Pyramidal-shaped Cells Research Articles

Orthosiphon stamineus Proteins Alleviate Hydrogen Peroxide Stress in SH-SY5Y Cells.

The neuroprotective potential of Orthosiphon stamineus leaf proteins (OSLPs) has never been evaluated in SH-SY5Y cells challenged by hydrogen peroxide (H2O2). This work thus aims to elucidate OSLP neuroprotective potential in alleviating H2O2 stress. OSLPs at varying concentrations were evaluated for cytotoxicity (24 and 48 h) and neuroprotective potential in H2O2-induced SH-SY5Y cells (24 h). The protective mechanism of H2O2-induced SH-SY5Y cells was also explored via mass-spectrometry-based label-free quantitative proteomics (LFQ) and bioinformatics. OSLPs (25, 50, 125, 250, 500, and 1000 µg/mL; 24 and 48 h) were found to be safe. Pre-treatments with OSLP doses (250, 500, and 1000 µg/mL, 24 h) significantly increased the survival of SH-SY5Y cells in a concentration-dependent manner and improved cell architecture—pyramidal-shaped cells, reduced clumping and shrinkage, with apparent neurite formations. OSLP pre-treatment (1000 µg/mL, 24 h) lowered the expressions of two major heat shock proteins, HSPA8 (heat shock protein family A (Hsp70) member 8) and HSP90AA1 (heat shock protein 90), which promote cellular stress signaling under stress conditions. OSLP is, therefore, suggested to be anti-inflammatory by modulating the “signaling of interleukin-4 and interleukin-13” pathway as the predominant mechanism in addition to regulating the “attenuation phase” and “HSP90 chaperone cycle for steroid hormone receptors” pathways to counteract heat shock protein (HSP)-induced damage under stress conditions.

Transcription factor programming of human ES cells generates functional neurons expressing both upper and deep layer cortical markers.

Human neurodegenerative disorders affect specific types of cortical neurons. Efficient protocols for the generation of such neurons for cell replacement, disease modeling and drug screening are highly warranted. Current methods for the production of cortical neurons from human embryonic stem (ES) cells are often time-consuming and inefficient, and the functional properties of the generated cells have been incompletely characterized. Here we have used transcription factor (TF) programming with the aim to induce rapid differentiation of human ES cells to layer-specific cortical neurons (hES-iNs). Three different combinations of TFs, NEUROGENIN 2 (NGN2) only, NGN2 plus Forebrain Embryonic Zinc Finger-Like Protein 2 (FEZF2), and NGN2 plus Special AT-Rich Sequence-Binding Protein 2 (SATB2), were delivered to human ES cells by lentiviral vectors. We observed only subtle differences between the TF combinations, which all gave rise to the formation of pyramidal-shaped cells, morphologically resembling adult human cortical neurons expressing cortical projection neuron (PN) markers and with mature electrophysiological properties. Using ex vivo transplantation to human organotypic cultures, we found that the hES-iNs could integrate into adult human cortical networks. We obtained no evidence that the hES-iNs had acquired a distinct cortical layer phenotype. Instead, our single-cell data showed that the hES-iNs, similar to fetal human cortical neurons, expressed both upper and deep layer cortical neuronal markers. Taken together, our findings provide evidence that TF programming can direct human ES cells towards cortical neurons but that the generated cells are transcriptionally profiled to generate both upper and deep layer cortical neurons. Therefore, most likely additional cues will be needed if these cells should adopt a specific cortical layer and area identity.

Isolation, culture optimization and functional characterization of stem cell neurospheres from mouse neonatal olfactory bulb and epithelium.

The olfactory epithelium contains basal cells with stem cell characteristics, which have the capacity to differentiate throughout life into olfactory receptor neurons (ORNs). Here we investigate the in vitro characteristics of stem cells taken from the olfactory bulb (OB) and the olfactory epithelium (OE) of neonatal TIS21 knock-in mice. The major aim of the study was the generation of olfactory neurospheres (ONS) derived from OB and OE of neonatal mice as a tool to further analyze the elementary processes of ORN development. Our data showed that the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF) leads to a significant increase in number of ONS derived from OB but not from OE. The differentiation of ONSs led to the formation of different neuronal cell types, in particular to bipolar-shaped cells as well as putative pyramidal-neurons, astrocytes and oligodendrocytes. Immunohistochemical staining confirmed the presence of astrocytes and neurons in both types of ONSs. In order to investigate the functionality of the neurons we performed calcium imaging and patch-clamp experiments. Calcium imaging experiments revealed that the application of high potassium concentration provokes calcium transients. No excitable properties, neither sodium currents nor action potentials, were observed for the bipolar-shaped cells derived from OB and OE neurospheres, which means that these types of cells morphologically defined as putative neuronal cells, were not physiologically active. Interestingly, patch-clamp recordings performed in the pyramidal-shaped cells of OB neurospheres showed sodium and potassium currents as well as action potentials. Our study will help to establish further models in the field of olfactology.

Pyramidal and Granule Cells Distribution through Hippocampal Fields: An Index for Sensory Information Processing

Background: This work aims at investigating the histology of hippocampus formation as structural model of information processing. The study addressed the question whether the pattern of cellular type distribution within hippocampal fields could be used as support of information processing in the hippocampus. Method: Pyramidal-shaped neurons presenting both cytoplasm and nucleus outlined clearly were measured systematically on brain slides, using a light microscope connected to a microcomputer equipped with a scanner software for measuring particles. Morphological types of cells were identified following class sizes and their distribution determined through hippocampal fields. Results: A battery of statistical tests: Sturges’ classification, class sizes distribution around overall mean, Bartlett’s sphericity test, principal components analysis (PCA) followed by correlations matrix analysis and ANOVA allowed two cellular groups to be identified in the hippocampus: large and small pyramidal-shaped cells. Conclusion: The results show that sensory information processing in the hippocampus could be built on two classes of pyramidal neurons that differed anatomically with probably different physiological functions. The study suggests combination ensembles clustering large and small pyramidal cells at different rates, as fundamental signaling units of the hippocampus.

Paneth cells

Paneth cells

Quantitative study of the developmental changes in calcium‐permeable AMPA receptor‐expressing neurons in the rat somatosensory cortex

The distribution of cells expressing calcium-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (CP-AMPARs) in the somatosensory cortex of rats at different developmental stages was studied using a kainate-stimulated Co(2+)-labeling assay in a quantitative manner. The applicability of this assay for identifying CP-AMPAR-expressing cells was first verified using cultured rat cortical neurons by means of fluorescence Ca(2+) imaging and pharmacological tools. Cells positively identified by the Co(2+)-labelinig assay resided primarily in the marginal zone and subplate of young fetuses and became more widely distributed throughout the cortex as the fetus matured. The majority, >80%, of these Co(2+)-positive cells were neurons, exhibiting immunoreactivity with the neuronal marker NeuN. The proportion of neurons that were Co(2+)-positive increased from approximately 25% to approximately 60% as the rat fetus grew into adulthood. In contrast, less than 20% of nonneuronal cells were Co(2+)-positive. Of the Co(2+)-positive neurons, 15%-31% exhibited GABA immunoreactivity and nonpyramidal-shaped cell bodies; these were presumably GABAergic neurons. Most of the remaining non-GABAergic/Co(2+)-positive neurons had pyramidal-shaped cell bodies and were presumably excitatory principle neurons. Around 70% of GABAergic neurons in the cortex were Co(2+)-positive. Furthermore, in the cortex of neonatal rats the Co(2+)-positive neurons were found to be more susceptible to kainate toxicity than the Co(2+)-negative cells. The Co(2+)-positive neurons in the subplate of neonatal rats were more vulnerable to kainate toxicity than their counterparts in the remaining cortical areas. Together, the widespread distribution and distinct susceptibility to excitotoxicity of CP-AMPAR-expressing neurons suggest that they play various important roles in the development and physiology of the rat cerebral cortex.

Organization of endogenous opioids in the rostral agranular insular cortex of the rat

The rostral agranular insular cortex (RAIC) of rats has opioid receptors and has been implicated in the analgesic and reinforcing effects of opiates. To help in understanding the function of endogenous opioids in this structure, we sought to identify and describe the opioid peptides intrinsic to the RAIC by using immunohistochemical methods. Immunolabeling for proopiomelanocortin (POMC), the precursor to beta-endorphin, and endomorphin 1 and 2 on sectioned rat forebrain revealed limited labeling consisting of individual varicose fibers. Immunolabeling for prodynorphin and enkephalin revealed numerous immunopositive cell bodies and fibers with distribution and morphology unique to each. Prodynorphin-immunopositive cell bodies consisted of two types: large, lightly labeled, pyramidal-shaped cell bodies in lamina V and more intensely labeled, small, ovoid cell bodies scattered in other lamina. Axonal fibers immunolabeled for prodynorphin varied in size and were found in all lamina. Immunolabeling for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) was rarely found in dynorphin-containing cell bodies (6%, 10/167) but was visible within a subpopulation of axons. Enkephalin immunolabeling was detected within a single morphological subpopulation of nonpyramidal neurons located predominantly in lamina II/III, 30% (33/109) of which were also GABA immunopositive. Axons immunolabeled for enkephalin were also abundant in lamina II/III. These results suggest that dynorphin and enkephalin peptides are the predominant endogenous opioids in the RAIC and their distinct distributions suggest divergent functional roles. The localization of prodynorphin immunoreactivity to pyramidal cells suggests the possibility that this neuropeptide may be used in RAIC projection neurons, whereas enkephalin distribution was more characteristic of a role in local networks.

Long-lived retrograde fluorescent labeling of corticospinal neurons in the living animal

For pathophysiological studies, it is advantageous to label specific neuronal populations in living animals. This study aimed to establish a method for stable and long-lasting fluorescent labeling of corticospinal neurons in the living animal. The two fluorescent dyes Fluoro-Red and Fluoro-Green were injected in the cervical spinal cord of anesthetized newborn rats. After a recovery period, treated rats were returned to the mother. After 24 h and 14 days, fixed brain sections revealed wide-spread fluorescence in elongated or pyramidal-shaped cell profiles in a discrete internal cortical layer, consistent with layer V pyramidal cells. Labeled neurons displayed spontaneous synaptic activity using the slice patch clamp method. These results suggest that these dyes are effective tools for pathophysiological and slice patch clamp studies focused on specific neuron groups.

Heterotopia in microcephaly induced by cytosine arabinoside: hippocampus in the neocortex.

Pregnant mice were injected intraperitoneally with cytosine arabinoside (Ara-C) on days 13.5 and 14.5 of pregnancy. The brains of their offspring were studied histologically and histochemically. In addition to dysgenic microcephaly, nodular structures consisting of cells with a relatively homogeneous morphology were observed in the depths of the cerebral cortex. The cell clusters were first seen around postnatal day 4, and had a cellular continuity with the disarrayed pyramidal cell layer in the CA 1 region of the hippocampus. Golgi-Cox staining showed a number of pyramidal-shaped cells in the clusters. Morphologically, they resembled the pyramidal neurons of the hippocampus. Immunohistochemical examination, using anti-serotonin or anti-tyrosine hydroxylase antibodies, also indicated similarities between the cell clusters and the pyramidal cell layer. It is, therefore, proposed that the cell clusters consisted of heterotopic pyramidal cells of the hippocampus. A few synaptic structures could already be detected in the heterotopic cell clusters on postnatal day 3 by electron microscopy. This early establishment of synaptic contact with related neurons may have caused the heterotopic localization of the pyramidal cells.

Electrophysiological and morphological properties of neurons in the rat superior colliculus. I. Neurons in the intermediate layer.

To begin characterizing the neural elements underlying the dynamic properties of local circuits in the mammalian superior colliculus (SC), electrophysiological and morphological properties of individual neurons in the intermediate layer [stratum griseum intermediale (SGI)] were investigated using whole cell patch-clamp recording and intracellular staining with biocytin in slice preparations from young (17-22 days old) and adult rats (7-8 wk old). Voltage responses to depolarizing current pulses of 223 neurons recorded in young rats were classified into six subclasses: regular-spiking neurons (n = 113), interspike intervals during depolarizing current pulses were constant; late-spiking neurons (n = 48), initiation of repetitive firing was delayed markedly from the onset of depolarizing pulses because of a transient hyperpolarization caused by A-like currents; burst-spiking neurons (n = 29), transient burst firing due to low-threshold Ca(2+) channels were observed at the firing threshold level; fast-spiking neurons (n = 19), constant repetitive firings at frequencies >100 Hz were observed for the duration of the depolarizing pulse; neurons with marked spike frequency adaptation (n = 11), interspike intervals more than doubled due to spike frequency adaptation during depolarizing pulses; and neurons with rapid spike inactivation (n = 3), spike amplitude rapidly reduced, width increased during depolarizing pulses, and spiking was terminated after generating a few spikes. In response to hyperpolarizing current pulses, two different types of inward rectification were observed; time-dependent inward rectification by hyperpolarization-activated current (I(h); n = 29) and time-independent inward rectification (n = 111). Morphological analysis showed that neurons expressing time-dependent inward rectification by I(h) had large somata, extended divergent dendrites dorsally into the superficial layers, and projected axons ventrally and sometimes dorsally, all characteristic features of wide-field vertical cells. Other neurons exhibited heterogeneous morphological properties, such as multipolar, fusiform, horizontal, or pyramidal-shaped cells. In adult rats, a total of 44 neurons showed similar electrophysiological properties except for the last type. These results indicate that the local circuits of the SC include neurons with at least five different firing properties and two different rectification properties; each with distinct electrophysiological and morphological characteristics that may be correlated with the functional output of the SC.

Electrophysiological diversity of pyramidal-shaped neurons at the granule cell layer/hilus border of the rat dentate gyrus recorded in vitro.

In the rat dentate gyrus, pyramidal-shaped cells located on the border of the granule cell layer and the hilus are one of the most common types of gamma-aminobutyric acid (GABA)-immunoreactive neurons. This study describes their electrophysiological characteristics. Membrane properties, patterns of discharge, and synaptic responses were recorded intracellularly from these cells in hippocampal slices. Each cell was identified as pyramidal-shaped by injecting the marker Neurobiotin intracellularly (n = 17). In several respects the membrane properties of the sampled cells were similar to "fast-spiking" cells (putative inhibitory interneurons) that have been described in other areas of the hippocampus. For example, input resistance was high (mean 91.3 megohms), the membrane time constant was short (mean 7.7 ms), and there was a large afterhyperpolarization following a single action potential (mean 10.5 mV at resting potential). However, the action potentials of most pyramidal-shaped cells were not as brief (mean 1.2 ms total duration) as those of most previously described fast-spiking cells. Many pyramidal-shaped neurons had strong spike frequency adaptation relative to other fast-spiking cells. Although these latter two characteristics were apparent in the majority of the sampled cells, there were exceptional pyramidal-shaped neurons with fast action potentials and weak adaptation, demonstrating the electrophysiological variability of pyramidal-shaped cells. Responses to outer molecular layer stimulation were composed primarily of excitatory postsynaptic potentials (EPSPs) rather than inhibitory postsynaptic potentials (IPSPs), and were usually small (EPSPs evoked at threshold were often less than 2 mV), and brief (less than 30 ms). There was variability, because in a few cells EPSPs evoked at threshold were much larger. However, regardless of EPSP amplitude, suprathreshold stimulation (up to 4 times the threshold stimulus strength) rarely evoked more than one action potential in any cell. The results suggest that stimulation of perforant path axons produces limited excitatory synaptic responses in pyramidal-shaped neurons. This may be one of the reasons why they are relatively resistant to prolonged perforant path stimulation. The pyramidal-shaped neurons located at the base of the granule cell layer have been associated historically with a basket plexus around granule cell somata, and have been called pyramidal "basket" cells. However, basket-like endings were rare and axon collaterals outside the granule cell layer as the outer molecular layer and the central hilus, and antidromic action potentials could be recorded in some cells in response to weak stimulation of these areas. Taken together with the electrophysiological variability, the results indicate that these cells are physiologically heterogeneous.

Immunohistochemical localization of neural visinin-like Ca 2+-binding protein 2 in adult rat brain

The distribution of neural visinin-like Ca 2+-binding protein 2 (NVP2) in adult rat brain was analyzed by immunoblot and immunohistochemical methods. NVP2 immunoreactivity was expressed intensely in the pyramidal cells of the CA1 and 2 regions of Ammon's horn, the granule cells of the dentate gyrus and the pyramidal cells of cerebral cortex layers II to VI, moderately in the pyramidal-shaped cells of the anterior olfactory nucleus and large spindle-shaped cells of the globus pallidus, and weakly in neurons in the nucleus accumbens and the anterior and dorsomedial thalamus. In most cell types, NVP2 immunoreactivity was located in the cytoplasm and plasma membrane of the cell bodies and dendrites.

A novel slow (< 1 Hz) oscillation of neocortical neurons in vivo: depolarizing and hyperpolarizing components

We describe a novel slow oscillation in intracellular recordings from cortical association areas 5 and 7, motor areas 4 and 6, and visual areas 17 and 18 of cats under various anesthetics. The recorded neurons (n = 254) were antidromically and orthodromically identified as corticothalamic or callosal elements receiving projections from appropriate thalamic nuclei as well as from homotopic foci in the contralateral cortex. Two major types of cells were recorded: regular-spiking (mainly slow-adapting, but also fast-adapting) neurons and intrinsically bursting cells. A group of slowly oscillating neurons (n = 21) were intracellularly stained and found to be pyramidal-shaped cells in layers III-VI, with luxuriant basal dendritic arbors. The slow rhythm appeared in 88% of recorded neurons. It consisted of slow depolarizing envelopes (lasting for 0.8-1.5 sec) with superimposed full action potentials or presumed dendritic spikes, followed by long-lasting hyperpolarizations. Such sequences recurred rhythmically at less than 1 Hz, with a prevailing oscillation between 0.3 and 0.4 Hz in 67% of urethane-anesthetized animals. While in most neurons (approximately 70%) the repetitive spikes superimposed on the slow depolarization were completely blocked by slight DC hyperpolarization, 30% of cells were found to display relatively small (3-12 mV), rapid, all-or-none potentials after obliteration of full action potentials. These fast spikes were suppressed in an all-or-none fashion at Vm more negative than -90 mV. The depolarizing envelope of the slow rhythm was reduced or suppressed at a Vm of -90 to -100 mV and its duration was greatly reduced by administration of the NMDA blocker ketamine. In keeping with this action, most (56%) neurons recorded in animals under ketamine and nitrous oxide or ketamine and xylazine anesthesia displayed the slow oscillation at higher frequencies (0.6-1 Hz) than under urethane anesthesia (0.3-0.4 Hz). In 18% of the oscillating cells, the slow rhythm mainly consisted of repetitive (15-30 Hz), relatively short-lasting (15-25 msec) IPSPs that could be revealed by bringing the Vm at more positive values than -70 mV. The long-lasting (approximately 1 sec) hyperpolarizing phase of the slow oscillation was best observed at the resting Vm and was reduced at about -100 mV. Simultaneous recording of another cell across the membrane demonstrated synchronous inhibitory periods in both neurons. Intracellular diffusion of Cl- or Cs+ reduced the amplitude and/or duration of cyclic long-lasting hyperpolaryzations.(ABSTRACT TRUNCATED AT 400 WORDS)

The embryonic form of neural cell surface molecule (E-NCAM) in the rat hippocampus and its reexpression on glial cells following kainic acid- induced status epilepticus

The neural cell adhesion molecule (NCAM) changes at the cell surface during development, from highly sialylated forms (embryonic or E-NCAM) to three size classes of less sialylated proteins with apparent molecular mass of 180, 140, and 120 kDa (adult NCAM). In the nervous system, E-NCAM has been localized in developing tissues, where it is thought to play a role in the structuring of neuronal groups and tissue pattern formation. In the present study a monoclonal antibody that specifically detects E-NCAM was used in immunoblot and immunohistochemical procedures. In developing rat hippocampus, E-NCAM cell expression was found to change according to a precise pattern and persisted until 1 month after birth. It was closely associated with the mossy fiber system, an area known for its sprouting propensity. In adult rats, although immunoreactivity considerably decreases and becomes undetectable by immunoblot analysis, E-NCAM was still found to be associated with a few pyramidal-shaped cells in the innermost part of the dentate gyrus. In order to acquire some insight into potential histogenetically plastic functions of E-NCAM, in another series of experiments adult rats were treated with kainic acid, a powerful excitotoxic and convulsant glutamate analog eliciting status epilepticus. When these animals were examined for E-NCAM expression, an intense labeling was found associated with glial-like cells, particularly in the hippocampal formation, and corresponding approximately to the reactive gliosis, as confirmed by staining with anti-glial fibrillary acidic protein antibodies. This expression was detectable from about 3 d following kainic acid administration and persisted for at least 12 weeks; it developed according to an observable spatiotemporal distribution pattern. In animals submitted to amygdala kindling, a nonlesional model of secondarily generalized epilepsy, no such reexpression of E-NCAM was observed. Our observations imply that polysialylation may be a means of identifying neuronal structures capable of plasticity in the CNS. Moreover, intense reexpression of E-NCAM could be a marker of reactive gliosis following brain damage.

Outward currents of single hippocampal cells obtained from the adult guinea‐pig.

1. Neurones were isolated from the hippocampus of adult guinea-pigs by enzymatic and mechanical treatment. The electrophysiological properties of these cells were examined immediately after dissociation by intracellular recordings using low-resistance electrodes (2-5 M omega). 2. Pyramidal-shaped cells were identified visually. Intracellular recordings showed that these cells have input resistances ranging from 200 to 1300 M omega. Passive voltage responses to hyperpolarizing current injection were fitted by single exponentials decaying with time constants ranging from 15 to 60 ms. This suggests that the electrotonic structure of these cells is compact such that injected current elicited isopotential intracellular responses. 3. Outward currents activated by depolarization were examined in these cells using voltage-clamp techniques. The amplitude and the time course of the outward currents were profoundly affected by the holding potential. For cells held at -50 mV or more positive, depolarizing steps produced a slowly rising outward current. At holding potentials negative to -55 mV depolarizing pulses produced an additional early transient outward current followed by a slowly rising component which decayed gradually during sustained depolarizations. 4. The outward currents were separated by their kinetic properties and their sensitivity to cobalt (Co2+), tetraethylammonium (TEA) and 4-aminopyridine (4-AP). 5. The transient current peaked within 6 ms of the onset of depolarizing pulses. It decayed exponentially with a time constant of 20-40 ms. The amplitude of the current activated by a fixed depolarization increased gradually as the duration or the amplitude of the hyperpolarizing pre-pulse increased. The current activated by a fixed depolarization reached its half-maximal level when the hyperpolarizing pre-pulse was at -83 mV. 6. 4-AP exerted two actions on the transient current. Firstly, the time constant of the falling phase decreased by about a factor of two. Secondly, the current was blocked in a time- and voltage-dependent manner: the block increased when the hyperpolarizing pre-pulse lengthened. TEA, up to 10 mM, did not affect the amplitude of the transient current. Co2+ suppressed this current. The effects of Co2+ consisted of a shift to the positive direction of the voltage dependence of the current. 7. The delayed currents can be divided into Ca2+-dependent and Ca2+-independent components. The component persistent in the Co2+ solution (K-current) decayed slowly with maintained depolarization (time constant greater than 3 s).(ABSTRACT TRUNCATED AT 400 WORDS)

Neuronal uptake and laminar distribution of tritiated aspartate, glutamate, gamma-aminobutyrate and glycine in the prestriate cortex of squirrel monkeys: Correlation with levels of cytochrome oxidase activity and their uptake in area 17

The neuronal uptake and laminar distribution of cortically injected tritium-labeled gamma-aminobutyrate (GABA), aspartic acid, glutamate and glycine was examined in the prestriate cortex of squirrel monkeys. The intent of this investigation was not to examine the role of these amino acids as neurotransmitters, but to correlate the distribution of tritium-labeled neurons with their levels of cytochrome oxidase activity. A comparison of the number of these labeled neurons was made between the metabolically active “puff” and the less active “nonpuff” regions. In addition, these results were contrasted with the findings in area 17. With each tritiated amino acid tested, labeled neurons that had either high or low levels of cytochrome oxidase activity were present in all laminae. However, the density of labeled neurons varied between lamina for a given amino acid as well as between different amino acids. While many neurons that were cytochrome oxidase-reactive were also tritium-labeled, cytochrome oxidase activity was not a prerequisite for the sequestering of tritium label. In fact, many of the labeled neurons exhibited relatively low levels of cytochrome oxidase activity. Similar to area 17, few aspartate- or glutamate-labeled neurons were present in laminae II–III. The number of labeled neurons for both amino acids increased in laminae IV–VI, with the greatest increase observed in laminae V–VI. Gamma-aminobutyrate-labeled neurons were more prevalent in laminae I and upper II than in the other laminae, whereas in area 17, a greater proportion of the labeled neurons were found in laminae V–VI. With the exception of the uppermost laminae, where GABA-labeled neurons were more abundant, the number of glycine-labeled neurons was significantly greater throughout most laminae than with the other amino acids examined. The density of glycine-labeled neurons in lamina IV, however, was significantly less than the number observed in lamina III even though lamina III was farther away from the injection site which was at the boundary between laminae V–VI. Glycine-labeled neurons were, on average, larger than those labeled with any other amino acid. Similar to area 17, more GABA- and glycine-labeled neurons were observed within the puff regions than in nonpuff regions. No puff/nonpuff differences were observed in the distribution of leucine-injected controls. Labeled neurons for each amino acid included stellate-, fusiform- and pyramidal-shaped cells, each of varying sizes. However, outside the intensely labeled injection sites, no GABA-labeled pyramidal cells were observed. With each amino acid tested, the cytochrome oxidase-reactive/tritium-labeled neurons were larger than their unreactive counterparts. Laminar size variations in labeled neurons occurred between laminae, as well as between the amino acids. In general, the labeled neurons found in lamina IV were smaller than in the other laminae. As in area 17, GABA- and glycine-labeled neurons had distinct uptake patterns, which in turn were different from the pattern exhibited by both aspartate and glutamate.

In vitro characterization of neurons in the ventral part of the nucleus tractus solitarius. I. Identification of neuronal types and repetitive firing properties.

1. An in vitro brain stem slice preparation from adult guinea pigs was used to determine the properties of neurons located in the ventral part of the nucleus tractus solitarius (NTS), an area associated with the dorsal respiratory group. Based upon their morphology and their repetitive firing properties, three classes of ventral NTS neurons, termed types I, II, and III, were observed. 2. Type I neurons were multipolar with pyramidal-shaped cell bodies. These neurons responded to prolonged depolarizations from a resting level of -50 mV with a discrete, high-frequency burst of spikes, which rapidly adapted to a low steady-state level. When depolarized from levels more negative than -65 mV, the initial burst was diminished. 3. Type II neurons were multipolar with fusiform-shaped cell bodies. Type II neurons responded to depolarizations from -50 mV with an initial high spike frequency, which gradually adapted to a steady-state level. When depolarized from levels more negative than -60 mV, these neurons displayed a delay between the onset of the stimulus and the first spike. This delay has been termed "delayed excitation." The expression of delayed excitation was modulated by both the size and duration of hyperpolarizing prepulses that preceded depolarization. 4. Type III neurons were multipolar with spherical shaped-cell bodies. In response to depolarizations from -50 mV, these neurons displayed high-frequency firing with little adaptation. The repetitive firing properties of type III neurons were not modulated by hyperpolarization. 5. Bulbospinal neurons in the ventral NTS were identified using retrograde transport of rhodamine-labeled latex beads injected into the region of the phrenic motor nucleus at spinal cord levels C4 through C6. Only type I and type II neurons were labeled in the ventral NTS (0.2-1.0 mm rostral to the obex). Both contralateral and ipsilateral projections were observed. Contralaterally, type I and II neurons were evenly distributed. Ipsilaterally, however, type II neurons accounted for two-thirds of the labeled neurons. 6. Type I and II neurons had similar input resistances and time constants: 97.0 +/- 17.6 M omega and 14.4 +/- 2.2 ms (n = 5) for type I and 107.0 +/- 11.2 M omega and 13.7 +/- 1.6 ms for type II (n = 5).(ABSTRACT TRUNCATED AT 400 WORDS)

Correlation between cytochrome oxidase staining and the uptake and laminar distribution of tritiated aspartate, glutamate, γ-aminobutyrate and glycine in the striate cortex of the squirrel monkey

The cellular uptake and laminar distribution of tritium-labeled γ-aminobutyrate, aspartate, glutamate and glycine were examined in the primary visual cortex of squirrel monkeys. The purpose was to correlate the distribution of these labeled neurons with their level of cytochrome oxidase activity, particularly in laminae II–III (puffs) and adjacent non-puff regions. In general, tritium-labeled neurons that had either high or low levels of cytochrome oxidase activity were present in all laminae with each amino acid tested; however, their density varied between laminae and with the amino acid injected. Specifically, in laminae II–III, very few neurons were labelled with either of the putative excitatory amino acids (asparate and glutamate). An increased uptake for both was observed in lamina IVC, with the greatest increase for each occurring in laminae V and VI. Significantly more neurons in each lamina were labeled with the putative inhibitory transmitters (γ-aminobutyrate and glycine) than with either aspartate or glutamate. γ-Aminobutyrate-labeled neurons were more prevalent in lamina II than III, and an increase in labeling was observed in laminae IV–VI, with the most prominent increase found in laminae V and VI. Glycine-labeled neurons were larger, more uniformly distributed and more abundant throughout all cortical laminae than those labeled with the other amino acids. Significantly more γ-aminobutyrate- and glycine-labeled neurons were found in the puff regions than in the non-puff areas. No difference was found between puff and non-puff regions for the tritium-labeled leucine controls. Labeled neurons included stellate, fusiform and pyramidal-shaped cells of varying sizes; however, γ-aminobutyrate-labeled pyramidal ceils were not observed outside of the intense injection site. Large glycine-labeled cytochrome-oxidase-reactive pyramidal cells (24–32 μm in diameter) were present at the boundary between laminae V and VI. In addition, a row of large glycine-labeled, fusiform neurons were present in lamina IVB. With each amino acid injected, the tritium-labeled neurons that were darkly reactive for cytochrome oxidase were, on average, larger than the tritium-labeled neurons that were only lightly reactive for cytochrome oxidase. Thus, each of the four amino acids tested had its unique pattern of distribution in the primate striate cortex. Whether one or all of them served as neurotransmitter(s) for distinct neuronal groups is beyond the scope of this study. Glycine, in particular, might be used in part or in whole for metabolic purposes. While many labeled neurons were highly reactive for cytochrome oxidase, not all reactive neurons took up all amino acids. The correlation appeared to be specific for types as well as locations of neurons.

The neuronal composition of area 17 of rat visual cortex. I. The pyramidal cells.

The pyramidal cells in area 17 of rat visual cortex have been examined by light microscopy using Golgi preparations and semithin plastic sections, and by electron microscopy. Pyramidal cells have cell bodies in layers II-VIa. The pyramidal cells in the lower portion of layer II/III are typical examples of this neuronal type in that they have pyramidal-shaped cell bodies, apical dendrites which ascend to layer I, and a skirt of basal dendrites. The pyramidal cells in upper layer II/III are similar in form but have shorter apical dendrites, while the most superficial pyramidal cells lack apical dendrites and instead have two or more primary dendrites that emanate from the upper surface of their somata. In layer V the pyramidal cells are of two sizes, medium and large, and both have a typical morphology, although the larger neurons have thicker apical dendrites and better-developed axon hillocks than the medium-sized pyramids. The medium-sized pyramidal cells of layer V outnumber the large ones to a ratio of 2.5:1. In layer IV a few typical medium-sized pyramidal cells are present, but the majority are small and can be regarded as star pyramids for they have dendrites radiating in all directions. No clearly identified spiny stellate cells have been encountered in layer IV. The pyramidal cells of layer VIa are also small, and most of them have apical dendrites which only ascend as far as layer IV. In addition to these varieties, both inverted and horizontally inclined pyramidal cells have been encountered. In electron micrographs it is apparent that although all of the pyramidal cells have symmetric axosomatic synapses, the frequency with which these synapses occur varies. The cell bodies of the various forms of pyramidal cells do not show a standard cytology. The medium-sized pyramidal cells of layer II/III usually have rounded nuclei, while the nuclei of the small pyramidal cells of layers IV and VIa are somewhat more irregular, and the large pyramidal cells of layer V have deeply indented nuclear envelopes. The appearance of the perikaryal cytoplasm also varies. The larger pyramidal cells have numerous mitochondria and well-developed Nissl bodies in their perikaryal cytoplasm, but the smaller cells have much-less-pronounced mitochondria and their rough endoplasmic reticulum is only organized into stacks at the bases of dendrites. Pyramidal cells account for about 87% of profiles of neuronal cell bodies with nuclei in layer II/III, 90% in layer IV, 89% in layer V, and 97% in layer VIa.

The neuronal composition of area 17 of rat visual cortex. II. The nonpyramidal cells.

In the preceding article the characteristics of the various types of pyramidal cells present in area 17 of rat visual cortex were described (Peters and Kara, '85). In the present article the nonpyramidal cell population of this cortex is considered. It is known from Golgi preparations that in layers II-VIa there are bipolar cells, smooth or sparsely spinous multipolar and bitufted cells with either unmyelinated local plexus or myelinated axons, and chandelier cells. Each of these cell types has been previously examined in Golgi-electron microscopic preparations. The question now being asked is whether the information about the characteristics of these different types of nonpyramidal cells derived from the Golgi-electron microscopic studies can be used to identify the cell bodies of nonpyramidal cells in tissue prepared for conventional electron microscopy. If this can be done then the neuronal composition of area 17 can be determined. It has been found that the cell bodies of bipolar cells can be readily identified because they are elongate and have nuclei with a vertical infolding and few axosomatic synapses, which are of both the symmetric and asymmetric varieties. Evidence is presented to show that there are two types of bipolar cells, small ones and large ones, the large ones being distinguished by their well-developed endoplasmic reticulum in which the cisternae are arranged parallel to the cell surface. Bipolar cells account for 6% of the neuronal profiles in layer II/III, 3% in layer IV, 5% in layer V, and 2% in layer VIa. The cell bodies of other types of nonpyramidal cells in layers II-VIa cannot be distinguished from each other in thin sections, because recognition of the different cell types depends upon the characteristics and distribution of their dendrites and axons. However, it is evident that in this group of neurons there are some with small cell bodies and others with large cell bodies, and in both size groups there are varieties of neurons which can be recognized from the characteristics of their perikaryal cytoplasm. All of these neurons have both symmetric and asymmetric axosomatic synapses. The greatest number of these nonpyramidal cells which are not bipolar in form is found within layer II/III, where they account for 7% of all neuronal profiles. These neurons comprise 4% of all neuronal profiles in layer IV, 6% in layer V, and 2% in layer VIa. Layers I and VIb contain only nonpyramidal cells, but these are different from the ones in layers II-VIa.(ABSTRACT TRUNCATED AT 400 WORDS)