David F. Donnelly

Maturation of carotid chemoreceptor sensitivity to hypoxia: in vitro studies in the newborn rat.

1. A preparation was developed to record single‐fibre chemoreceptor afferent activity from carotid bodies of newborn and adult rats in vitro. The response to severe hypoxia was studied as a function of developmental age in four age groups: 1‐2, 4‐7, 10‐15 days and adult (25‐30 days). 2. During superfusion with HEPES‐saline at room PO2 and at 26 or 35 degrees C, afferent chemoreceptor activity could be recorded in all age groups. No significant difference was found among groups in baseline discharge frequency at 26 or 35 degrees C. 3. All chemoreceptors responded to anoxia (PO2 congruent to 0 Torr) with a rapid increase in discharge frequency. At 35 degrees C, peak discharge frequency of single chemoreceptor afferents was significantly greater in the adult (15.7 +/‐ 1.6 Hz, mean +/‐ S.E.M., n = 18) and 10‐15 days (11.2 +/‐ 4.2, n = 8) compared to rats of 1‐2 days (4.3 +/‐ 0.7, n = 14) and 4‐7 days of age (3.9 +/‐ 1.0, n = 7). 4. At 2 min into the anoxia period, all chemoreceptor activities were reduced from their peak discharge levels. At 35 degrees C, this decrease was significantly greater in the adult compared to the newborn. 5. During the period of decreased activity during anoxia, the chemoreceptor discharge could not be increased by inter‐stream injection of acetylcholine (100 micrograms) or dopamine (100 micrograms), although these drugs were effective in increasing discharge rate prior to hypoxia. 6. We conclude that: (1) postnatal maturation of chemoreceptor sensitivity to hypoxia is present in vitro, (2) maturation occurs between the first and second week after birth in the rat, and (3) the decrease in activity during prolonged anoxia is not greater in the newborn compared to the adult. Thus, maturational changes occur in the sensitivity of the glomus cell‐nerve ending complex to hypoxia.

Developmental changes in hypoxia-induced catecholamine release from rat carotid body, in vitro.

1. Developmental changes in free tissue catecholamine levels were studied using Nafion‐coated, carbon fibre electrodes placed in rat carotid bodies, in vitro. Simultaneously, single fibre chemoreceptor afferent activity was recorded from the sinus nerve. Five age groups were examined: 1, 2, 6, 10 and 20‐30 days of age. 2. Using fast‐scan voltammetry, similar current peaks were observed during exposure to exogenous dopamine and during superfusion with hypoxic saline. This suggests that changes in carbon fibre electrode current are due to an increase in free tissue catecholamines. 3. Baseline catecholamine levels were significantly less in the 1‐6 day age groups compared to 10 day and 20‐30 day rats. 4. During 1 min of hypoxia the peak concentration of tissue catecholamine was significantly less in the 1 day compared to the 2 day age groups, and these were less than in 10 day and 20‐30 day rats. 5. Peak nerve response during hypoxia increased with age from 4.5 +/‐ 0.6 Hz in the 1 day to 10.5 +/‐ 1.6 Hz in the 6 day and to 15.5 +/‐ 2.2 Hz in the 20‐30 day rats. 6. We conclude that (1) resting free tissue catecholamine levels increase with age in the newborn period, (2) hypoxia causes enhanced catecholamine release, and (3) the magnitude of the release increases in the postnatal period as does the nerve activity.

Altered functional properties of satellite glial cells in compressed spinal ganglia

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.

Hypoglossal motoneuron responses to pulmonary and superior laryngeal afferent inputs

In decerebrate, paralyzed cats ventilated with a cycle-triggered pump, the inspiratory discharges of the hypoglossal (whole nerve or single fibers), phrenic, and recurrent laryngeal nerves were compared, and the effects of pulmonary and superior laryngeal afferent inputs were observed. During lung inflations in phase with neural inspiration, hypoglossal and recurrent laryngeal activities differed from phrenic with respect to (a) burst onset times: both preceded the phrenic; (b) overall pattern: phrenic, augmenting; hypoglossal, decrementing; recurrent laryngeal, plateau-like. When inflation was withheld, the phrenic pattern was not markedly changed, but both hypoglossal and recurrent laryngeal became augmenting; the marked increase of hypoglossal activity (both whole nerve and single fiber) indicated strong inhibition by lung afferents. Superior laryngeal electrical stimulation evoked excitation of the contralateral phrenic (latency 4.1 msec) and the ipsilateral whole hypoglossal (latency 5.3 msec), followed by bilateral inhibitions (durations 20-30 msec); most hypoglossal fibers showed only inhibition. We conclude that, although both hypoglossal and phrenic outputs are driven by the inspiratory pattern generator(s), their promotor systems differ with respect to influences from central and peripheral inputs.

Cortical δ-opioid receptors potentiate K+ homeostasis during anoxia and oxygen-glucose deprivation

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 δ-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-Ala2, D-Leu5]-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.

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.

Modulation of glomus cell membrane currents of intact rat carotid body.

1. In order to understand better the relationship between sinus nerve chemoreceptor activity and changes in glomus cell membrane current, both were measured simultaneously in rat carotid bodies in vitro. Mean membrane resistance of intact glomus cells was 1327 +/‐ 140 M omega (n = 104, mean +/‐ S.E.M.) and membrane capacitance was 7.9 +/‐ 0.8 pF (n = 28). 2. Over the course of 15 min following the start of whole‐cell recording, outward current increased by 169 +/‐ 48% (n = 19), but there was no significant change in holding current or membrane resistance. Reversal potential of the tail current was not changed over this time period. Current run‐up was not affected by addition of ATP, Ca2+, okadaic acid or H‐7 to the pipette fluid. 3. Brief hypoxia (30‐45 s duration, 0 mmHg at nadir) caused a rapid increase in nerve activity, but, on average, no significant change in cell holding current, or resistance. Outward current slightly decreased during hypoxia but failed to recover in the post‐hypoxia period. 4. Tetraethylammonium (20 mM), and 4‐aminopyridine (1 mM) reduced the outward current to 54 +/‐ 7 and 66 +/‐ 3% of control, respectively, but basal nerve activity was unchanged and the nerve response to hypoxia remained intact. 5. These results suggest that hypoxia modulation of glomus cell K+ current is not the primary initiating factor in the nerve response to brief periods of hypoxia in the rat carotid body.

Time course of alterations in pre- and post-synaptic chemoreceptor function during developmental hyperoxia

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.

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.

Ventilatory and carotid body chemoreceptor responses to purinergic P2X receptor antagonists in newborn rats

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.