Eating during the biological night is associated with nausea.

OBJECTIVES: This study assessed whether there was a time-of-day effect on nausea reports in participants during studies employing circadian protocols. METHODS: Visual-analog-scales of nausea ratings were recorded from 34 participants (18-70years; 18 women) during forced desynchrony studies, where meals were scheduled at different circadian phases. Subjective nausea reports from a further 81 participants (18-35years; 36 women) were recorded during constant routine studies, where they ate identical isocaloric hourly snacks for 36-40 hours. RESULTS: Feelings of nausea varied by circadian phase in the forced desynchrony studies, peaking during the biological night. Nausea during the constant routine was reported by 27% of participants, commencing 2.9 ± 5.2 hours after the midpoint of usual sleep timing, but was never reported to start in the evening (4-9 PM). CONCLUSIONS: Nausea occurred more often during the biological night and early morning hours. This timing is relevant to overnight and early morning shift workers and suggests that a strategy to counteract that is to pay careful attention to meal timing.

Late isocaloric eating increases hunger, decreases energy expenditure, and modifies metabolic pathways in adults with overweight and obesity.

Late eating has been linked to obesity risk. It is unclear whether this is caused by changes in hunger and appetite, energy expenditure, or both, and whether molecular pathways in adipose tissues are involved. Therefore, we conducted a randomized, controlled, crossover trial (ClinicalTrials.gov NCT02298790) to determine the effects of late versus early eating while rigorously controlling for nutrient intake, physical activity, sleep, and light exposure. Late eating increased hunger (p < 0.0001) and altered appetite-regulating hormones, increasing waketime and 24-h ghrelin:leptin ratio (p < 0.0001 and p = 0.006, respectively). Furthermore, late eating decreased waketime energy expenditure (p = 0.002) and 24-h core body temperature (p = 0.019). Adipose tissue gene expression analyses showed that late eating altered pathways involved in lipid metabolism, e.g., p38 MAPK signaling, TGF-ß signaling, modulation of receptor tyrosine kinases, and autophagy, in a direction consistent with decreased lipolysis/increased adipogenesis. These findings show converging mechanisms by which late eating may result in positive energy balance and increased obesity risk.

Daytime eating prevents mood vulnerability in night work.

Shift workers have a 25 to 40% higher risk of depression and anxiety partly due to a misalignment between the central circadian clock and daily environmental/behavioral cycles that may negatively affect mood and emotional well-being. Hence, evidence-based circadian interventions are required to prevent mood vulnerability in shift work settings. We used a stringently controlled 14-d circadian paradigm to assess mood vulnerability during simulated night work with either daytime and nighttime or daytime-only eating as compared with simulated day work (baseline). Simulated night work with daytime and nighttime eating increased depression-like mood levels by 26.2% (p-value adjusted using False Discovery Rates, pFDR = 0.001; effect-size r = 0.78) and anxiety-like mood levels by 16.1% (pFDR = 0.001; effect-size r = 0.47) compared to baseline, whereas this did not occur with simulated night work in the daytime-only eating group. Importantly, a larger degree of internal circadian misalignment was robustly associated with more depression-like (r = 0.77; P = 0.001) and anxiety-like (r = 0.67; P = 0.002) mood levels during simulated night work. These findings offer a proof-of-concept demonstration of an evidence-based meal timing intervention that may prevent mood vulnerability in shift work settings. Future studies are required to establish if changes in meal timing can prevent mood vulnerability in night workers.

Impact of chronic sleep restriction on sleep continuity, sleep structure, and neurobehavioral performance.

Chronic sleep restriction (CSR) has been associated with adverse effects including cognitive impairment and increased risk of diabetes and cardiovascular disease. Yet, sleep restriction therapy is an essential component of most behavioral treatments for insomnia. Moreover, little is known about the impact of CSR on sleep continuity and structure in healthy people whose need for sleep is satiated. We investigated the impact of CSR on sleep continuity and structure in nine healthy participants. They had 4 nights of sleep extension, 2 nights of post-extension sleep, 21 nights of CSR (5/5.6-hour time-in-bed), and 9 nights of recovery sleep. Compared to postextension sleep, during CSR sleep duration was reduced by 95.4 ±â€…21.2 min per night, Slow-Wave Activity was significantly increased, and sleep was more consolidated. During recovery, sleep duration was increased by 103.3 ±â€…23.8 min compared to CSR, and the CSR-induced increase in Slow-Wave Activity persisted, particularly after the 5-hour exposure. Yet, we found that sustained vigilant attention was not fully recovered even after nine nights of recovery sleep. Our results suggest that CSR improves traditional metrics of sleep quality and may have a persistent impact on sleep depth, which is consistent with the reported benefits on sleep continuity and structure of sleep restriction therapy. However, these improvements in traditional metrics of sleep quality were associated with deterioration rather than improvement in neurobehavioral performance, demonstrating that sleep duration should be included in assessments of sleep quality. These results have implications for the long-term use of sleep restriction in the behavioral treatment of insomnia. Clinical Trial Registration: Impact of Chronic Circadian Disruption vs. Chronic Sleep Restriction on Metabolism (https://clinicaltrials.gov/ct2/show/; #NCT02171273).

Chronic circadian disruption on a high-fat diet impairs glucose tolerance.

BACKGROUND: Nearly 14% of Americans experience chronic circadian disruption due to shift work, increasing their risk of obesity, diabetes, and other cardiometabolic disorders. These disorders are also exacerbated by modern eating habits such as frequent snacking and consumption of high-fat foods. METHODS: We investigated the effects of recurrent circadian disruption (RCD) on glucose metabolism in C57BL/6 mice and in human participants exposed to non-24-h light-dark (LD) schedules vs. those on standard 24-h LD schedules. These LD schedules were designed to induce circadian misalignment between behaviors including rest/activity and fasting/eating with the output of the near-24-h central circadian pacemaker, while minimizing sleep loss, and were maintained for 12 weeks in mice and 3 weeks in humans. We examined interactions of these circadian-disrupted schedules compared to control 24-h schedules with a lower-fat diet (LFD, 13% in mouse and 25-27% in humans) and high-fat diet (HFD, 45% in mouse and 45-50% in humans). We also used young vs. older mice to determine whether they would respond differently to RCD. RESULTS: When combined with a HFD, we found that RCD caused significant weight gain in mice and increased body fat in humans, and significantly impaired glucose tolerance and insulin sensitivity in both mice and humans, but this did not occur when RCD was combined with a LFD. This effect was similar in both young and older mice. CONCLUSION: These results in both humans and a model organism indicate that circadian disruption has an adverse effect on metabolism among individuals eating a high-fat Western-style diet, even in the absence of significant sleep loss, and suggest that reducing dietary fat may protect against the metabolic consequences of a lifestyle (such as shift work) that involves chronic circadian disruption.

Proof-of-principle demonstration of endogenous circadian system and circadian misalignment effects on human oral microbiota.

Circadian misalignment-the misalignment between the central circadian "clock" and behavioral and environmental cycles (including sleep/wake, fasting/eating, dark/light)-results in adverse cardiovascular and metabolic effects. Potential underlying mechanisms for these adverse effects include alterations in the orogastrointestinal microbiota. However, it remains unknown whether human oral microbiota has endogenous circadian rhythms (i.e., independent of sleep/wake, fasting/eating, and dark/light cycles) and whether circadian misalignment influences oral microbiota community composition. Healthy young individuals [27.3 ± 2.3 years (18-35 years), 4 men and 2 women, body-mass index range: 18-28 kg/m2 ] were enrolled in a stringently controlled 14-day circadian laboratory protocol. This included a 32-h constant routine (CR) protocol (endogenous circadian baseline assessment), a forced desynchrony protocol with four 28-h "days" under ~3 lx to induce circadian misalignment, and a post-misalignment 40-h CR protocol. Microbiota assessments were performed on saliva samples collected every 4 h throughout both CR protocols. Total DNA was extracted and processed using high-throughput 16S ribosomal RNA gene amplicon sequencing. The relative abundance of specific oral microbiota populations, i.e., one of the five dominant phyla, and three of the fourteen dominant genera, exhibited significant endogenous circadian rhythms. Importantly, circadian misalignment dramatically altered the oral microbiota landscape, such that four of the five dominant phyla and eight of the fourteen dominant genera exhibited significant circadian misalignment effects. Moreover, circadian misalignment significantly affected the metagenome functional content of oral microbiota (inferred gene content analysis), as indicated by changes in specific functional pathways associated with metabolic control and immunity. Collectively, our proof-of-concept study provides evidence for endogenous circadian rhythms in human oral microbiota and show that even relatively short-term experimental circadian misalignment can dramatically affect microbiota community composition and functional pathways involved in metabolism and immune function. These proof-of-principle findings have translational relevance to individuals typically exposed to circadian misalignment, including night shift workers and frequent flyers.

Daytime eating prevents internal circadian misalignment and glucose intolerance in night work.

Night work increases diabetes risk. Misalignment between the central circadian "clock" and daily behaviors, typical in night workers, impairs glucose tolerance, likely due to internal misalignment between central and peripheral circadian rhythms. Whether appropriate circadian alignment of eating can prevent internal circadian misalignment and glucose intolerance is unknown. In a 14-day circadian paradigm, we assessed glycemic control during simulated night work with either nighttime or daytime eating. Assessment of central (body temperature) and peripheral (glucose and insulin) endogenous circadian rhythms happened during constant routine protocols before and after simulated night work. Nighttime eating led to misalignment between central and peripheral (glucose) endogenous circadian rhythms and impaired glucose tolerance, whereas restricting meals to daytime prevented it. These findings offer a behavioral approach to preventing glucose intolerance in shift workers.

Chronic Sleep Restriction While Minimizing Circadian Disruption Does Not Adversely Affect Glucose Tolerance.

Insufficient sleep, which has been shown to adversely affect metabolism, is generally associated with prolonged exposure to artificial light at night, a known circadian disruptor. There is growing evidence suggesting that circadian disruption adversely affects metabolism, yet few studies have attempted to evaluate the adverse metabolic effects of insufficient sleep while controlling for circadian disruption. We assessed postprandial glucose and insulin responses to a standard breakfast meal in healthy adults (n = 9) who underwent 3 weeks of chronic sleep restriction (CSR) in a 37-day inpatient study while minimizing circadian disruption by maintaining the same duration of light exposure each study day. We compared these results to findings from an earlier inpatient study which used a forced desynchrony (FD) protocol to assess the influence of 3 weeks of CSR combined with recurrent circadian disruption (RCD) on glycemic control in healthy adults (n = 21). CSR combined with RCD resulted in significantly elevated postprandial plasma glucose levels (p < 0.0001), while CSR with minimized circadian disruption had no adverse glycemic effects after 3 weeks of exposure (EXP). These results suggest that one mechanism by which sleep restriction impacts metabolism may be via concurrent circadian disruption.

3D-Printed Personal Protective Face Shields During the COVID-19 Pandemic: A Survey of Canadian Frontline Workers.

Background During the coronavirus disease 2019 pandemic, three-dimensional (3D) printing was utilized to rapidly produce face shields for frontline workers in response to an acute shortage of personal protective equipment (PPE). In this study, we examine the perceived utility and performance of 3D-printed (3DP) face shields through a survey of frontline workers in Ontario, Canada. Methodology Frontline workers who received community-produced 3DP face shields from the Canadian initiative "3DPPE GTHA" (March-December 2020) were invited to participate in the study. The survey response rate was 54.3%. Of 63 respondents, 39 were patient-facing and 24 were community-facing frontline workers. Participants were asked to rate performance measures in 10 categories on a five-point Likert scale. Data were categorized by organization and frontline worker type, and a t-test was used to determine statistically significant differences among subgroups. Results The mean preference for 3DP face shields among respondents was 3.2 out of 5 (95% confidence interval [CI]: 2.1-4.3). Community-facing respondents reported significantly greater overall utility scores for 3DP face shields (3.58, 95% CI: 3.38-3.79) compared to respondents working in a patient-facing profession (2.95, 95% CI: 2.77-3.13; p < 0.05). However, no differences were reported in portability and compatibility with other PPE. Respondents from organizations with large service volumes reported significantly lower overall utility scores (2.67, 95% CI: 2.44-2.89) than respondents in organizations with smaller service volumes (3.45, 95% CI: 3.28-3.62; p < 0.05). Conclusions Community-facing frontline workers and those from smaller service volume organizations endorse higher utility for 3DP face shields than patient-facing frontline workers. Despite this, frontline workers generally rate 3DP face shields positively. 3DP face shields are a viable option for personal and community use and can be used to supplement supply in a community setting.

Sleep Restriction With Circadian Disruption Negatively Alter Bone Turnover Markers in Women.

PURPOSE: The purpose of this work is to determine whether an uncoupling of bone turnover markers (BTMs) occurs in women exposed to the combination of sleep restriction with circadian disruption (SRCD), as previously reported in men. METHODS: Four bone biomarkers (N-terminal propeptide of type I procollagen [P1NP] and osteocalcin = bone formation; C-telopeptide [CTX] = bone resorption; sclerostin = bone formation inhibitor) were measured in bihourly samples over 24 hours at baseline and after approximately 3 weeks of sleep restriction (~5.6 hours of sleep/24 hours) with concurrent circadian disruption (SRCD, recurring 28-hour "day" in dim light). Maximum likelihood estimation in a repeated-measures model was used to assess the effects of SRCD and age on bone biomarkers. RESULTS: Five women were young (22 ±â€…2.8 years) and four were older (58 ±â€…1.8 years). Baseline bone biomarker levels did not differ by age (all P ≥ .07). Bone formation markers were lower after SRCD (estimate ±â€…SEE, ΔP1NP = -9.5 ±â€…2.8 µg/L, P = .01; Δosteocalcin = -2.3 ±â€…0.9 ng/mL, P = .04). The P1NP decline was greater in young women (ΔP1NP = -12.9 ±â€…3.7 µg/L, P = .01). After SRCD, CTX was significantly higher in young women (0.182 ±â€…0.069 ng/mL, P = .04) but did not change in older women. CONCLUSIONS: These pilot data are similar to previous findings in men and suggest that SRCD negatively altered bone metabolism in women by decreasing markers of bone formation and, in young women, increasing a marker of bone resorption. If sustained, this pattern of BTM uncoupling may lead to bone loss and lower bone mineral density.

Effects of Shift Work on the Eating Behavior of Police Officers on Patrol.

Recent studies indicate that the timing of food intake can significantly affect metabolism and weight management. Workers operating at atypical times of the 24-h day are at risk of disturbed feeding patterns. Given the increased risk of weight gain, obesity and metabolic syndrome in shift working populations, further research is required to understand whether their eating behavior could contribute to these increased metabolic risks. The objective of this study was to characterize the dietary patterns of police officers across different types of shifts in their natural environments. Thirty-one police officers (six women; aged 32.1 ± 5.4 years, mean ± SD) from the province of Quebec, Canada, participated in a 28- to 35-day study, comprising 9- to 12-h morning, evening, and night shifts alternating with rest days. Sleep and work patterns were recorded with actigraphy and diaries. For at least 24 h during each type of work day and rest day, participants logged nutrient intake by timestamped photographs on smartphones. Macronutrient composition and caloric content were estimated by registered dieticians using the Nutrition Data System for Research database. Data were analyzed with linear mixed effects models and circular ANOVA. More calories were consumed relative to individual metabolic requirements on rest days than both evening- and night-shift days (p = 0.001), largely sourced from increased fat (p = 0.004) and carbohydrate (trend, p = 0.064) intake. Regardless, the proportions of calories from carbohydrates, fat, and protein did not differ significantly between days. More calories were consumed during the night, between 2300 h and 0600 h, on night-shift days than any other days (p < 0.001). Caloric intake occurred significantly later for night-shift days (2308 h ± 0114 h, circular mean ± SD) than for rest days (1525 h ± 0029 h; p < 0.01) and was dispersed across a longer eating window (13.9 h ± 3.1 h vs. 11.3 h ± 1.8 h, mean ± SD). As macronutrient proportions were similar and caloric intake was lower, the finding of later meals times on night-shift days versus rest days is consistent with emerging hypotheses that implicate the biological timing of food intake-rather than its quantity or composition-as the differentiating dietary factor in shift worker health.

Impact of Circadian Disruption on Cardiovascular Function and Disease.

The circadian system, that is ubiquitous across species, generates ∼24 h rhythms in virtually all biological processes, and allows them to anticipate and adapt to the 24 h day/night cycle, thus ensuring optimal physiological function. Epidemiological studies show time-of-day variations in adverse cardiovascular (CV) events, and controlled laboratory studies demonstrate a circadian influence on key markers of CV function and risk. Furthermore, circadian misalignment, that is typically experienced by shift workers as well as by individuals who experience late eating, (social) jet lag, or circadian rhythm sleep-wake disturbances, increases CV risk factors. Therefore, understanding the mechanisms by which the circadian system regulates CV function, and which of these are affected by circadian disruption, may help to develop intervention strategies to mitigate CV risk.

Human Resting Energy Expenditure Varies with Circadian Phase.

There is emerging evidence that circadian misalignment may alter energy expenditure, leading to obesity risk among those with irregular schedules [1-5]. It has been reported that energy expenditure is affected by the timing of sleep, exercise, and meals [6]. However, it is unclear whether the circadian system also modulates energy expenditure, independent of behavioral state and food intake. Here, we used a forced desynchrony protocol to examine whether fasted resting energy expenditure (REE) varies with circadian phase in seven participants. This protocol allowed us to uncouple sleep-wake and activity-related effects from the endogenous circadian rhythm, demonstrating that REE varies by circadian phase. REE is lowest at circadian phase ∼0°, corresponding to the endogenous core body temperature (CBT) nadir in the late biological night, and highest at circadian phase ∼180° in the biological afternoon and evening. Furthermore, we found that respiratory quotient (RQ), reflecting macronutrient utilization, also varies by circadian phase. RQ is lowest at circadian phase ∼240° and highest at circadian phase ∼60°, which corresponds to biological morning. This is the first characterization of a circadian profile in fasted resting energy expenditure and fasted respiratory quotient (with rhythmic profiles in both carbohydrate and lipid oxidation), decoupled from effects of activity, sleep-wake cycle, and diet in humans. The rhythm in energy expenditure and macronutrient metabolism may contribute to greater weight gain in shift workers and others with irregular schedules.

Supramammillary glutamate neurons are a key node of the arousal system.

Basic and clinical observations suggest that the caudal hypothalamus comprises a key node of the ascending arousal system, but the cell types underlying this are not fully understood. Here we report that glutamate-releasing neurons of the supramammillary region (SuMvglut2) produce sustained behavioral and EEG arousal when chemogenetically activated. This effect is nearly abolished following selective genetic disruption of glutamate release from SuMvglut2 neurons. Inhibition of SuMvglut2 neurons decreases and fragments wake, also suppressing theta and gamma frequency EEG activity. SuMvglut2 neurons include a subpopulation containing both glutamate and GABA (SuMvgat/vglut2) and another also expressing nitric oxide synthase (SuMNos1/Vglut2). Activation of SuMvgat/vglut2 neurons produces minimal wake and optogenetic stimulation of SuMvgat/vglut2 terminals elicits monosynaptic release of both glutamate and GABA onto dentate granule cells. Activation of SuMNos1/Vglut2 neurons potently drives wakefulness, whereas inhibition reduces REM sleep theta activity. These results identify SuMvglut2 neurons as a key node of the wake-sleep regulatory system.

Bone Turnover Markers After Sleep Restriction and Circadian Disruption: A Mechanism for Sleep-Related Bone Loss in Humans.

Context: Sleep abnormalities are associated with low bone mineral density. Underlying mechanisms are unknown. Objective: Investigate the impact of sleep restriction with circadian disruption on bone biomarkers. Design: Intervention study. Participants and Methods: Four bone biomarkers [C-terminal cross-linked telopeptide of type I collagen (CTX) = bone resorption, N-terminal propeptide of type I procollagen (P1NP) = bone formation, sclerostin and fibroblast growth factor 23 = osteocyte function] were measured in bihourly serum samples over 24 hours at baseline and after ∼3 weeks of sleep restriction (5.6 hours sleep/24 hours) with concurrent circadian disruption (recurring 28-hour "day" in dim light) in 10 men (age groups: 20 to 27 years, n = 6; 55 to 65 years, n = 4). The effects of sleep/circadian disruption and age on bone biomarker levels were evaluated using maximum likelihood estimation in a mixed model for repeated measures. Results: P1NP levels were lower after intervention compared with baseline (P < 0.001); the decrease in P1NP was greater for younger compared with older men (28.0% vs 18.2%, P < 0.001). There was no change in CTX (Δ = 0.03 ± 0.02 ng/mL, P = 0.10). Sclerostin levels were higher postintervention in the younger men only (Δ = 22.9% or 5.64 ± 1.10 pmol/L, P < 0.001). Conclusions: These data suggest that 3 weeks of circadian disruption with concurrent sleep restriction can lead to an uncoupling of bone turnover wherein bone formation is decreased but bone resorption is unchanged. Circadian disruption and sleep restriction may be most detrimental to bone in early adulthood.

Projections from the subparaventricular zone define four channels of output from the circadian timing system.

The subparaventricular zone of the hypothalamus (SPZ) is the main efferent target of neural projections from the suprachiasmatic nucleus (SCN) and an important relay for the circadian timing system. Although the SPZ is fairly homogeneous cytoarchitecturally and neurochemically, it has been divided into distinct functional and connectional subdivisions. The dorsal subdivision of the SPZ (dSPZ) plays an important role in relaying signals from the SCN controlling body temperature rhythms, while the ventral subdivision (vSPZ) is critical for rhythms of sleep and locomotor activity (Lu et al. [] J Neurosci 21:4864-4874). On the other hand, the medial part of the SPZ receives input mainly from the dorsomedial SCN, whereas the lateral SPZ receives input from the ventrolateral SCN and the retinohypothalamic tract (Leak and Moore [] J Comp Neurol 433:312-334). We therefore investigated whether there are corresponding differences in efferent outputs from these four quadrants of the SPZ (dorsolateral, ventrolateral, dorsomedial, and ventromedial) by a combination of anterograde and retrograde tracing. We found that, while all four subdivisions of the SPZ share a similar backbone of major projection pathways to the septal region, thalamus, hypothalamus, and brainstem, each segment of the SPZ has a specific set of targets where its projections dominate. Furthermore, we observed intra-SPZ projections of varying densities between the four subdivisions. Taken together, this pattern of organization suggests that the circadian timing system may have several parallel neural outflow pathways that provide a road map for understanding how they subserve different functions.

Sympathetic input modulates, but does not determine, phase of peripheral circadian oscillators.

The circadian clock in the suprachiasmatic nucleus (SCN) maintains phase synchrony among circadian oscillators throughout the organism. Environmental light signals entrain the SCN, but timed, limited meal access acts as an overriding time cue for several peripheral tissues. We present data from a peripheral oscillator, the submaxillary salivary gland, in which temporal restriction of meals fails to entrain gene expression. In day-fed rats, submaxillary gland rhythms in expression of the clock gene Period1 (Per1) stay entrained to the light cycle (peaking at night) or become arrhythmic. This result suggests that feeding cues compete weakly with light cycle cues to set the phase of clock genes in this tissue. Since the submaxillary glands receive sympathetic innervation originating in the SCN, which relays light cycle cues to other oscillators, we attempted to assess the role of this neural input in phase control of submaxillary Per1 expression. We sympathetically denervated the submaxillary glands before subjecting rats to daytime-restricted feeding. After denervation, Per1 rhythms in all submaxillary glands shifted phase 180 degrees and entrained to daytime feeding. These results support the hypothesis that peripheral oscillators may receive multiple signals contributing to their phase of entrainment. Sympathetic efferents from the SCN can relay light cycle information, while other external cues may reach tissues through other efferents or nonneural pathways. In an abnormal, disruptive regimen such as daytime-restricted feeding, these different signals compete. Arrhythmicity may result if one signal is not clearly dominant. Elimination of the dominant signal (e.g., surgical sympathectomy) may allow a secondary signal to control phase.

Coexpression of vesicular glutamate transporter-3 and gamma-aminobutyric acidergic markers in rat rostral medullary raphe and intermediolateral cell column.

Markers of serotonergic, gamma-aminobutyric acid (GABA)-ergic (glutamic acid decarboxylase, 67 kDa isoform; GAD-67), and glutamatergic transmission (vesicular glutamate transporter 3; VGLUT3) have been detected in presumed sympathetic premotor neurons of the medullary raphe, a region that controls sympathetic tone to brown fat, skin blood vessels, and heart. In this study, the degree of coexpression of these markers was examined in raphe neurons by simultaneous histological detection of tryptophan hydroxylase (TrpOH) immunoreactivity with GAD-67 mRNA and VGLUT3 mRNA. Over half (52%) of the VGLUT3 mRNA-positive neurons expressed one or both of the other markers. The proportion of VGLUT3 neurons containing at least one of the other two markers was even higher (89%) for VGLUT3 spinally projecting neurons. VGLUT3 neurons containing markers for both serotonin and GABA were especially numerous (50-72%, depending on rostrocaudal level) within the marginal layer of raphe pallidus and the parapyramidal region. The dual GABAergic and glutamatergic nature of some bulbospinal raphe neurons was suggested by the presence of nerve terminals immunoreactive (ir) for both VGLUT3 and GABA in the intermediolateral cell column (IML) as detected by electron microscopy. VGLUT3-ir terminals formed approximately equal numbers of symmetric and asymmetric synapses onto presumed preganglionic neurons (nitric oxide synthase-ir profiles) or GABA-ir dendrites in IML, and terminals immunoreactive for both VGLUT3 and GABA always formed symmetric synapses. These data suggest that medullary raphe VGLUT3 neurons could inhibit sympathetic outflow and that their spinal targets include both preganglionic neurons and GABAergic interneurons.