Characterizing applications, exposure risks, and hazard communication for engineered nanomaterials in construction.

BACKGROUND: Engineered nanomaterials (ENMs) may pose health risks to workers. Objectives were to characterize ENM applications in construction, identify exposure scenarios, and evaluate the quality of safety data sheets (SDSs) for nano-enabled construction products. METHODS: SDSs and product data were obtained from a public database of nano-enabled construction products. Descriptive statistics were calculated for affected trades, product categories, and types of ENMs. A sample of SDSs (n = 33) was evaluated using modified criteria developed by NIOSH researchers. Bulk analysis via transmission electron microscopy characterized nanoparticles in a subset of products. RESULTS: Companies report using >50 ENMs in construction products. ENM composition could not be determined via SDSs for 38.1% of the 907 products examined. Polymers and metal oxides tied for most frequently reported ENMs (n = 87, 9.6%). Nano silica, graphene, carbon nanotubes, and silver nanoparticles were also frequently reported. Most of the products were paints and coatings (n = 483, 53.3%), followed by pre-market additives, cementitious materials, insulation, and lubricants. Workers in twenty construction trades are likely to handle nano-enabled products, these particularly encompass cement and brick masons, painters, laborers, carpenters, glaziers, and insulators. A wide range of exposure scenarios were identified. SDSs were classified as satisfactory (18%), in need of improvement (12%), or in need of significant improvement (70%). Bulk analyses revealed discrepancies between actual ENM composition and those in SDSs. DISCUSSION AND CONCLUSION: There has been significant progress investigating risks to construction workers posed by ENMs, but SDSs need major improvements. This study provides new insights on the use of ENMs in construction, exposure risks, and hazard communication.

Occupational exposure risk during spraying of biocidal paint containing silver nanoparticles.

The study assessed potential to exceed occupational exposure limits while spraying paint with and without a silver nanoparticle biocidal additive. A tradesperson performed the tasks in a sealed chamber with filtered air supply. Integrated air sampling entailed transmission electron microscopy with energy dispersive X-ray analysis, direct-reading of particle number concentrations, and determination of silver mass concentration by NIOSH Method 7300. Silver nanoparticles were primarily embedded in paint spray droplets but also observed as isolated particles. Using an α-level of 0.05, median nanoparticle number concentrations did not differ significantly when spraying conventional vs. biocidal paint, although statistically significant differences were observed at specific particle size ranges <100 nm. The geometric mean concentration of total silver while spraying biocidal paint (n = 6) was 2.1 µg/m3 (95% CI: 1.5-2.8 µg/m3), and no respirable silver was detected (<0.50 µg/m3). The results address a lack of silver nanoparticle exposure data in construction and demonstrate the feasibility of a practical sampling approach. Given similar conditions, the measurements suggest a low probability of exceeding a proposed silver nanoparticle exposure limit of 0.9 µg/m3 as an airborne 8-hr time-weighted average respirable mass concentration. A full workday of exposure to respirable silver at the highest possible level in this study (<0.50 µg/m3) would not exceed the exposure limit, although limitations in comparing short task-based exposures to an 8-hr exposure limit must be noted. There was airflow in the study chamber, whereas exposure levels could increase over time in work environments lacking adequate ventilation. Potential to exceed the exposure limit hinged upon the respirable fraction of the paint mist, which could vary by material and application method. Additional research would improve understanding of silver nanoparticle exposure risks among construction trades, and biological responses to these exposures. Given the potential for exposure variability on construction jobsites, safety and health professionals should be cognizant of methods to assess and control silver nanoparticle exposures.

Change in prevalence of asbestos-related disease among sheet metal workers 1986 to 2016.

BACKGROUND: A medical screening program began in 1986 for sheet metal workers exposed to asbestos, primarily while working alongside insulators applying spray-on asbestos materials, a practice banned in 1973. Exposure continues during maintenance, renovation, and repair. METHODS: Radiographic abnormalities among 26 397 sheet metal workers examined from 1986 to 2016 were analyzed by year of entry into the trade. Logistic regression was used to examine risk factors for parenchymal and pleural abnormalities among the overall study population and among the subcohort who entered the trade after 1973. RESULTS: Prevalence of parenchymal disease was 17.4% for those starting work before 1950 compared with 0.8% for those starting work after 1973 (adjusted prevalence odds ratio [pOR] = 26.65, 95% confidence interval [CI] = 18.46-38.46). For each calendar year after 1973, entering the trade 1 year later was associated with an estimated 12.7% decreased odds of acquiring asbestos-related disease (adjusted pOR = 0.873, 95% CI = 0.832-0.916). CONCLUSION: Sheet metal workers who began work after the US implemented environmental and occupational regulations develop asbestos-related disease at much reduced rates, consistent with regulatory projections made for nonmalignant asbestos-related disease by the Occupational Safety and Health Administration at the time. Cancer remains a concern among this cohort, and lung cancer screening recommendations should consider year of entry into the trade. This study highlights the importance of regulatory intervention and of continued surveillance.

Heat-related deaths among construction workers in the United States.

BACKGROUND: Heat is a severe hazard for construction workers and may be worsening with global warming. This study sought to explore heat-related deaths among U.S. construction workers and a possible association with climate change. METHODS: Heat-related deaths in the Census of Fatal Occupational Injuries from 1992 to 2016 were analyzed. Denominators estimated from the Current Population Survey were matched with demographic and occupational categories in rate calculations. Statistical tests were used to examine heat-related deaths in relation to time, geographic region, and temperature. RESULTS: Construction workers, comprising 6% of the total workforce, accounted for 36% (n = 285) of all occupational heat-related deaths from 1992 to 2016 in the U.S. Mean temperatures from June to August increased gradually over the study period. Increasing summer temperatures from 1997 to 2016 were associated with higher heat-related death rates (r = 0.649; 95% confidence interval: 0.290, 0.848). Compared to all construction workers (risk index = 1), statistically significant elevated risk of heat-related death was found among Hispanics (1.21), in particular workers born in Mexico (1.91). Occupations with a high risk index included cement masons (10.80), roofers (6.93), helpers (6.87), brick masons (3.33), construction laborers (1.93) and heating, air conditioning, and refrigeration mechanics (1.60). CONCLUSIONS: U.S. construction workers are at a high risk of heat-related death, and this risk has increased with climate change over time. Effective workplace interventions, enhanced surveillance, and improved regulations and enforcement should accompany broader efforts to combat global warming. The construction industry can help reduce global warming through increased implementation of green building principles.

Exposure to airborne nano-titanium dioxide during airless spray painting and sanding.

The objectives of the study were to measure and characterize exposure to airborne nanoscale titanium dioxide during airless spraying and sanding of a nano-enabled paint, and to evaluate the effectiveness of dust capture methods in reducing airborne nanoparticle concentrations. A tradesperson performed the work activities in an environmentally controlled chamber. Samples were collected in the tradesperson's breathing zone and in surrounding areas to assess bystander exposure. Filter-based samples were analyzed using gravimetric methods, scanning electron microscopy, and energy dispersive spectroscopy. Differential particle count data were obtained by means of a scanning mobility particle sizer. Local exhaust ventilation provided statistically significant reductions of airborne nanoparticle concentrations during sanding. Sanding the paint after drying with a handheld power sander generated relatively low levels of airborne titanium dioxide. In contrast, task-based exposure measurements collected during the initial airless spray application of the nano-enabled paint suggested a potential for occupational exposures to exceed the time-weighted average exposure limit for ultrafine titanium dioxide recommended by the National Institute for Occupational Safety and Health. Painters applying nano-enabled coatings may have little recourse but to rely, in some instances, on lower tiers of the hierarchy of controls, such as personal protective equipment. In light of these findings, employers and industrial hygienists should characterize exposures and implement the hierarchy of controls to ensure painters are sufficiently protected.

Inhalation exposure during spray application and subsequent sanding of a wood sealant containing zinc oxide nanoparticles.

Nano-enabled construction products have entered into commerce. There are concerns about the safety of manufactured nanomaterials, and exposure assessments are needed for a more complete understanding of risk. This study assessed potential inhalation exposure to ZnO nanoparticles during spray application and power sanding of a commercially available wood sealant and evaluated the effectiveness of local exhaust ventilation in reducing exposure. A tradesperson performed the spraying and sanding inside an environmentally-controlled chamber. Dust control methods during sanding were compared. Filter-based sampling, electron microscopy, and real-time particle counters provided measures of exposure. Airborne nanoparticles above background levels were detected by particle counters for all exposure scenarios. Nanoparticle number concentrations and particle size distributions were similar for sanding of treated versus untreated wood. Very few unbound nanoparticles were detected in aerosol samples via electron microscopy, rather nano-sized ZnO was contained within, or on the surface of larger airborne particles. Whether the presence of nanoscale ZnO in these aerosols affects toxicity merits further investigation. Mass-based exposure measurements were below the NIOSH Recommended Exposure Limit for Zn, although there are no established exposure limits for nanoscale ZnO. Local exhaust ventilation was effective, reducing airborne nanoparticle number concentrations by up to 92% and reducing personal exposure to total dust by at least 80% in terms of mass. Given the discrepancies between the particle count data and electron microscopy observations, the chemical identity of the airborne nanoparticles detected by the particle counters remains uncertain. Prior studies attributed the main source of nanoparticle emissions during sanding to copper nanoparticles generated from electric sander motors. Potentially contrary results are presented suggesting the sander motor may not have been the primary source of nanoparticle emissions in this study. Further research is needed to understand potential risks faced by construction workers exposed to mixed aerosols containing manufactured nanomaterials. Until these risks are better understood, this study demonstrates that engineering controls can reduce exposure to manufactured nanomaterials; doing so may be prudent for protecting worker health.

An analysis of permanent work disability among construction sheet metal workers.

BACKGROUND: Causes of permanent work disability in the sheet metal industry are not well characterized. METHODS: Pension records were used to compare causes of disability among sheet metal workers and the U.S. working population. Subgroup analysis examined the major causes of sheet metal worker disability. RESULTS: Musculoskeletal disorders (MSDs), circulatory disease, and injuries were leading causes of sheet metal worker disability (47.2%, 13.7%, 10.9% of awards, respectively). Award distribution differed from the U.S. working population (P < 0.0001); MSDs and injuries accounted for higher proportions of sheet metal worker awards, particularly at spine, shoulder, and knee. CONCLUSIONS: Higher proportions of awards caused by MSD or injury among sheet metal workers may reflect higher rates of work-related injuries and MSDs, a high likelihood of disability with construction work given the same impairment, or higher prevalence of other conditions in the general population. Prevention requires task-specific ergonomic innovations and proven participatory interventions.

Regulation of visceral sympathetic tone by A5 noradrenergic neurons in rodents.

The ventrolateral pons contains the A5 group of noradrenergic neurons which regulate the circulation and probably breathing. The present experiments were designed to identify these neurons definitively in vivo, to examine their response to chemoreceptor stimuli (carotid body stimulation and changes in brain pH) and to determine their effects on sympathetic outflow. Bulbospinal A5 neurons, identified by juxtacellular labelling in anaesthetized rats, had a slow regular discharge, were vigorously activated by peripheral chemoreceptor stimulation with cyanide, but only mildly activated by hyperoxic hypercapnia (central chemoreceptor stimulation). The caudal end of the A5 region also contained neurons with properties reminiscent of retrotrapezoid neurons. These cells lacked a spinal axon and were characterized by a robust response to CO2. The pH sensitivity of A5 neurons, examined in brain slices from neonatal (postnatal days 6­10) tyrosine hydroxylase (TH)-GFP transgenic mice, was about 10 times smaller than that of similarly recorded retrotrapezoid neurons. Selective stimulation of the A5 neurons in rats using channelrhodopsin optogenetics (A5 TH neurons represented 66% of transfected cells) produced fivefold greater activation of the renal nerve than the lumbar sympathetic chain. In summary, adult A5 noradrenergic neurons are vigorously activated by carotid body stimulation. This effect presumably contributes to the increase in visceral sympathetic nerve activity elicited by acute hypoxia. A5 neurons respond weakly to hypercapnia in vivo or to changes in pH in slices suggesting that their ability to sense local variations in brain pH or Pco2 is limited.

Photostimulation of retrotrapezoid nucleus phox2b-expressing neurons in vivo produces long-lasting activation of breathing in rats.

The retrotrapezoid "nucleus" (RTN), located in the rostral ventrolateral medullary reticular formation, contains a bilateral cluster of approximately 1000 glutamatergic noncatecholaminergic Phox2b-expressing propriobulbar neurons that are activated by CO(2) in vivo and by acidification in vitro. These cells are thought to function as central respiratory chemoreceptors, but this theory still lacks a crucial piece of evidence, namely that stimulating these particular neurons selectively in vivo increases breathing. The present study performed in anesthetized rats seeks to test whether this expectation is correct. We injected into the left RTN a lentivirus that expresses the light-activated cationic channel ChR2 (channelrhodopsin-2) (H134R mutation; fused to the fluorescent protein mCherry) under the control of the Phox2-responsive promoter PRSx8. Transgene expression was restricted to 423 +/- 38 Phox2b-expressing neurons per rat consisting of noncatecholaminergic and C1 adrenergic neurons (3:2 ratio). Photostimulation delivered to the RTN region in vivo via a fiberoptic activated the CO(2)-sensitive neurons vigorously, produced a long-lasting (t(1/2) = 11 s) increase in phrenic nerve activity, and caused a small and short-lasting cardiovascular stimulation. Selective lesions of the C1 cells eliminated the cardiovascular response but left the respiratory stimulation intact. In rats with C1 cell lesions, the mCherry-labeled axon terminals originating from the transfected noncatecholaminergic neurons were present exclusively in the lower brainstem regions that contain the respiratory pattern generator. These results provide strong evidence that the Phox2b-expressing noncatecholaminergic neurons of the RTN region function as central respiratory chemoreceptors.

Botzinger expiratory-augmenting neurons and the parafacial respiratory group.

In neonatal rat brains in vitro, the rostral ventral respiratory column (rVRC) contains neurons that burst just before the phrenic nerve discharge (PND) and rebound after inspiration (pre-I neurons). These neurons, called parafacial respiratory group (pfRG), have been interpreted as a master inspiratory oscillator, an expiratory rhythm generator or simply as neonatal precursors of retrotrapezoid (RTN) chemoreceptor neurons. pfRG neurons have not been identified in adults, and their phenotype is unknown. Here, we confirm that the rVRC normally lacks pre-I neurons in adult anesthetized rats. However, we show that, during hypercapnic hypoxia, a population of rVRC expiratory-augmenting (E-AUG) neurons consistently develops a pre-I discharge. These cells reside in the Bötzinger region of the rVRC, they express glycine-transporter-2, and their axons arborize throughout the VRC. Hypoxia triggers an identical pre-I pattern in retroambigual expiratory bulbospinal neurons, but this pattern is not elicited in Bötzinger expiratory-decrementing neurons, Bötzinger inspiratory neurons, RTN neurons, and blood pressure-regulating neurons. In conclusion, under hypoxia in vivo, abdominal expiratory premotor neurons of adult rats develop a pre-I pattern reminiscent of that observed in neonate brainstems in vitro. In the rVRC of adult rats, pre-I cells include selected rhythmogenic neurons (glycinergic Bötzinger neurons) but not RTN chemoreceptors. We suggest that the pfRG may not be an independent rhythm generator but a heterogeneous collection of E-AUG neurons (glycinergic Bötzinger neurons, possibly facial motor and premotor neurons), the discharge of which becomes preinspiratory under specific experimental conditions resulting from, in part, a prolonged and intensified activity of postinspiratory neurons.

Activation of the retrotrapezoid nucleus by posterior hypothalamic stimulation.

The retrotrapezoid nucleus (RTN) contains chemically defined neurons (ccRTN neurons) that provide a pH-regulated excitatory drive to the central respiratory pattern generator. Here we test whether ccRTN neurons respond to stimulation of the perifornical hypothalamus (PeF), a region that regulates breathing during sleep, stress and exercise. PeF stimulation with gabazine increased blood pressure, phrenic nerve discharge (PND) and the firing rate of ccRTN neurons in isoflurane-anaesthetized rats. Gabazine produced an approximately parallel upward shift of the steady-state relationship between ccRTN neuron firing rate and end-tidal CO(2), and a similar shift of the relationship between PND and end-tidal CO(2). The central respiratory modulation of ccRTN neurons persisted after gabazine without a change in pattern. Morphine administration typically abolished PND and reduced the discharge rate of most ccRTN neurons (by 25% on average). After morphine administration, PeF stimulation activated the ccRTN neurons normally but PND activation and the central respiratory modulation of the ccRTN neurons were severely attenuated. In the same rat preparation, most (58%) ccRTN neurons expressed c-Fos after exposure to hypercapnic hyperoxia (6-7% end-tidal CO(2); 3.5 h; no hypothalamic stimulation) and 62% expressed c-Fos under hypocapnia (approximately 3% end-tidal CO(2)) after PeF stimulation. Under baseline conditions (approximately 3% end-tidal CO(2), hyperoxia, no PeF stimulation) few (11%) ccRTN neurons expressed c-Fos. In summary, most ccRTN neurons are excited by posterior hypothalamic stimulation while retaining their normal response to CNS acidification. ccRTN neurons probably contribute both to the chemical drive of breathing and to the feed-forward control of breathing associated with emotions and or locomotion.

Photostimulation of channelrhodopsin-2 expressing ventrolateral medullary neurons increases sympathetic nerve activity and blood pressure in rats.

To explore the specific contribution of the C1 neurons to blood pressure (BP) control, we used an optogenetic approach to activate these cells in vivo. A lentivirus that expresses channelrhodopsin-2 (ChR2) under the control of the catecholaminergic neuron-preferring promoter PRSx8 was introduced into the rostral ventrolateral medulla (RVLM). After 2-3 weeks, ChR2 was largely confined to Phox2b-expressing neurons (89%). The ChR2-expressing neurons were non-GABAergic, non-glycinergic and predominantly catecholaminergic (54%). Photostimulation of ChR2-transfected RVLM neurons (473 nm, 20 Hz, 10 ms, 9 mW) increased BP (15 mmHg) and sympathetic nerve discharge (SND; 64%). Light pulses at 0.2-0.5 Hz evoked a large sympathetic nerve response (16 x baseline) followed by a silent period (1-2 s) during which another stimulus evoked a reduced response. Photostimulation activated most (75%) RVLM baroinhibited neurons sampled with 1/1 action potential entrainment to the light pulses and without accommodation during 20 Hz trains. RVLM neurons unaffected by either CO(2) or BP were light-insensitive. Bötzinger respiratory neurons were activated but their action potentials were not synchronized to the light pulses. Juxtacellular labelling of recorded neurons revealed that, of these three cell types, only the cardiovascular neurons expressed the transgene. In conclusion, ChR2 expression had no discernable effect on the putative vasomotor neurons at rest and was high enough to allow precise temporal control of their action potentials with light pulses. Photostimulation of RVLM neurons caused a sizable sympathoactivation and rise in blood pressure. These results provide the most direct evidence yet that the C1 neurons have a sympathoexcitatory function.

TASK channels determine pH sensitivity in select respiratory neurons but do not contribute to central respiratory chemosensitivity.

Central respiratory chemoreception is the mechanism by which the CNS maintains physiologically appropriate pH and PCO2 via control of breathing. A prominent hypothesis holds that neural substrates for this process are distributed widely in the respiratory network, especially because many neurons that make up this network are chemosensitive in vitro. We and others have proposed that TASK channels (TASK-1, K(2P)3.1 and/or TASK-3, K(2P)9.1) may serve as molecular sensors for central chemoreception because they are highly expressed in multiple neuronal populations in the respiratory pathway and contribute to their pH sensitivity in vitro. To test this hypothesis, we examined the chemosensitivity of two prime candidate chemoreceptor neurons in vitro and tested ventilatory responses to CO2 using TASK channel knock-out mice. The pH sensitivity of serotonergic raphe neurons was abolished in TASK channel knock-outs. In contrast, pH sensitivity of neurons in the mouse retrotrapezoid nucleus (RTN) was fully maintained in a TASK null background, and pharmacological evidence indicated that a K+ channel with properties distinct from TASK channels contributes to the pH sensitivity of rat RTN neurons. Furthermore, the ventilatory response to CO2 was completely retained in single or double TASK knock-out mice. These data rule out a strict requirement for TASK channels or raphe neurons in central respiratory chemosensation. Furthermore, they indicate that a non-TASK K+ current contributes to chemosensitivity of RTN neurons, which are profoundly pH-sensitive and capable of driving respiratory output in response to local pH changes in vivo.

Selective lesion of retrotrapezoid Phox2b-expressing neurons raises the apnoeic threshold in rats.

Injection of the neurotoxin saporin-substance P (SSP-SAP) into the retrotrapezoid nucleus (RTN) attenuates the central chemoreflex in rats. Here we ask whether these deficits are caused by the destruction of a specific type of interneuron that expresses the transcription factor Phox2b and is non-catecholaminergic (Phox2b(+)TH(-)). We show that RTN contains around 2100 Phox2b(+)TH(-) cells. Injections of SSP-SAP into RTN destroyed Phox2b(+)TH(-) neurons but spared facial motoneurons, catecholaminergic and serotonergic neurons and the ventral respiratory column caudal to the facial motor nucleus. Two weeks after SSP-SAP, the apnoeic threshold measured under anaesthesia was unchanged when fewer than 57% of the Phox2b(+)TH(-) neurons were destroyed. However, destruction of 70 +/- 3.5% of these cells was associated with a dramatic rise of the apnoeic threshold (from 5.6 to 7.9% end-expiratory P(CO(2))). In anaesthetized rats with unilateral lesions of around 70% of the Phox2b(+)TH(-) neurons, acute inhibition of the contralateral intact RTN with muscimol instantly eliminated phrenic nerve discharge (PND) but normal PND could usually be elicited by strong peripheral chemoreceptor stimulation (8/12 rats). Muscimol had no effect in rats with an intact contralateral RTN. In conclusion, the destruction of the Phox2b(+)TH(-) neurons is a plausible cause of the respiratory deficits caused by injection of SSP-SAP into RTN. Two weeks after toxin injection, 70% of these cells must be killed to cause a severe attenuation of the central chemoreflex under anaesthesia. The loss of an even greater percentage of these cells would presumably be required to produce significant breathing deficits in the awake state.

Expression of Phox2b by brainstem neurons involved in chemosensory integration in the adult rat.

Central congenital hypoventilation syndrome is caused by mutations of the gene that encodes the transcription factor Phox2b. The syndrome is characterized by a severe form of sleep apnea attributed to greatly compromised central and peripheral chemoreflexes. In this study, we analyze whether Phox2b expression in the brainstem respiratory network is preferentially associated with neurons involved in chemosensory integration in rats. At the very rostral end of the ventral respiratory column (VRC), Phox2b was present in many VGlut2 (vesicular glutamate transporter 2) mRNA-containing neurons. These neurons were functionally identified as the respiratory chemoreceptors of the retrotrapezoid nucleus (RTN). More caudally in the VRC, many fewer neurons expressed Phox2b. These cells were not part of the central respiratory pattern generator (CPG), because they were typically cholinergic visceral motor neurons or catecholaminergic neurons (presumed C1 neurons). Phox2b was not detected in serotonergic neurons, in the A5, A6, and A7 noradrenergic cell groups nor within the main cardiorespiratory centers of the dorsolateral pons. Phox2b was expressed by many solitary tract nucleus (NTS) neurons including those that relay peripheral chemoreceptor information to the RTN. These and previous observations by others suggest that Phox2b is expressed by an uninterrupted chain of neurons involved in the integration of peripheral and central chemoreception (carotid bodies, chemoreceptor afferents, chemoresponsive NTS neurons projecting to VRC, RTN chemoreceptors). The presence of Phox2b in this circuit and its apparent absence from the respiratory CPG could explain why Phox2b mutations disrupt breathing automaticity during sleep without causing major impairment of respiration during waking.

Galanin is a selective marker of the retrotrapezoid nucleus in rats.

The rat retrotrapezoid nucleus (RTN) contains CO(2)-activated neurons that contribute to the central chemoreflex and to breathing automaticity. These neurons have two known markers, the transcription factor Phox2b and vesicular glutamate transporter 2 (VGLUT2). Noncatecholaminergic galanin-immunoreactive (ir) neurons within a region of the lower brainstem that seems identical to what is currently defined as the RTN have been previously described. Here we ask whether these galanin-expressing neurons are the same cells as the recently characterized CO(2)-sensitive neurons of the RTN. By using in situ hybridization, we found that pre-pro-galanin (PPGal) mRNA is expressed by an isolated cluster of neurons that is co-extensive with the RTN as defined by a population of strongly Phox2b-ir neurons devoid of tyrosine hydroxylase (Phox2b(+)/TH(-) neurons). This bilateral structure contains about 1,000 PPGal mRNA-positive neurons in the rat. The PPGal mRNA-positive neurons were Phox2b(+)/TH(-) and as susceptible to destruction by the toxin [Sar(9), Met (O(2))(11)]-substance P as the rest of the RTN Phox2b(+)/TH(-) cells of the RTN. CO(2)-activated neurons were recorded in the RTN of anesthetized rats and were labeled with biotinamide. Many of those cells (7/17, 41%, five rats) contained PPGal-mRNA. In conclusion, galanin mRNA is a very specific marker of the glutamatergic Phox2b(+)/TH(-) neurons of the RTN, but galanin mRNA identifies only half of these putative central respiratory chemoreceptors.

Acid sensitivity and ultrastructure of the retrotrapezoid nucleus in Phox2b-EGFP transgenic mice.

The retrotrapezoid nucleus (RTN) contains noncholinergic noncatecholaminergic glutamatergic neurons that express the transcription factor Phox2b (chemically coded or "cc" RTN neurons). These cells regulate breathing and may be central chemoreceptors. Here we explore their ultrastructure and their acid sensitivity by using two novel BAC eGFP-Phox2b transgenic mice (B/G, GENSAT JX99) in which, respectively, 36% and 100% of the cc RTN neurons express the transgene in complete or partial anatomical isolation from other populations of eGFP neurons. All but one of the eGFP-labeled RTN neurons recorded in these mice were acid activated in slices. These cells contained VGLUT2 mRNA, and 50% contained preprogalanin mRNA (determined by single-cell PCR in the B/G mouse). Two neuronal subgroups were revealed, which differed in discharge rate at pH 7.3 (type I approximately 2; type II approximately 4 Hz) and the degree of alkalization that silenced the cells (type I 7.4-7.6, type II 7.8-8.0). Medial to the RTN, C1 neurons recorded in a tyrosine hydroxylase-GFP mouse were pH insensitive between pH 6.9 and pH 7.5. Ultrastructural studies demonstrated that eGFP-labeled RTN neurons were surrounded by numerous capillaries and were often in direct contact with glial cells, pericytes, and the basement membrane of capillaries. Terminals contacting large proximal eGFP dendrites formed mainly symmetric, likely inhibitory, synapses. Terminals on more distal eGFP dendrites formed preferentially asymmetric, presumably excitatory, synapses. In sum, C1 cells are pH insensitive, whereas cc RTN neurons are uniformly acid sensitive. The RTN neurons receive inhibitory and excitatory synaptic inputs and may have unfettered biochemical interactions with glial cells and the local microvasculature.

Inhibitory input from slowly adapting lung stretch receptors to retrotrapezoid nucleus chemoreceptors.

The retrotrapezoid nucleus (RTN) contains CO(2)-activated interneurons with properties consistent with central respiratory chemoreceptors. These neurons are glutamatergic and express the transcription factor Phox2b. Here we tested whether RTN neurons receive an input from slowly adapting pulmonary stretch receptors (SARs) in halothane-anaesthetized ventilated rats. In vagotomized rats, RTN neurons were inhibited to a variable extent by stimulating myelinated vagal afferents using the lowest intensity needed to inhibit the phrenic nerve discharge (PND). In rats with intact vagus nerves, RTN neurons were inhibited, also to a variable extent, by increasing positive end-expiratory pressure (PEEP; 2-6 cmH(2)O). The cells most sensitive to PEEP were inhibited during each lung inflation at rest and were instantly activated by stopping ventilation. Muscimol (GABA-A agonist) injection in or next to the solitary tract at area postrema level desynchronized PND from ventilation, eliminated the lung inflation-synchronous inhibition of RTN neurons and their steady inhibition by PEEP but did not change their CO(2) sensitivity. Muscimol injection into the rostral ventral respiratory group eliminated PND but did not change RTN neuron response to either lung inflation, PEEP increases, vagal stimulation or CO(2). Generalized glutamate receptor blockade with intracerebroventricular (i.c.v.) kynurenate eliminated PND and the response of RTN neurons to lung inflation but did not change their CO(2) sensitivity. PEEP-sensitive RTN neurons expressed Phox2b. In conclusion, RTN chemoreceptors receive an inhibitory input from myelinated lung stretch receptors, presumably SARs. The lung input to RTN may be di-synaptic with inhibitory pump cells as sole interneurons.

GABAergic pump cells of solitary tract nucleus innervate retrotrapezoid nucleus chemoreceptors.

The retrotrapezoid nucleus (RTN) contains central respiratory chemoreceptors that are inhibited by activation of slowly adapting pulmonary stretch receptors (SARs). Here we examine whether RTN inhibition by lung inflation could be mediated by a direct projection from SAR second-order neurons (pump cells). Pump cells (n = 56 neurons, 13 rats) were recorded in the nucleus of solitary tract (NTS) of halothane-anesthetized rats with intact vagus nerves. Pump cells had discharges that coincided with lung inflation as monitored by the tracheal pressure. Their activity increased when end-expiratory pressure was raised and stopped instantly when ventilation was interrupted in expiration. Many pump cells could be antidromically activated from RTN (12/36). Nine of those were labeled with biotinamide. Of these nine cells, eight contained glutamic acid decarboxylase 67 (GAD67) mRNA and seven were found to reside in the lower half of the interstitial subnucleus of NTS (iNTS). Using the retrograde tracer cholera toxin-B, we confirmed that neurons located in or close to iNTS innervate RTN (two rats). Many such neurons contained GAD67 mRNA and a few contained glycine transporter2 (GLYT2) mRNA. Anterograde tract tracing with biotinylated dextranamide (four rats) applied to iNTS also confirmed that this region innervates RTN by a predominantly GABAergic projection. This work confirms that many rat NTS pump cells are located in and around the interstitial subnucleus at area postrema level. We demonstrate that a GABAergic subset of these pump cells innervates the RTN region. We conclude that these inhibitory neurons probably contact RTN chemoreceptors and mediate their inhibition by lung inflation.

Peripheral chemoreceptor inputs to retrotrapezoid nucleus (RTN) CO2-sensitive neurons in rats.

The rat retrotrapezoid nucleus (RTN) contains pH-sensitive neurons that are putative central chemoreceptors. Here, we examined whether these neurons respond to peripheral chemoreceptor stimulation and whether the input is direct from the solitary tract nucleus (NTS) or indirect via the respiratory network. A dense neuronal projection from commissural NTS (commNTS) to RTN was revealed using the anterograde tracer biotinylated dextran amine (BDA). Within RTN, 51% of BDA-labelled axonal varicosities contained detectable levels of vesicular glutamate transporter-2 (VGLUT2) but only 5% contained glutamic acid decarboxylase-67 (GAD67). Awake rats were exposed to hypoxia (n = 6) or normoxia (n = 5) 1 week after injection of the retrograde tracer cholera toxin B (CTB) into RTN. Hypoxia-activated neurons were identified by the presence of Fos-immunoreactive nuclei. CommNTS neurons immunoreactive for both Fos and CTB were found only in hypoxia-treated rats. VGLUT2 mRNA was detected in 92 +/- 13% of these neurons whereas only 12 +/- 9% contained GAD67 mRNA. In urethane-chloralose-anaesthetized rats, bilateral inhibition of the RTN with muscimol eliminated the phrenic nerve discharge (PND) at rest, during hyperoxic hypercapnia (10% CO(2)), and during peripheral chemoreceptor stimulation (hypoxia and/or i.v. sodium cyanide, NaCN). RTN CO(2)-activated neurons were recorded extracellularly in anaesthetized intact or vagotomized rats. These neurons were strongly activated by hypoxia (10-15% O(2); 30 s) or by NaCN. Hypoxia and NaCN were ineffective in rats with carotid chemoreceptor denervation. Bilateral injection of muscimol into the ventral respiratory column 1.5 mm caudal to RTN eliminated PND and the respiratory modulation of RTN neurons. Muscimol did not change the threshold and sensitivity of RTN neurons to hyperoxic hypercapnia nor their activation by peripheral chemoreceptor stimulation. In conclusion, RTN neurons respond to brain P(CO(2)) presumably via their intrinsic chemosensitivity and to carotid chemoreceptor activation via a direct glutamatergic pathway from commNTS that bypasses the respiratory network. RTN neurons probably contribute a portion of the chemical drive to breathe.