Search

In this study, we explore how two different prompt types within an online computer-based inquiry learning environment enhance 392 7th grade students’ explanations of evolution with three teachers. In the elaborating prompt condition, students are prompted to write explanations that support the accepted theory of evolution. In the competing prompt condition, students are prompted to write explanations that differentiate two views of evolution associated with Darwin and Lamarck. Data sources included a pretest and posttest, an embedded item, observations, logged teacher guidance, and teacher interviews. Findings show similar pretest to posttest gains in students’ understanding of evolution for both conditions, but this pattern was not uniform across all three teachers. For one teacher, students who received competing theory prompts produced significantly higher gains than those who received elaborating theory prompts. A closer look at embedded student work reveals a higher degree of teacher participation (i.e., grading and guidance) than for the other teachers. Our findings illustrate how helping students distinguish between competing scientific claims can support learning in an inquiry unit, but may require a higher degree of teacher participation and reinforcement. We discuss the implications of these findings for enhancing students’ scientific explanations. © 2016 Wiley Periodicals, Inc. J Res Sci Teach 53: 1341–1363, 2016.

Carotid body (CB) glomus cells depolarize in response to hypoxia, causing a [Ca(2+)](i) increase, at least in part, through activation of voltage-dependent channels. Recently, Turner et al. (2013) showed that mouse glomus cells with knockout of TASK1/3(-/-) channels have near-normal [Ca(2+)](i) response to hypoxia. Thus, we postulated that TRP channels may provide an alternate calcium influx pathway which may be blocked by the TRP channel antagonist, 2-APB (2-aminoethoxydiphenylborane). We confirmed that 2-APB inhibited the afferent nerve response to hypoxia, as previously reported (Lahiri S, Patel G, Baby S, Roy A (2009) 2-APB mediated effects on hypoxic calcium influx in rat carotid body glomus cells. FASEB 2009, Abstract, LB157; Kumar P, Pearson S, Gu Y (2006) A role for TRP channels in carotid body chemotransduction? FASEB J 20:A12-29). To examine the mechanism for this inhibition, we examined dissociated rat CB glomus cells for [Ca(2+)](i) responses to hypoxia, anoxia (with sodium dithionite), 20 mM K(+), NaSH, NaCN, and FCCP in absence/presence of 2-APB (100 μM). Also the effect of 2-APB on hypoxia and/or anoxia were investigated on NADH and mitochondria (MT) membrane potential. Our findings are as follows: (1) 2-APB significantly blocked the [Ca(2+)](i) increase in response to hypoxia and anoxia, but not the responses to 20 mM K(+). (2) The [Ca(2+)](i) responses NaSH, NaCN, and FCCP were significantly blocked by 2-APB. (3) Hypoxia-induced increases in NADH/NAD(+) and MT membrane depolarization were not effected by 2-APB. Thus TRP channels may provide an important pathway for calcium influx in glomus cells in response to hypoxia.

The metabolic hypothesis of carotid body chemoreceptor hypoxia transduction proposes an impairment of ATP production as the signal for activation. We hypothesized that mitochondrial complex IV blockers and hypoxia would act synergistically in exciting afferent nerve activity. Following a pre-treatment with low dosage sodium cyanide (10-20μM), the hypoxia-induced nerve response was significantly reduced along with hypoxia-induced catecholamine release. However, in isolated glomus cells, the intracellular calcium response was enhanced as initially predicted. This suggests a cyanide-mediated impairment in the step between the glomus cell intracellular calcium rise and neurotransmitter release from secretory vesicles. Administration of a PKC blocker largely reversed the inhibitory actions of cyanide on the neural response. We conclude that the expected synergism between cyanide and hypoxia occurs at the level of glomus cell intracellular calcium but not at downstream steps due to a PKC-dependent inhibition of secretion. This suggests that at least one regulatory step beyond the glomus cell calcium response may modulate the magnitude of chemoreceptor responsiveness., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Chronic post-natal hyperoxia reduces the hypoxic ventilatory response by reducing the carotid body sensitivity to acute hypoxia as demonstrated by a reduced afferent nerve response, reduced calcium response of carotid body glomus cells and reduced catecholamine secretion in response to acute hypoxia. The present study examined whether hyperoxia alters the electrophysiological characteristics of glomus cells. Rats were treated with hyperoxia for 1 week starting at P1 or P7 and for 2 weeks starting at P1 followed by harvesting and dissociation of their carotid bodies for whole cell, perforated-patch recording. As compared to glomus cells from normoxia animals, hyperoxia treated cells showed a significant reduction in the magnitude of depolarization in response to hypoxia and anoxia, despite little change in the depolarizing response to 20 mM K(+). Resting cell membrane potential in glomus cells from rats exposed to hyperoxia from P1 to P15 and studied at P15 was slightly depolarized compared to other treatment groups and normoxia-treated cells, but conductance normalized to cell size was not different among groups. We conclude that postnatal hyperoxia impairs carotid chemoreceptor hypoxia transduction at a step between hypoxia sensing and membrane depolarization. This occurs without a major change in baseline electrophysiological characteristics, suggesting altered signaling or alterations in the relative abundance of different leak channel isoforms., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Carotid body chemoreceptors increase their action potential (AP) activity in response to a decrease in arterial oxygen tension and this response increases in the post-natal period. The initial transduction site is likely the glomus cell which responds to hypoxia with an increase in intracellular calcium and secretion of multiple neurotransmitters. Translation of this secretion to AP spiking levels is determined by the excitability of the afferent nerve terminals that is largely determined by the voltage-dependence of activation of Na(+) channels. In this review, we examine the biophysical characteristics of Na(+) channels present at the soma of chemoreceptor afferent neurons with the assumption that similar channels are present at nerve terminals. The voltage dependence of this current is consistent with a single Na(+) channel isoform with activation around the resting potential and with about 60-70% of channels in the inactive state around the resting potential. Channel openings, due to transitions from inactive/open or closed/open states, may serve to amplify external depolarizing events or generate, by themselves, APs. Over the first two post-natal weeks, the Na(+) channel activation voltage shifts to more negative potentials, thus enhancing the amplifying action of Na(+) channels on depolarization events and increasing membrane noise generated by channel transitions. This may be a significant contributor to maturation of chemoreceptor activity in the post-natal period., (Copyright © 2012 Elsevier B.V. All rights reserved.)

ATP, acting through P2X(2)/P2X(3) receptor-channel complexes, plays an important role in carotid body chemoexcitation in response to natural stimuli in the rat. Since the channels are permeable to calcium, P2X activation by ATP should induce changes in intracellular calcium ([Ca(2+)](i)). Here, we describe a novel ex vivo approach using fluorescence [Ca(2+)](i) imaging that allows screening of retrogradely labeled chemoafferent neurons in the petrosal ganglion of the rat. ATP-induced [Ca(2+)](i) responses were characterized at postnatal days (P) 5-8 and P19-25. While all labeled cells showed a brisk increase in [Ca(2+)](i) in response to depolarization by high KCl (60 mM), only a subpopulation exhibited [Ca(2+)](i) responses to ATP. ATP (250-1,000 μM) elicited one of three temporal response patterns: fast (R1), slow (R2), and intermediate (R3). At P5-8, R2 predominated and its magnitude was attenuated 44% by the P2X(1) antagonist, NF449 (10 μM), and 95% by the P2X(1)/P2X(3)/P2X(2/3) antagonist, TNP-ATP (10 μM). At P19-25, R1 and R3 predominated and their magnitudes were attenuated 15% by NF449, 66% by TNP-ATP, and 100% by suramin (100 μM), a nonspecific P2 purinergic receptor antagonist. P2X(1) and P2X(2) protein levels in the petrosal ganglion decreased with development, while P2X(3) protein levels did not change significantly. We conclude that the profile of ATP-induced P2X-mediated [Ca(2+)](i) responses changes in the postnatal period, corresponding with changes in receptor isoform expression. We speculate that these changes may participate in the postnatal maturation of chemosensitivity.

Previous work demonstrated that hyperoxia (30-60% O(2)) exposure in the post-natal period reduces the ventilatory response to acute hypoxia and this impairment may continue considerably beyond the period of hyperoxia exposure. Previous work from our laboratory demonstrated that 1-2 weeks of hyperoxia (60% O(2)) starting between P1 and P14: reduced the single chemoreceptor unit response to hypoxia, reduced the rise in glomus cell calcium caused by acute hypoxia and reduced hypoxia-induced catecholamine release (Donnelly 05, Donnelly 09). The present study asked whether the impairment extended to hypoxia-induced membrane depolarization, an earlier step in the transduction cascade. Perforated patch, whole-cell recordings were obtained from rat glomus cells exposed to hyperoxia from P0-P8 or P8-P15 and age-matched control groups. In both cases, hypoxia-induced membrane depolarization was significantly less in the hyperoxia treated groups compared to controls, while depolarization to 20 mM K(+) was not significantly affected. Resting membrane potential and input resistance were also not different in the hyperoxia treated groups. Whole carotid body quantitative real time PCR showed that TASK-1, TASK-3 and L-type Ca(2+) channel expression was significantly down-regulated at Hyper 8-15 compared to controls. We conclude that 1 week of postnatal hyperoxia during the early and late stage of CB maturation impairs organ function by affecting the coupling between hypoxia and glomus cell depolarization. This may be caused by altered expression of TASK1, TASK3 or L-type Ca(2+) channel gene expression. We speculate that an identification of cellular changes caused by hyperoxia may yield unique insights to the mechanism of oxygen sensing by the carotid bodies.

Carotid body (CB) glomus cells respond to hypoxia by releasing neurotransmitters, such as ATP, which are believed to stimulate excitatory receptors on apposed nerve endings of the carotid sinus nerves as well as bind to autoreceptors on the glomus cell membrane to modulate response magnitude. The CB response to hypoxia is small at birth and increases during postnatal maturation in mammals. As ATP has been shown to inhibit the glomus cell response to hypoxia via an autoreceptor mechanism, we hypothesized that ATP-mediated inhibition may vary with age and play a role in postnatal development of the hypoxia response magnitude. The effects of ATP on CB glomus cell intracellular calcium ([Ca(2+)](i)) responses to hypoxia were studied at two ages, P0-1 and P14-18. The inhibitory effect of ATP or a stable ATP analog on the glomus cell response to hypoxia was greater in newborn rats compared to the more mature age group. Use of selective P2Y receptor agonists and antagonists suggests that the inhibitory effect of ATP on the glomus cell [Ca(2+)](i) response to hypoxia may be mediated by a P2Y12 receptor. Thus, developmental changes in ATP-mediated glomus cell inhibition may play a role in carotid chemoreceptor postnatal maturation.

Chronic compression (CCD) of the dorsal root ganglion (DRG) is a model of human radicular pain produced by intraforaminal stenosis and other disorders affecting the DRG, spinal nerve, or root. Previously, we examined electrophysiological changes in small-diameter lumbar level 3 (L3) and L4 DRG neurons treated with CCD; the present study extends these observations to medium-sized DRG neurons, which mediate additional sensory modalities, both nociceptive and non-nociceptive. Whole-cell patch-clamp recordings were obtained from medium-sized somata in the intact DRG in vitro. Compared with neurons from unoperated control animals, CCD neurons exhibited a decrease in the current threshold for action potential generation. In the CCD group, current densities of TTX-resistant and TTX-sensitive Na(+) current were increased, whereas the density of delayed rectifier voltage-dependent K(+) current was decreased. No change was observed in the transient or "A" current after CCD. We conclude that CCD in the mouse produces hyperexcitability in medium-sized DRG neurons, and the hyperexcitability is associated with an increased density of Na(+) current and a decreased density of delayed rectifier voltage-dependent K(+) current.

Carotid bodies mediate hypoxia sensing for the respiratory system and increase their sensitivity in the post-natal period. The present study examined the characteristics and developmental change of fast Na(+) currents of chemoreceptor afferent neurons. Rat carotid bodies (P2-P19) were harvested intact with the petrosal ganglia and whole-cell recordings obtained from petrosal somas whose axons projected to the carotid body. The magnitude of Na(+) current increased in the post-natal period in parallel with increased conduction velocity and somal size. Voltage-dependence of activation significantly shifted towards negative potentials but no significant change occurred in the voltage dependence of inactivation or the slope factors for activation or inactivation. The leftward shift in activation increased slowly or non-inactivating currents around resting potential which increases afferent neuron excitability, a result confirmed in current clamp recordings. These results suggest that a developmental shift in Na(+) current activation plays a role in chemoreceptor maturation by enhancing excitability of the afferent neuron., (Copyright © 2011. Published by Elsevier B.V.)

A post-natal increase in carotid body (CB) hypoxia responsiveness occurs at the level of carotid sinus nerve activity, intracellular calcium, cell membrane depolarization and hypoxic inhibition of O(2)-sensitive background K(+) conductance. TASK-1, TASK-1/3 and TASK-3 are functionally expressed in CB glomus cells, with TASK-1/3 providing the major part of the O(2)-sensitive TASK-like background K(+) conductance. Here we report the effects of graded hypoxia on TASK-like channel activity in CB glomus cells from rats aged 0 to 1, 6 to 7 and 16 to 18 days; the time frame of postnatal CB functional maturation. TASK was active in nearly all cell-attached patches and TASK activity during normoxia did not differ across ages. Hypoxia produced a progressive decrease in channel opening frequency with graded decreases in O(2) level and also produced glomus cell depolarization, as assessed by the shift in reversal potential of TASK single channel current. Hypoxic inhibition of TASK activity was least at P0-P1 and increased with age mainly between 6-7 and 16-18 days. The O(2)-sensitive TASK activity was significantly greater in glomus cells from P16 to P18 when compared to cells from P0 to P1 day old rats. These results support the hypothesis that postnatal carotid body functional maturation is due, at least in part, to changes in the sensitivity of TASK to the hypoxic signals generated in glomus cells., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Carotid body glomus (CB) cells express different types of K(+) channels such as TASK, BK, and Kv channels, and hypoxia has been shown to inhibit these channels. Here we report the presence of a ∼72-pS channel that has not been described previously in CB cells. In cell-attached patches with 150 mM K(+) in the pipette and bath solutions, TASK-like channels were present (∼15 and ∼36-pS). After formation of inside-out patches, a 72-pS channel became transiently active in ∼18% of patches. The 72-pS channel was K(+)-selective, inhibited by 2-4 mM ATP and 10-100 μM glybenclamide. The 72-pS channel was observed in CB cells isolated from newborn, 2-3 week and 10-12 week-old rats. Reverse transcriptase-PCR and immunocytochemistry showed that Kir6.1, Kir6.2, SUR1 and SUR2 were expressed in CB glomus cells as well as in non-glomus cells. Acute hypoxia (∼15 mmHg O(2)) inhibited TASK-like channels but failed to activate the 72-pS channel in cell-attached CB cells. K(+) channel openers (diazoxide, pinacidil, levcromakalim), sodium cyanide and removal of extracellular glucose also did not activate the 72-pS channel in the cell-attached state. The hypoxia-induced elevation of intracellular [Ca(2+)] was unchanged by glybenclamide or diazoxide. NaCN-induced increase in [Ca(2+)] was not affected by 10 μM glybenclamide but inhibited by 100 μM glybenclamide. Acute glucose deprivation did not elevate [Ca(2+)] in the presence or absence of glybenclamide. These results show that an ATP-sensitive K(+) channel is expressed in the plasma membrane of CB cells, but is not activated by short-term metabolic inhibition. The functional relevance of the 72-pS channel remains to be determined., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We investigated the effects of chronic compression (CCD) of the L3 and L4 dorsal root ganglion (DRG) on pain behavior in the mouse and on the electrophysiological properties of the small-diameter neuronal cell bodies in the intact ganglion. CCD is a model of human radicular pain produced by intraforaminal stenosis and other disorders affecting the DRG, spinal nerve, or root. On days 1, 3, 5, and 7 after the onset of compression, there was a significant decrease from preoperative values in the threshold mechanical force required to elicit a withdrawal of the foot ipsilateral to the CCD (tactile allodynia). Whole cell patch-clamp recordings were obtained, in vitro, from small-sized somata and, for the first time, in the intact DRG. Under current clamp, CCD neurons exhibited a significantly lower rheobase compared with controls. A few CCD but no control neurons exhibited spontaneous action potentials. CCD neurons showed an increase in the density of TTX-resistant and TTX-sensitive Na(+) current. CCD neurons also exhibited an enhanced density of voltage-dependent K(+) current, due to an increase in delayed rectifier K(+) current, without a change in the transient or "A" current. We conclude that CCD in the mouse produces a model of radicular pain, as we have previously demonstrated in the rat. While the role of enhanced K(+) current remains to be elucidated, we speculate that it represents a compensatory neuronal response to reduce ectopic or aberrant levels of neuronal activity produced by the injury.

Carotid body chemoreceptors transduce a decrease in arterial oxygen tension into increased sinus nerve action potential (AP) activity which undergoes a maturational increase in the post-natal period. MaxiK-channels channels are proposed to play a major role in organ function based on their maturation-dependent expression in glomus cells and inhibition by acute hypoxia. To better resolve the role of this channel, single-unit AP activity of rat chemoreceptor neurons was recorded, in vitro, during a progressive decrease in oxygen from normoxia (∼150 Torr) to moderate hypoxia (∼60 Torr). Blockade of MaxiK channels with charybdotoxin (100 nM) in both older (P16-P18) and younger (P2-P3) animals resulted in no significant change in AP activity, but increased nerve conduction speed in the older animals. In dissociated glomus cells, charybdotoxin slightly enhanced the intracellular calcium response to acute hypoxia at both ages. We conclude that MaxiK channels play little or no role in mediating the response to acute, moderate hypoxia, either in the newborn or older animal., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Chronic postnatal hyperoxia blunts the hypoxic ventilatory response (HVR) in rats, an effect that persists for months after return to normoxia. To determine whether decreased carotid body O(2) sensitivity contributes to this lasting impairment, single-unit chemoafferent nerve and glomus cell calcium responses to hypoxia were recorded from rats reared in 60% O(2) through 7d of age (P7) and then returned to normoxia. Single-unit nerve responses were attenuated by P4 and remained low through P7. After return to normoxia, hypoxic responses were partially recovered within 3d and fully recovered within 7-8d (i.e., at P14-15). Glomus cell calcium responses recovered with a similar time course. Hyperoxia altered carotid body mRNA expression for O(2)-sensitive K(+) channels TASK-1, TASK-3, and BK(Ca), but only TASK-1 mRNA paralleled changes in chemosensitivity (i.e., downregulation by P7, partial recovery by P14). Collectively, these data do not support a role for reduced O(2) sensitivity of individual chemoreceptor cells in long-lasting reduction of the HVR after developmental hyperoxia., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Adenosine triphosphate, acting through purinergic P2X receptors, has been shown to stimulate ventilation and increase carotid body chemoreceptor activity in adult rats. However, its role during postnatal development of the ventilatory response to hypoxia is yet unknown. Using whole body plethysmography, we measured ventilation in normoxia and in moderate hypoxia (12% fraction of inspired O₂, 20 min) before and after intraperitoneal injection of suramin (P2X₂ and P2X₃ receptor antagonist, 40 mg/kg) in 4-, 7-, 12-, and 21-day-old rats. Suramin reduced baseline breathing (∼20%) and the response to hypoxia (∼30%) in all rats, with a relatively constant effect across ages. We then tested the effect of the specific P2X₃ antagonist, A-317491 (150 mg/kg), in rats aged 4, 7, and 21 days. As with suramin, A-317491 reduced baseline ventilation (∼55%) and the hypoxic response (∼40%) at all ages studied. Single-unit carotid body chemoreceptor activity was recorded in vitro in 4-, 7-, and 21-day-old rats. Suramin (100 μM) and A-317491 (10 μM) significantly depressed the sinus nerve chemosensory discharge rate (∼80%) in normoxia (Po₂ ∼150 Torr) and hypoxia (Po₂ ∼60 Torr), and this decrease was constant across ages. We conclude that, in newborn rats, P2X purinergic receptors are involved in the regulation of breathing under basal and hypoxic condition, and P2X₃-containing receptors play a major role in carotid body function. However, these effects are not age dependent within the age range studied.

In vivo electrophysiological recordings from cell bodies of primary sensory neurons are used to determine sensory function but are commonly performed blindly and without access to voltage- (patch-clamp) electrophysiology or optical imaging. We present a procedure to visualize and patch-clamp the neuronal cell body in the dorsal root ganglion, in vivo, manipulate its chemical environment, determine its receptive field properties, and remove it either to obtain subsequent molecular analyses or to gain access to deeper lying cells. This method allows the association of the peripheral transduction capacities of a sensory neuron with the biophysical and chemical characteristics of its cell body., ((c) 2010 Elsevier B.V. All rights reserved.)

Carotid body chemoreceptors transduce a decrease in arterial oxygen tension into increased action potential (AP) activity on the sinus nerve, which increases the drive to breathe. The mechanism by which AP activity increases is unresolved, but acetylcholine (ACh), acting through nicotinic receptors, is postulated to be a major contributor to nerve excitation based partly on the demonstration that pharmacological antagonism of nicotinic receptors reduces the afferent nerve response in some studies. However, most previous studies relied on indirect measures of chemoreceptor activity or utilized a recording configuration that is sensitive to AP morphology in addition to AP frequency. In the present study, single-unit AP activity was recorded from the soma of rat chemoreceptor neurons in vitro. The nicotinic blocker mecamylamine (50 microM) ablated the excitatory actions of exogenous ACh and increased, rather than decreased, AP activity during moderate hypoxia. At higher dosage (500 microM) AP height was reduced, conduction velocity slowed, and conduction failure occurred, especially during hypoxia, producing the appearance of a decreased response to hypoxia. Recovery from mecamylamine block was slow (>10 min). In contrast to mecamylamine, suramin, a P2X receptor blocker, reversibly inhibited the response to hypoxia, suggesting relatively free diffusion of drugs to the glomus cell/nerve synaptic site. These results strongly suggest that ACh acting through nicotinic receptors does not mediate excitatory transmission in rat carotid body and that previous results demonstrating such a role may have been partially influenced by changes in AP morphology or conduction failure.

The cell bodies of sensory neurons in the dorsal root ganglion (DRG) are enveloped by satellite glial cells (SGCs). In an animal model of intervertebral foraminal stenosis and low-back pain, a chronic compression of the DRG (CCD) increases the excitability of neuronal cell bodies in the compressed ganglion. The morphological and electrophysiological properties of SGCs were investigated in both CCD and uninjured, control lumbar DRGs. SGCs responded within 12 h of the onset of CCD as indicated by an increased expression of glial fibrillary acidic protein (GFAP) in the compressed DRG but to lesser extent in neighboring or contralateral DRGs. Within 1 week, coupling through gap junctions between SGCs was significantly enhanced in the compressed ganglion. Under whole-cell patch clamp recordings, inward and outward potassium currents, but not sodium currents, were detected in individual SGCs. SGCs enveloping differently sized neurons had similar electrophysiological properties. SGCs in the compressed vs. control DRG exhibited significantly reduced inwardly rectifying potassium currents (Kir), increased input resistances and positively shifted resting membrane potentials. The reduction in Kir was greater for nociceptive medium-sized neurons compared to non-nociceptive neurons. Kir currents of SGCs around spontaneously active neurons were significantly reduced 1 day after compression but recovered by 7 days. These data demonstrate rapid alterations in glial membrane currents and GFAP expression in close temporal association with the development of neuronal hyperexcitability in the CCD model of neuropathic pain. However, these alterations are not fully sustained and suggest other mechanisms for the maintenance of the hyperexcitable state., ((c) 2009 Wiley-Liss, Inc.)

Postnatal hyperoxia exposure reduces the carotid body response to acute hypoxia and produces a long-lasting impairment of the ventilatory response to hypoxia. The present work investigated the time course of pre- and post-synaptic alterations following exposure to hyperoxia (Fl(O2) = 0.6) for 1, 3, 5, 8 and 14 days (d) starting at postnatal day 7 (P7) as compared to age-matched controls. Hyperoxia exposure for 1d enhanced the nerve response and glomus cell calcium response to acute hypoxia, but exposure for 3-5d caused a significant reduction in both. Hypoxia-induced catecholamine release and nerve conduction velocity were significantly decreased by 5d hyperoxia. We conclude that hyperoxia exerts pre-synaptic (glomus cell calcium and secretory responses) and post-synaptic (afferent nerve excitability) actions to initially enhance and then reduce the chemoreceptor response to acute hypoxia. The parallel changes in glomus cell calcium response and nerve response suggest causality between the two and that environmental hyperoxia can affect the coupling between acute hypoxia and glomus cell calcium regulation.

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.

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.

Peripheral arterial chemoreceptors, particularly the carotid body chemoreceptors, are the primary sites for the detection of hypoxia and reflexly increase ventilatory drive and behavioral arousal during hypoxic or asphyxial events. Newborn infants are at risk for hypoxic and asphyxial events during sleep, yet, the strength of the chemoreceptor responses is low or absent at birth and then progressively increases with early postnatal development. This review summarizes the available data showing that even though the "oxygen sensor" in the glomus cells has not been unequivocally identified, it is clear that development affects many of the other properties of the chemoreceptor unit (glomus cell, afferent nerve fibers and neurotransmitter profile at the synapse) that are necessary and essential for the propagation of the "sensing" response, and exposure to hypoxia, hyperoxia and nicotine can modify normal development of each of the components leading to altered peripheral chemoreceptor responses.

The mechanism by which action potentials (APs) are generated in afferent nerve fibers in the carotid body is unknown, but it is generally speculated to be release of an excitatory transmitter and synaptic depolarizing events. However, previous results suggested that Na(+) channels in the afferent nerve fibers play an important role in this process. To better understand the potential mechanism by which Na(+) channels may generate APs, a mathematical model of chemoreceptor nerve fibers that incorporated Hodgkin-Huxley-type Na(+) channels with kinetics of activation and inactivation, as determined previously from recordings of petrosal chemoreceptor neurons, was constructed. While the density of Na(+) channels was kept constant, spontaneous APs arose in nerve terminals as the axonal diameter was reduced to that in rat carotid body. AP excitability and pattern were similar to those observed in chemoreceptor recordings: 1) a random pattern at low- and high-frequency discharge rates, 2) a high sensitivity to reductions in extracellular Na(+) concentration, and 3) a variation in excitability that increased with AP generation rate. Taken together, the results suggest that an endogenous process in chemoreceptor nerve terminals may underlie AP generation, a process independent of synaptic depolarizing events.

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.

Carotid body chemoreceptors respond to a decrease in arterial partial pressure of O(2) with an increase in sinus nerve action potential (AP) activity which initiates a number of protective reflexes. The spike generation process is unresolved but is generally considered to be caused by a synaptic depolarizing potential (SDP) in the nerve endings caused by release of an excitatory transmitter from the glomus cell, which is a secretory cell that is presynaptic to the nerve terminals. To detect the purported SDPs, stimulating electrodes were placed at sites within the carotid body from which orthodromic APs could be evoked at low threshold currents. The probability of AP generation as a function of stimulus current was fitted well to a Boltzmann distribution. Subthreshold electrical stimuli which were expected to summate with subthreshold SDPs, failed, in all instances, to evoke APs at the expected probability. When the stimulus was gated to the occurrence of a spontaneous AP, no change in electrical threshold was observed as the delay between the spontaneous AP and electrical stimulus was increased, despite the presumed disappearance of an SDP in the post-AP period. Decreases in spontaneous AP generation rate, caused by hyperoxia, were associated with only slight changes in the mean orthodromic stimulus threshold, but with a significant increase in slope of the Boltzmann function, suggesting a decrease in the variance of nerve terminal excitability during hyperoxia. These results suggest that AP generation is not due to SDP events; rather, AP generation is likely to be due to a process endogenous to the nerve terminals that modulates the variability of nerve terminal excitability.

Central neurons are extremely vulnerable to hypoxic/ischemic insult, which is a major cause of neurologic morbidity and mortality as a consequence of neuronal dysfunction and death. Our recent work has shown that delta-opioid receptor (DOR) is neuroprotective against hypoxic and excitotoxic stress, although the underlying mechanisms remain unclear. Because hypoxia/ischemia disrupts ionic homeostasis with an increase in extracellular K(+), which plays a role in neuronal death, we asked whether DOR activation preserves K(+) homeostasis during hypoxic/ischemic stress. To test this hypothesis, extracellular recordings with K(+)-sensitive microelectrodes were performed in mouse cortical slices under anoxia or oxygen-glucose deprivation (OGD). The main findings in this study are that (1) DOR activation with [D-Ala(2), D-Leu(5)]-enkephalinamide attenuated the anoxia- and OGD-induced increase in extracellular K(+) and decrease in DC potential in cortical slices; (2) DOR inhibition with naltrindole, a DOR antagonist, completely abolished the DOR-mediated prevention of increase in extracellular K(+) and decrease in DC potential; (3) inhibition of protein kinase A (PKA) with N-(2-[p-bromocinnamylamino]-ethyl)-5-isoquinolinesulfonamide dihydrochloride had no effect on the DOR protection; and (4) inhibition of protein kinase C (PKC) with chelerythrine chloride reduced the DOR protection, whereas the PKC activator (phorbol 12-myristate 13-acetate) mimicked the effect of DOR activation on K(+) homeostasis. These data suggest that activation of DOR protects the cortex against anoxia- or ODG-induced derangement of potassium homeostasis, and this protection occurs via a PKC-dependent and PKA-independent pathway. We conclude that an important aspect of DOR-mediated neuroprotection is its early action against derangement of K(+) homeostasis during anoxia or ischemia.

Amiodarone, lamotrigine, and phenytoin, common antiarrhythmic and antiepileptic drugs, inhibit a persistent sodium current in neurons (I(NaP)). Previous results from our laboratory suggested that I(NaP) is critical for functionality of peripheral chemoreceptors. In this study, we determined the effects of therapeutic levels of amiodarone, lamotrigine, and phenytoin on peripheral chemoreceptor and ventilatory responses to hypoxia. Action potentials (APs) of single chemoreceptor afferents were recorded using suction electrodes advanced into the petrosal ganglion of an in vitro rat peripheral chemoreceptor complex. AP frequency (at Po(2) approximately 150 Torr and Po(2) approximately 90 Torr), conduction time, duration, and amplitude were measured before and during perfusion with therapeutic dosages of the drug or vehicle. Hypoxia-induced catecholamine secretion within the carotid body was measured using amperometry. With the use of whole body plethysmography, respiration was measured in unanesthesized rats while breathing room air, 12% O(2), and 5% CO(2), before and after intraperitoneal administration of amiodarone, lamotrigine, phenytoin, or vehicle. Lamotrigine (10 microM) and phenytoin (5 microM), but not amiodarone (5 microM), decreased chemoreceptor AP frequency without affecting other AP parameters or magnitude of catecholamine secretion. Similarly, lamotrigine (5 mg/kg) and phenytoin (10 mg/kg) blunted the hypoxic but not the hypercapnic ventilatory response. In contrast, amiodarone (2.5 mg/kg) did not alter the ventilatory response to hypoxia or hypercapnia. We conclude that lamotrigine and phenytoin at therapeutic levels impair peripheral chemoreceptor function and ventilatory response to acute hypoxia. These are consistent with I(NaP) serving an important function in AP generation and may be clinically important in the care of patients using these drugs.

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.

Systemic hypoxia in mammals is sensed and transduced by the carotid body into increased action potential (AP) frequency on the sinus nerve, resulting in increased ventilation. The mechanism of hypoxia transduction is not resolved, but previous work suggested that fast Na(+) channels play an important role in determining the rate and timing of APs (Donnelly, DF, Panisello JM, and Boggs D. J Physiol. 511: 301-311, 1998). We speculated that Na(+) channel activity between APs, termed persistent Na(+) current (I(NaP)), is responsible for AP generation that and riluzole and phenytoin, which inhibit this current, would impair organ function. Using whole cell patch clamp recording of intact petrosal neurons with projections to the carotid body, we demonstrated that I(NaP) is present in chemoreceptor afferent neurons and is inhibited by riluzole. Furthermore, discharge frequencies of single-unit, chemoreceptor activity, in vitro, during normoxia (Po(2) 150 Torr) and during acute hypoxia (Po(2) 90 Torr) were significantly reduced by riluzole concentrations at or above 5 microM, and by phenytoin at 100 microM, without significant affect on nerve conduction time, AP magnitude (inferred from extracellular field), and AP duration. The effect of both drugs appeared solely postsynaptic because hypoxia-induced catecholamine release in the carotid body was not altered by either drug. The respiratory response of unanesthetized, unrestrained 2-wk-old rats to acute hypoxia (12% inspired O(2) fraction), which was measured with whole body plethysmography, was significantly reduced after treatment with riluzole (2 mg/kg ip) and phenytoin (20 mg/kg ip). We conclude that I(NaP) is present in chemoreceptor afferent neurons and serves an important role in peripheral chemoreceptor function and, hence, in the ventilatory response to hypoxia.

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.

Carotid body chemoreceptors undergo significant maturational changes in the post-natal period over a period of days to weeks. This is likely initiated by the rise in Pa(O2) at the time of birth and reflects the changing value of "normoxia" from 25 Torr to near 100 Torr. Chemoreceptors in the newborn period have a lower absolute discharge frequency and the dynamic response to acute hypoxia is less compared to the adult. This maturation change appears due to changes occurring presynaptically to the afferent nerve fibers. Hypoxia-induced secretion from the glomus cell (catecholamine and other constituents of dense cored vesicles) is enhanced whilst constitutive (non-hypoxia-dependent) release is reduced with age. On the post-synaptic side, the number of afferent synaptic sites increases four- to five-fold in the post-natal period and there may be an increase in afferent nerve excitability. Both of these changes are subject to environmental perturbations in which post-natal exposure to chronic hypoxia or hyperoxia leads to significantly reduced organ sensitivity and function. Thus, developmental changes and environmental factors may significantly change the ability of an animal to detect and respond to hypoxic insults, perhaps leading to periods of heightened vulnerability to hypoxic stresses.

Hyperoxia in the immediate perinatal period, but not in adult life, is associated with a life-long impairment of the ventilatory response to acute hypoxia. This effect is attributed to a functional impairment of peripheral chemoreceptors, including a reduction in the number of chemoreceptor afferent fibers and a reduction in "whole nerve" afferent activity. The purpose of the present study was to assess the activity levels of single chemoreceptor units in the immediate posthyperoxic period to determine whether functional impairment extended to single chemoreceptor units and whether the impairment was only induced by hyperoxia exposure in the immediate postnatal period. Two groups of rat pups were exposed to 60% inspired O2 fraction for 2 wk at ages 0-14 days and 14-28 days, at which time single-unit activities were isolated and recorded in vitro. Compared with control pups, hyperoxia-treated pups had a 10-fold reduction in baseline (normoxia) spiking activity. Peak unit responses to 12, 5, and 0% O2 were reduced and nerve conduction time was significantly slower in both hyperoxia-treated groups compared with control groups. We conclude that 1) hyperoxia greatly reduces single-unit chemoreceptor activities during normoxia and acute hypoxia, 2) the treatment effect is not limited to the immediate newborn period, and 3) at least part of the impairment may be due to changes in the afferent axonal excitability.

Carotid body chemoreceptors respond to a decrease in arterial oxygen tension by increasing spiking activity on the sinus nerve. Our understanding of the oxygen-transducing ability of the organ arose from studies in the 1930s intended to understand how metabolic poisons stimulated breathing. Since that time, an intimate link between energy state and hypoxia sensing has been assumed and forms the basis of the metabolic hypothesis of oxygen sensing. This hypothesis is supported by studies demonstrating a loss of mitochondrial potential in carotid body cells at oxygen tensions that cause no change in cells from other tissues. Although the nature of the coupling between mitochondrial function and nerve excitation remains unresolved, experimental evidence supports roles for (1) release of mitochondrial calcium stores, (2) modulation of membrane channels that are linked to mitochondrial complexes I and IV, and (3) generation of signaling intermediates, such as reactive oxygen species (ROS) from complex I and III of the electron transport chain. If the mitochondrion is the oxygen-sensing site for peripheral chemoreceptors, then there exists the potential ability to manipulate, perhaps pharmacologically, the sensing function by alterations in expression of uncoupler proteins or chemicals that can alter the affinity of cytochrome oxidase for oxygen. Such manipulation may be useful for the treatment of hypoventilation syndromes or high altitude accommodation.

A chronic compression of the DRG (CCD) produces cutaneous hyperalgesia and an enhanced excitability of neuronal somata in the compressed ganglion. The hyperpolarization-activated current (I(h)), present in the somata and axons of DRG neurons, acts to induce a depolarization after a hyperpolarizing event and, if upregulated after CCD, may contribute to enhanced neuronal excitability. Whole-cell patch-clamp recordings were obtained from acutely dissociated, retrogradely labeled, cutaneous, adult rat DRG neurons of medium size. Neurons were dissociated from L4 and L5 control DRGs or DRGs that had each been compressed for 5-7 d by L-shaped rods inserted into the intervertebral foramina. I(h), consisting of a slowly activating inward current during a step hyperpolarization, was recorded from every labeled, medium-sized neuron and was blocked by 1 mm cesium or 15 microm ZD7288. Compared with control, CCD increased the current density and rate of activation significantly without changing its reversal potential, voltage dependence of activation, or rate of deactivation. Because I(h) activation provides a depolarizing current to the neuron, thus enhancing neuronal excitability, our results are consistent with the hypothesis that I(h) contributes to hyperalgesia after CCD-induced nerve injury.

Substance P has been proposed to be an important neurotransmitter in the carotid body with the neurokinin 1 (NK1) receptor, mediating excitation between the glomus cells and afferent nerve endings. In order to better understand the role of substance P, this study examined chemoreceptor afferent activity, in vitro, and tissue catecholamine levels and release in adult, wild-type mice and mice lacking the gene for the NK1 receptor (NK1-KO). Groups did not differ significantly in body weight, carotid body dopamine content or carotid body norepinephrine content. In wild-type mice, single unit activity increased from 0.59 +/- 0.14 Hz to 19.78 +/- 2.27 Hz during superfusion with strong hypoxia (PO2 approximately 25 Torr). Chemoreceptor activity in NK1-KO mice, increased from 0.71 +/- 0.23 to 21.50 +/- 3.62 Hz, and neither baseline or peak frequencies were significantly different from the wild-type group. Less severe hypoxia (PO2 approximately 45 torr), evoked peak activities of 12.50 +/- 1.88 and 10.64 +/- 3.72 Hz in wild-type and NK1-KO mice, which were also not significantly different. In response to severe hypoxia, free-tissue catecholamine increased to 4.92 +/- 0.85 microm in wild-type mice and 4.26 +/- 0.63 microm in NK1-KO mice, which were also not significantly different. It may therefore be concluded that loss of NK1 receptors has little effect on chemoreceptor function in the mouse, and thus they play, at best, a minor role in the hypoxic chemoreception process.

Carotid body chemoreceptors transduce a decrease in arterial oxygen tension into an increase in spiking activity on the sinus nerve, and this response increases with postnatal age over the first week or two of life. Previous work from our laboratory has suggested a major role of axonal Na(+) channels in the initiation of afferent spiking activity. Using RT-PCR of the petrosal ganglia we identified Na(+) channel TTX-S isoforms Na(v)1.1, Na(v)1.6, and Na(v)1.7 and the TTX-resistant (TTX-R) isoforms Na(v)1.8 and Na(v)1.9 at high levels. Electrophysiologic recordings (at 3 ages: 3 days, 9 days, 18-20 days) of neurons that project to the carotid body exhibited predominantly fast-inactivating sodium currents, with a bimodal recovery from inactivation at -80 mV (fast component approximately 8 ms; slow component approximately 90 ms). Developmental age had little effect with no change in peak current density (approximately 1.4 nA/pF) and was associated with a slight, but significant increase in the speed of recovery from inactivation at -140 and -120 mV but not at other potentials. Assuming that the same Na(+) channel complement is present at the nerve terminal as at the soma, the association of a sensory modality (chemoreception) with a relatively uniform Na(+) channel profile suggests that the rapid kinetics of TTX-S channels may be essential for some aspects of chemoreceptor function beyond mediating simple axonal conduction.

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.

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.

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.

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.

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.

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.

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.

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.