"Turning up the heat": role of neurotrophic batokines in the postnatal maturation and remodeling of brown adipose tissue in deer mice.

Activation of brown adipose tissue (BAT) thermogenesis impacts energy balance and must be tightly regulated. Several neurotrophic factors, expressed in BAT of adult laboratory rodents, have been implicated in remodeling the sympathetic neural network to enhance thermogenesis [e.g., nerve growth factor (NGF), neuregulin-4 (NRG4), and S100b]. Here, we compare, to our knowledge, for the first time, the relative roles of three neurotrophic "batokines" in establishing/remodeling innervation during postnatal development and adult cold stress. We used laboratory-reared Peromyscus maniculatus, which rely heavily on BAT-based thermogenesis for survival in the wild, beginning between postnatal days (P) 8 and 10. BAT sympathetic innervation was enhanced from P6 to P10, and exogenous NGF, NRG4, and S100b stimulated neurite outgrowth from P6 sympathetic neurons. Endogenous BAT protein stores and/or gene expression of NRG4, S100b, and calsyntenin-3ß (which may regulate S100b secretion) remained high and constant during development. However, endogenous NGF was low and ngf mRNA was undetectable. Conditioned media (CM) from cultured P10 BAT slices stimulated neurite outgrowth from sympathetic neurons in vitro, which was inhibited by antibodies against all three growth factors. P10 CM had significant amounts of secreted NRG4 and S100b protein, but not NGF. By contrast, BAT slices from cold-acclimated adults released significant amounts of all three factors relative to thermoneutral controls. These data suggest that although neurotrophic batokines regulate sympathetic innervation in vivo, their relative contributions differ depending on the life stage. They also provide novel insights into the regulation of BAT remodeling and BAT's secretory role, both of which are critical to our understanding of mammalian energy homeostasis.NEW & NOTEWORTHY In altricial Peromyscus mice, the developmental shift to endothermy accompanies the establishment of the brown adipose tissue sympathetic neural network. Cultured slices of neonatal BAT secreted high quantities of two predicted neurotrophic batokines: S100b and neuregulin-4, but surprisingly low levels of the classic neurotrophic factor, NGF. Despite low NGF, neonatal BAT-conditioned media was highly neurotrophic. Cold-exposed adults use all three factors to dramatically remodel BAT, suggesting that BAT-neuron communication is life-stage dependent.

Tibial bone forces can be monitored using shoe-worn wearable sensors during running.

Tibial bone stress injury is a common overuse injury experienced by runners, which results from repetitive tissue forces. Wearable sensor systems (wearables) that monitor tibial forces could help understand and reduce injury incidence. However, there are currently no validated wearables that monitor tibial bone forces. Previous work using simulated wearables demonstrated accurate tibial force estimates by combining a shoe-worn inertial measurement unit (IMU) and pressure insole with a trained algorithm. This study aimed assessed how accurately tibial bone forces could be estimated with existing wearables. Nine recreational runners ran at a series of different speeds and slopes, and with various stride patterns. Shoe-worn IMU and insole data were input into a trained algorithm to estimate peak tibial force. We found an average error of 5.7% in peak tibial force estimates compared with lab-based estimates calculated using motion capture and a force instrumented treadmill. Insole calibration procedures were essential to achieving accurate tibial force estimates. We concluded that a shoe-worn, multi-sensor system is a promising approach to monitoring tibial bone forces in running. This study adds to the literature demonstrating the potential of wearables to monitor musculoskeletal forces, which could positively impact injury prevention, and scientific understanding.

Pulmonary neuroepithelial bodies are polymodal airway sensors: evidence for CO2/H+ sensing.

Pulmonary neuroepithelial bodies (NEB) in mammalian lungs are thought to function as airway O2 sensors that release serotonin (5-HT) in response to hypoxia. Direct evidence that NEB cells also respond to airway hypercapnia/acidosis (CO2/H(+)) is presently lacking. We tested the effects of CO2/H(+) alone or in combination with hypoxia on 5-HT release from intact NEB cells in a neonatal hamster lung slice model. For the detection of 5-HT release we used carbon fiber amperometry. Fluorescence Ca(2+) imaging method was used to assess CO2/H(+)-evoked changes in intracellular Ca(2+). Exposure to 10 and 20% CO2 or pH 6.8-7.2 evoked significant release of 5-HT with a distinct rise in intracellular Ca(2+) in hamster NEBs. This secretory response was dependent on the voltage-gated entry of extracellular Ca(2+). Moreover, the combined effects of hypercapnia and hypoxia were additive. Critically, an inhibitor of carbonic anhydrase (CA), acetazolamide, suppressed CO2/H(+)-mediated 5-HT release. The expression of mRNAs for various CA isotypes, including CAII, was identified in NEB cells from human lung, and protein expression was confirmed by immunohistochemistry using a specific anti-CAII antibody on sections of human and hamster lung. Taken together our findings provide strong evidence for CO2/H(+) sensing by NEB cells and support their role as polymodal airway sensors with as yet to be defined functions under normal and disease conditions.

Potential roles of ATP and local neurons in the monitoring of blood O2 content by rat aortic bodies.

Aortic bodies are arterial chemoreceptors presumed to monitor blood O2 content by unknown mechanisms, in contrast to their well-studied carotid body counterparts, which monitor PO2 and /pH. We recently showed that rat aortic body chemoreceptors (type I cells), located at the left vagus-recurrent laryngeal nerve bifurcation, responded to PO2 and PCO2 /pH in a manner similar to carotid body type I cells. These aortic bodies are uniquely associated with a group of local neurons, which are also sensitive to these stimuli. Here, we hypothesized that these local neurons may contribute to monitoring blood O2 content. During perforated patch recordings, ATP, known to be released from (carotid body) type I cells and red blood cells during hypoxia, induced inward currents and excited ≈ 45% of local neurons (EC50 ≈ 1 µm), mainly via heteromeric P2X2/3 purinoceptors. While ATP also induced a rise in intracellular [Ca(2+)] in a subpopulation of these neurons, almost all of them responded to nicotinic cholinergic agonists. During paired recordings, several juxtaposed neurons showed strong bidirectional electrical coupling, suggesting a local co-ordination of electrical activity. Perfusion with Evans Blue dye resulted in labelling of aortic body paraganglia, suggesting they have ready access to circulatory factors, e.g. ATP released from red blood cells during hypoxia. When combined with confocal immunofluorescence, the dye-labelled regions coincided with areas containing tyrosine hydroxylase-positive type I cell clusters and P2X2-positive nerve endings. We propose a working model whereby local neurons, red blood cells, ATP signalling and low blood flow contribute to the unique ability of the aortic body to monitor blood O2 content.

Low glucose sensitivity and polymodal chemosensing in neonatal rat adrenomedullary chromaffin cells.

Glucose is the primary metabolic fuel in mammalian fetuses, yet mammals are incapable of endogenous glucose production until several hours after birth. Thus, when the maternal supply of glucose ceases at birth there is a transient hypoglycemia that elicits a counterregulatory surge in circulating catecholamines. Because the innervation of adrenomedullary chromaffin cells (AMCs) is immature at birth, we hypothesized that neonatal AMCs act as direct glucosensors, a property that could complement their previously established roles as hypoxia and acid hypercapnia sensors. During perforated-patch, whole cell recordings, low glucose depolarized and/or excited a subpopulation of neonatal AMCs; in addition, aglycemia (0 mM glucose) caused inhibition of outward K(+) current, blunted by the simultaneous activation of glibenclamide-sensitive K(ATP) channels. Some cells were excited by each of the three metabolic stimuli, i.e., aglycemia, hypoxia (Po(2) ∼30 mmHg), and isohydric hypercapnia (10% CO(2); pH = 7.4). Using carbon fiber amperometry, aglycemia and hypoglycemia (3 mM glucose) induced robust catecholamine secretion that was sensitive to nickel (50 µM and 2 mM) and the L-type Ca(2+) channel blocker nifedipine (10 µM), suggesting involvement of both T-type and L-type voltage-gated Ca(2+) channels. Fura-2 measurements of intracellular Ca(2+) ([Ca(2+)] (i)) revealed that ∼42% of neonatal AMCs responded to aglycemia with a significant rise in [Ca(2+)] (i). Approximately 40% of these cells responded to hypoxia, whereas ∼25% cells responded to both aglycemia and hypoxia. These data suggest that together with hypoxia and acid hypercapnia, low glucose is another important metabolic stimulus that contributes to the vital asphyxia-induced catecholamine surge from AMCs at birth.

A rotenone-sensitive site and H2O2 are key components of hypoxia-sensing in neonatal rat adrenomedullary chromaffin cells.

In the perinatal period, adrenomedullary chromaffin cells (AMC) directly sense PO2 and secrete catecholamines during hypoxic stress, and this response is lost in juvenile ( approximately 2 week-old) chromaffin cells following postnatal innervation. Here we tested the hypothesis that a rotenone-sensitive O2-sensor and ROS are involved in the hypoxic response of AMC cultured from neonatal and juvenile rats. In whole-cell recordings, hypoxia (PO2=5-15 mm Hg) inhibited outward current in neonatal AMC; this response was reversed by exogenous H2O2 and mimicked and occluded by intracellular catalase (1000 units/ml), as well as the antioxidants, N-acetyl-L-cysteine (NAC; 50 microM) and Trolox (200 microM). Acute hypoxia decreased ROS levels and stimulated ATP secretion in these cells, as measured by luminol and luciferin-luciferase chemiluminescence, respectively. Of several mitochondrial electron transport chain (ETC) inhibitors tested, only rotenone, a complex I blocker, mimicked and occluded the effects of hypoxia on outward current, cellular ROS, and ATP secretion. Succinate donors, which act as complex II substrates, reversed the effects of hypoxia and rotenone in neonatal AMC. In contrast, in hypoxia-insensitive juvenile AMC, neither NAC nor rotenone stimulated ATP secretion though they both caused a decrease in ROS levels. We propose that O2-sensing by neonatal AMC is mediated by decreased ROS generation via a rotenone-sensitive site that is coupled to outward current inhibition and secretion. Interestingly, juvenile AMC display at least two modifications, i.e. an uncoupling of the O2-sensor from ROS regulation, and an apparent insensitivity of outward current to decreased ROS.

Oxygen sensing in neuroepithelial and adrenal chromaffin cells.

Oxygen sensing and initiation of appropriate physiological responses to hypoxia are crucial for survival. The molecular identity of the sensor has generally sparked considerable interest and controversy in O2-sensitive cells. In mammals, pulmonary neuroepithelial bodies (NEBs) and adrenal chromaffin cells (AMCs) are O2 sensitive, particularly during the transition from intrauterine to air-breathing life. In NEBs, there is good evidence that the O2 sensor is a plasma membrane-bound NADPH oxidase which during hypoxia, signals K+ channel inhibition, membrane depolarization and neurosecretion via changes in reactive oxygen species (ROS) (e.g. H2O2). Accordingly, hypoxic sensitivity is lost in NEBs from transgenic mice deficient in the gp91(phox) subunit of NADPH oxidase; it is, however, retained in neonatal AMCs from these transgenic mice. A search for the O2 sensor in neonatal rat AMCs suggests a role for the mitochondrial electron transport chain. For example, the complex I blocker, rotenone (1 microM), mimics hypoxia in causing K+ channel inhibition and ATP secretion, and occludes hypoxic sensitivity. The evidence is consistent with hypoxia and rotenone acting via a decrease in ROS. In contrast, the complex IV blocker cyanide (2 mM) did not mimic the effects of hypoxia. We propose thatchanges in ROS serve as a common link between the O2 sensor and secretion in perinatal NEBs and chromaffin cells. However, the subcellular localization of the O2 sensor appears to be different between these two cell types.

Biophysical characterization of whole-cell currents in O2-sensitive neurons from the rat glossopharyngeal nerve.

In this study we use nystatin perforated-patch and conventional whole-cell recording to characterize the biophysical properties of neuronal nitric oxide synthase (nNOS)-expressing paraganglion neurons from the rat glossopharyngeal nerve (GPN), that are thought to provide NO-mediated efferent inhibition of carotid body chemoreceptors. These GPN neurons occur in two populations, a proximal one near the bifurcation of the GPN and the carotid sinus nerve, and a more distal one located further along the GPN. Both populations were visualized in whole mounts by vital staining with the styryl pyridinium dye, 4-Di-2-ASP (D289). Following isolation in vitro, proximal and distal neurons had similar input resistances (mean: 1.5 and 1.6 GOmega, respectively), input capacitances (mean: 25.0 and 27.4 pF, respectively), and resting potentials (mean: -53.9 and -53.3 mV, respectively). All neurons had similar voltage-dependent currents composed of: tetrodotoxin (TTX)-sensitive Na+ currents (IC50 approximately 0.2 microM), prolonged and transient Ca2+ currents, and delayed rectifier-type K+ currents. Threshold activation for the Na+ currents was approximately -30 mV and they were inactivated within 10 ms. Inward Ca2+ currents consisted of nifedipine-sensitive L-type, omega-agatoxin IVA-sensitive P/Q-type, omega-conotoxin GVIA-sensitive N-type, SNX-482-sensitive R-type, and Ni2+-sensitive, but SNX-482-insensitive, T-type channels. The voltage-dependent outward K+ currents were sensitive to tetraethylammonium (TEA; 10 mM) and 4-aminopyridine (4-AP; 2 mM). Exposure to a chemosensory stimulus, hypoxia (PO2 range: 80-5 Torr), caused a dose-dependent decrease in K+ current which persisted in the presence of TEA and 4-AP, consistent with the involvement of background K+ channels. Under current clamp, GPN neurons generated TTX-sensitive action potentials, and in spontaneously active neurons, hypoxia caused membrane depolarization and an increase in firing frequency. These properties endow GPN neurons with an exquisite ability to regulate carotid body chemoreceptor function during hypoxia, via voltage-gated Ca2+-entry, activation of nNOS, and release of NO.

O2 sensing by recombinant TWIK-related halothane-inhibitable K+ channel-1 background K+ channels heterologously expressed in human embryonic kidney cells.

Hypoxic inhibition of K+ channels provides a link between low O2 and cell function, and in glossopharyngeal neurons hypoxic inhibition of a TWIK-related halothane-inhibitable K+ channel-1 (THIK-1)-like background K+ channel regulates neuronal function. In the present study, we examined directly the O2 sensitivity of recombinant THIK-1 channels, expressed in human embryonic kidney (HE293) cells. THIK-1 expression conferred a moderately outwardly rectifying halothane-inhibited and arachidonic acid-potentiated K+ current and invoked a strongly hyperpolarized resting membrane potential. Endogenous K+ currents in untransfected cells were unaffected by either agent. Hypoxia (P(O2), 20 mmHg) reversibly inhibited THIK-1 currents and caused membrane depolarization, effects that were occluded by halothane. Neither the mitochondrial complex I inhibitors rotenone, myxothiazol and sodium cyanide, nor the NADPH oxidase inhibitors diphenylene iodonium and phenylarsine oxide, were effective in inhibiting the O2-sensitivity of THIK-1. Thus, hypoxic inhibition of THIK-1 occurs by a mechanism dissimilar to that which regulates the activity of other members of the background K+ channel family. Given the O2 sensitivity of THIK-1 channels and their abundant expression in the CNS, we raise for the first time the possibility of a physiological and/or pathological role for these channels during brain ischemia.

Whole-cell currents in two subpopulations of cultured rat petrosal neurons with different tetrodotoxin sensitivities.

In this study we use whole-cell recording to characterize at least two distinct populations of cultured neurons from perinatal rat petrosal or petrosal/jugular ganglia based on differential sensitivity of the transient inward Na+ current to tetrodotoxin. These ganglia supply chemoreceptor and baroreceptor afferents which mediate several cardiovascular reflexes. Approximately 50% of the neurons sampled had Na+ currents that were virtually unaffected by bath addition of tetrodotoxin (0.5-2.0 microM) but were abolished by choline substitution for external Na+. The majority of the remaining neurons had Na+ currents that were rapidly and reversibly blocked by 500 nM tetrodotoxin. A few cells had both tetrodotoxin-resistant and tetrodotoxin-sensitive Na+ currents. All neurons had similar voltage-activated Ca2+ and K+ currents. The inward Ca2+ current had no obvious fast transient or T-type component and appeared to be due mainly to the presence of long-lasting L-type Ca2+ channels. The outward currents consisted largely of a delayed rectifying K+ current (IKdr) and a Ca(2+)-activated K+ current (IKca), but no obvious fast transient K+ current (IA) was observed. Exposure to a chemosensory stimulus, hypoxia (PO2 approximately 20 Torr), had no effect on these neurons, in contrast to the pronounced decrease in K+ current it produces in cultured glomus cells, the presumed chemoreceptors and normal targets for a subset of petrosal neurons in vivo. Current-clamp recordings indicated that some neurons gave single spikes while others gave multiple spikes in response to long-depolarizing stimuli. No correlation between spiking behaviour and tetrodotoxin-sensitivity was observed. Thus, cultures enriched in petrosal neurons contain subpopulations with differential sensitivities to tetrodotoxin. Since many of these neurons innervate a single chemosensory target organ, the carotid body, it is of interest to know whether one or both subtypes can form functional synapses with glomus cells of the carotid body and mediate a chemoreceptor reflex.

A fluorescent microscopic study of the development of rat touch domes and their Merkel cells.

The ability of the fluorescent dye quinacrine to label epidermal Merkel cells was used to study the development of touch domes (Haarscheiben) in rat skin. In embryonic and early postnatal pups, sites of touch dome primordia were reliably located within strips of separated trunk epidermis by the occurrence of discrete clusters of fluorescent cells scattered across the basal cell layer. As in excised adult domes, most of these fluorescent (Merkel) cells lay caudal to the emerging tylotrich hair follicle and the cell cluster formed an annular or crescentric disc. Though all the touch domes that comprise the adult population appear to contain labelled Merkel cells by postnatal day 4, the number of these cells per dome continues to increase some 3-4 fold after birth to reach the average adult number (ca 90 cells) on the dorsal trunk around the fifth week. At about this time the adult size (ca 250 micron) of the dorsal Haarscheiben is also reached, as estimated by the length of the long axis of the fluorescent disc. The rostro-caudal orientation of this long axis varied from dome to dome, usually in the range 0-45 degrees clockwise or counterclockwise, and a functional correlate of this is suggested. The density of labelled dome primordia is higher on the dorsal than on the ventral trunk from as early as embryonic day E20-21 and remains higher although the density of domes decreases steadily across the skin during postnatal growth of the animal. This study utilizes a novel fluorescent labelling technique that is applicable to microscopic studies on the development of Merkel cells within their epithelial locations, in this instance the rat touch domes. Although the full population of domes is established early in development, the Merkel cells within a dome continue to increase in number for several weeks after physiological function is known to appear in this structure.

Reinnervation of the rat touch dome restores the Merkel cell population reduced after denervation.

By using the fluorescent dye quinacrine as a marker for the Merkel cells in the rat touch dome, we previously showed that a sustained denervation of the dome causes a rapid and persistent loss of about 60% of its Merkel cells [Nurse, Macintyre and Diamond (1984) Neuroscience 11, 521-533]. We now show that if the sensory nerves to the skin are crushed (or cut) in 2-week old pups and allowed to regenerate, the Merkel cell population within touch domes shows a biphasic response; there is an initial loss of Merkel cells associated with the early phase of denervation, followed by an increase, associated with the phase of reinnervation. Physiological tests revealed that many (though not all) domes within initially deafferented skin had become functionally reinnervated and had their Merkel cell numbers either wholly or partially restored some 40-100 days post operatively. In one case an adult reinnervated dome, that appeared normal physiologically and by its complement of quinacrine fluorescent (Merkel) cells, also had normal histological features in toluidine blue sections and normally innervated Merkel cells in the electron microscope. These results, based on the use of quinacrine to visualize the Merkel cell population in the touch dome, suggest that sensory nerves may induce the differentiation of new Merkel cells in domes where these cells have become reduced after denervation.

A quantitative study of the time course of the reduction in Merkel cell number within denervated rat touch domes.

By using the fluorescent dye quinacrine as a marker for the Merkel cells in rat touch domes we have shown that denervation results in a progressive reduction in the number of these cells to a level that remains relatively constant at about 40% of that present at the time of denervation. The time-course of quinacrine fluorescent cell changes after denervation could be described by assuming that (i) there are two populations of quinacrine fluorescent cells, one labile and the other stable, and (ii) the labile population is the one most sensitive to denervation and disappears exponentially with a half-time of ca 10 days. It appeared that this time-course of decay of the labile quinacrine fluorescent cells was the same whether the denervation was performed during the period of postnatal development studied (at 7 and 14 days), when normally Merkel cells are rapidly added to the dome, or later (at 35 and 60 days) when the adult population is virtually established. Correlative electron microscopic studies confirmed that quinacrine fluorescent cell counts reflect fairly accurately the Merkel cell population in denervated domes. These quantitative findings based on the use of quinacrine to visualize the entire Merkel cell population of touch domes show that the normal development and maintenance of these cells are trophically dependent on sensory nerves, although a subpopulation may persist even in long-term denervated domes. In addition, the similarity of the first order rate constant for the decay of quinacrine fluorescent cells after denervation and for the normal generation of quinacrine fluorescent cells suggests that the labile Merkel cell population is one that turns over continuously in the normally innervated touch dome.

Carotid body chemoreceptors in dissociated cell culture.

Carotid body (CB) glomus or type 1 cells act as peripheral chemoreceptors which detect changes in arterial PO(2), PCO(2), and pH and help maintain homeostasis via the reflex control of ventilation. Over the last approximately 12 years significant progress has been made towards understanding chemotransduction mechanisms using freshly isolated or cultured type 1 cells. The latter preparation allows several powerful experimental manipulations (e.g., co-culture with sensory neurons) resulting in significant advances in our understanding of CB chemoreception. Here, we review several properties of type 1 cells after several days to weeks in culture. Typically, cultured type 1 cells grow in monolayer clusters enveloped by glial-like, type II, or sustentacular cells, which are immunopositive for the glial marker, glial fibrillary acid protein (GFAP). These cells can undergo DNA synthesis, evidenced by uptake of bromodeoxyuridine (BrdU), and show a limited capacity for cell division. Mitosis and survival of type 1 cells can be regulated by oxygen tension and/or growth factors (e.g., bFGF, insulin). In the rat, type 1 cells are immunopositive for several monoaminergic markers, including tyrosine hydroxylase (TH), dopamine transporter (DAT), and 5-HT. They also express cholinergic markers (e.g., vesicular acetylcholine transporter; VAChT), the highly conserved synaptic vesicle protein (SV2), and gap junctional proteins including Connexin 32 (Cx32). Moreover, in long-term culture ( approximately 2 weeks) they retain expression of O(2)-sensitive, TASK-1-like, and Ca(2+)-dependent (BK), K(+) channels as revealed by immunocytochemistry or RT-PCR analysis of mRNA extracted from type 1 clusters after removal from the culture surface.

Basic FGF localization in rat carotid body: paracrine role in O2 -chemoreceptor survival.

Exposure of perinatal rat carotid body (CB) O2-chemoreceptors to basic fibroblast growth factor (bFGF) or hypoxia in vitro increases mitotic activity. Using double-label immunofluorescence, we localized bFGF and its receptor (FGFR) to tyrosine hydroxylase-positive (TH+) chemoreceptors in vitro; bFGF immunoreactivity also localized to chemoreceptors in CB tissue sections. Mitotic activity, measured as percentage TH+ cells that took up bromodeoxyuridine, was relatively constant ( approximately 29%) in normoxic (20% O2) cultures grown with or without bFGF neutralizing antibody (nAb). However, the number of surviving chemoreceptors was significantly reduced in nAb-treated cultures. Under chronic hypoxia (6% O2), the presence of nAb significantly reduced chemoreceptor survival to approximately 70% of control, without affecting mitotic activity. Thus, autocrine/ paracrine actions of endogenous bFGF may help promote CB chemoreceptor survival.

Nicotinic acetylcholine sensitivity of rat petrosal sensory neurons in dissociated cell culture.

Using whole-cell, patch-clamp techniques we investigated acetylcholine (ACh) sensitivity of dissociated sensory neurons from rat petrosal ganglia after 4 h-14 days in vitro. In approx. 68% of petrosal neurons (PN; n = 109) ACh, applied by fast perfusion or pressure ejection from a 'puffer' pipette, caused a rapid depolarization associated with a conductance increase. Under voltage clamp near the resting potential (approx. - 60 mV), ACh induced a hexamethonium-sensitive, inward current (IACh), mimicked by nicotine application, suggesting the presence of neuronal nicotinic acetylcholine receptors (nAChR). The reversal potential of IACh occurred near 0 mV (n = 4), a region where the I-V curve displayed a prominent rectification. The dose-response relation for IACh versus ACh concentration was fitted by the Hill equation with EC50 = approx. 33.9 microM and Hill coefficient = approx. 1.6. The activation phase of IACh was well fitted by a single exponential with mean (+/- S.E.M.) time constant of 102 +/- 82 ms (n = 6); the desensitization phase of IACh was best fitted by the sum of two exponentials, with time constant of 870 +/- 210 ms (n = 6) and 8576 +/- 1435 ms (at -70 mV). Fluctuation analysis yielded an apparent single-channel conductance of 21.6 +/- 10 pS (mean +/- S.E.M.; n = 4). These data indicate that a major subpopulation of sensory neurons in visceral petrosal ganglia of the rat express nAChR. Thus, if similar receptors are present on corresponding nerve terminals, they could mediate fast afferent excitation in response to ACh released at peripheral targets, e.g., the chemosensory carotid body.

Does endogenous 5-HT mediate spontaneous rhythmic activity in chemoreceptor clusters of rat carotid body?

Spontaneous voltage fluctuations often occurred during perforated-patch recordings from clustered rat carotid body (CB) chemoreceptors in vitro. This activity sometimes appeared as rhythmic-like spikes, when cluster size exceeded approximately 20 cells. Spike discharge could be augmented or induced by hypoxia or 5-HT (2-10 microM) application, and inhibited by the 5-HT receptor blocker, ketanserin (50-100 microM). Thus, endogenous 5-HT may contribute to spontaneous firing within CB receptor clusters via autocrine/paracrine mechanisms.

Effects of pHi and pHe on membrane currents recorded with the perforated-patch method from cultured chemoreceptors of the rat carotid body.

In this study we investigated the effects of intracellular pH (pHi) and extracellular pH (pHe) on whole-cell currents in cultured glomus cells of the rat carotid body and small, intensely fluorescent (SIF) cells of sympathetic ganglia. The use of the perforated-patch recording technique along with established methods of cytoplasmic acidification allowed us to carry out this study without greatly disturbing the cell's endogenous pH regulatory mechanisms. A reversible decrease in the outward K+ current (20-30%) was observed during acid loading of glomus (and SIF cells) using the K+/H+ ionophore nigericin (3 microM) and acetate (20 mM). A reversible decrease in the inward Na+ current was also observed in both cell types during nigericin application. Application of amiloride (0.1 mM) to the bathing solution inhibited recovery of the K+ current from an acid load implicating the Na+/H+ antiporter as a mechanism involved in pH homeostasis in glomus cells. A reversible decrease in K+ and Na+ currents was also observed during changes in pHe from 7.4 to 6.5. The effects of pHi on membrane currents, Ca2+ levels, and neurotransmitter release are discussed in the context of the role of glomus cells as primary transducers of chemosensory stimuli in arterial blood.

Electrophysiological characterization of 5-HT receptors on rat petrosal neurons in dissociated cell culture.

The petrosal ganglion supplies chemoafferent pathways via the glossopharyngeal (IXth) nerve to peripheral targets which release various neurotransmitters including serotonin (5-HT). Here, we combined rapid 5-HT application with patch clamp, whole-cell recording to investigate whether 5-HT receptors are expressed on isolated petrosal neurons (PN), cultured from 7-12 day-old rat pups. In responsive cells, the dominant effect of 5-HT was a rapid depolarization associated with a conductance increase in approximately 43% of the neurons (53/123); however, in a minority population ( approximately 6%; 8/123), 5-HT caused membrane depolarization associated with a conductance decrease. In the former group, 5-HT produced a transient inward current (I5-HT) in neurons voltage-clamped near the resting potential ( approximately -60 mV); the effect was mimicked by the 5-HT3 receptor-specific agonist, 2-methyl-5-HT, suggesting it was mediated by 5-HT3 receptors. Further, I5-HT was selectively inhibited by the 5-HT3 receptor-specific antagonist MDL72222 (1-10 microM), but was unaffected by either 5-HT1/5-HT2 receptor antagonist, spiperone, or by 5-HT2 receptor-specific antagonist, ketanserin (50-100 microM). I5-HT displayed moderate inward rectification and had a mean reversal potential (+/-S.E.M.) of -4.3+/-6.6 mV (n=6). Application of 5-HT (dose range: 0.1-100 microM) produced a dose-response curve that was fitted by the Hill equation with EC50= approximately 3.4 microM and Hill coefficient= approximately 1.6 (n=8). The activation phase of I5-HT (10 microM 5-HT at -60 mV) was well fitted by a single exponential with mean (+/-S.E.M.) time constant of 45+/-30 ms (n=6). The desensitization phase of I5-HT was best fitted by a single exponential with mean (+/-S.E.M.) time constant of 660+/-167 ms (n=6). Fluctuation analysis yielded an apparent mean single-channel conductance (+/-S.E.M) of 2.7+/-1.5 pS (n=4) at -60 mV. In the minority ( approximately 6%) population of neurons which responded to 5-HT with a conductance decrease, the depolarization was blocked by the 5-HT2 receptor antagonist, ketanserin (50 microM). Taken together, these results suggest that 5-HT3 receptors are the major subtype expressed by rat petrosal neurons, and therefore are candidates for facilitating chemoafferent excitation in response to 5-HT released from peripheral targets.

Contrasting effects of HEPES vs HCO3(-)-buffered media on whole-cell currents in cultured chemoreceptors of the rat carotid body.

In this study we compared the effects of physiological bicarbonate/CO2-buffered media (BBM) with the commonly used N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid (HEPES)-buffered media (HBM) on whole-cell currents in cultured rat arterial chemoreceptors (i.e. glomus cells) using the perforated-patch technique. Two separate effects were observed on switching from HBM to BBM. First, in the majority of cells tested (31 of 36) there was an increase in the leakage conductance (ca. 5 fold) and a concomitant increase in channel noise, which in preliminary studies appears to arise from the opening of large-conductance anion channels. Second, there was a reversible decrease in voltage-activated outward K+ current which we attribute to cytoplasmic acidification, catalysed by carbonic anhydrase in glomus cells.