Intracellular recordings from rat thalamic VL neurons: a study combined with intracellular staining

Summary Intracellular recordings from thalamic neurons receiving cerebellar inputs were performed under urethane anesthesia in the rat. A total of 64 neurons were recorded intracellularly with micropipettes filled with 4% biocytin solution (dissolved in 0.5 M K-acetate), and cerebellar-induced EPSPs (CN-EPSPs), the membrane resistance and firing properties were analyzed with intracellular current injections. The mean latency of CN-EPSPs was 1.9 ± 0.8 ms and the mean input resistance measured in 10 neurons was 17.6 ± 5.0 M. Thirty-two out of 35 stained neurons were analysed morphologically; 28 of these neurons were located in the VL, and 26 received CN-EPSPs. Their somata were round or polygonal in shape and the mean size was 22.5 × 15.2 m. They had radially extending spinous dendrites, and the mean radii of the dendritic fields were 214.7 m in the frontal and 171.4 m in the sagittal planes. These morphological features were similar to those observed in the sensory relay nucleus of the thalamus.     

Effects of stimulation of the primary auditory cortex upon colliculogeniculate neurons in the inferior colliculus of the cat

Electrical stimulation of the primary auditory cortex (AI) of the cat was found to evoke EPSPs, IPSPs or EPSP-IPSP sequences in colliculogeniculate (CG) neurons in the inferior colliculus (IC) which responded antidromically to stimulation of the medial geniculate nucleus. The CG neurons responding to the AI stimulation with short-latency EPSPs (1.0-1.4 msec) were located in the dorsomedial portion of the central nucleus of the IC. On the other hand, latencies of IPSPs elicited in CG neurons by AI stimulation ranged from 2.0 to 4.5 msec.     

Role of intracellular Mg2+ in the activation of muscarinic K+ channel in cardiac atrial cell membrane

Effects of intracellular Mg²⁺ in the activation of a muscarinic K⁺ channel were examined in single atrial cells, using patch-recording techniques. In cell-attached patch recordings, acetylcholine (ACh) or adenosine (Ado), present in the pipette, activated a specific population of K⁺ channels. In inside-out patches, openings of the K⁺ channel by ACh or Ado diminished and did not resume until Mg²⁺ was added to the perfusate which contained GTP or GTP-S, a non-hydrolyzable GTP analogue. Channel openings caused by GTP faded by removing Mg²⁺, while GTP-S-induced openings persisted steadily even when both Mg²⁺ and GTP-S were removed. In contrast to the case of GTP-induced channel openings, the GTP-S-induced openings were not inhibited by the A protomer of pertussi toxin with NAD. From these observations, we concluded: 1) Intracellular Mg²⁺ is essential for GTP to activate the GTP-binding protein. 2) Deactivation of the N protein may be caused by hydrolysis of GTP to GDP. This process may not require Mg²⁺. 3) During the activation by GTP analogues, the N protein may be dissociated into its subunits.     

Intracellular signalling involved in modulating human endothelial barrier function

The endothelium dynamically regulates the extravasation of hormones, macromolecules and other solutes. In pathological conditions, endothelial hyperpermeability can be induced by vasoactive agents, which induce tiny leakage sites between the cells, and by cytokines, in particular vascular endothelial growth factor, which increase the exchange of plasma proteins by vesicles and intracellular pores. It is generally believed that the interaction of actin and non-muscle myosin in the periphery of the endothelial cell, and the destabilization of endothelial junctions, are required for endothelial hyperpermeability induced by vasoactive agents. Transient short-term hyperpermeability induced by histamine involves Ca2+/calmodulin-dependent activation of the myosin light chain (MLC) kinase. Prolonged elevated permeability induced by thrombin in addition involves activation of the small GTPase RhoA and Rho kinase, which inhibits dephosphorylation of MLC. It also involves the action of other protein kinases. Several mechanisms can increase endothelial barrier function, depending on the tissue affected and the cause of hyperpermeability. They include blockage of specific receptors, and elevation of cyclic AMP by agents such as beta2-adrenergic agents. Depending on the vascular bed, nitric oxide and cyclic GMP can counteract or aggravate endothelial hyperpermeability. Finally, inhibitors of RhoA activation and Rho kinase represent a potentially valuable group of agents with endothelial hyperpermeability-reducing properties.     

An electrophysiological action of acetylcholinesterase independent of its catalytic site

Summary Acetylcholinesterase (AChE) is released from the cell bodies and/or dendrites of dopaminergic neurones in the substantia nigra. Extracellular AChE can modify both the electrical activity of dopaminergic nigral neurones and the associated motor behaviour of the animal. These effects seem to be unrelated to hydrolysis of acetylcholine, but the underlying cellular mechanisms of these actions of AChE are unknown. The possible non-cholinergic action of AChE on the membrane properties of dopaminergic neurones was thus investigated by intracellular recording from midbrain slices in vitro. Application of AChE resulted in a marked hyperpolarization of the membrane accompanied by a decrease in input resistance, sometimes preceded by a period of spontaneous firing. Butyrylcholinesterase (BuChE) was without effect. AChE pre-treated with an irreversible inhibitor (Soman) of its enzymic activity caused similar changes to those seen following administration of untreated AChE. It is concluded that AChE can modify the membrane properties of nigrostriatal neurones in a way that is independent of its ability to hydrolyse acetylcholine. This novel biological property of AChE provides a possible mechanism by which this neurosecretory protein could modulate the functioning of the neurones from which it is secreted and suggests that other non-cholinergic actions of AChE might exist.     

Behavioral modulation of neuronal activity in monkey striate cortex: excitation in the absence of active central fixation

Summary Two rhesus monkeys were trained to fixate a small fixation point for randomly varying periods of time using the dimming paradigm. During single unit recording in the striate cortex the fixation point was turned off for a few hundred milliseconds in the presence of a peripheral stimulus. During the disappearance of the fixation point the peripheral stimulus could dim and because of that, in some trials the monkey's saccade to the stimulus right after the offset of the fixation point. In other trials of the same task the monkeys suppressed the execution of a saccade without missing the dimming of the stimulus. During the monkeys performance of this task, striate cortex neurons display an increase of activity after fixation point offset in the presence of a receptive field stimulus, independent of the monkey's decision to look at it or not. Its occurrence can be interpreted as a sign of the monkey having interrupted active fixation of the fixation point and/or having shifted its visual attention towards the peripheral target without necessarily executing a saccade to it. This work was supported by the Deutsche Forschungsgemeinschaft This work was supported by the Deutsche Forschungsgemeinschaft This work was supported by the Deutsche Forschungsgemeinschaft     

Ultrastructural observations on beaded α-motoneuron dendrites

Beaded dendrites of 1α-motoneurons intracellularly labelled with horseradish peroxidase (HRP) were studied ultrastructurally in eight adult cats. For comparison, adjacent unlabelled beaded dendrites of unknown origin were also included in the study. Electron microscopy revealed no signs of degeneration or poor fixation according to common criteria. With the exception of the HRP-reaction product no difference in structure was observed between labelled and unlabelled beaded dendrites. Both the beads and their interconnecting segments were postsynaptic to boutons of normal appearance containing spherical (S-type boutons) or flattened vesicles (F-type boutons). The values for synaptic covering and synaptic packing density of the beaded dendritic regions, which usually were located in the periphery of the dendritic trees, were clearly lower than values obtained previously for cell bodies and proximal dendrites of a-motoneurons.     

Acidic pH enhances the invasive behavior of human melanoma cells

As a consequence of poor perfusion and elevated acid production, the extracellular pH (pH^ex) of tumors is generally acidic. Despite this, most in vitro experiments are still performed at the relatively alkaline pH^ex of 7.4. This is significant, because slight changes in pH^ex can have profound effects on cell phenotype. In this study we examined the effects of mildly acidic conditions on the in vitro invasive potential of two human melanoma cell lines: the highly invasive C8161, and poorly invasive A375P. We observed that culturing of either cell line at acidic pH (6.8) caused dramatic increases in both migration and invasion, as measured with the Membrane Invasion Culture System (MICS). This was not due to a direct effect of pH on the invasive machinery, since cells cultured at normal pH (7.4) and tested at acidic pH did not exhibit increased invasive potential. Similarly, cells cultured at acidic pH were more aggressive than control cells when tested at the same medium pH. These data indicate that culturing of cells at mildly acidic pH induces them to become more invasive. Since acid pH will affect the intracellular pH (pHⁱⁿ) and intracellular calcium ([Ca²⁺]ⁱⁿ), we examined the effect of these parameters on invasion. While changes in [Ca²⁺]ⁱⁿwere not consistent with invasive potential, the changes in pHⁱⁿ were. While these conditions decrease the overall amount of gelatinases A and B secreted by these cells, there is a consistent and significant increase in the proportion of the activated form of gelatinase B.     

Growth inhibition of Mycobacterium bovis by IFN-{gamma} stimulated macrophages: regulation by endogenous tumor necrosis factor-{alpha} and by IL-10

Murine bone marrow-derived macrophages (BMM) are able to inhibitthe intracellular growth of Mycobacterium bovis and Mycobacteriumtuberculosis H37Rv after activation with recomblnant (r) IFNand growth inhibition is mediated by reactive nitrogen intermediates(RNI) derived from L-arglnlne. We now demonstrate that tumornecrosis factor (TNF)- acts as an endogenous cofactor in theinduction of mycobacterial growth inhibition. TNF- was producedby BMM stimulated with rlFN- and infected with mycobacterla,and a specific antlserum to TNF- Inhibited rlFN--lnduced productionof RNI as well as growth inhibition of M. bovis. IL-10, a cytokinewhich suppresses antlmycobacterial macrophage functions, wasalso produced by BMM activated with hFN- and infected with M.bovis. IFN--induced production of TNF- and of reactive nitrogenintermediates as well as mycobacterial growth inhibition wereinhibited by exogenous IL-10, but only when given prior to IFN-stimulation. We conclude that the outcome of mycobacterial infectionis regulated by a coordinate interplay between IFN-, TNF- andIL-10.     

Intracellular cytokine expression in whole blood preparations from normals and patients with atopic dermatitis

In recent years, the importance of characterizing the role of cytokines in a wide range of clinical conditions has resulted in development of new methods to assess cytokine expression in clinical samples. The use of anti-cytokine MoAbs and flow cytometry to detect cytokines intracellularly at the single-cell level has the potential to quantify cytokine production in different diseases. For this technique to be useful in a clinical setting, rapid throughput of clinical samples and a cheap, reliable assay would be required, therefore the development of the above technique using unseparated whole blood samples would be advantageous. Using this technique, only one study to date (Maino et al., 1996) has used unseparated whole blood as the source of cells for detecting intracellular cytokines. In clinical practice, whole blood may be optimal, since this most closely approximates conditions in vivo: as no purification of blood mononuclear cells is required, very little blood is needed to detect a number of cytokines simultaneously in various lymphocyte subpopulations, and the assay can be applied to samples from infants and children. In this study we describe an intracellular cytokine assay using unseparated whole blood from normals. In activated CD8⁻ T cells, IL-2 and interferon-gamma (IFN-γ) were optimally induced after 10 h stimulation with phorbol 12-myristate acetate (PMA)/ionomycin, and in CD8⁺ T cells IL-2 was optimally induced after 10 h and IFN-γ after 6 h. The levels of IL-2 and IFN-γ in CD8⁺ and CD8⁻ T cells in four healthy individuals were consistent on four occasions over a 3-month period. In a large group of 34 normal subjects, there was considerable heterogeneity in CD3/IL-2⁺ (range 9.7–41.3) and CD3/IFN-γ⁺ cells (10.1–44), expressed as a percentage of total lymphocytes. In patients with atopic dermatitis (n = 5) there was a significantly decreased percentage of CD3⁺/CD8⁺ peripheral blood T cells expressing IFN-γ and an increased percentage of CD3⁺/CD8⁻ T cells expressing IL-4 compared with non-atopic dermatitis controls (n = 5). Possible applications of this technique are discussed.