In SOD1 transgenic rat model of ALS, breathing capacity is preserved until late in disease progression despite profound respiratory motor neuron (MN) cell death. To explore mechanisms preserving breathing capacity, we assessed inspiratory EMG activity in diaphragm and external intercostal T2 (EIC2) and T5 (EIC5) muscles in anesthetized SOD1 rats at disease end-stage (20% decrease in body mass). We hypothesized that despite significant phrenic motor neuron loss and decreased phrenic nerve activity, diaphragm electrical activity and trans-diaphragmatic pressure (Pdi) are maintained to sustain ventilation. We alternatively hypothesized that EIC activity is enhanced, compensating for impaired diaphragm function. Diaphragm, EIC2 and EIC5 muscle EMGs and Pdi were measured in urethane-anesthetized, spontaneously breathing female SOD1 rats versus wild-type littermates during normoxia (arterial PO ~90mmHg, PCO ~45mmHg), maximal chemoreceptor stimulation (MCS: 10.5% O/7% CO), spontaneous augmented breaths and sustained tracheal occlusion. Phrenic MNs were counted in C3-5; T2 and T5 ventrolateral MNs were counted. In end-stage SOD1 rats, 29% of phrenic MNs survived (vs. wild-type), yet integrated diaphragm EMG amplitude was normal. Nevertheless, maximal Pdi decreased ~30% vs. wild type (p<0.01) and increased esophageal to gastric pressure ratio (p<0.05), consistent with persistent diaphragm weakness. Despite major T2 and T5 MN death, integrated EIC2 (100% greater than wild type) and EIC5 (300%) EMG amplitudes were increased in mutant rats during normoxia (p<0.01), possibly compensating for decreased Pdi. Thus, despite significant phrenic MN loss, diaphragm EMG activity is maintained; in contrast, Pdi was not, suggesting diaphragm dysfunction. Presumably, increased EIC EMG activity compensated for persistent diaphragm weakness. These adjustments contribute to remarkable preservation of breathing ability despite major respiratory motor neuron death and diaphragm dysfunction.

Circadian rhythms are endogenous and entrainable daily patterns of physiology and behavior. Molecular mechanisms underlie circadian rhythms, characterized by an ~24-h pattern of gene expression of core clock genes. Although it has long been known that breathing exhibits circadian rhythms, little is known concerning clock gene expression in any element of the neuromuscular system controlling breathing. Furthermore, we know little concerning gene expression necessary for specific respiratory functions, such as phrenic motor plasticity. Thus, we tested the hypotheses that transcripts for clock genes ( Bmal1, Clock, Per1, and Per2) and molecules necessary for phrenic motor plasticity ( Htr2a, Htr2b, Bdnf, and Ntrk2) oscillate in regions critical for phrenic/diaphragm motor function via RT-PCR. Tissues were collected from male Sprague-Dawley rats entrained to a 12-h light-dark cycle at 4 zeitgeber times (ZT; n = 8 rats/group): ZT5, ZT11, ZT17, and ZT23; ZT0 = lights on. Here, we demonstrate that 1) circadian clock genes ( Bmal1, Clock, Per1, and Per2) oscillate in regions critical for phrenic/diaphragm function, including the caudal medulla, ventral C3–C5 cervical spinal cord, and diaphragm; 2) the clock protein BMAL1 is localized within CtB-labeled phrenic motor neurons; 3) genes necessary for intermittent hypoxia-induced phrenic/diaphragm motor plasticity ( Htr2b and Bdnf) oscillate in the caudal medulla and ventral C3–C5 spinal cord; and 4) there is higher intensity of immunofluorescent BDNF protein within phrenic motor neurons at ZT23 compared with ZT11 ( n = 11 rats/group). These results suggest local circadian clocks exist in the phrenic motor system and confirm the potential for local circadian regulation of neuroplasticity and other elements of the neural network controlling breathing.

Plasticity is a fundamental property of the neural system controlling breathing. One key example of respiratory motor plasticity is phrenic long-term facilitation (pLTF), a persistent increase in phrenic nerve activity elicited by acute intermittent hypoxia (AIH). pLTF can arise from distinct cell signaling cascades initiated by serotonin versus adenosine receptor activation, respectively; these signaling cascades interact via powerful cross-talk inhibition. Here, we demonstrate that the daily rest versus active phase and the duration of hypoxic episodes within an AIH protocol have profound impact on the magnitude and even mechanism of pLTF due to shifts in the serotonin/adenosine balance. Using the historical “standard” AIH protocol (3, 5 min moderate hypoxic episodes), we demonstrate that pLTF magnitude is unaffected by exposure in the mid-active versus mid-rest phase, yet the mechanism driving pLTF shifts from serotonin-dominant during mid-rest to adenosine-dominant in the mid-active phase. This mechanistic “flip” results from combined influences of hypoxia-evoked adenosine release and normal cycles in basal spinal adenosine between the rest versus active phase. Since AIH consisting of shorter hypoxic episodes but the same cumulative duration of hypoxia (15, 1 min episodes) elicits less adenosine release during hypoxic episodes, mid-rest pLTF is amplified due to a diminished adenosine constraint to serotonin-driven plasticity; on the other hand, this same 15 × 1 AIH protocol delivered in the mid-active phase suppresses serotonin-dominant pLTF due to elevated background adenosine levels but low hypoxia-evoked adenosine release. These findings demonstrate the importance of the serotonin/adenosine balance in regulating the amplitude and even mechanism of AIH-induced pLTF. Since AIH is emerging as a promising therapeutic modality to restore respiratory and non-respiratory movements in people with spinal cord injury or ALS, knowledge of how time-of-day and hypoxic episode duration impact the serotonin/adenosine balance and the magnitude and mechanism of pLTF has profound biological, experimental and translational implications.

Visual features associated with a task and those that predict noxious events both prompt selectively heightened visuocortical responses. Conflicting views exist regarding how the competition between a task‐related and a threat‐related feature is resolved when they co‐occur in time and space. Utilizing aversive classical Pavlovian conditioning, we investigated the visuocortical representation of two simultaneously presented, fully overlapping visual stimuli. Isoluminant red and green random dot kinematogram (RDK) stimuli were flickered at distinct tagging frequencies (8.57 Hz, 12 Hz) to elicit distinguishable steady‐state visual evoked potentials (ssVEPs). Occasional coherent motion events prompted a motor response (task) or predicted a noxious noise (threat). These events occurred either in the green (task cue), the red (threat cue), or in both RDKs simultaneously. In the initial habituation phase, participants responded to coherent motion of the green RDK with a key press, but no loud noise was presented at any time. Here, selective amplification was seen for the task‐relevant (green) RDK, and interference was observed when both RDKs simultaneously showed coherent motion. Upon pairing the threat cue with the noxious noise in the subsequent acquisition phase, the threat cue‐evoked ssVEP (red RDK) was also amplified, but this amplification did not interact with amplification of the task cue or alter the behavioral or visuocortical interference effect observed during simultaneous coherent motion. Although competing feature conjunctions resulted in interference in the visual cortex, the acquisition of a bias toward an individual threat‐related feature did not result in additional cost effects.

16Visual features that are associated with a task and those that predict noxious events both prompt 17 selectively heightened visuocortical responses. Conflicting views exist regarding how the 18 competition between a task-related and a threat-related feature is resolved when they co-occur in 19 time and space. Utilizing aversive differential Pavlovian conditioning, we investigated the 20 visuocortical representation of two simultaneously presented, fully overlapping visual stimuli. 21 Stimuli were isoluminant red and green random dot kinematograms (RDKs) which flickered at 22 two tagging frequencies (8.57 Hz, 12 Hz) to elicit distinguishable steady-state visual evoked 23 potentials (ssVEPs). Occasional coherent motion events prompted a motor response or predicted 24 a noxious noise. These events occurred either in the green (task cue), the red (threat cue), or in 25 both RDKs simultaneously. In an initial habituation phase, participants responded to coherent 26 motion of the green RDK with a key press, but no loud noise was presented at any time. Here, 27 selective amplification was seen for the task-relevant (green) RDK, but interference was 28 observed when both RDKs simultaneously showed coherent motion. Upon pairing the threat cue 29 with the noxious noise in the subsequent acquisition phase, the threat cue-evoked ssVEP (red 30 RDK) was also amplified, but this amplification did not interact with amplification of the task 31 cue, and did not alter the behavioral or visuocortical interference effect seen during simultaneous 32 coherent motion. Results demonstrate that although competing feature conjunctions result in 33 interference in visual cortex, the acquisition of a bias towards an individual threat-related feature 34 does not result in additional cost effects. 35 36 37 Cortical competition between task and threat cues 3 Significance statement 38 Selectively perceiving and adaptively responding to cues associated with danger are fundamental 39 functions of the vertebrate brain. In humans, their disruption or dysregulation is at the core of 40 many psychiatric diagnoses, including fear, anxiety, post-traumatic syndromes, and mood 41 disorders. The present study examined the competitive interactions between the prioritization of 42 threat cues and a concurrent cognitive task, to characterize how the human attention system 43 manages limited resources in the presence of threat. Results showed that the selection of an 44 individual feature signaling imminent threat is not at the cost of concurrent attention 45 performance, even when threat and task stimuli overlapped in space. Findings support recent 46 models of emotion/attention interactions that emphasize flexible, feature-based allocation of 47 resources to biologically relevant stimuli. 48 49 Cortical competition between task and threat cues 4 Introduction 50 The visual system receives dense sensory information, continually exceeding the limited 51 capacity of visual cognition. In response to this challenge, the human brain has evolved 52 mechanisms...