Nerve growth factor acutely potentiates synaptic transmission in vitro and induces dendritic growth in vivo on adult neurons in airway parasympathetic ganglia

Elevated levels of nerve growth factor (NGF) and NGF-mediated neural plasticity may have a role in airway diseases such as asthma and chronic obstructive pulmonary disease (COPD). Although NGF is known to affect sensory and sympathetic nerves, especially during development, little is known regarding its effect on parasympathetic nerves, especially on adult neurons. The purpose of this study was to analyze the acute and chronic effects of NGF on the electrophysiological and anatomical properties of neurons in airway parasympathetic ganglia from adult guinea pigs. Using single cell recording, direct application of NGF caused a lasting decrease in the cumulative action potential afterhyperpolarization (AHP) and increased the amplitude of vagus nerve-stimulated nicotinic fast excitatory postsynaptic potentials. Neuronal responsiveness to nicotinic receptor stimulation was increased by NGF, which was blocked by the tyrosine kinase inhibitor, K-252a, implicating neurotrophin-specific (Trk) receptors. Neurotrophin-3 and brain-derived neurotrophic factor had no effect on the synaptic potentials, AHP, or nicotinic response; inhibition of cyclooxygenase with indomethacin inhibited the effect of NGF on the cumulative AHP. Forty-eight hours after in vivo application of NGF to the trachealis muscle caused an increase in dendritic length on innervating neurons. These results are the first to demonstrate that NGF increases the excitability of lower airway parasympathetic neurons, primarily through enhanced synaptic efficacy and changes to intrinsic neuron properties. NGF also had dramatic effects on the growth of dendrites in vivo. Such effects may indicate a new role for NGF in the regulation of parasympathetic tone in the diseased or inflamed lower airways.     

Peptidergic synaptic transmission in sympathetic ganglia

Biologically active peptides have been localized in neuronal cell bodies, axons, and synaptic boutons of sympathetic ganglia; some of these peptides may be neurotransmitters. For example, substances immunologically similar to substance P and luteinizing hormone-releasing hormone appear to be released from nerve terminals in sympathetic ganglia. In each case, the postsynaptic action of the peptide lasts for several minutes and is accompanied by a combination of decreases and increases in the membrane conductance that are voltage dependent. These peripheral peptidergic synapses may be models for peptidergic transmission in the central nervous system where detailed analysis is more difficult.     

Ureteric ganglia in the guinea pig

We studied the ureter of guinea pigs to ascertain whether there are ganglia and ganglion neurons directly associated with this structure. We consistently observed ureteric ganglia and individual neurons in the adventitia of the ureter or very close to it. There were on average 87 neurons per ureter, and they were scattered over the entire length of the tube.     

Selective pharmacological blocking of synaptic transmission through the parasympathetic ganglia: Effects on the cardiovascular system

In acute experiments on rats and dogs, compounds IEM-1556 and IEM-1678, the blockers of transmission through the parasympathetic ganglia, reduced the negative chronotropic effect of stimulation of the vagus nerve (VN), while practically not changing the heart rate (HR). In chronic experiments on dogs, these compounds increased the HR, substantially reduced the respiratory heart arrhythmia, did not change the arterial blood pressure (AP), and reduced the chronotropic effects of VN stimulation. IEM-1556 exerted more strong and long-lasting blocking effects on vagal heart control than IEM-1678 did, but in anesthetized animals could evoke a drop in the AP. Acetylcholine, if administered during the action of the above compounds, inhibited heart activity. It is concluded that both IEM-1678 and IEM-1556 are selective parasympatholytics (although IEM-1556 may produce a side effect). The above compounds block synaptic transmission through the intracardiac parasympathetic ganglia and do not affect neuro-effector transmission in the heart.Neirofiziologiya/Neurophysiology, Vol. 28, No. 2/3, pp. 151–159, March–June, 1996.     

Neuronal firing in guinea pig neocortical slices surviving in vitro during adenosine-induced blockade of synaptic transmission

Background firing activity was recorded in guinea pig neocortical slices maintained using extracellular techniques. Between 30 and 40% of neurons continued to generate action potentials, although at a reduced rate, when synaptic disruption had been induced by adenosine or adenosine 5-monophosphate action. These cells were classed as endogenously active. No connection could be shown between neuronal firing pattern and capacity for autonomous generation of action potentials. The remaining neurons tested remained inactive after synaptic disruption, but regained their capacity for spontaneous firing following washout. The activity of these cells was classified as exogenous (or the result of synaptic excitation induced by other neurons in the same slice). The majority of cells with a highly regular discharge pattern initially stopped discharging during synaptic blockade and resumed their activity following washout. This would suggest that a miniature excitatory circuit with 30–140 msec cycles operates in these slices.Institute of Biological Physics, Academy of Sciences of the USSR, Pushchino. Translated from Neirofiziologiya, Vol. 19, No. 6, pp. 816–824, November–December, 1987.     

Regulation of epithelial transport in the jejunal mucosa of the guinea pig by neurokinins

This study characterizes the actions of the neurokinins and calcitonin-gene related peptide (CGRP) on electrolyte transport across the mucosa of the guinea pig jejunum in vitro in a modified Ussing chamber. By following changes in short circuit current (Isc) induced by substance P (SP) and neurokinins A & B (NKA & NKB) in the presence and absence of tetrodotoxin (TTX) and atropine, it was established that two distinct neurokinin receptors are involved in the regulation of electrolyte transport. NKA preferentially activates a neuronal receptor since the actions of this neurokinin were inhibited by both TTX and atropine. SP, whose actions were reduced to a lesser extent by TTX and atropine, is considered to activate preferentially a receptor on the epithelial cells. The third neurokinin, NKB, appears to act non-selectively on both the neuronal and epithelial receptors. CGRP, which per se did not affect Isc, markedly potentiated the increases in Isc induced by SP and NKB, and thus acts synergistically with the epithelial neurokinin receptor. These results suggest that two distinct neurokinin receptors (the NK-1 and the NK-2) regulate epithelial transport in the jejunal mucosa of the guinea pig, and furthermore indicate that at least one of the peptides found in enteric nerves (i.e. CGRP) modulates the actions of neurokinins on epithelial cells.     

Effects of PGE2 in guinea pig colonic myenteric ganglia

PGE(2) is a proinflammatory mediator that can influence many cell types. This study was conducted to determine whether PGE(2) alters the electrical activity of distal colonic myenteric neurons, because colitis is typically associated with altered motility and changes in neural signaling may be involved. The electrical properties of intact myenteric neurons were evaluated with intracellular microelectrodes. Acute application of PGE(2) elicited a prolonged depolarization in both AH and S neurons with little effect on input resistance or electrical excitability. PGE(2) effects were suppressed by tetrodotoxin (TTX) or neurokinin (NK) receptor antagonists, indicating that PGE(2) acts directly and indirectly to depolarize colonic neurons. PGE(2)-evoked depolarization was concentration dependent (approximately 3 microM EC(50)) and was attenuated by the E prostanoid (EP)1/2 receptor antagonist, AH-6809. When preparations were maintained for 48 h in the presence of the stable PGE(2) analog PGE(2)-ethanolamide (10 microM), neurons exhibited a significant membrane depolarization and enhanced excitability. These results suggest that PGE(2) can play a role in altered motility in colitis by evoking changes in the electrical properties of myenteric neurons.     

Protective role of epithelium in the guinea pig airway

We developed an in vitro system to assess the role of the epithelium in regulating airway tone using the intact guinea pig trachea (J. Appl. Physiol. 64: 466-471, 1988). This method allows us to study the response of the airway when its inner epithelial surface or its outer serosal surface is stimulated independently. Using this system we evaluated how the presence of intact epithelium can affect pharmacological responsiveness. We first examined responses of tracheae with intact epithelium to histamine, acetylcholine, and hypertonic KCl when stimulated from the epithelial or serosal side. We then examined the effect of epithelial denudation on the responses to these agonists. With an intact epithelium, stimulation of the inner epithelial side always caused significantly smaller changes in diameter than stimulation of the outer serosal side. After mechanical denudation of the epithelium, these differences were almost completely abolished. In the absence of intact epithelium, the trachea was 35-fold more sensitive to histamine and 115-fold more sensitive to acetylcholine when these agents were applied to the inner epithelial side. In addition, the presence of an intact epithelium almost completely inhibited any response to epithelial side challenge with hypertonic KCl. These results indicate that the airway epithelial layer has a potent protective role in airway responses to luminal side stimuli, leading us to speculate that changes in airway reactivity measured in various conditions including asthma may result in part from changes in epithelial function.     

Effects of beta-agkistrodotoxin on synaptic transmission in toad sympathetic ganglia]

The effects of beta-agkistrodotoxin (beta-AgTX) on synaptic transmission of the toad sympathetic ganglia were investigated by intracellular recording techniques. Superfusion of beta-AgTX (30 microgram/ml, 5-15 min) reversibly inhibited the cholinergic fast excitatory postsynaptic potential (f-EPSP, n = 16) and the fast components of acetylcholine (ACh) potential induced by micropressure administration of ACh (n = 24). Comparison of beta-AgTX effect in the same cell group showed significantly different inhibition rates on f-EPSP (77.2 +/- 27.7%) and ACh potential (25.5 +/- 17.5%) (n = 6, P less than 0.01, F test). During application of beta-AgTX (30 or 50 micrograms/ml) for 15-30 min, no detectable change was found in non-cholinergic late slow EPSPs (n = 22). The results suggest that beta-AgTX selectively inhibits the cholinergic transmission of the toad sympathetic ganglia by both presynaptic and postsynaptic mechanism.     

Selective pharmacological blockade of synaptic transmission in parasympathetic pathways to the heart in rats

The blocking effects of newly synthesized compounds N-decyltropine bromide (IEM-1556) and its derivatives N-methyltropine iodide (IEM-1893) and N-hexyltropine iodide (IEM-1848), possessing aliphatic radicals of different length, on the vagus-induced reduction of the heart rate were studied in acute experiments on rats. The effects produced by these compounds on the level of arterial blood pressure and synaptic transmission in the superior cervical ganglion (SCG) of the rat were studied and compared with those produced by classical ganglion-blocking agents (hexamethonium and trimetaphan) and muscle relaxants (pancuronium and decamethonium). IEM-1556 much more effectively blocked the vagus-induced reduction of the heart rate than other tested blocking agents, and, in contrast to the classical ganglion-blocking agents and decamethonium, did not reduce the arterial blood pressure. In addition, IEM-1556 was less effective than hexamethonium in blocking synaptic transmission through the SCG. IEM-1893 and IEM-1848 demonstrated lower efficacy in blocking SCG transmission, if compared with that of IEM-1556. The results suggest that IEM-1556 is a highly selective blocking agent for parasympathetic versus sympathetic pathways, and its selectivity is determined by the presence of a decyl aliphatic radical in its molecule.Neirofiziologiya/Neurophysiology, Vol. 27, No. 5/6, pp. 323–330, September–December, 1995.     

Adenosine selectively inhibits noncholinergic transmission in guinea pig bronchi

The neuromodulatory action of adenosine and ATP was investigated in isolated guinea pig bronchial strip chain preparations contracted with electrical field stimulation. The tissues were placed in organ baths containing physiological salt solution and stimulated at 8-Hz frequency, 0.5-ms pulse duration, and 30 V (approximately 100 mA) for 5 s. Electrical field stimulation evoked a biphasic contraction of bronchial muscle, consisting of an initial contraction followed by a sustained contraction, which was mediated by intramural cholinergic and noncholinergic nerve stimulations, respectively. Adenosine, at concentrations greater than M, caused a concentration-dependent inhibition in the height of the noncholinergically mediated contraction, accompanied by a very weak inhibition on the cholinergically mediated response. ATP (10(-5) to 3 x 10(-3) M) also produced a similar inhibitory effect on the noncholinergically mediated contraction, but the inhibitory potency was less than that of adenosine. The inhibitory response to adenosine was enhanced by the pretreatment with dipyridamole (2 x 10(-6) M) but antagonized with aminophylline (10(-5) M). Contractions of bronchial muscle evoked by exogenous acetylcholine (2 x 10(-6) M) or substance P (2 x 10(-7) M) were significantly inhibited by the adenosine (3 x 10(-4) M) pretreatment. These data suggest that in isolated guinea pig bronchi adenosine selectively inhibits noncholinergic neurotransmission through prejunctional P1-purinoceptors.     

Anesthesia protocol for hyperpnea-induced airway obstruction in the guinea pig

Guinea pigs (GP; Cavia porcellus) are used extensively as an experimental animal model in a wide range of disciplines including respiratory physiology. Guinea pigs are difficult to anesthetize, and many investigators use paralytic agents to eliminate spontaneous respiratory movements; however, strict federal regulations and institutional policies governing use of paralytic agents are few. We report an anesthesia protocol, using the injectable anesthetic agents sodium pentobarbital (SP) and xylazine (XYL) for the GP that induces consistent anesthesia while eliminating use of paralytic agents. Sixty percent of the calculated SP dose (45 mg/kg of body weight) was given for anesthesia induction, followed by 50% of the calculated XYL dose (7 mg/kg) 15 min later. Depth of anesthesia was monitored by response to toe pinch, ECG, and spontaneous respiratory movements. The animals were given additional boosts of SP (5 to 15% of the original dose, i.p. or i.v.) if a change in anesthesia depth was noted. Thirty-one animals completed the hyperpnea-induced bronchoconstriction (HIB) study with no fatalities. Using this protocol, we collected consistent, repeatable, and reliable data without use of propranolol or skeletal muscle paralytics. We believe that this protocol is not restricted to the GP and could be adapted for use in other terminal experiments.