Layer VIb Research Articles

Synaptic organization of serotonin-immunoreactive fibers in primary visual cortex of the macaque monkey.

The macaque neocortex is very densely innervated by serotonin-containing fibers. The highest density of these fibers is in primary sensory regions such as the primary visual cortex. By using an antibody against serotonin, we analyzed the distribution and morphology of serotonin-immunoreactive fibers and synapses in the primary visual cortex of the adult cynomolgus monkey. In addition, we quantified the laminar distribution of labeled varicosities and the distances between varicosities in single fibers. While serotonin-immunoreactive fibers are found in all cortical layers, at least three bands of heightened density of innervation were readily recognized that were coincident with 1) layer IIIB to IVC alpha, 2) layer VA, and 3) layer VIB. Layer IVC alpha of area 17 contained more varicosities per unit area than any other sublayer. There was a high degree of variability in the intervaricosity distances along single fibers; more than half were longer than 10 microns. At the electron microscopic level, synaptic contacts were also observed throughout the entire thickness of area 17, with the highest frequency in layer IV. The labeled varicosities were packed with electron-lucent synaptic vesicles and formed synaptic complexes with small, but conspicuous, post-synaptic densities. Dendritic shafts were the most common postsynaptic target of the labeled synapses. Among these characteristically slender post-synaptic shafts, profiles with structural features of both spiny and smooth dendrites were observed. The small diameter of most of the postsynaptic dendrites indicated that distal dendrites were preferentially contacted by serotonin-immunoreactive varicosities. Although direct identification of the postsynaptic neurons will be required for complete characterization of this circuitry, the distribution of serotonin-immunoreactive varicosities suggests that serotoninergic interactions in the primary visual cortex of the cynomolgus monkey are directed predominantly at the distal dendrites of granular and infragranular neurons rather than at targets in the supragranular layers.

Regional and laminar density of the dopamine innervation in adult rat cerebral cortex

The topographie distribution and density of the dopamine innervation in adult rat cerebral cortex were investigated by means of a recently improved radioautographic procedure for the light microscopic visualization and counting of monoamine axonal varicosities. Dopamine terminals were specifically labeled by high-affinity uptake in whole cerebral hemisphere slices incubated for 15 min at 35°C with 10 −6M tritiated dopamine in the presence of 10 −4M pargyline and 5 × 10 −6M desipramine. The slices were subsequently fixed, embedded in Epon and processed for light microscope radioautography as large 4-μm-thick (whole hemisphere) or smaller, semi-thin sections (selected areas). In radioautographs of serial semi-thin sections exposed for various periods of time, the number of labeled axonal varicosities reached a plateau after 12–14 days of exposure. Counts on such sections of increasing thickness allowed to calculate a correcting factor to transform numbers obtained from 4-μm-thick sections into their equivalent for a tissue thickness of 0.5 μm from which all varicosities were detected. The number of labeled varicosities could then be expressed per mm 3 of tissue after measuring their mean caliper diameter in electron microscope radioautographs. As visualized at 3 transverse levels representing most of the major cytoarchitectonic divisions of cerebral cortex, two novel aspects were recognized in the topographic distribution of dopamine terminals: (1) the presence of a dopamine innervation in layer VIb of the frontal, parietal, temporal and occipital neocortex, and (2) a significant contingent of dopamine varicosities within the deep and not only upper layers of supragenual cingulate cortex. A fair number of dopamine varicosities were also detected in the upper layers of the dorsomedial frontal area, in the retrosplenial and adjacent occipital cortex as well as in the ventral subiculum and field CA1 of the hippocampus. As measured in 10 sectors representing different cortical regions, the highest density of dopamine innervation was found in the supragenual cingulate cortex (1.7 × 10 6) and particularly in its layers II and III (3.1 × 10 6). A slightly lower density was measured in the anteromedian “prefrontal” cortex (1.0 × 10 6). The rostrorhinal and the perirhinal cortex showed moderate dopamine innervation (3.0 and 5.5 × 10 5) with varicosities in every layer. The piriform and the posterior entorhinal cortex were also moderately and ubiquitously innervated (2.5 and 3.0 × 10 5). In the dorsofrontal, parietal and temporal neocortex, overall dopamine innervation was of low density (5.0−6.0 × 10 4) and concentrated in layer VI(2 × 10 5 varicosities/mm 3). The primary visual and the lateral occipital neocortex had the lowest density, with terminals also confined to layer VI(4.0 × 10 4). Together with recent immunocytochemical observations on developing or adult rat cortex, these results justify some revision of current views regarding the organizational features of the mesocortical dopamine system.

Cogeneration of retrogradely labeled corticocortical projection and GABA-immunoreactive local circuit neurons in cerebral cortex

The times of origin of cortico-cortical projection neurons and local circuit neurons in rat visual cortex were determined. The birthdates of the projection neurons were assessed using a technique that combined retrograde labeling with lectin-bound horseradish peroxidase and tritiated thymidine autoradiography. The birthdates of some cortical local circuit neurons were determined by combining GABA immunocytochemistry with [ 3H]thymidine autoradiography. Double-labeled neurons (those with retrograde or immunoreactive label in their perikarya and autoradiographic silver grains over their nuclei) were born during the third week of gestation. Projection and local circuit neurons born on gestational day 14, 15, 17, 19 or 20 were located primarily in layer VIb, VIa, V, III or II, respectively. Thus, both populations of neurons are generated by parallel and concurrent inside-to-outside patterns.

Acetylcholinesterase-immunoreactive axonal network in monkey visual cortex.

Immunocytochemistry with antibodies to human erythrocyte acetylcholinesterase (AChE) was used to demonstrate axons containing this enzyme in the visual cortex (area 17) of macaques. AChE-positive fibers were most dense in layers I, IIIB, IV, and VIB of Hassler and Wagner (I, IVA, IVC, and VIB of Brodmann) with three strata of more sparsely distributed fibers (layers II-IIIA, IIIC, and V-VIA of Hassler and Wagner [II-III, IVB, and V-VIA of Brodmann]). Layer I contained the most dense plexus of AChE-positive axons, oriented horizontally. Horizontally oriented axons were also located in layers IIIB, IIIC, IV, and V of Hassler and Wagner (IVA, IVB, IVC, and V of Brodmann). Layer VIB contained axons of variable orientation, apparently afferent to cortex. Moderately stained neuronal perikarya were occasionally encountered in layer VIB and superficial white matter, but no intensely stained neurons were seen.

A comparative study of two populations of acetylcholine-sensitive neurons in rat somatosensory cortex

The properties of two populations of acetylcholine-sensitive neurons, defined by their different laminar distribution (group A, layers V and upper VIa; group B, layer VIb and underlying white matter) were studied in the SmI cortex of urethane-anesthetized rats. The spontaneous activity of group B neurons was significantly lower than in group A. The proportion of neurons excited by iontophoretic acetylcholine, the duration and the pharmacological properties of the excitations were the same in both groups. The dose-response curves obtained for sets of neurons of both groups, recorded with the same electrode, had comparable slopes. Carbachol had a greater potency than acetylcholine in both groups. In contrast, the latency of the response to acetylcholine was significantly shorter in group B. The amount of current required to increase the spontaneous discharge rate of a given neuron by 200% was lower in group B than in group A. These data suggest that the relative change in firing frequency induced by a given cholinergic input is likely to be larger in group B than in group A. Finally, neuronal excitations could still be obtained in both groups when acetylcholine was applied 300 μm, but not 600 μm, from the cell body, suggesting that the spatial distribution of acetylcholine receptors is similar in groups A and B.

Acetylcholinesterase-containing neurons of layer VIb in immature neocortex: possible component of an early formed intrinsic cortical circuit.

In this ontogenetic study the neurons of layer VIb of rodent somatosensory cortex have been characterized using acetylcholinesterase (AchE) histochemistry, Golgi, and electron microscopic techniques. Already, at birth, the neurons were found to be AchE-rich. They contain presumed AchE reaction product within their granular endoplasmic reticulum. These cells send fine axons upwards towards the subpial layer where they terminate in a dense, AchE-rich, fiber plexus. In chronically undercut cortex, AchE staining persists in layer VIb neurons and in the subpial fiber plexus. These observations continue to support the view that there is an intrinsic neuronal circuit connecting layer VI with I in immature neocortex. The findings also raise the possibility that the putative circuit is cholinergic.

On the striate area of the human isocortex. A Golgi- and pigmentarchitectonic study.

The distribution pattern of the pigment in various layers of the primary visual cortex of man is described by means of a method for the stereomicroscopical demonstration of neurolipofuscins in sections up to 1,000 mum thick. This paper presents the striate area as being composed of 12 cellular layers, which can be clearly delineated by a successive examination of both the Nissland the pigment preparations. Layers I, II, and III resemble more or less the corresponding laminae of other isocortical fields. The pyramids within the deep portion of layer III are unusually small and additionally are intermingled with thorny stellate cells which are a characteristic component of layer IV. Accordingly, this band of cells is called IIIc-IVa. The subjacent layers IVb and IVcalpha are poorly pigmented. In Nissl-preparations layer IVb, which contains only a few cells, is followed by a broad band of cells tightly packed together, which includes layers IVcalpha, IVcbeta, IVd, and Va. These layers cannot be reliably delineated in Nissl-preparations but appear in pigment preparations as distinct and sharply limited laminae. Particularly, layer IVcbeta, which is built up by minute thorny stellate cells, is marked by coarse and intensely stained lipofuscin granules, whereas IVd is mainly populated by minute pyramids which are almost devoid of pigment deposits. A remarkably narrowed layer Va, the pyramids of which store a medium number of lipofuscin grains, completes this series (IVcalpha, IVcbeta, IVd, Va). The cell-sparse layer Vb is almost devoid of pigment granules. Layer VIa has specialized pyramidal cells containing finely grained pigment. The relatively broad layer VIb shows loosely arranged cells poorly endowed with lipofuscin granules.