Fluoresceinated peanut agglutinin (PNA) is a marker for live O(2) sensing glomus cells in rat carotid body.

Experiments using live dissociated carotid body (CB) cells for patch clamping, [Ca(++)](i) or other measurements require positive identification of the cell being recorded. At present, cell morphology is usually employed, but several cell types within the carotid body evidence similar morphologic characteristics. Therefore, we sought to develop a method utilizing a vital dye to identify glomus cells before and during experiments that require live cells, such as patch clamp studies. It was previously reported that the binding sites for peanut agglutinin (PNA) were highly expressed by all neuroendocrine-derivatives of the sympathoadrenal neural crest, including glomus cells, small, intensely fluorescent cells, PC-12 cells, and adrenal chromaffin cells in situ (katz et al. 1995). By utilizing the binding characteristics of galactose-specific lectin peanut agglutinin (PNA) on the outer cell membrane, we tested the possibility that the fluoresceinated PNA may preferentially bind to CB glomus cells. The results to date show: (1) Rhodamine tagged PNA (Rhod-PNA) binds to the live dissociated glomus cells in less than one hour incubation and can be visualized in superfused cells; (2) Rhod-PNA labeled cells are perfectly matched with tyrosine hydroxylase (TH) positive glomus cells; (3) Rhod-PNA did not interfere with Fura-2 for Ca(++) imaging; (4) Rhod-PNA bound to glomus cells in [Ca(++)](i) studies does not affect O(2) response of glomus cells. Thus fluoresceinated PNA may be a useful marker for live CB glomus studies, without adversely affecting their physiologic response.

Time-dependence of hyperoxia-induced impairment in peripheral chemoreceptor activity and glomus cell calcium response.

In mammals, transient exposure to hyperoxia for a period of weeks during perinatal life leads to impairment of the ventilatory response to acute hypoxia, which may persist long beyond the duration of the hyperoxia exposure. The impairment of the ventilatory response to hypoxia is due to hyperoxia-induced reduction of carotid chemoreceptor sensitivity to hypoxia. We previously demonstrated that hyperoxia exposure in rats, from birth to two weeks of age, profoundly reduced carotid chemoreceptor single axonal responses to acute hypoxia challenge. However, the time course and mechanisms of this impairment are not known. Therefore, we investigated the effect of hyperoxia (FiO(2) = 0.6) on neonatal rats after 1, 3, 5, 8, and 14 days of exposure, starting at postnatal day 7. Carotid chemoreceptor single unit activities, nerve conduction time and glomus cell calcium responses to acute hypoxia were recorded in vitro. After 1 day in hyperoxia, single unit spiking rate in response to acute hypoxia was increased compared to controls. After 5 days in hyperoxia, the spiking response to acute hypoxia was significantly reduced compared to controls, nerve conduction time was lengthened and the glomus cell calcium response to acute hypoxia was reduced compared to controls. We conclude that perinatal exposure to hyperoxia, in rats, impairs the glomus cell calcium response (pre-synaptic) and the afferent nerve excitability (post-synaptic). The time course indicates that hyperoxia exerts these effects within days.

Hypoxia transduction by carotid body chemoreceptors in mice lacking dopamine D(2) receptors.

Hypoxia-induced dopamine (DA) release from carotid body (CB) glomus cells and activation of postsynaptic D(2) receptors have been proposed to play an important role in the neurotransmission process between the glomus cells and afferent nerve endings. To better resolve the role of D(2) receptors, we examined afferent nerve activity, catecholamine content and release, and ventilation of genetically engineered mice lacking D(2) receptors (D(2)(-/-) mice). Single-unit afferent nerve activities of D(2)(-/-) mice in vitro were significantly reduced by 45% and 25% compared with wild-type (WT) mice during superfusion with saline equilibrated with mild hypoxia (Po(2) approximately 50 Torr) or severe hypoxia (Po(2) approximately 20 Torr), respectively. Catecholamine release in D(2)(-/-) mice was enhanced by 125% in mild hypoxia and 75% in severe hypoxia compared with WT mice, and the rate of rise was increased in D(2)(-/-) mice. We conclude that CB transduction of hypoxia is still present in D(2)(-/-) mice, but the response magnitude is reduced. However, the ventilatory response to acute hypoxia is maintained, perhaps because of an enhanced processing of chemoreceptor input by brain stem respiratory nuclei.

MCP-1 enhances excitability of nociceptive neurons in chronically compressed dorsal root ganglia.

Previous experimental results from our laboratory demonstrated that monocyte chemoattractant protein-1 (MCP-1) depolarizes or increases the excitability of nociceptive neurons in the intact dorsal root ganglion (DRG) after a chronic compression of the DRG (CCD), an injury that upregulates neuronal expression of both MCP-1 and mRNA for its receptor CCR2. We presently explore the ionic mechanisms underlying the excitatory effects of MCP-1. MCP-1 (100 nM) was applied, after CCD, to acutely dissociated small DRG neurons with nociceptive properties. Under current clamp, the proportion of neurons depolarized was similar to that previously observed for CCD-treated neurons in the intact ganglion, although the magnitude of depolarization was greater. MCP-1 induced a decrease in rheobase by 44 +/- 10% and some cells became spontaneously active at resting potential. Action potential width at a voltage equal to 10% of the peak height was increased from 4.94 +/- 0.23 to 5.90 +/- 0.47 ms. In voltage clamp, MCP-1 induced an inward current in 27 of 50 neurons held at -60 mV, which increased with concentration over the range of 3 to 300 nM (EC(50) = 45 nM). The MCP-1-induced current was not voltage dependent and had an estimated reversal potential of -27 mV. In addition, MCP-1 inhibited a voltage-dependent, noninactivating outward current, presumably a delayed rectifier type K(+) conductance. We conclude that MCP-1 enhances excitability in CCD neurons by, at least, two mechanisms: 1) activation of a nonvoltage-dependent depolarizing current with characteristics similar to a nonselective cation conductance and 2) inhibition of a voltage-dependent outward current.

Effects of a chronic compression of the dorsal root ganglion on voltage-gated Na+ and K+ currents in cutaneous afferent neurons.

A chronic compression of the dorsal root ganglion (CCD) produces ipsilateral cutaneous hyperalgesia that is associated with an increased excitability of neuronal somata in the compressed ganglion, as evidenced by spontaneous activity and a lower rheobase. We searched for differences in the properties of voltage-gated Na+ and K+ currents between somata of CCD- and control (unoperated) rats. CCD was produced in adult rats by inserting two rods through the intervertebral foramina, one compressing the L4, and the other, the ipsilateral, L5 dorsal root ganglion (DRG). After 5-9 days, DRG somata were dissociated and placed in culture for 16-26 h. Cutaneous neurons of medium size (35-45 microm), Fluorogold-labeled from the hindpaw, were selected for whole cell patch-clamp recording of action potentials and ion currents. In comparison with control neurons, CCD neurons had steady-state activation curves for TTX-sensitive (TTX-S) Na+ currents that were shifted in the hyperpolarizing direction, and CCD neurons had enhanced TTX-resistant (TTX-R) current. CCD neurons also had smaller, fast-inactivating K+ currents (Ka) at voltages from -30 to 50 mV. The reduction in Ka, the hyperpolarizing shift in TTX-S Na+ current activation, and the enhanced TTX-R Na+ current may all contribute to the enhanced neuronal excitability and thus to the pain and hyperalgesia associated with CCD.

Developmental aspects of oxygen sensing by the carotid body.

The carotid body chemoreceptors, the major hypoxia sensory organs for the respiratory system, undergo a significant increase in their hypoxia responsiveness in the postnatal period. This is manifest by a higher level of afferent nerve activity for a given level of arterial oxygen tension. The mechanism for the enhanced sensitivity is unresolved, but most work has focused on the glomus cell, a secretory cell apposed to the afferent nerve ending and believed to be the site of hypoxia transduction. The glomus cell secretory response to hypoxia increases postnatally, and this is correlated with an enhanced calcium rise in response to hypoxia and an increase in oxygen-sensitive potassium currents. These changes are sensitive to the level of hypoxia in the postnatal period, and significant impairment of organ function is observed with postnatal hypoxia as well as postnatal hyperoxia. Although many questions remain, especially with regard to the coupling of glomus cells to nerve endings, the use of cellular and molecular techniques should offer resolution in the near future.

Single-unit recordings of arterial chemoreceptors from mouse petrosal ganglia in vitro.

A preparation was developed that allows for the recording of single-unit chemoreceptor activity from mouse carotid body in vitro. An anesthetized mouse was decapitated, and each carotid body was harvested, along with the sinus nerve, glossopharyngeal nerve, and petrosal ganglia. After exposure to collagenase/trypsin, the cleaned complex was transferred to a recording chamber where it was superfused with oxygenated saline. The ganglia was searched for evoked or spontaneous unit activity by using a glass suction electrode. Single-unit action potentials were 57 +/- 10 (SE) (n = 16) standard deviations above the recording noise, and spontaneous spikes were generated as a random process. Decreasing superfusate PO(2) to near 20 Torr caused an increase in spiking activity from 1. 3 +/- 0.4 to 14.1 +/- 1.9 Hz (n = 16). The use of mice for chemoreceptor studies may be advantageous because targeted gene deletions are well developed in the mouse model and may be useful in addressing unresolved questions regarding the mechanism of chemotransduction.

Developmental changes in chemoreceptor nerve activity and catecholamine secretion in rabbit carotid body: possible role of Na+ and Ca2+ currents.

In order to better understand the post-natal increase in peripheral chemoreceptor responsiveness to hypoxia, chemoreceptors of newborn (1-2 days) and older (10-12 days, 30 days, adult) rabbits were isolated and superfused, in vitro. The free tissue catecholamine concentration was measured using carbon-fiber voltammetry and pauci-fiber nerve activity was recorded from the sinus nerve during stimulation (4 min) with graded hypoxia or increased potassium. Both the peak catecholamine and peak nerve responses to stimulation with 10% and 0% oxygen increased with age, particularly between 10 and 30 days of age. In contrast, peak nerve and peak catecholamine responses to increased potassium did not significantly change with age. For a better understanding of how responsiveness increases with age, the fast Na+ and the Ca2+ currents were measured from isolated glomus cells of newborn and older rabbits, but the magnitude of the currents when normalized to membrane area was not significantly different between ages. We conclude that: (1) rabbit chemoreceptors mature in the newborn period (10-30 days) and part of this maturation is an increase in catecholamine secretion, (2) maturation of hypoxia transduction primarily occurs in steps prior to depolarization since potassium-evoked responses were not affected, and (3) an increase in the magnitude of glomus cell fast Na+ or Ca2+ currents is not a likely mechanism for the maturational change, but changes in the oxygen sensitivity of these currents cannot be excluded.

Developmental changes in membrane properties of chemoreceptor afferent neurons of the rat petrosal ganglia.

Carotid body chemoreceptors increase their responsiveness to hypoxia in the postnatal period, but the mechanism for this increase is unresolved. The purpose of the present study was to examine developmental changes in cellular characteristics of chemoreceptor afferent neurons in the petrosal ganglia with the underlying hypothesis that developmental changes occur and may account for the developmental increase in chemoreceptor responsiveness. Chemoreceptor complexes (carotid body, sinus nerve, glossopharyngeal nerve, and petrosal ganglia) were harvested from rats, aged 3-40 days, and intracellular recordings were obtained from petrosal ganglion neurons using sharp electrode impalement. All chemoreceptor neurons across ages were C fibers with conduction velocities <1 m/s and generated repetitive action potentials with depolarization. Resting membrane potential was -61.3 +/- 0.9 (SE) mV (n = 78) and input resistance was 108 +/- 6 MOmega and did not significantly change with age. Cell capacitance was 32.4 +/- 1.7 pF and did not change with age. Rheobase averaged 0.21 +/- 0.02 nA and slightly increased with age. Action potentials were followed by an afterhyperpolarization of 12.4 +/- 0.6 mV and time constant 6.9 +/- 0.5 ms; only the time constant decreased with age. These results, obtained in rat, demonstrate electrophysiologic characteristics which differ substantially from that previously described in cat chemoreceptor neurons. In general developmental changes in cell characteristics are small and are unlikely to account for the developmental increase in chemoreceptor responsiveness with age.

K+ currents of glomus cells and chemosensory functions of carotid body.

The mechanism by which the carotid body senses hypoxia is not resolved, but the glomus cell, a secretory cell apposed to the afferent nerve endings, is believed to play an essential role. It is proposed that hypoxia causes glomus cell depolarization, leading to activation of voltage-gated calcium influx and enhanced secretion of an excitatory transmitter. The initial step, hypoxia induced depolarization, may be mediated by several candidate K+ channels which are sensitive to hypoxia, including: (1) a transient, voltage-dependent current; (2) a calcium and voltage dependent current; and (3) a non-voltage dependent, leak K+ current. If these channels represent the initial step in the hypoxia transduction cascade then it would be expected that K+ channel blocking agents would mimic the hypoxia response, leading to glomus cell secretion and increased nerve activity. This has been tested for the first two channels which are sensitive to classical K+ channel blocking agents, and, in general, results have not borne out this prediction. At present, the pharmacology of the leak K+ channel is not determined, and the experiment has not been undertaken. Thus, at present, hypoxic inhibition to a K+ channel in the glomus cell may initiate chemotransduction but there are many unanswered questions, especially the failure of K+ channel blocking agents to emulate the hypoxic response.

Chemotransduction by carotid body chemoreceptors is dependent on bicarbonate currents.

Previous studies have demonstrated that bicarbonate enhances the speed and magnitude of the carotid body chemoreceptor response to hypoxia. We hypothesized that this enhancement is associated with enhanced hypoxia-induced catecholamine (CAT) secretion from glomus cells. Single-fiber nerve activity and free tissue catecholamine (carbon fiber microvoltammetry) were measured in rat carotid body, in vitro. The peak CAT and nerve responses during 1 min anoxia were larger in the presence of bicarbonate than in its absence (peak CAT: 16.7 +/- 2.7 vs. 5.1 +/- 1.1 microM; peak nerve: 28.2 +/- 1.6 vs. 16.7 +/- 1.4 Hz). Bicarbonate particularly enhanced the responses to moderate hypoxia (PO2 approximately 80 Torr) which caused no secretion or increased nerve activity in the absence of bicarbonate, but caused significant stimulation in the presence of bicarbonate (peak nerve = 15.2 Hz; peak CAT = 8.6 microM). The bicarbonate effect was not due to alterations in intracellular pH since it was not blocked by exchanger blockers (DIDS) or mimicked by acidification of the medium. However, anion channel blockade by 9-AC or DPC reduced anoxia-induced CAT secretion in the presence of bicarbonate. We conclude that bicarbonate greatly enhances stimulus/secretion coupling in glomus cells, probably through modulation of an anion current carried by bicarbonate.

Effect of sodium perturbations on rat chemoreceptor spike generation: implications for a Poisson model.

1. The sensitivity of arterial chemoreceptor spike generation to reductions in excitability was examined using rat chemoreceptors in vitro. Axonal excitability was reduced by reducing extracellular sodium concentration ([Na+]o) by 10-40% or by applying low doses of tetrodotoxin (TTX). 2. In normoxia and in hypoxia, an isosmotic reduction in [Na+]o caused a proportional decrease in single-fibre, spiking nerve activity. For a 20% reduction in [Na+]o, nerve activity decreased to 54 +/- 7% of control in normoxia and 41 +/- 5% in hypoxia. 3. Low doses of TTX (25-50 nM) caused a similar decrease in spiking frequency, but this response was variable amongst fibres, with some fibres unaffected by TTX. 4. A reduction in [Na+]o by 20% caused a slowing of conduction velocity, measured using an electrical stimulus delivered to an electrode placed in the carotid body. Threshold current for spike generation was increased by about 2.7 +/- 1.4%. Threshold current increased by 6.5 +/- 3.7% following a 40% reduction in [Na+]o. 5. The spike generation process was modelled as a Poisson process in which depolarizing events summate and give rise to an action potential. The experimental data were best fitted to a high order process characterized by a large number of events and high event threshold. 6. This result is not consistent with depolarization events caused by episodic transmitter release, but suggests that afferent spike generation is an endogenous process in the afferent nerve fibres, perhaps linked to random channel activity or to thermal noise fluctuations.

Patch clamp recording from the intact dorsal root ganglion.

A method for patch-clamp recording from intact dorsal root ganglion (DRG) cells in rat is described. The L4 and L5 DRGs with sciatic nerve attached were excised from rats (10-15 days old) and placed in a recording chamber after removing the ganglion sheath and dissolving the connective tissue with dilute collagenase. The somata of individual cells were exposed by gentle surface cleaning through a perfusion micropipette. Somata were classified as Abeta, Adelta or C based on the cell size and the shape of the action potential (AP). Under current clamp, axonal conduction velocity (CV) was calculated from the distance between a stimulating electrode and the center of the ganglion divided by the latency of the AP elicited by stimulation of the sciatic nerve. CVs ranged from 0.2-0.8 m/s for C cells, 0.8-2.4 for Adelta and 3.2-5.0 for A/beta cells. AP threshold occurred at a significantly more positive potential in C cells than in Adelta and Abeta cells. Under voltage clamp, sodium currents were recorded from C cells. Both TTX-resistant (TTX-R) and TTX-sensitive (TTX-S currents) were demonstrated in the present study. The results demonstrate the feasibility of patch-clamp recording from intact, identified DRG cells in vitro.

Integration of bronchomotor and ventilatory responses to chemoreceptor stimulation in developing sheep.

We analyzed the changes induced by central chemoreceptor stimulation on the lung resistances and phrenic neurogram of anesthetized newborn (3-6 days, n = 9) and 9 week old lambs (n = 3). Starting from hypocapneic apnea, 5% CO2 inhalation evoked a reversible increase in total lung resistance in both newborn and 9 week old lambs (median = 112%). The resistance increase preceded phrenic breathing and was greater for the peripheral (233%) than for the central airways (57%), independent of age. Increases in lung and airway resistance caused by CO2 were reversed totally by atropine and only partially by apnea-producing doses of fentanyl. Our results demonstrate that parasympathetic outflow to the sheep airways is already driven by central chemoreceptor inputs during the newborn period. Even at this early age, bronchomotor responses to central chemoreceptor stimulation are more prominent in the peripheral than in the central airways and exhibit a lower threshold for activation and less sensitivity to opioid inhibition than phrenic responses.

Axotomy increases the excitability of dorsal root ganglion cells with unmyelinated axons.

Axotomy increases the excitability of dorsal root ganglion cells with unmyelinated axons. J. Neurophysiol. 78: 2790-2794, 1997. To better understand the neuronal mechanism of neuropathic pain, the effect of axotomy on the excitability of dorsal root ganglion (DRG) cells with unmyelinated axons (C cells) was investigated. Whole cell patch-clamp recordings were performed on intact DRG cells with intact axons or with axons transected 7-12 days earlier. C cells were identified by 1) soma size, 2) action potential morphology, 3) conduction velocity, and 4) in some cases, injection of Fast Blue into the injured nerve fibers. Axotomy reduced (more negative) action potential threshold but did not significantly change resting membrane potential, action potential duration, or maximal depolarization rate. Axotomy significantly increased the peak sodium current measured under voltage-clamp conditions. In Fast Blue-labeled (injured) cells, the tetrodotoxin (TTX)-sensitive current was enhanced while the TTX-resistant current was reduced. These results suggest that axotomy increased the excitability of C cells, possibly because of a preferential increase in expression of TTX-sensitive sodium currents.

Are oxygen dependent K+ channels essential for carotid body chemo-transduction?

The mechanism by which the carotid body senses hypoxia and causes an increase in spiking activity on the sinus nerve is not well resolved. Most experimental attention is focused on the glomus cell, a secretory cell which is apposed to the afferent nerve endings and which is the presumed site of oxygen sensing. It is proposed that hypoxia causes glomus cell depolarization by inhibiting an oxygen-sensitive K+ current. This leads to depolarization, activation of voltage-gated calcium influx and enhanced secretion of an excitatory transmitter. At present, 4 candidate oxygen-sensitive K+ currents have been identified based on patch-clamp studies of isolated glomus cells. Recent experiments using intact carotid bodies have been undertaken to identify which current is most likely to mediate the hypoxia response. Three of the four currents are sensitive to K+ channel blocking agents (TEA, 4-AP and charybdotoxin), yet all these agents failed to mimic hypoxia, neither stimulating chemoreceptor nerve activity nor enhancing catecholamine secretion. Thus, the fourth current, a leak current which is insensitive to these agents is the most likely candidate for mediating glomus cell depolarization, but the drug-sensitivity of this current is not yet known which precludes a direct test of this speculation.

Chemoreceptor nerve excitation may not be proportional to catecholamine secretion.

Enhanced catecholamine secretion from the carotid body glomus cells is hypothesized to play an essential role in mediating the peripheral chemoreceptor response to hypoxia. To test aspects of this hypothesis, the relationship between catecholamine secretion and nerve activity was examined during repetitive hypoxia stimuli and after catecholamine depletion with reserpine. Single-fiber afferent serve activity was measured along with an estimate of free tissue catecholamine by using Nafion-coated carbon-fiber microelectrodes placed in rat carotid bodies in vitro. Baseline and stimulated nerve and catecholamine levels were quantified during repetitive stimulation (anoxia of 1-min duration; PO2 = 0 Torr at nadir, repeated each 200 s). Peak stimulated catecholamine progressively decreased from 26.4 +/- 2.6 microM for the first stimulus to 7.5 +/- 0.9 microM for the fifth stimulus (n = 15), but peak nerve activity was much less affected (23.0 +/- 1.9 Hz, first trial; 19.9 +/- 1.4 Hz, fifth trial). An exposure to moderate hypoxia (approximately 80 Torr) before the repetitive anoxia stimuli produced catecholamine levels comparable to those obtained during repetitive anoxia, but peak nerve activity was significantly less (22.5 +/- 3.4 vs. 12.7 +/- 2.1 Hz). Pretreatment with reserpine (1 mg/100 g) resulted in a large reduction in the average hypoxia-induced catecholamine response (1.4 +/- 0.3 microM, n = 9), but peak nerve activity was not different from nontreated controls. These results demonstrate an independence between carotid body catecholamine secretion and nerve activity, suggesting that nerve excitation is, at least, partially mediated through pathways independent of granule secretion.