseolus vulgaris-leucoagglutinin (PHA-L) has been used as an anterograde axonal marker for light microscopy. This report describes the use of this lectin as an anterograde marker for electron microscopy. PHA-L was injected into mouse SmI cortex or ventrobasal thalamus. Using standard immunohistochemical techniques, the transported lectin was tagged with antibody, which was then visualized with avidin-biotin-horseradish peroxidase binding. Light microscopy demonstrated anterograde transport to predicted cortical regions. With the electron microscope, labeled axon terminals were seen forming asymmetric synapses with spines, dendrites and cell bodies.