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.

Optical frequency averaging of light.

The use of averaging has long been known to reduce noise in statistically independent systems that exhibit similar levels of stochastic fluctuation. This concept of averaging is general and applies to a wide variety of physical and man-made phenomena such as particle motion, shot noise, atomic clock stability, measurement uncertainty reduction, and methods of signal processing. Despite its prevalence in use for reducing statistical uncertainty, such averaging techniques so far remain comparatively undeveloped for application to light. We demonstrate here a method for averaging the frequency uncertainty of identical laser systems as a means to narrow the spectral linewidth of the resulting radiation. We experimentally achieve a reduction of frequency fluctuations from 40 Hz to 28 Hz by averaging two separate laser systems each locked to a fiber resonator. Only a single seed laser is necessary here as acousto-optic modulation is used to enable independent control of the second path. This technique of frequency averaging provides an effective solution to overcome the linewidth constraints of a single laser alone, particularly when limited by fundamental noise sources such as thermal noise, irrespective of the spectral shape of noise.

Cooling of an integrated Brillouin laser below the thermal limit.

Photonically integrated resonators are promising as a platform for enabling ultranarrow linewidth lasers in a compact form factor. Owing to their small size, these integrated resonators suffer from thermal noise that limits the frequency stability of the optical mode to ∼100 kHz. Here, we demonstrate an integrated stimulated Brillouin scattering (SBS) laser based on a large mode-volume annulus resonator that realizes an ultranarrow thermal-noise-limited linewidth of 270 Hz. In practice, yet narrower linewidths are required before integrated lasers can be truly useful for applications such as optical atomic clocks, quantum computing, gravitational wave detection, and precision spectroscopy. To this end, we employ a thermorefractive noise suppression technique utilizing an auxiliary laser to reduce our SBS laser linewidth to 70 Hz. This demonstration showcases the possibility of stabilizing the thermal motion of even the narrowest linewidth chip lasers to below 100 Hz, thereby opening the door to making integrated microresonators practical for the most demanding future scientific endeavors.

Involving Stakeholders as Communication Partners in Research Dissemination Efforts.

Moving evidence into practice requires the support of stakeholders, who are critical actors in the research process. Yet, research teams need strategies for determining who these stakeholders are, what their roles should be, and how to involve them in research and dissemination activities. In this Perspective, we discuss steps for identifying, categorizing, and including stakeholders in the research process, as a precursor to involving them as communication partners in research dissemination efforts. Effectively communicating the results of research is critical for increasing stakeholders' buy-in for the adoption and sustainment of this evidence. However, this communication is best if it comes from the end-users themselves, the stakeholders, who have a specified involvement in the research process. Combining elements from dissemination, implementation, and management science literature, we identify specific tools and strategies for researchers to (1) understand the roles of various stakeholders potentially impacted by their work, and (2) recognize the specific communication activities these stakeholders could be engaged in, to support the dissemination of research findings. We present a 3-Step Plan for identifying, categorizing, and involving stakeholders in the research process in a way that will lead to their role as communication partners when results are ready to be disseminated widely.

From implementation to sustainment: A large-scale adverse event disclosure support program generated through embedded research in the Veterans Health Administration.

In 2008, the Veterans Health Administration published a groundbreaking policy on disclosing large-scale adverse events to patients in order to promote transparent communication in cases where harm may not be obvious or even certain. Without embedded research, the evidence on whether or not implementation of this policy was generating more harm than good among Veteran patients was unknown. Through an embedded research-operations partnership, we conducted four research projects that led to the development of an evidence-based large-scale disclosure toolkit and disclosure support program, and its implementation across VA healthcare. Guided by the Consolidated Framework for Implementation Research, we identified specific activities corresponding to planning, engaging, executing, reflecting and evaluating phases in the process of implementation. These activities included planning with operational leaders to establish a shared research agenda; engaging with stakeholders to discuss early results, establishing buy-in of our efforts and receiving feedback; joining existing operational teams to execute the toolkit implementation; partnering with clinical operations to evaluate the toolkit during real-time disclosures; and redesigning the toolkit to meet stakeholders' needs. Critical lessons learned for implementation success included a need for stakeholder collaboration and engagement, an organizational culture involving a strong belief in evidence, a willingness to embed researchers in clinical operation activities, allowing for testing and evaluation of innovative practices, and researchers open to constructive feedback. At the conclusion of the research, VA operations worked with the researchers to continue to support efforts to spread, scale-up and sustain toolkit use across the VA healthcare system, with the final goal to establish long-term sustainability.

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.

Impact of laser frequency noise on high-extinction optical modulation.

In present literature on integrated modulation and filtering, limitations in the extinction ratio are dominantly attributed to a combination of imbalance in interfering wave amplitude, instability of control signals, stray light (e.g., in the cladding), or amplified spontaneous emission from optical amplifiers. Here we show that the existence of optical frequency noise in single longitudinal mode lasers presents an additional limit to the extinction ratio of optical modulators. A simple frequency-domain model is used to describe a linear optical system's response in the presence of frequency noise, and an intuitive picture is given for systems with arbitrary sampling time. Understanding the influence of frequency noise will help guide the design choices of device and system engineers and offer a path toward even higher-extinction optical modulators.

Compact and high-precision wavemeters using the Talbot effect and signal processing.

Precise knowledge of a laser's wavelength is crucial for applications from spectroscopy to telecommunications. Here, we present a wavemeter that operates on the Talbot effect. Tone parameter extraction algorithms are used to retrieve the frequency of the periodic signal obtained in the sampled Talbot interferogram. Theoretical performance analysis based on the Cramér-Rao lower bound as well as experimental results are presented and discussed. With this scheme, we experimentally demonstrate a compact and high-precision wavemeter with below 10 pm single-shot estimation uncertainty under the 3-σ criterion around 780 nm.

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.

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.

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.

Investigating Clinical Benefits of a Novel Sleep-Focused Mind-Body Program on Gulf War Illness Symptoms: A Randomized Controlled Trial.

OBJECTIVE: Mind-Body Bridging (MBB) has been shown to be effective for improving disturbed sleep. In this prospective randomized controlled trial, we evaluated the efficacy of sleep-focused MBB compared with sleep education control (SED) for improving sleep in previously deployed Gulf War veterans. METHODS: US military service members with sleep and physical health complaints who were deployed in 1990-1991 were randomized to receive three weekly sessions of either MBB (n = 33) or SED (n = 27) between 2012 and 2015. The primary outcome of Medical Outcomes Study Sleep Scale was completed at baseline, weekly during treatment, postintervention, and 3-month follow-up. Secondary outcome measures for posttraumatic stress disorder, depression, fatigue, quality of life, symptom severity, and mindfulness were completed at baseline, postintervention and 3-month follow-up. Salivary samples were collected at five time points per day at each visit for cortisol and α-amylase assessment. Clinician-administered assessments of sleep and co-occurring conditions were conducted at baseline and postintervention. RESULTS: MBB was significantly more efficacious than SED in reducing disturbed sleep at follow-up (F(1,180.54) = 4.04, p = .046). In addition, self-reported posttraumatic stress disorder (F(1,56.42) = 4.50, p = .038) for the treatment effect, depression (F(1,93.70) = 4.44, p = .038), and fatigue symptoms (F(1,68.58) = 3.90, p = .050) at follow-up improved in MBB compared with those in SED. Consistently higher percentages of veterans in MBB reported improvements of sleep, pain, and composite sleep/general co-occurring symptoms at the postclinical evaluation, as compared with veterans in SED. Finally, the mean waking level of salivary α-amylase in the MBB declined to a greater extent than that in the SED, at follow-up (F(1,88.99) = 3.78, p = .055), whereas no effects were found on cortisol. CONCLUSIONS: Sleep-focused MBB can improve sleep and possibly also co-occurring symptoms in Gulf War veterans. CLINICAL TRIAL REGISTRATION: Clinicaltrials.gov, NCT01543997.

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.

Mortality among sheet metal workers participating in a respiratory screening program.

BACKGROUND: The Sheet Metal Occupational Health Institute Trust (SMOHIT) established a screening program in 1985 to examine the health hazards of the sheet metal industry in the U.S. and Canada. METHODS: 17,345 individuals with over 20 years in the trade and who participated in the program were followed for causes of death between 1986 and 2010. Both SMRs and Cox proportional hazards models investigated predictors of death due to lung cancer, mesothelioma, and chronic obstructive pulmonary disease (COPD). RESULTS: Significant excess mortality was seen for mesothelioma and asbestosis. Controlling for smoking, a strong trend for increasing lung cancer risk with increasing chest x-ray profusion >0/0 was observed. With an profusion score <1/0, FEV1 /FVC <80% was associated with lung cancer risk. COPD risk increased with increasing profusion score. CONCLUSIONS: This study demonstrates asbestos-related diseases among workers with largely indirect exposures and an increased lung cancer risk with low ILO scores.

Two sessions of sleep-focused mind-body bridging improve self-reported symptoms of sleep and PTSD in veterans: A pilot randomized controlled trial.

OBJECTIVE: Sleep disturbance is highly prevalent among veterans. As an alternative to sleep medications with their undesirable side effects, nonpharmacological mind-body interventions may be beneficial for sleep management in primary care. The aim of this pilot study was to investigate whether a novel mind-body intervention, mind-body bridging (MBB), focusing on sleep, could improve self-reported sleep disturbance and comorbid symptoms in veterans. METHODS: This pilot study was a randomized controlled trial at the Veterans Affairs Salt Lake City Health Care System in which 63 veterans with self-reported sleep disturbance received MBB or an active sleep education control. Both interventions were conducted in two sessions, once per week. Patient-reported outcomes included the following: primary-Medical Outcomes Study (MOS) Sleep Survey, MOS Short Form-36V; secondary-Center for Epidemiological Studies-Depression, PTSD Check List-Military, Five-Factor Mindfulness Questionnaire. RESULTS: At both Week 1 (1 week after the first session) and post-intervention assessments, while sleep disturbance decreased in both groups, MBB performed significantly better than did the control group. Furthermore, self-reported PTSD symptoms improved in MBB, while they remained unchanged in the control. Overall mindfulness increased in MBB, while it remained unchanged in the control. CONCLUSIONS: This study provides preliminary evidence that a brief sleep-focused MBB could be a promising intervention for sleep and potentially other comorbid symptoms (e.g., PTSD). MBB could help patients develop awareness skills to deal with sleep-related symptoms. Integration of MBB into primary care settings may enhance care of patients with sleep disturbance and co-morbid symptoms.

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 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.

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.

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.