Kinetic properties and adrenergic control of TREK-2-like channels in rat medial prefrontal cortex (mPFC) pyramidal neurons.

TREK-2-like channels were identified on the basis of electrophysiological and pharmacological tests performed on freshly isolated and enzymatically/mechanically dispersed pyramidal neurons of the rat medial prefrontal cortex (mPFC). Single-channel currents were recorded in cell-attached configuration and the impact of adrenergic receptors (α1, α2, ß) stimulation on spontaneously appearing TREK-2-like channel activity was tested. The obtained results indicate that noradrenaline decreases the mean open probability of TREK-2-like channel currents by activation of ß1 but not of α1- and α2-adrenergic receptors. Mean open time and channel conductance were not affected. The system of intracellular signaling pathways depends on the activation of protein kinase A. We also show that adrenergic control of TREK-2-like channel currents by adrenergic receptors was similar in pyramidal neurons isolated from young, adolescent, and adult rats. Immunofluorescent confocal scans of mPFC slices confirmed the presence of the TREK-2 protein, which was abundant in layer V pyramidal neurons. The role of TREK-2-like channel control by adrenergic receptors is discussed.

Muscarinic receptor control of pyramidal neuron membrane potential in the medial prefrontal cortex (mPFC) in rats.

Damage to the cholinergic input to the prefrontal cortex has been implicated in neuropsychiatric disorders. Cholinergic endings release acetylcholine, which activates nicotinic and/or G-protein-coupled muscarinic receptors. Muscarinic receptors activate transduction systems, which control cellular effectors that regulate the membrane potential in medial prefrontal cortex (mPFC) neurons. The mechanisms responsible for the cholinergic-dependent depolarization of mPFC layer V pyramidal neurons in slices obtained from young rats were elucidated in this study. Glutamatergic and GABAergic transmission as well as tetrodotoxin (TTX)-sensitive Na(+) and voltage-dependent Ca(++) currents were eliminated. Cholinergic receptor stimulation by carbamoylcholine chloride (CCh; 100 µM) evoked depolarization (10.0 ± 1.3 mV), which was blocked by M1/M4 (pirenzepine dihydrochloride, 2 µM) and M1 (VU 0255035, 5 µM) muscarinic receptor antagonists and was not affected by a nicotinic receptor antagonist (mecamylamine hydrochloride, 10 µM). CCh-dependent depolarization was attenuated by extra- (20 µM) or intracellular (50 µM) application of an inhibitor of the ßγ-subunit-dependent transduction system (gallein). It was also inhibited by intracellular application of a ßγ-subunit-binding peptide (GRK2i, 10µM). mPFC pyramidal neurons express Nav1.9 channels. CCh-dependent depolarization was abolished in the presence of antibodies against Nav1.9 channels in the intracellular solution and augmented by the presence of ProTx-I toxin (100 nM) in the extracellular solution. CCh-induced depolarization was not affected by the following reagents: intracellular transduction system blockers, including U-73122 (10 µM), chelerythrine chloride (5 µM), SQ 22536 (100 µM) and H-89 (2 µM); channel blockers, including Ba(++) ions (200 µM), apamin (100 nM), flufenamic acid (200 µM), 2-APB (200 µM), SKF 96365 (50 µM), and ZD 7288 (50 µM); and a Na(+)/Ca(++) exchanger blocker, benzamil (20 µM). We conclude that muscarinic M1 receptor-dependent depolarization in mPFC pyramidal neurons is evoked by the activation of Nav1.9 channels and that the signal transduction pathway involves G-protein ßγ subunits.

Effect of cyclic adenosine monophosphate on the G protein-dependent inward rectifier K(+)-like channel current in medial prefrontal cortex pyramidal neurons.

Cyclic adenosine monophosphate (cAMP) levels in medial prefrontal cortex (mPFC) pyramidal neurons are altered in neuropsychiatric disorders. cAMP is a component of the transduction pathways involved in the control of ionic channels by metabotropic receptors. The purpose of this study was to determine whether cAMP modifies the activity of the G protein-dependent inward rectifier K(+) (GIRK)-like channel current in the mPFC pyramidal neurons of 3-week-old rats. Channel currents were recorded in a patch clamp cell-attached configuration. Membrane-permeable adenylyl cyclase activator forskolin (10 µM) and membrane-permeable protein kinase A (PKA) activator 8-Br-cAMP (100 µM) were found to significantly decrease the open probability (Po) of the GIRK-like channels. Conversely, selective protein kinase A inhibitors: H-89 (10 µM) and KT5720 (0.5 µM) increased the open probability of the GIRK-like channels. Also, the effect of forskolin was tested after preincubation of the neurons with the PKA inhibitor (KT5720). The application of forskolin, despite PKA inhibition, significantly decreased the Po of the GIRK-like channels. This finding suggested that GIRK-like channel current activity might also be inhibited by cAMP in a PKA-independent manner. A compound, 8CPT-2Me-cAMP (10 µM), which is a specific activator of the Epac protein, which in turn is another intracellular target of cAMP, was also found to inhibit GIRK-like channel activity. We conclude that the constitutive activity of neuronal GIRK-like channel currents is inhibited by cAMP. We suggest that PKA and Epac might be components of the transduction pathway between cAMP and the GIRK channels.

D(1) dopaminergic control of G protein-dependent inward rectifier K(+) (GIRK)-like channel current in pyramidal neurons of the medial prefrontal cortex.

Pyramidal neurons of the medial prefrontal cortex (mPFC) exhibit dopamine-dependent prolonged depolarization, which may lead to persistent activity. Persistent activation of prefrontal cortex neurons has been proposed to underlie the working memory process. The purpose of our study was to test the hypothesis that activation of D(1) dopamine receptors leads to inhibition of G protein-dependent inward rectifier K(+) (GIRK) channels, thereby supporting the prolonged depolarization of mPFC pyramidal neurons. Experiments were performed on 3-week-old rats. GIRK-like channel currents recorded from pyramidal neurons showed the following properties at -75 mV: open probability (NPo), 2.5+/-0.3 x 10(-3); mean open time, 0.53+/-0.05 ms; and conductance, 29.9+/-1.6 pS (n=60). The channel currents were strongly inward-rectified. GIRK channel currents were reversibly inhibited by the D(1) agonists SKF 38393 (10 microM) and SKF 81297 (10 microM). This inhibition was abolished by prior application of a dopamine receptor antagonist and by application of the membrane-permeable protein kinase C inhibitors chelerythrine chloride (3 microM) and calphostin C (10 microM). It was also found that the application of D(1) dopamine receptor agonists or GIRK channel inhibitors evoked depolarization of mPFC pyramidal neurons in rats. Moreover, prior application of a GIRK channel blocker eliminated the depolarizing effect of D(1) agonists. We conclude that activation of D(1) dopamine receptors may lead to inhibition of GIRK channel currents that may, in turn, lead to the prolonged depolarization of mPFC pyramidal neurons in juvenile rats.

Kinetic properties of voltage-gated Na+ currents in rat muscular sympathetic neurons with and without adenosine triphosphate and guanosine triphosphate in intracellular solution.

Voltage-gated Na+ currents were recorded from anatomically identified postganglionic muscular sympathetic neurons without and with ATP and GTP in the intracellular solution. The main findings of the study were that cells without ATP and GTP in the intracellular solution express a higher amplitude and greater density of voltage-gated Na+ current, and their Na+ current activates faster and also inactivates faster time dependently. The current is also steady-state inactivated to a lesser degree and recovers from inactivation more slowly in cells without added ATP and GTP. These findings suggest that the presence of ATP and GTP, substrates for channel phosphorylation, changes the kinetic properties of Na+ currents.

Voltage-dependent K+ currents in rat cardiac dorsal root ganglion neurons.

We have assessed the expression and kinetics of voltage-gated K(+) currents in cardiac dorsal root ganglion (DRG) neurons in rats. The neurons were labelled by prior injection of a fluorescent tracer into the pericardial sack. Ninety-nine neurons were labelled: 24% small (diameter<30 microm), 66% medium-sized (diameter 30 microm>.48 microm) and 10% large (>48 microm) neurons. Current recordings were performed in small and medium-sized neurons. The kinetic and pharmacological properties of K(+) currents recorded in these two groups of neurons were identical and the results obtained from these neurons were pooled. Three types of K(+) currents were identified:a) I(As), slowly activating and slowly time-dependently inactivating current, with V(1/2) of activation -18 mV and current density at +30 mV equal to 164 pA/pF, V(1/2) of inactivation at -84 mV. b) I(Af) current, fast activating and fast time-dependently inactivating current, with V(1/2) of activation at two mV and current density at +30 mV equal to 180 pA/pF, V(1/2) of inactivation at -26 mV. At resting membrane potential I(As) was inactivated, whilst I(Af), available for activation. The I(As) currents recovered faster from inactivation than I(Af) current. 4-Aminopiridyne (4-AP) (10 mM) and tetraethylammonium (TEA) (100 mM) produced 98% and 92% reductions of I(Af) current, respectively and 27% and 66% of I(As) current, respectively. c) The I(K) current that did not inactivate over time. Its V(1/2) of activation was -11 mV and its current density equaled 67 pA/pF. This current was inhibited by 95% (100 mM) TEA, whilst 4-AP (10 mM) produced its 23% reduction. All three K(+) current components (I(As), I(Af) and I(K)) were present in every small and medium-sized cardiac DRG neuron. We suggest that at hyperpolarized membrane potentials the fast reactivating I(As) current limits the action potential firing rate of cardiac DRG neurons. At depolarised membrane potentials the I(Af) K(+) current, the reactivation of which is very slow, does not oppose the firing rate of cardiac DRG neurons.

Voltage-dependent Ca2+ currents in rat cardiac dorsal root ganglion neurons.

This study presents the kinetic and pharmacological properties of voltage-gated Ca(2+) currents in anatomically defined cardiac dorsal root ganglion (DRG) neurons in rats. The neurons were labelled by prior injection of fluorescent tracer Fast Blue into the pericardial sack. There were three distinct groups of neurons with respect to cell size: small (27% of total; cell capacitance <30 pF), medium (65% of total; capacitance 30-80 pF) and large neurons (8% of total; capacitance >80 pF). The properties of Ca(2+) currents were tested in small and medium-sized neurons. In large neurons currents could not be adequately controlled and were not analysed. Ca(2+) currents did not completely inactivate during 100 ms depolarising voltage steps. The activation thresholds in small (-36.9+/-1.3 mV) and medium (-39.0+/-1.3 mV) size neurons were similar. Current densities were 105.8 pA/pF in small and 97.4 pA/pF in large neurons and also did not differ. The kinetic properties of activation and inactivation did not differ between small and medium-sized cardiac DRG neurons. At membrane potentials between -50 and -60 mV (the expected resting membrane potential in these neurons) 55 to 70% of Ca(2+) currents in small and medium-sized neurons were available for activation. Both, small and medium-sized neurons expressed similar proportions of L (7.5%), N (25%) and P/Q (36%) type Ca(2+) currents. We conclude that small and medium-sized cardiac DRG neurons are homogeneous with respect to the expression and properties of voltage-gated Ca(2+) currents. Voltage-gated Ca(2+) currents probably play an important role in action potential generation in cardiac DRG neurons due to their availability for activation at resting membrane potential, their high density and voltage threshold close to the threshold for voltage-gated Na(+) currents.

Kinetic and pharmacological properties of Ca(2+) currents in postganglionic sympathetic neurones projecting to muscular and cutaneous effectors.

Voltage-gated Ca(2+) channels are expressed in neurones and greatly influence neuronal activity by activating Ca(2+)-dependent K(+) channels. The whole cell patch-clamp technique was used to compare the kinetic and pharmacological properties of voltage-dependent Ca(2+) currents in two groups of sympathetic neurones identified by the fluorescent tracer Fast Blue: putative muscular sympathetic neurones (MSN) and putative cutaneous sympathetic neurones (CSN). The tracer was injected into the muscular part of the diaphragm (to mark MSN) and into the skin of the ear (to mark CSN). The capacitance of MSN (23.0 pF) was larger than the capacitance of CSN (12.6 pF). The maximum current in MSN (1.3 nA) was also larger than in CSN (0.93 nA). However, the current density was larger in CSN (77. 3 pA/pF) than in MSN (57.7 pA/pF) and the current activation rate was faster in CSN (0.27 nA/ms) than in MSN (0.19 nA/ms). V(1/2) and slope factors of activation and inactivation were not significantly different for MSN and CSN. The majority of Ca(2+) current was available for activation in both categories of neurones at resting membrane potential. Ca(2+) currents in MSN and CSN were blocked by nifedipine (7.0 and 3.6%, respectively), omega-Agatoxin-IVA (23.0 and 25.6%, respectively) and omega-conotoxin-GVIA (67.0 and 65.1%, respectively). We found that CSN are twice as small, have higher Ca(2+) current density and their Ca(2+) activation rate is faster in comparison to MSN. Such properties may lead to faster rise of Ca(2+) concentration in the cytoplasm of the CSN comparing to MSN and more effectively dampen their activity due to more effective activation of Ca(2+)-dependent K(+) current. Both kinds of neurones express high proportion of N and P/Q Ca(2+) current.

[Anatomic and functional identification of sensory neurons in neurophysiologic studies]. / Identyfikacja anatomiczna i funkcjonalna neuronów obwodowych w badaniach neurofizjologicznych.

The electrophysiological studies indicate that peripheral autonomic and primary sensory neurones display a wide variety of ionic currents. The experiments aimed at biophysical and pharmacological analysis of ionic currents are frequently performed on isolated neurones devoid of axones and dendrites. Consequently the ionic currents recordings are limited to the cell soma. The function of somatic ionic channels is largely unknown. We suggest that the functional meaning of the somatic ionic currents can be facilitated by analysis currents properties in functionally identified neurones. Examples are given of different biophysical and pharmacological currents properties in cardiac, glandular, cutaneous and muscular sympathetic neurones. It is concluded that the resting and reflex activity in different categories of sympathetic neurones may be profoundly affected by the biophysical properties of ionic channels expressed in their soma.

Quantitative differences between kinetic properties of Na(+) currents in postganglionic sympathetic neurones projecting to muscular and cutaneous effectors.

The activity of muscular and cutaneous sympathetic neurones has been shown to be differentially regulated. The differences may partially stem from the different ionic channel expression and current kinetics in these neurones, particularly that of Na(+) channels, which play a critical role in action potential generation and modulation of neuronal excitability. The whole cell patch-clamp technique was used to compare the kinetic properties of Na(+) currents in two groups of sympathetic neurones identified by the fluorescent tracer Fast Blue: putative muscular sympathetic neurones (PMSN) and putative cutaneous sympathetic neurones (PSSN). The tracer was injected into the muscular part of the diaphragm (to mark PMSN) and into the skin of the ear (to mark PSSN). Both kinds of neurones expressed fast activating, fast inactivating, voltage dependent and TTX sensitive Na(+) currents. However, the electrical characteristics of the cells were markedly different: (1) The capacitance of PMSN (21.7 pF) was larger than PSSN (12.7 pF). Maximum current in PMSN (3.1 nA) was also larger than in PSSN (2.0 nA). Calculated current density was smaller in PMSN (148.0 pA/pF) than in PSSN (181.1 pA/pF). Slope conductance was larger in PMSN compared to PSSN (102.7 nS and 73.6 nS respectively). (2) V(1/2) of activation for PMSN (-20.9 mV) was more negative than the potential recorded for PSSN (-16.7 mV); the slope factors were not different. (3) V(1/2) for inactivation was more negative for PMSN than for PSSN (-66.3 vs. -60.8 mV); again, the slope factors for inactivation were not different. (4) The rate of recovery from inactivation could be described by the sum of two exponential functions. In PMSN the fast and slow recovery exponential factors tau(f) and tau(s) were 12.6 (66%) and 83.9 (34%) ms, while in PSSN they were shorter and equalled 8.2 (62%) and 41.9 (38%) ms, respectively. We conclude that the Na(+) currents of PMSN and PSSN have different kinetic properties.

Properties of Na+ currents in putative submandibular and cardiac sympathetic postganglionic neurones.

This study was performed to compare the kinetic properties of Na+ currents in putative salivary and cardiac postganglionic sympathetic neurones isolated from the superior cervical and stellate ganglia, respectively. Neurones were labelled with a fluorescent tracer-Fast Blue, injected into the submandibular gland (in the case of salivary neurones) and into the pericardial cavity or left ventricular wall (in the case of cardiac neurones). Voltage-dependent Na+ current was then isolated and recorded from labelled cells. The major findings of this study were: (1) Peak Na+ current was larger in salivary than in cardiac neurones (5.7 nA vs. 2.4 nA; for 30 mM Na+ in extra- and 15 mM in the intracellular solution). (2) The somata of salivary neurones were twice as large as those of cardiac neurones, as indicated by the values of their membrane capacitance (36 pF vs. 18 pF). (3) There was a greater Na+ current density (169 pA/pF vs. 128 pA/pF) in salivary than in cardiac neurones. (4) Recovery from inactivation was faster in salivary neurones with 90% recovery time being 93 ms for salivary and 144 ms in cardiac neurones. (5) Half-activation times were voltage-dependent and consistently longer for salivary than for cardiac neurones. (6) Remaining parameters, such as current threshold, maximum current voltage and kinetics of steady-state inactivation did not significantly differ in salivary compared to cardiac neurones.

Stellate neurones innervating the rat heart express N, L and P/Q calcium channels.

The aim of the study was to investigate the kinetic properties and identify the subtypes of Ca2+ currents in the cardiac postganglionic sympathetic neurones of rats. Neurones were labelled with a fluorescent tracer--Fast-Blue, injected into the pericardial cavity. Voltage-dependent Ca2+ currents were recorded from dispersed stellate ganglion cells that showed Fast Blue labelling. Only high threshold voltage-dependent Ca2+ currents were found in the somata of cardiac sympathetic neurones. Their maximum amplitude, mean cell capacitance and current density were respectively: 0.67 nA, 19.3 pF and 36.4 pA/pF (n = 21). The maximum Ca2+ conductance was 51.3 nS (n = 14). Half activation voltage equalled +11.0 mV and the slope factor for conductance 11.1 (n = 14). As tested with a 10 s pre-pulse, the Ca2+ current began to inactivate at -80 mV. Half inactivation voltage and slope factor for steady-state inactivation were -36.6 mV and 14.1 (n = 9), respectively. Saturating concentration of L channel blocker (nifedipine), N channel blocker (omega-conotoxin-GVIA), P/Q channel blocker (omega-Agatoxin-IVA) and N/P/Q channel blocker (omega-conotoxin-MVIIC) reduced the total Ca2+ current by 26.8% (n = 7), 57.1% (n = 12), 25.9% (n = 6) and 69.4% (n = 6), respectively. These results show that the somata of cardiac postganglionic cardiac sympathetic neurones contain significant populations of N, L and P/Q high threshold Ca2+ channels.

Distribution of compound potentials recorded from the surface of isolated stellate ganglia following stimulation of postganglionic branches, in rats and cats.

The experiments were performed on 9 cat and 18 rat isolated stellate ganglia. Rats and cats were anesthetized with alpha-glucochloralose or urethane, respectively. The ganglia, isolated with their branches, were transferred to a recording chamber and constantly superfused with artificial extracellular fluid bubbled with 95% O2 and 5% CO2. Branches of the ganglion were one by one placed in suction electrodes and stimulated. Antidromic evoked potentials were systematically recorded from numerous points on the ganglion surface. The area under the curve of the negative wave of each recorded potential was considered proportional to the number of neurons located in the vicinity of the recording electrode, projecting to the stimulated nerve. We have found that: (1) cardiac sympathetic neurons are located in the lower, caudal half of the ganglia; (2) vertebral sympathetic neurons occupy the cranial, upper half of the ganglia; (3) neurons with axons in the ansae are positioned near the point of exit of the respective ansa from the ganglion; (4) localization of neurons projecting to the same branches is very similar on both sides--right and left; (5) this localization is also similar in rats compared to cats.

Properties of postganglionic sympathetic fibers isolated from the right recurrent laryngeal nerve.

The pattern of response of 45 single postganglionic sympathetic axons dissected from the right recurrent laryngeal nerve was examined in chloralose-anesthetized cats. Both vagoaortic nerves were cut, and both sinus nerves were left intact. Each neuron, based on the presence of cardiac and respiratory rhythmicities in its resting activity and reaction to systemic hypoxia (10% O2 in N2 for 2 min), was classified into one of three classes. Class I neurons (n = 29, 64%) were activated during systemic hypoxia and had a pronounced cardiac and inspiration-related rhythmicity in their resting activity. Class II neurons (n = 12,27%) were inhibited during systemic hypoxia, and their cardiac and respiratory rhythmicities were either negligible or totally absent. Class III neurons (n = 4,9%), similarly to class I, had a pronounced cardiac and inspiratory rhythmicity but were not affected by systemic hypoxia. The systemic hypoxia was always accompanied by an increase in blood pressure. We conclude that class I and possibly class III neurons innervate the arteries of upper airways. We also discuss the possibility that class II neurons are responsible for regulating the smooth muscles of upper airways.

Functional classification of afferent phrenic nerve fibres and diaphragmatic receptors in cats.

1. Single afferent fibres with receptive fields in the diaphragm (272 units) dissected from the right phrenic nerve were classified according to the following properties: reaction to contraction of the diaphragm, resting activity, conduction velocity, location and properties of receptive fields, and reaction to injection of bradykinin and lactic acid into the internal thoracic artery. Nine additional fibres dissected from the phrenic nerve had receptive fields outside the diaphragm. The experiments were performed on chloralose-anaesthetized cats. 2. Ninety-six fibres (36%) had high resting activity when unloaded by contraction of the diaphragm, had low-threshold receptive fields in the muscle and were mostly group II and III fibres. They probably innervated muscle spindles. 3. Eighty-eight fibres (32%) were vigorously activated by contraction of the diaphragm. They had low-threshold receptive fields located in the musculotendinous border and central tendon. Their conduction velocity was in the range for group II and III fibres. We infer that they may innervate tendon organs. 4. Eighty-eight fibres (32%) were slightly affected or not affected by diaphragmatic contraction. They had low- and high-threshold receptive fields located mostly in the muscular part of the diaphragm, and negligible resting activity. Most of them were group III and IV afferent fibres and were activated when bradykinin and lactic acid were applied to their receptive fields. Possibly these low- and high-threshold receptors innervated diaphragmatic ergo- and nociceptors, respectively. 5. Sensory outflow from the diaphragm was found to be somatotopically organized, so that fibres with receptive fields in the sternocostal portion were predominantly located in the upper phrenic nerve root, and those with lumbar receptive fields were in the lower root. 6. It is concluded that the phrenic nerve contains fibres from several distinct classes of sensory receptors: muscle spindles, tendon organs, ergoceptors and nociceptors. The sensory diaphragmatic outflow to the spinal cord is somatotopically organized.

Pulmonary chemoreflex and phrenic sympathetic efferents.

The pulmonary chemoreflex components such as reactions of phrenic sympathetic neuron (PhSN) activity, phrenic nerve activity, heart rate and blood pressure were tested in chloralose-anesthetized, paralyzed cats. 10 micrograms to 160 micrograms phenylbiguanide (PBG) in 0.9% NaCl was injected into the pulmonary circulation. PBG injected into the right atrium (in 11 of 19 experiments) and into the pulmonary artery (in 5 of 8 experiments), evoked short-latency (1-1.4 sec) dose-dependent increase in PhSN activity accompanied by increase in blood pressure, and followed by decrease in these two variables. In all experiments, activity of the phrenic nerve was depressed, and bradycardia occurred after PBG injection. All responses to PBG injections into the pulmonary artery were abolished following bilateral vagotomy. In the same procedure related to the right atrium after vagotomy, the increases in PhSN activity and blood pressure were also abolished, although a decrease in heart rate, PhSN activity and in the amplitude of phrenic nerve discharges together with an increase in their frequency persisted. Our results suggest that short-latency increase in PhSN activity is a component of pulmonary chemoreflex.

Reflex carotid body chemoreceptor control of phrenic sympathetic neurons.

The reflex reaction of phrenic sympathetic neurons to stimulation of carotid body chemoreceptors was tested in chloralose-anesthetized and paralyzed cats with both vago-aortic nerves cut. During systemic hypoxia (animals ventilated with 10% O2 in N2) the sympathetic phrenic nerve activity increased from 100% in the control to 269%. This increase was markedly attenuated after cutting both sinus nerves. Reflex excitatory response in phrenic sympathetic neurons with the latency of 150 msec was evoked by electrical stimulation of the right carotid sinus nerve (3 pulses of 0.2 msec, 333 Hz). The central transmission time of the reflex was about 90 msec. Injecting 0.1 ml of 1 M NaHCO3 saturated with CO2 (in order to activate carotid body chemoreceptors) into the right or left carotid sinus, evoked excitatory responses in sympathetic neurons regardless of the side. The stimulation of carotid body chemoreceptors also increased somatic phrenic nerve activity. The three methods applied to the stimulation of carotid body chemoreceptors produced increase of phrenic nerve sympathetic activity.

Properties of postganglionic sympathetic neurons with axons in phrenic nerve.

The aim of the study was to test the reflex and resting properties of postganglionic sympathetic neurons with axons located in the right phrenic nerve. The experiments have been performed on chloralose-anesthetized cats with both vago-aortic nerves cut. The somata or the postganglionic sympathetic neurons were located in the stellate ganglion. Axons of these neurons passed through the upper and lower phrenic nerve roots and through the phrenic nerve itself. The presence of cardiac and respiratory rhythmicities was detected in the activity of the phrenic postganglionic sympathetic neurons. Hyperventilation, which abolished burst discharges of the phrenic nerve, decreased the sympathetic activity by 14%. Systemic hypoxia (ventilating the animals for 2 min with 8% O2 in N2) increased the sympathetic activity threefold. The results of our experiments suggest that axons of the sympathetic neurons located in the right phrenic nerve could possibly be diaphragmatic muscle vasoconstrictors.

Properties of postganglionic sympathetic neurons with axons in the right thoracic vagus.

The resting and reflex-evoked activities of single postganglionic sympathetic neurons with axons in the right thoracic vagus were tested in chloralose-anaesthetized cats. The properties of a majority of neurons were found to be similar. Cardiac- and inspiration-related rhythmicities were present in the resting activity of sympathetic neurons. Their resting activity was not affected by hyperventilation which abolished phrenic nerve discharges. Systemic hypoxia (2 min; 8% O2 in N2) increased the activity of the neurons more effectively in the deafferented state than when both sinus nerves remained intact. Injection of 0.1 ml 1 M sodium bicarbonate saturated with CO2, which activates peripheral chemoreceptors in the right or left carotid sinus, usually evoked a decrease in sympathetic activity in animals with both sinus nerves intact. We concluded that activation of peripheral chemoreceptors may inhibit the activity of the sympathetic neurons with axons in the right thoracic vagus. We suggest that the described sympathetic neurons may be a functionally homogeneous population which may innervate the conducting system of the heart. The close localization of sympathetic and parasympathetic axons in the vagus nerve may facilitate sympathetic-parasympathetic interaction at the level of their endings in the heart.

Spinal segmental sympathetic outflow to cervical sympathetic trunk, vertebral nerve, inferior cardiac nerve and sympathetic fibres in the thoracic vagus.

The spinal segmental localization of preganglionic neurons which convey activity to the sympathetic nerves, i.e. vertebral nerve, right inferior cardiac nerve, sympathetic fibres in the thoracic vagus and cervical sympathetic trunk, was determined on the right side in chloralose anaesthetized cats. For that purpose the upper thoracic white rami were electrically stimulated with a single pulse, suprathreshold for B and C fibres, and the evoked responses were recorded in the sympathetic nerves. The relative preganglionic input from each segment of the spinal cord to the four sympathetic nerves was determined from the size of the evoked responses. It was found that each sympathetic nerve receives a maximum preganglionic input from one segment of the spinal cord (dominant segment) and that the preganglionic input gradually decreased from neighbouring segments. The spinal segmental preganglionic outflow to the cervical sympathetic trunk, thoracic vagus, right inferior cardiac nerve and vertebral nerve gradually shifted from the most rostral to the most caudal spinal cord segments. In some cases, a marked postganglionic component was found in the cervical sympathetic trunk. It was evoked by preganglionic input from the same spinal cord segments which transmitted activity to the vertebral nerve. These results indicate that there is a fixed relation between the spinal segmental localization of preganglionic neurons and the branch of the stellate ganglion receiving the input from these neurons.