Whole cell recordings from respiratory neurons in the medulla of brainstem-spinal cord preparations isolated from newborn rats

In brainstem-spinal cord preparations isolated from newborn rats, a whole cell recording technique was applied to record membrane potentials of inspiratory (Insp) and pre-inspiratory (Pre-I) neurons in the ventrolateral medulla. Labelling of these respiratory neurons with Lucifer Yellow allowed analysis of their locations and morphology. Intracellular membrane potentials from 25 Insp neurons were recorded. Average resting membrane potential was –49 mV (n=25) and input resistance was 306 M. Insp neurons were classified into three types from the patterns of synaptic potentials. Type I neurons (n=11) had a high probability of excitatory postsynaptic potentials (EPSPs) in the pre- and post-inspiratory phases. Type II neurons (n=7) showed abrupt transition to the burst phase from the resting potential level without increased EPSPs in the preinspiratory phase. Type III neurons (n=7) were hyperpolarized by inhibitory postsynaptic potentials (IPSPs) in the pre- and post-inspiratory phases. These Insp neurons, located in the ventrolateral medulla 80–490m from the ventral surface, were 10–30 m in diameter, and had various soma shapes (pyramidal, spherical or fusiform). Intracellular membrane potentials from 24 Pre-I neurons were recorded. The average resting membrane potential was –45 mV (n=24), and the input resistance was 320 M. Typical Pre-I neurons showed fairly great depolarization accompanied by action potentials during their burst phase and repolarization during the inspiratory phase. Most Pre-I neurons appeared to have a high level of synaptic activity. These cells were located in the ventrolateral medulla 50–440 m below the ventral surface and had pyramidal or fusiform somas of 10–25 m in diameter. Stimulation of the ipsilateral IXth, Xth roots or the spinal cord (C3 level) induced orthodromic responses in most Insp or Pre-I neurons. An antidromic action potential was induced in only one Pre-I neuron by stimulation at the ipsilateral C3 level. Many Insp or Pre-I neurons had dendrites that terminated close to the ventral surface of the medulla. The present study revealed postsynaptic activity of respiratory neurons in the rostral ventrolateral medulla, which is consistent with the excitatory and inhibitory synaptic connections from Pre-I neurons to Insp neurons, and inhibitory synaptic connections for Insp neurons to Pre-I neurons.     

Functional organization of inferior area 6 in the macaque monkey

Summary The functional properties of neurons located in the rostral part of inferior area 6 were studied in awake, partially restrained macaque monkeys. The most interesting property of these neurons was that their firing correlated with specific goal-related motor acts rather than with single movements made by the animal. Using the motor acts as the classification criterion we subdivided the neurons into six classes, four related to distal motor acts and two related to proximal motor acts. The distal classes are: Grasping-with-the-hand-and-the-mouth neurons, Grasping-with-the-hand neurons, Holding neurons and Tearing neurons. The proximal classes are: Reaching neurons and Bringing-to-the-mouth-or-to-the-body neurons. The vast majority of the cells belonged to the distal classes. A particularly interesting aspect of distal class neurons was that the discharge of many of them depended on the way in which the hand was shaped during the motor act. Three main groups of neurons were distinguished: Precision grip neurons, Finger prehension neurons, Whole hand prehension neurons. Almost the totality of neurons fired during motor acts performed with either hand. About 50% of the recorded neurons responded to somatosensory stimuli and about 20% to visual stimuli. Visual neurons were more difficult to trigger than the corresponding neurons located in the caudal part of inferior area 6 (area F4). They required motivationally meaningful stimuli and for some of them the size of the stimulus was also critical. In the case of distal neurons there was a relationship between the type of prehension coded by the cells and the size of the stimulus effective in triggering the neurons. It is proposed that the different classes of neurons form a vocabulary of motor acts and that this vocabulary can be accessed by somatosensory and visual stimuli.     

Activation of atrial muscarinic K+ channels by low concentrations ofβγ subunits of rat brain G protein

Effects of G protein subunits from rat brain on cardiac K⁺ channel was examined in single atrial cells of guinea-pig, using patch clamp techniques. We found that 10 pM concentration of rat brain subunits preparation could activate the atrial muscarine receptor-gated K⁺ channel (I_K.ACh). Neither the detergent, CHAPS, used to suspend nor the boiled preparation activated I_K.ACh. Furthermore, preincubation of subunits preparation in Mg²⁺-free solution, which easily inactivated -GTP-S, did not affect -activation of I_K.ACh. We concluded, therefore, that subunits themselves can activate I_K.ACh.Supported by the grants from the Ministry of Education, Culture and Science of Japan and from the Calcium Signal Workshop on Cardiovascular Systems     

Metabolic control of respiratory neuronal activity and the accompanying changes in breathing movements of the rabbit

Summary The property of the neuronal membrane to be permeable to metabolic modifiers of two regulatory enzymes has been utilized to manipulate the spike activity of inspiratory (I) and expiratory-inspiratory (EI) neurons of the bulbar respiratory centre. The neurons have been classified according to their response to lung distension or collapse (- or -type) and to hyperventilation (tonic firing denoted by +, cessation of activity by –). Using extracellular microelectrodes for single unit recording, the medulla oblongata was superfused with a metabolite-containing CSF. The various neuronal sub-types exhibited a differential activating or inhibitory response to one or several metabolic effectors. For example I ⁺ units were activated by 5 mM glucose-6-phosphate (G-6-P) and 3.5 mM 3-phosphoglycerate (3-PGA), which both inhibited I ⁺ neurons, while 5 mM AMP inhibited I ⁺ much more strongly than I ⁺ cells. The spike density of I ^– and I ^– neurons was increased in the presence of 2.5 mM fructose-6-phosphate and 3.5–5 mM AMP, but became reduced by G-6-P. In contrast, 3 mM fructose-1,6-diphosphate and 5 mM 3-PGA activated the I ^– but inhibited the I ^– neurons. The EI units were characteristically activated by 10 mM citrate, which inhibited all I-type neurons. Activations of the I and I neurons led to an accelerated respiratory rate and a higher tidal volume, while the opposite was true for EI neurons. Intravenous injection of metabolites could not duplicate the striking effects under local applications.Supported by the Deutsche Forschungsgemeinschaft, Grant Ch 25/1.     

A comparative immunohistological study of cerebellar enolases

Summary A comparative immunohistological study of the neurone-specific enolase and enolase, demonstrates the exclusive neuronal localization of enolase and its absence from glial cells. In contrast, enolase is located in astroglial cells. The validity of enolase as a neuronal marker and enolase as an astrocytic marker, is confirmed both by a double labelling technique, using antibodies to and to revealed with fluorescence or peroxidase in the same tissue sections, and by immunoelectronmicroscopy.     

Ribonuclease activity of the “myofibrillary” fraction of normal and autolyzed muscle tissue

The separation in a sucrose gradient of the myofibrillary fraction of normal and autolyzed muscle tissue gave 4 components. During post-mortem destruction of the tissue there was observed a slight decrease of the myofibrillary fraction yield and also certain changes in the distribution of protein between different components. Under the selected conditions RNase activity was found in all 4 components. During the course of autolysis enzymatic activity increased in the whole myofibrillary fraction, as well as in the lysosomal-mitochondrial components of myofibrils.Research Laboratory, Ministry of Health of the USSR, Moscow. Translated from Buylleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 5, pp. 533–537, May, 1978.     

Neuron Activity in the Prefrontal Cortex of the Brain in Rats with Different Typological Characteristics in Conditions of Emotional Stimulation

Male Wistar rats were separated according to the emotional resonance method (groups of animals avoiding (altruists) and not avoiding (egotists) the pain cries of partner rats) and neuron activity in the prefrontal areas of the cortex was studied in the right and left hemispheres. Assessments were made of changes in the frequency of nerve cell spike activity (in relation to the baseline activity of neurons in sated animals) in rats subjected to one day of food deprivation and after electrical stimulation of emotionally positive (lateral hypothalamus) and negative (tegmentum of the midbrain) brain structures and after exposure to the pain cries of partner rats. The results of these experiments revealed a series of differences in the cell activities of the two groups of rats. In conditions of hunger, the discharge frequency in the altruists was higher than that in egotists. Cortical neuron responses to positive stimulation were greater than those to negative stimulation in rats of both groups. Intracerebral stimulation produced significantly greater increases in discharge frequency in neurons of both prefrontal areas of the cortex in altruists than in egotists. In both groups of rats, neurons in the right hemisphere responded to emotionally negative stimulation with significantly greater activation than cells in the left hemisphere, while activity in the left hemisphere was greater in conditions of emotionally positive stimulation. Altruists showed significantly greater neuron responses during exposure to pain cries from victim rats in both the right and left hemispheres. The responses of egotists to victim cries were not significantly different from baseline activity levels.     

Effect of muscular work on the motor function of the stomach and duodenum

Summary The effect of muscle work on the gastric and duodenal motor function was studied in dogs.It was observed that the static stress inhibited the hunger contractions of the stomach and duodenum; the inhibitory effect disappeared as the stress ceased. The static muscle work effect was more pronounced in hunger contractions of the stomach than in those of the duodenum. Under the influence of static stress, the periodic hunger contractions of the stomach varied more extensively than its acid and digestive contractions.Presented by Active Member Acad. Med. Sci. USSR V. N. Chernigovsky     

The significance of respiration for thoracic duct flow in relation to other driving forces of lymph flow

Experiments were performed to study the effect of respiratory intrathoracic pressure changes upon thoracic duct lymph propulsion as compared to other forces driving lymph flow in anaesthetized and artificially ventilated dogs. The effect of an open bilateral pneumothorax upon thoracic duct flow and protein composition was determined at rest, with passive limb movement and during saline infusion. The effect of hyperventilation was also tested.Thoracic duct flow was 30 l/min/kg, 45 l/min/kg and 60 l/min/kg at rest, with passive limb movement and saline infusion, respectively. These flows were decreased by opening the pneumothorax by 11 l/min/kg, 12 l/min/kg and 8 l/min/kg, respectively, and returned to the control level after the thorax was closed. The lymph protein concentration and lymph albumin to globulin ratio were not changed significantly. During hyperventilation, lymph flow was increased and showed a retarded decrease after hyperventilation had ceased. Lymph protein composition was not changed significantly by hyperventilation.The data confirm that lymph is propelled in anaesthetized dogs by respiratory intrathoracic pressure changes. The significance of this respiratory pump decreases, when lymph flow is increased by activation of the tissue pump or vis a tergo. Consequently, the respiratory pump may be assumed to play a secondary role in lymph propulsion in the conscious state when the other forces driving lymph flow are more predominant.Presented in part at the 48th meeting of the Deutsche Physiologische Gesellschaft [18]Supported by the Deutsche Forschungsgemeinschaft     

Reversions to respiratory competence of omnipotent sup45 suppressor mutants may be caused by secondary sup45 mutations

The molecular nature of the sup45 respiratory deficient omnipotent suppressor, and of three reversions to respiratory competence which removed the suppressor effect of the initial mutation, was examined. All reversions were caused by secondary sup45 mutations which indicates a direct connection between sup45 respiratory and translational functions. Computer analysis showed the local changes of Sup45 protein characteristics in the suppressor strain and revertants in comparison to the wild-type protein. The distribution of mutant sites in relation to evolutionary conserved, and tentatively functional, regions in the Sup45 protein is discussed.     

Restricted Vβ gene usage of tumour-infiltrating T lymphocytes in primary gastric malignant B-cell lymphoma

Ten cases of primary gastric malignant lymphoma (PGL) were investigated by immunohistochemical and molecular genetic analysis. These cases were diagnosed histopathologically as follicular small cleaved cell type (1 case), diffuse small cleaved cell type (3 cases) and diffuse large cell type (6 cases) based on the WF (Working Formulation) classification. Seven cases classified as small cleaved or diffuse large cell type belong to low (4 cases) or high (3 cases) grade MALT lymphoma according to Isaacson's classification. All PGL belonged to B lineage cells according to immunohistochemical study and immunoglobulin rearrangements. Rearrangements of TCR chain genes were observed in four of the ten cases. The possibility that the TCR rearrangements were caused by tumour-infiltrating T-cells (TILs) was supported by the following observations: the tumours did not show T- and B-cell biphenotype, TCR exhibited functional VDJ rearrangement and V usage pattern was not a neoplastic type. Analysis of the repertoire of the TCR chain in TILs revealed a common usage of V2 in the above four cases, and furthermore, predominant usage of a particular chain composed of V2-D2.1-J2.3 was observed in one of the four cases. These results indicate that the TILs of PGL have a restricted TCR repertoire.     

Structure of the intramural nerves of the rat bladder

The bladder of adult female rats receives 16,000 axons (i.e., is the target of that many ganglion neurons) of which at least half are sensory. In nerves containing between 40 and 1200 axons cross-sectional area is proportional to number of axons; >99% of axons are unmyelinated. A capsule forms a seal around nerves and ends abruptly where nerves, after branching, contain 10 axons. A single blood vessel is present in many of the large nerves but never in nerves of <600 axons. The number of glial cells was estimated through the number of their nuclei. There is a glial nucleus profile every 76 axonal profiles. Each glial cell is associated with many axons and collectively covers 1,000 m of axonal length. In all nerves a few axonal profiles contain large clusters of vesicles independent of microtubules. The axons do not branch; they alter their relative position along the nerve; they vary in size along their length; none has a circular profile. All the axons are fully wrapped by glial cells and never contact each other. The volume of axons is larger than that of glial cells (55%–45%), while the surface of glial cell is twice as extensive as that of axons; there are 2.27 m² of axolemma and 4.60 m² of glial cell membrane per gram of nerve. Of the mitochondria of a nerve 3/4 are in axons and 1/4 in glial cells.     

A Proposed Heuristic for Communicating Heritability Estimates to the General Public, with Obesity as an Example

It is well established that many continuously distributed traits have a heritable component. However, it is often difficult to communicate to the general public the meaning of quantitative estimates of heritability. To address this problem, the present paper introduces a heuristic for communicating heritability to nonscientific audiences. This heuristic involves adopting an extremely simplified model of inheritance and artificially (and somewhat arbitrarily) defining a cutoffs of low environmental risk and affectation status. Using body weight and obesity as an example, we present a table which gives estimates of the proportion of obese persons who are genetically obese assuming varying levels of environmental risk for obesity and relative body weight scores for defining obesity. The resulting statistic may prove useful for lay audiences in understanding a heritability estimate.     

Synergistic effects of tumour necrosis factor-α and interferon-γ on feline haemopoietic progenitors

Pathogenic mechanisms that underlie feline leukaemia virus subgroup-C (FeLV-C) induced erythroid aplasia are unknown. FeLV-C infection is associated with higher serum levels of interferon- (IFN-) and tumour necrosis factor- (TNF-), which may act synergistically to cause haemopoietic suppression. In the present studies, the synergistic effects of TNF- and IFN- on feline bone marrow progenitors in vitro were evaluated. Bone marrow mononuclear cells from specific-pathogen-free cats were exposed to TNF- (100 and 200 pg/ml) and IFN- (100 or 200 units/ml), alone or in combination, for 2 h before plating for clonal assays of colony forming units. Our results show that TNF- and IFN- in combination caused marked suppression of feline colony forming units-erythroid (CFU-E), burst forming units-erythroid (BFU-E), and colony forming units-fibroblasts (CFU-F), whereas colony forming units-granulocyte/macrophage (CFU-GM) were minimally affected. The same concentrations of TNF- and IFN- alone had minimal effects on CFU-E, BFU-E and CFU-F. These results suggest that TNF- and IFN- may play a significant role in regulating haemopoiesis in cats and may be involved in the pathogenesis of erythroid aplasia in cats infected with feline leukaemia virus.     

Die Kreislaufanpassung bei Hyperthyreose

Zusammenfassung Es wird über Herzkatheteruntersuchungen an 8 Patienten mit Hyperthyreose berichtet. Zwei Fälle entsprechen in reiner Form der geläufigen Vorstellung des sog. High Output-Zustandes, während die übrigen 6 Fälle verschiedene Varianten der Kreislaufanpassung an die hyperthyreotische Stoffwechsellage darstellen. Die Mechanismen, durch welche solche Anpassungen zustande kommen, werden analysiert. Folgende Reaktionstypen wurden beobachtet: Klassicher High Output ohne Venendrucksteigerung, High Output mit Venendrucksteigerung, relativer High Output mit einseitiger Venendrucksteigerung, normales Herzminutenvolumen bei normalem Venendruck. Auf die Frage der Abgrenzung von Kompensation und Dekompensation des Kreislaufs bei Hyperthyreose wird kurz eingegangen.     

Density heterogeneities of hepatitis C virus in human sera due to the binding of β-lipoproteins and immunoglobulins

Heterogeneities in the density of hepatitis C virus RNA-carrying material (HCV-RNA-CM) found in human sera (1.03–1.20 g/cm³) are attributed to the binding of low-density lipoproteins and/or of IgG. In some sera HCV-RNA-CM seems to be nearly totally bound to -lipoproteins and cannot be precipitated by anti-IgG (); in others more than 95% of HCV-RNA-CM is bound to IgG and cannot be precipitated by anti--lipoprotein. Furthermore, there are sera from which HCV-RNA-CM can be completely be precipitated by either anti--lipoprotein or anti-IgG (), pointing to a binding of the two serum proteins to the same HCV-RNA-CM. There are other sera from which HCV-RNA-CM can be partially precipitated by the one or the other antiserum, leaving behind fractions, which are bound to -lipoprotein or to IgG. HCV-RNA-CM cannot be precipitated from some sera either by anti--lipoprotein or by anti-IgG ().     

Nicotinic acetylcholine receptor-ion channels involved in synaptic currents in bullfrog sympathetic ganglion cells and effects of atropine

The nicotinic acetylcholine receptor-ion channels (AChR channels) of the bullfrog sympathetic ganglion cells were studied with a two-electrode voltage clamp technique. The decay phase of the fast excitatory postsynaptic current (fast e.p.s.c.) in B-type neurones followed a double exponential function whose time constants were 3.2 and 8.0 ms at –60 mV and increased with membrane hyperpolarization. Likewise, the decay phase of the fast e.p.s.c. in C-type neurones was double-exponential with time constants of 4.4 and 12.3 ms (at –60 mV). The miniature e.p.s.c. in B-type neurones also decayed with a double exponential function (2.7 and 15.4 ms at –100 mV). Analysis of acetylcholine-induced current fluctuations revealed the power spectral density distribution of a double Lorentzian function which yielded the time constants of elementary events [_noise(f) and _noise(s): 1.7 and 29.7 ms, respectively, at –100 mV] and the averaged elementary conductance (: 7.8 pS).The amplitude of fast e.p.s.c. and the time constant of the fast component of its decay phase decreased during the initial (acute) phase (within 15 min) of the action of atropine (3 M), but recovered during the later (chronic) phase (more than 30 min after application) of the action. The slow component was affected by atropine in a manner similar to the fast component during the acute phase.During the chronic phase, however, the slow time constant recovered and exceeded the control value. Furthermore, this prolongation remained for at least 1 h after the removal of atropine. _noise(s) during the chronic phase of the action was also prolonged to 50.3 ms, while [_noise(f) (2.1 ms) was similar to the control value. The amplitude and quantal size of the fast excitatory postsynaptic potential were also decreased during the acute phase of atropine action, and recovered during the chronic phase. Interestingly, they were reduced transiently during the course of removal of atropine from the bath.These results revealed that the decay phases of the fast e.p.s.c. and miniature e.p.s.c. have two components which are explained either by the existence of two types of AChR channels having different open times or by a single type having three states with rate constants of certain relationships in the bullfrog sympathetic ganglion cells and suggested that their open forms are blocked by atropine only transiently and later desensitized to the blocking action.     

On the regulation of the expressed L-type calcium channel by cAMP-dependent phosphorylation

The Ca²⁺ channel subunits _1C-a and _1C-b were stably expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293 cells. The peak Ba²⁺ current (I _Ba) of these cells was not affected significantly by internal dialysis with 0.1 mM cAMP-dependent protein kinase inhibitor peptide (mPKI), 25 M cAMP-dependent protein kinase catalytic subunit (PKA), or a combination of 25 M PKA and 1 M okadaic acid. The activity of the _1C-b channel subunit expressed stably in HEK 293 cells was depressed by 1 M H 89 and was not increased by superfusion with 5 M forskolin plus 20 M isobutylmethylxanthine (IBMX). The _1C-a·₂·₂/ complex was transiently expressed in HEK 293 cells; it was inhibited by internal dialysis of the cells with 1 M H 89, but was not affected by internal dialysis with mPKI, PKA or microcystin. Internal dialysis of cells expressing the _1C-a·₂·₂/ channel with 10 M PKA did not induce facilitation after a 150-ms prepulse to +50 mV. The Ca²⁺ current (I _Ca) of cardiac myocytes increased threefold during internal dialysis with 5 M PKA or 25 M microcystin and during external superfusion with 0.1 M isoproterenol or 5 M forskolin plus 50 M IBMX. These results indicate that the L-type Ca²⁺ channel expressed is not modulated by cAMP-dependent phosphorylation to the same extent as in native cardiac myocytes.     

Three-dimensional reconstruction of transmitter-identified central neurons by “en bloc” immunofluorescence histochemistry and confocal scanning microscopy

Summary A new method for three-dimensional reconstruction of transmitter-identified neurons is presented which involves en bloc immunofluorescence histochemistry and confocal scanning microscopy. The technique was applied to different types of neurons in the rat brain and lamprey spinal cord. Thick sections or tissue blocs (50–200 m thick) were incubated with antisera against neuropeptides or monoaminergic markers, followed by fluorescent secondary antibodies. Three-dimensional reconstructions were obtained by scanning the preparations in sequential focal planes with a thin laser beam, while sampling the emitted light in each focal plane. The method is convenient and can be applied to a wide variety of neuron types. The reconstructions obtained are accurate since the optical serial sections of the specimen are perfectly aligned, and optic disturbances such as halo phenomena do not occur.     

A simple method for measurement of inulin in nanoliter samples using glass capillaries

Summary A simple method using glass capillaries instead of microcuvettes for measurement of inulin in nanoliter samples is given. Inulin was determined with anthron reagent (5 or 10 nl samples +3 l anthron reagent). Glass capillary tubes (o.d.=1 mm, i.d.=0.68 mm, length=150 mm) in which the chemical reaction took place during incubation at 56°C were directly introduced into the optical system of a Zeiss spectrophotometer PMQ II with sphere attachment and objective.Extinction was measured vertically to the axis of the capillary. The changes of extinction of 20 different capillaries with the blank at different positions was only 1.13×10^–3. The exactness of measurement in the concentration range of 100 200 400 750 1500 3000 mg-% inulin was for 5nl/3 l: 19.8 11.0 6.7 4.7 3.0 2.2%. 10nl/3 l: 13.0 8.4 5.1 3.9%.This method of measurement may also be applicable for other colorimetric reactions with nanoliter samples.This work was supported by Fonds zur Förderung der wissenschaftlichen Forschung.