Daniel Weinreich

Subtypes of vagal afferent C‐fibres in guinea‐pig lungs

An ex vivo, vagally innervated, lung preparation was used to address the hypothesis that vagal C‐fibres comprise at least two distinct phenotypes. Histological and extracellular electrophysiological experiments revealed that vagal C‐fibres innervating the pulmonary system are derived from cell bodies situated in two distinct vagal sensory ganglia. The jugular (superior) ganglion neurones project C‐fibres to both the extrapulmonary airways (larynx, trachea and bronchus) and the lung parenchymal tissue. By contrast, C‐fibres from nodose (inferior) neurones innervate primarily structures within the lungs. Histologically, nodose neurones projecting lung C‐fibres were different from the jugular neurones in that they were significantly less likely to express neurokinins. The nerve terminals within the lungs of both nodose and jugular C‐fibres responded with action potential discharge to capsaicin and bradykinin application, but only the nodose C‐fibre population responded with action potential discharge to the P2X selective receptor agonist α,β‐methylene‐ATP. Whole cell patch clamp recording of capsaicin‐sensitive nodose and jugular ganglion neurones retrogradely labelled from the lung tissue revealed that, like the nerve terminals, lung specific nodose C‐fibre neurones express functional P2X receptors, whereas lung specific jugular C‐fibres do not. The data support the hypothesis that both neural crest‐derived neurones (jugular ganglia) and placode‐derived neurones (nodose ganglia) project C‐fibres in the vagus, and that these two C‐fibre populations represent distinct phenotypes.

Electrophysiological properties and chemosensitivity of guinea pig nodose ganglion neurons in vitro

Conventional intracellular recording techniques were employed to obtain information on the electrophysiological and pharmacological characteristics of C-type neurons in the guinea pig nodose ganglia. Approximately 90% of the cell bodies gave rise to axons with conduction velocities consistent with C-fibers (0.9-1.1 m/s). The average resting membrane potential and input impedence was about -60 mV and 45 M sigma, respectively. Orthodromic electrical stimulation of the vagus nerve 20-30 mm caudal to the ganglion produced overshooting action potentials in the nodose neurons. The falling phase of the action potential was followed by a transient (50-300 ms) fast hyperpolarization (AHPfast). In 20% of C-type neurons the AHPfast was followed by a slowly developing, long-lasting afterhyperpolarization (AHPslow) that limited the ability of the neuron to fire action potentials at high frequency. The AHPslow magnitude was dependent on the number of spikes, had a reversal potential of -87 mV, and was abolished by 100 microM cadmium chloride, suggesting that it is produced by a calcium-dependent potassium current. In about 30% of the nodose neurons, hyperpolarizing current steps from resting potential produced a time- and voltage-dependent anomalous rectification in the electrotonic potential. External cesium (1 mM), but not barium (100 microM) reversibly blocked this effect. Single-electrode voltage-clamp measurements revealed a slowly developing inward current in these neurons that grows in magnitude with step hyperpolarizations from resting potential, and has an estimated reversal potential of about -44 mV. These properties suggest that this current is analogous to IH observed in many peripheral and central neurons. Autacoids including serotonin, histamine, several prostanoids, peptidoleukotriene, and bradykinin, were examined for their ability to affect the excitability of the nodose neurons. Serotonin was the only autacoid capable of depolarizing the membrane potential to action potential firing threshold. The serotonin-induced membrane depolarization was associated with a significant increase in input conductance. Histamine depolarized the membrane potential of the C-type neurons in 28/30 neurons. Bradykinin, prostacyclin, and leukotriene C4 were found to cause membrane depolarizations in a subset (73%, 31%, and 50%, respectively) of nodose neurons. The AHPslow was virtually abolished by bradykinin, prostacyclin, and in a subset of neurons, leukotriene C4. Inhibition of the AHPslow was accompanied by a change in the accommodative properties of the neurons, reflected by the increased frequency at which the neuron could successfully elicit repetitive action potentials.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunologically induced neuromodulation of guinea pig nodose ganglion neurons

The influence of specific antigen challenge on the excitability of C-cells in nodose ganglia isolated from actively sensitized guinea pigs was evaluated using intracellular recording techniques. Antigen (ovalbumin) caused a significant depolarization (approximately 8 mV) of the resting membrane potential. Antigen exposure had differing effects on the membrane input impedance; decreasing it in 15 neurons, increasing it in 6 neurons, and having no effect in 8 neurons. About 20% of guinea pig nodose C-cells reveal a long-lasting after-spike hyperpolarization (AHPslow). Antigen challenge reversibly blocked the AHPslow in 4 of 18 neurons studied in 18 ganglia. About 30% of the nodose ganglion neurons display a time- and voltage-dependent inward rectification at membrane potentials more negative than -75 mV. Exposing the ganglion to the sensitizing antigen consistently blocked this response in 8 of 8 neurons. Histological assessment of toluidine blue stained cells revealed that the nodose ganglion contained approximately 100 mast cells. Exposing the ganglion to ovalbumin stimulated mast cell degranulation, as measured by a decrease in number of stained cells, and evoked the release of histamine, PGD2, and immunoreactive peptidoleukotrienes from the tissue. The results support the hypothesis that endogenous inflammatory mediators released during the immediate hypersensitivity (allergic) reactions can modulate the excitability of primary C-fiber afferents. Mechanisms underlying antigen-induced neuromodulation of these neurons include depolarization of the resting membrane potential, changes in membrane resistance, blockade of a time- and voltage-dependent anomalous rectifier, and, in some cells, blockade of the AHPslow.

Inhibition of calcium‐dependent spike after‐hyperpolarization increases excitability of rabbit visceral sensory neurones.

1. Conventional intracellular recordings were made from rabbit nodose neurones in vitro. Prostaglandins D2 and E2, but not F2 alpha, produced a selective, concentration‐dependent (1‐100 nM) inhibition of a slow, Ca2+‐dependent spike after‐hyperpolarization (a.h.p.). Block of the slow a.h.p. was accompanied by an increased membrane resistance and a small (less than 10 mV) depolarization of the membrane potential. Inhibition of the slow a.h.p. produced no change in the voltage‐current relationship other than the increased membrane resistance. 2. In C neurones with slow a.h.p.s, trains of brief depolarizing current pulses (2 ms duration, 0.1‐10 Hz) could not elicit repetitive action potentials without failure at rates above 0.1 Hz. By contrast, C neurones without slow a.h.p.s could respond at stimulus frequencies up to 10 Hz. The frequency‐dependent spike firing ability of slow a.h.p. neurones was eliminated by inhibition of the slow a.h.p. 3. Action potentials were also evoked by intrasomatic injection of paired, depolarizing current ramps (1 nA/10 ms, 0.1‐5 s inter‐ramp interval). For neurones without a slow a.h.p., the current threshold and number of evoked spikes were the same for both ramps, and the ramps were nearly superimposable. In neurones with a slow a.h.p., the current threshold for the first spike in the second ramp was greatly increased (300‐500%) and the number of evoked spikes was reduced. Following inhibition of the slow a.h.p., the current threshold and number of evoked spikes was the same for both ramps. 4. Forskolin, a direct activator of the catalytic subunit of adenylate cyclase, also produced a concentration‐dependent inhibition of the slow a.h.p., with 50% block at 30 nM. Prostaglandin D2 and forskolin produced identical enhancement of excitability in C neurones and neither substance produced any effect on C neurones that could not be attributed to inhibition of the Ca2+‐dependent K+ conductance associated with the slow a.h.p. We propose that, in some visceral sensory neurones, the level of excitability is regulated by cyclic AMP‐mediated control of the slow a.h.p.

Endogenous histamine excites neurones in the guinea-pig superior cervical ganglion in vitro

1. Intracellular recordings were obtained from neurones in the guinea‐pig superior cervical ganglion (SCG) in vitro to study the electrophysiological effects of endogenously released histamine. 2. Guinea‐pigs were actively sensitized to the specific antigen, ovalbumin. SCG removed from these animals rapidly released a significant proportion of their endogenous histamine stores into the extracellular space upon exposure to the sensitizing antigen. Several observations indicated that the released histamine was derived from ganglionic mast cells. 3. The electrophysiological effects produced by antigen challenge in a neurone mimicked qualitatively and quantitatively those effects produced by exogenously applied histamine in the same neurone. Under current clamp the membrane effects of antigen and histamine included a transient depolarization, an increase in input resistance and transient blockade of a long‐duration component of the spike after‐hyperpolarization. In voltage clamp histamine and antigen produced an inward current and decreased membrane conductance. 4. Histamine H1, but not H2 or H3 receptor antagonists prevented the membrane depolarization to both histamine and antigen treatments. 5. These convergent biochemical, physiological and pharmacological data demonstrate that a sufficient quantity of endogenous histamine is released by an antigenic stimulus in SCG from sensitized guinea‐pigs to affect specific electrophysiological characteristics of neurones. Histamine may thus be involved in mediating interactions between the mammalian immune system and the peripheral sympathetic nervous system.

Advances in Vagal Afferent Neurobiology

Development and Plasticity The Embryology of Vagal Sensory Neurons, C.V. H. Baker Vagal Afferent Neurons: Neurotrophic Factors and Epigenetic Influences, C.J. Helke Vagal Sensory Ganglion Neurons Voltage-Gated Ion Channels in Vagal Afferent Neurons, J.H. Schild, K.D. Alfrey, and B.Y. Li Electrophysiological Studies of Target-Identified Vagal Afferent Cell Bodies, D. Weinreich Vagal Sensory Nerve Terminals Advances in Neural Tracing of Vagal Afferent Nerves andTerminals, T.L. Powley and R.J. Phillips Mechanotransduction by Vagal Tension Receptors in the Upper Gut, S.J.H. Brookes, V.P. Zagorodnyuk, and M. Costa Chemical Transduction in Vagal Afferent Nerve Endings, M.J. Carr Connection in the CNS Synaptic Transmission in the Nucleus Tractus Solitarius (NTS), A.C. Bonham and C.-Y. Chen Nitroxergic Modulation in the NTS: Implications for Cardiovascular Function, J.F.R. Paton, J. Deuchars, S. Wang, and S. Kasparov Monoaminergic Modulation in the Brainstem: Implication for Airway Function, M.A. Haxhiu, C.T. Moore, and P. Kc Organ Specific Afferent Nerve Bronchopulmonary Vagal Afferent Nerves, L.-Y. Lee and B.J. Undem Vagal Afferents Innervating the Gastrointestinal Tract, M.J. Beyak and D. Grundy Cardiac Vagal Afferent Nerves, H.D. Schultz Vagal Reflexes and Sensation Vagal Afferent Nerve Stimulated Reflexes in the GI Tract, J.N. Sengupta and R. Shaker Reflexes Initiated by Activation of the Vagal Afferent Nerves Innervating the Airways and Lungs, B.J. Canning and S.B. Mazzone Axon Reflex and Neurogenic Inflammation in Vagal Afferent Nerves, G. Piedimonte Vagal Afferents and Visceral Pain, W. Janig Electrical Stimulation of the Vagus Nerve for the Treatment of Epilepsy, S.C. Schachter Index

Ca(2+)‐induced Ca2+ release mediates Ca2+ transients evoked by single action potentials in rabbit vagal afferent neurones.

1. Standard intracellular recording techniques with ‘sharp’ micropipettes were used to evoke action potentials (APs) in acutely dissociated adult nodose neurones. 2. APs induced a transient increase in [Ca2+]i (a calcium transient), recorded with fura‐2, that was dependent upon [Ca2+]o and the number of APs. Over the range of one to sixty‐five APs, the relation between the amplitude of the calcium transient and the number of APs was well fitted by a rectangular hyperbola (chi 2 = 3.53, r = 0.968). From one to four APs, the calcium transient‐AP relation can be described by a line with a slope of 9.6 nM AP‐1 (r = 0.999). 3. Charge movement corresponding to Ca2+ influx evoked by a single AP was 39 +/‐ 2.8 pC (mean +/‐ S.E.M.) and did not change significantly during trains of one to thirty‐one APs (P < 0.05). 4. Caffeine (10 mM), a known agonist of the ryanodine receptor, produced an increase in [Ca2+]i. The caffeine‐induced rise in [Ca2+]i was attenuated (by > 90%) by lowering [Ca2+]o, and by ryanodine (10 microM), 2,5‐di(t‐butyl)hydroquinone (DBHQ, 10 microM), or thapsigargin (100 nM). 5. Neurones incubated with ryanodine, DBHQ or thapsigargin required at least eight APs to evoke a detectable calcium transient. These reagents did not significantly affect Ca2+ influx (P < 0.05). In the presence of these inhibitors, the calcium transient‐AP relation exhibited slopes of 1.2, 1.1 and 1.9 nM AP‐1 for ryanodine, DBHQ and thapsigargin, respectively. When compared with the slope of 9.6 nM AP‐1 in non‐treated neurones, it appears that Ca2+ influx produced by a single AP is amplified by ca 5‐ to 10‐fold.

Two pharmacologically distinct histamine receptors mediating membrane hyperpolarization on identified neurons of Aplysia californica

Two distinct hyperpolarizing responses are produced when histamine is iontophoretically applied onto the somal membranes of identified neurons within the cerebral ganglion of Aplysia: a biphasic response consisting of a rapid component (less than 5 sec) usually superimposed upon a slowly developing component; or a monophasic slowly developing response 5-20 sec in duration. The reversal potential values for the fast (typically -65 mV) and the slow (typically -89 mV) responses, and their shift to new values when the external potassium or chloride concentrations were altered, revealed that the fast and slow potentials are produced predominantly by conductance increases to chloride and potassium ions, respectively. The effects of histamine H1- and H2-receptor agonists and antagonists were studied to characterize the pharmacological properties of histamine receptors mediating these two ionically dissimilar hyperpolarizing responses. The slow potassium-dependent hyperpolarization could be mimicked by several histamine analogues; the most potent tested were the H1-receptor agonist, 2-methylhistamine, and the H2-receptor agonist, 4-methylhistamine. Neither of these agents mimicked the fast chloride-dependent histamine response. The slow potassium-dependent responses induced by histamine or histamine agonists were completely and reversibly blocked by the H2-receptor antagonist, cimetidine. By contrast, the slow potassium-dependent hyperpolarizations produced by iontophoretically applied acetylcholine or by dopamine to the same neurons were unaffected by cimetidine. Other H1 and H2 antagonists tested were either ineffective, or only partially blocked the slow hyperpolarizations in a non-selective manner. The fast chloride-dependent hyperpolarizations were not selectively antagonized by any of the H1 or H2 reagents tested, although they were effectively suppressed by tubocurarine and strychnine. These data indicate that two pharmacologically distinct histamine receptors mediate potassium- and chloride-dependent hyperpolarizations in Aplysia neurons. Neither of these receptors, however, could be classified as strictly H1 or H2 according to criteria presently used in non-neuronal tissues. The selectivity and reversibility of cimetidine indicate that this particular antihistaminic could be a valuable pharmacological tool for defining putative histaminergic synapses in Aplysia and perhaps other nervous systems.

Two calcium‐sensitive spike after‐hyperpolarizations in visceral sensory neurones of the rabbit.

Intracellular recordings were made from rabbit nodose neurones in vitro. Two temporally distinct spike after‐hyperpolarizations (a.h.p.s) were identified in a subpopulation of C‐type neurones. The fast a.h.p. after a single spike lasted no longer than 500 ms, while the slow a.h.p. persisted for seconds. Both a.h.p.s. were increased in amplitude in low K+ (0.56 mM) solutions and decreased in amplitude in high K+ (11.2 mM) solutions, and both were reversed at hyperpolarized membrane potentials. The slow a.h.p. was reduced in low Ca2+ (0.22 mM), in the presence of Ca2+ antagonists (Ni2+, 1 mM; Cd2+, 100 microM; or Co2+, 1 mM) and was enhanced in tetraethylammonium (5 mM). In approximately half of the cells tested, the fast a.h.p. was reduced in low Ca2+ and in the presence of the Ca2+ antagonists. In the remaining cells the fast a.h.p. was insensitive to these procedures. Prostaglandin (PGE1, 1‐10 micrograms/ml) reduced the slow a.h.p. in all cells tested. Neither the Ca2+‐sensitive nor the Ca2+‐insensitive fast a.h.p. was affected by the prostaglandin. It is concluded that there is a subpopulation of C‐type nodose neurones possessing a slow a.h.p. which is due to a Ca2+‐dependent K+ current. This subpopulation of neurones can further be divided on the basis of the presence of a Ca2+‐sensitive fast a.h.p. Furthermore, PGE1 pharmacologically separates the fast and slow a.h.p.s by selectively blocking the slow one. The blockage by the PGE1 is most probably not due to a reduction in Ca2+ influx.

Histamine H1 receptor activation blocks two classes of potassium current, IK(rest) and IAHP to excite ferret vagal afferents

1 Intracellular recordings were made in intact and acutely dissociated vagal afferent neurones (nodose ganglion cells) of the ferret to investigate the membrane effects of histamine. 2 In current‐clamp or voltage‐clamp recordings, histamine (10 μm) depolarized the membrane potential (10 ± 0.8 mV; mean ±s.e.m.; n= 27) or produced an inward current of 1.6 ± 0.35 nA (n= 27) in ∼80% of the neurones. 3 Histamine (10 μm) also blocked the post‐spike slow after‐hyperpolarization (AHPslow) present in 80% of these neurones (95 ± 3.2%; n= 5). All neurones possessing AHPslow in ferret nodose were C fibre neurones; all AHPslow neurones had conduction velocities ≤ 1 m s−1(n= 7). 4 Both the histamine‐induced inward current and the block of AHPslow were concentration dependent and each had an estimated EC50 value of 2 μm. These histamine‐induced effects were mimicked by the histamine H1 receptor agonist 2‐(2‐aminoethyl)thiazole dihydrochloride (10 μm) and blocked by the H1 antagonists pyrilamine (100 nm) or diphenhydramine (100 nm). Schild plot analysis of the effect of pyrilamine on the histamine‐induced inward current revealed a pA2 value of 9.7, consistent with that expected for an H1 receptor. Neither impromidine (10 μm) nor R(−)‐α‐methylhistamine (10 μm), selective H2 or H3 agonists, respectively, significantly affected the membrane potential, input resistance or AHPslow. 5 The reversal potential (Vrev) for the histamine‐induced inward current was −84 ± 2.1 mV (n= 4). The Vrev for the histamine response shifted in a Nernstian manner with changes in the extracellular potassium concentration. Alterations in the extracellular chloride concentration had no significant effect on the Vrev of the histamine response (n= 3). The Vrev for the AHPslow was –85 ± 1.7 mV (n= 4). 6 These results indicate that histamine increases the excitability of ferret vagal afferent somata by interfering with two classes of potassium current: the resting or ‘leak’ potassium current (IK(rest)) and the potassium current underlying a post‐spike slow after‐hyper‐polarization (IAHP). Both these effects can occur in the same neurone and involve activation of the same histamine receptor subtype, the histamine H1 receptor.