Prenatal intermittent hypoxia sensitizes the laryngeal chemoreflex, blocks serotoninergic shortening of the reflex, and reduces 5-HT3 receptor binding in the NTS in anesthetized rat pups.

We tested the hypothesis that exposure to intermittent hypoxia (IH) during pregnancy would prolong the laryngeal chemoreflex (LCR) and diminish the capacity of serotonin (5-hydroxytryptamine; 5-HT) to terminate the LCR. Prenatal exposure to IH was associated with significant prolongation of the LCR in younger, anesthetized, postnatal day (P) rat pups age P8 to P16 compared to control, room air (RA)-exposed rat pups of the same age. Serotonin microinjected into the NTS shortened the LCR in rat pups exposed to RA during gestation, but 5-HT failed to shorten the LCR in rat pups exposed to prenatal IH. Given these observations, we tested the hypothesis that prenatal hypoxia would decrease binding to 5-HT3 receptors in the nucleus of the solitary tract (NTS) where 5-HT acts to shorten the LCR. Serotonin 3 receptor binding was reduced in younger rat pups exposed to IH compared to control, RA-exposed rat pups in the age range P8 to P12. Serotonin 3 receptor binding was similar in older animals (P18-P24) regardless of gas exposure during gestation. The failure of the 5-HT injected into the NTS to shorten the LCR was correlated with a developmental decrease in 5-HT3 receptor binding in the NTS associated with exposure to prenatal IH. In summary, prenatal IH sensitized reflex apnea and blunted processes that terminate reflex apneas in neonatal rat pups, processes that are essential to prevent death following apneas such as those seen in babies who died of SIDS.

Activation of serotonergic neurons in the medullary caudal raphe shortens the laryngeal chemoreflex in anaesthetized neonatal rats.

NEW FINDINGS: What is the central question of this study? Does activation of serotonergic neurons in the caudal medullary raphe, some of which project to the nucleus of the solitary tract, shorten the laryngeal chemoreflex? What is the main finding and its importance? We found that serotonin originating from neurons in the caudal raphe acts through a 5-HT3 receptor located in the nucleus of the solitary tract to terminate reflex apnoea. Failure or deficiency of this arousal-related process is likely to be relevant to the pathogenesis of sudden infant death syndrome. Failure to terminate apnoea and arouse is likely to contribute to sudden infant death syndrome (SIDS). Serotonin is deficient in the brainstems of babies who have died of SIDS. We tested the hypothesis that activation of serotoninergic neurons in the caudal medullary raphe, some of which project to the nucleus of the solitary tract (NTS), would shorten the laryngeal chemoreflex (LCR). We studied anaesthetized neonatal rat pups between postnatal days 9 and 17. We injected 5-40 µl of water into the larynx to elicit the LCR and measured the duration of respiratory disruption. Microinjection of 50 nl of 100 µm AMPA into the caudal medullary raphe shortened the apnoeas (P < 0.001) and respiratory inhibition (P < 0.005) associated with the LCR. When 50 nl of 30 mm ondansetron, a 5-HT3 antagonist, was microinjected bilaterally into the NTS, AMPA microinjected into the caudal raphe no longer shortened the LCR. After bilateral microinjection of vehicle into the NTS, AMPA microinjection into the caudal raphe significantly shortened the LCR. AMPA, a glutamate receptor agonist, may activate many neurons within the caudal raphe, but blocking the 5-HT3 receptor-dependent responses in the NTS prevented the shortening of the LCR associated with AMPA microinjections into the caudal raphe. Thus, serotonin originating from neurons in the caudal raphe acts through a 5-HT3 receptor located in the NTS to terminate or shorten the LCR. Serotonin is deficient in the brainstems of babies who have died of SIDS, and deficient serotonergic termination of apnoea is likely to be relevant to the pathogenesis of SIDS.

Interleukin-1ß and interleukin-6 enhance thermal prolongation of the LCR in decerebrate piglets.

Thermal stress and prior upper respiratory tract infection are risk factors for the Sudden Infant Death Syndrome. The adverse effects of prior infection are likely mediated by interleukin-1ß (IL-1ß). Therefore, we examined the single and combined effects of IL-1ß and elevated body temperature on the duration of the Laryngeal Chemoreflex (LCR) in decerebrate neonatal piglets ranging in age from post-natal day (P) 3 to P7. We examined the effects of intraperitoneal (I.P.) injections of 0.3mg/Kg IL-1ß with or without I.P. 10mg/Kg indomethacin pretreatment on the duration of the LCR, and in the same animals we also examined the duration of the LCR when body temperature was elevated approximately 2°C. We found that IL-1ß significantly increased the duration of the LCR even when body temperature was held constant. There was a significant multiplicative effect when elevated body temperature was combined with IL-1ß treatment: prolongation of the LCR was significantly greater than the sum of independent thermal and IL-1ß-induced prolongations of the LCR. The effects of IL-1ß, but not elevated body temperature, were blocked by pretreatment with indomethacin alone. We also tested the interaction between IL-6 given directly into the nucleus of the solitary tract (NTS) bilaterally in 100ngm microinjections of 50µL and pretreatment with indomethacin. Here again, there was a multiplicative effect of IL-6 treatment and elevated body temperature, which significantly prolonged the LCR. The effect of IL-6 on the LCR, but not elevated body temperature, was blocked by pretreatment with indomethacin. We conclude that cytokines interact with elevated body temperature, probably through direct thermal effects on TRPV1 receptors expressed pre-synaptically in the NTS and through cytokine-dependent sensitization of the TRPV1 receptor. This sensitization is likely initiated by cyclo-oxygenase-2 dependent synthesis of prostaglandin E2, which is stimulated by elevated levels of IL-1ß or IL-6. Inflammatory sensitization of the LCR coupled with thermal prolongation of the LCR may increase the propensity for apnea and Sudden Infant Death Syndrome.

TRPV1 channels in the nucleus of the solitary tract mediate thermal prolongation of the LCR in decerebrate piglets.

Elevating body temperature or just the temperature of the dorsal medulla by approximately 2°C prolongs the laryngeal chemoreflex (LCR) in decerebrate neonatal piglets. We tested the hypothesis that transient receptor potential vanilloid 1 (TRPV1) receptors in the nucleus of the solitary tract (NTS) mediate thermal prolongation of the LCR. We studied the effect of a selective TRPV1 receptor antagonist on thermal prolongation of the LCR, and we tested the effect of a TRPV1 agonist on the duration of the LCR under normothermic conditions. We studied 37 decerebrate neonatal piglets between the ages of post-natal days 4 and 7. The TRPV1 receptor antagonist, 5'-iodoresiniferatoxin (65µM/L in 100nL), blocked thermal prolongation of the LCR when injected bilaterally into the region of the NTS. The TRPV1 agonist, resiniferatoxin (0.65-1.0mM/L in 100nL), prolonged the LCR after bilateral injection into the NTS even when the body temperature of each piglet was normal. The effect of the TRPV1 agonists could be blocked by treatment with the GABA(A) receptor antagonist, bicuculline, whether given intravenously (0.3mg/kg) or focally injected bilaterally into the NTS (10mM in 100nL). We conclude that TRPV1 receptors in the NTS mediate thermal prolongation of the LCR.

An adenosine A(2A) agonist injected in the nucleus of the solitary tract prolongs the laryngeal chemoreflex by a GABAergic mechanism in decerebrate piglets.

Hyperthermic prolongation of the laryngeal chemoreflex (LCR) in decerebrate piglets is prevented or reversed by GABA(A) receptor antagonists and adenosine A(2A) (Ad-A(2A)) receptor antagonists administered in the nucleus of the solitary tract (NTS). Therefore, we tested the hypothesis that enhanced GABA(A) activity and administration of the Ad-A(2A) agonist, CGS-21680, would prolong the LCR in normothermic conditions. We studied 46 decerebrate piglets ranging from 3 to 8 postnatal days of age. Focal injection into the NTS of 100 nl of 0.5 m nipecotic acid, a GABA reuptake inhibitor, significantly (P < 0.05) prolonged the LCR in normothermic conditions in 10 of 11 animals tested. Injecting 100 nl of 5-12.5 microm CGS-21680 unilaterally or bilaterally into the NTS also prolonged the LCR in normothermic conditions (n = 15), but the effect was smaller than that of unilateral injection of nipecotic acid. Systemic administration of the GABA(A) receptor antagonist, bicuculline, prevented the CGS-21680-dependent prolongation of the LCR in normothermic animals (n = 11). We conclude that thermal prolongation of the LCR depends on a thermally sensitive process or set of neurons in the NTS, which, when activated by elevated brain temperature, enhances adenosinergic and GABAergic function in the region of the NTS. These results emphasize the importance of a thermally sensitive integrative site in the dorsal medulla that, along with sites in the ventral medulla, determine the response to laryngeal chemoreflex stimulation.

Gestational cigarette smoke exposure and hyperthermic enhancement of laryngeal chemoreflex in rat pups.

Laryngeal chemoreflex (LCR) apnea occurs in infant mammals of many species in response to water or other liquids in the laryngeal lumen. The apnea can last for many seconds, sometimes leading to dangerous hypoxemia, and has therefore been considered as a possible mechanism in the Sudden Infant Death Syndrome (SIDS). We have found recently that this reflex is markedly prolonged in decerebrate piglets and anesthetized rat pups that are warmed 1-3 degrees C above their normal body temperatures. We intermittently exposed pregnant rats to cigarette smoke and examined the LCR in their four- to fifteen-day-old offspring under general anesthesia, with and without whole body warming. During warming, pups of gestationally smoke-exposed dams had significantly longer LCR-induced respiratory disruption than similarly warmed control pups. The results may be significant for the pathogenesis and/or prevention of SIDS as maternal cigarette smoking during human pregnancy and heat stress in infants are known risk factors for SIDS.

An adenosine A(2A) antagonist injected in the NTS reverses thermal prolongation of the LCR in decerebrate piglets.

Hyperthermia prolongs the laryngeal chemoreflex (LCR). Under normothermic conditions, adenosine antagonists shorten and adenosine A(2A) (Ad-A(2A)) agonists prolong the LCR. Therefore, we tested the hypothesis that SCH-58261, an Ad-A(2A) receptor antagonist, would prevent thermal prolongation of the LCR when injected unilaterally within the nucleus of the solitary tract (NTS). We studied decerebrate piglets aged 4-13 days. We elicited the LCR by injecting 0.1ml of water into the larynx and recorded integrated phrenic nerve activity. The laryngeal chemoreflex was prolonged when the body temperature of each piglet was raised approximately 2.5 degrees C, and SCH-58261 reversed the thermal prolongation of the LCR when injected into the NTS (n=13), but not when injected in the nucleus ambiguus (n=9). Injections of vehicle alone into the NTS did not alter the thermal prolongation of the LCR (n=9). We conclude that activation of adenosine receptors, perhaps located on GABAergic neurons in the NTS, contributes to thermal prolongation of the LCR.

Elevated body temperature exaggerates laryngeal chemoreflex apnea in decerebrate piglets.

We investigated the interaction between body temperature and the duration of the laryngeal chemoreflex (LCR) in decerebrate piglets. Elevating body temperature by approximately 2 degrees C prolongs the duration of the LCR and the length of apnea associated with the reflex. This thermal prolongation seems to arise within the nucleus of the solitary tract in the brainstem, and we believe the thermal effect is mediated by enhanced GABAergic neurotransmission.

Laryngeal apnea in rat pups: effects of age and body temperature.

In neonatal mammals of many species, including human infants, apnea and other reflex responses frequently arise from stimulation of laryngeal receptors by ingested or regurgitated liquids. These reflexes, mediated by afferents in the superior laryngeal nerves (SLNs), are collectively known as the laryngeal chemoreflex (LCR) and are suspected to be responsible for some cases of the sudden infant death syndrome (SIDS). The LCR is strongly enhanced by mild increases in body temperature in decerebrate piglets, a finding that is of interest because SIDS victims are often found in overheated environments. Because of the experimental advantages of studying reflex development and mechanisms in neonatal rodents, we have developed methods for eliciting laryngeal apnea in anesthetized rat pups and have examined the influence of mild hyperthermia in animals ranging in age from 3 to 21 days. We found that apnea and respiratory disruption, elicited either by intralaryngeal water or by electrical stimulation of the SLN, occurred at all ages studied. Raising body temperature by 2-3 degrees C prolonged the respiratory disturbance in response to either stimulus. This effect of hyperthermia was prominent in the youngest animals and diminished with age. We conclude that many studies of the LCR restricted to larger neonatal animals in the past can be performed in infant rodents using appropriate methods. Moreover, the developmental changes in the LCR and in the thermal modulation of the LCR seem to follow different temporal profiles, implying that distinct neurophysiological processes may mediate the LCR and thermal prolongation of the LCR.

Unilateral microdialysis of gabazine in the dorsal medulla reverses thermal prolongation of the laryngeal chemoreflex in decerebrate piglets.

The laryngeal chemoreflex (LCR) is elicited by water in the larynx and leads to apnea and respiratory disruption in immature animals. The LCR is exaggerated by the elevation of brain temperature within or near the nucleus of the solitary tract (NTS) in decerebrate piglets. Thermal prolongation of reflex apnea elicited by superior laryngeal nerve stimulation is reduced by systemic administration of GABA(A) receptor antagonists. Therefore, we tested the hypothesis that microdialysis within or near the NTS of gabazine, a GABA(A) receptor antagonist, would reverse thermal prolongation of the LCR. We examined this hypothesis in 21 decerebrate piglets (age 3-13 days). We elicited the LCR by injecting 0.1 ml of water into the larynx before and after each piglet's body temperature was elevated by approximately 2.5 degrees C and before and after 2-5 mM gabazine was dialyzed unilaterally and focally in the medulla. Elevated body temperature failed to prolong the LCR in one piglet, which was excluded from analysis. Elevated body temperature prolonged the LCR in all the remaining animals, and dialysis of gabazine into the region near the NTS (n = 10) reversed the thermal prolongation of the LCR even though body temperature remained elevated. Dialysis of gabazine in other medullary sites (n = 10) did not reverse thermal prolongation of the LCR. Gabazine had no consistent effect on baseline respiratory activity during hyperthermia. These findings are consistent with the hypothesis that hyperthermia activates GABAergic mechanisms in or near the NTS that are necessary for the thermal prolongation of the LCR.

GABAergic processes mediate thermal prolongation of the laryngeal reflex apnea in decerebrate piglets.

We tested the hypotheses that elevated body temperature would prolong reflex apnea following electrical stimulation of the superior laryngeal nerve (SLN) in decerebrate neonatal piglets and that thermal prolongation of reflex apnea after stimulation of the SLN depended on GABAergic mechanisms. These studies were conducted in 13 decerebrate piglets (age 3-15 days). The SLN was stimulated at approximately 1.5 times the threshold stimulus level for 10 s starting at the beginning of inspiration. We measured the duration of the apnea and respiratory disruption that followed SLN stimulation. Elevating body temperature prolonged the duration of the apnea and respiratory disruption that followed SLN stimulation, and treatment with antagonists of gama-aminobutyric acid A-type (GABAA) receptors reversed the thermal prolongation of reflex apnea and the period of respiratory disruption even though body temperature remained elevated. We conclude that elevated body temperature enhances or amplifies GABAergic mechanisms that prolong the respiratory inhibition following electrical stimulation of the SLN.

An essential role for endocytosis of rhodopsin through interaction of visual arrestin with the AP-2 adaptor.

Previously, we have identified a class of retinal degeneration mutants in Drosophila in which the normally transient interaction between arrestin2 (Arr2) and rhodopsin is stabilized and the complexes are rapidly internalized into the cell body by receptor-mediated endocytosis. The accumulation of protein complexes in the cytoplasm eventually results in photoreceptor cell death. We now show that the endocytic adapter protein AP-2 is essential for rhodopsin endocytosis through an Arr2-AP-2beta interaction, and mutations in Arr2 that disrupt its interaction with the beta subunit of AP-2 prevent endocytosis-induced retinal degeneration. We further demonstrate that if the interaction between Arr2 and AP-2 is blocked, this also results in retinal degeneration in an otherwise wild-type background. This indicates that the Arr2-AP-2 interaction is necessary for the pathology observed in a number of Drosophila visual system mutants, and suggests that regular rhodopsin turnover in wild-type photoreceptor cells by Arr2-mediated endocytosis is essential for photoreceptor cell maintenance.

TGF-beta 1 uses distinct mechanisms to inhibit IFN-gamma expression in CD4+ T cells at priming and at recall: differential involvement of Stat4 and T-bet.

TGF-beta1 plays a critical role in restraining pathogenic Th1 autoimmune responses in vivo, but the mechanisms that mediate TGF-beta1's suppressive effects on CD4(+) T cell expression of IFN-gamma expression remain incompletely understood. To evaluate mechanisms by which TGF-beta1 inhibits IFN-gamma expression in CD4(+) T cells, we primed naive wild-type murine BALB/c CD4(+) T cells in vitro under Th1 development conditions in the presence or the absence of added TGF-beta1. We found that the presence of TGF-beta1 during priming of CD4(+) T cells suppressed both IFN-gamma expression during priming as well as the development of Th1 effector cells expressing IFN-gamma at a recall stimulation. TGF-beta1 inhibited the development of IFN-gamma-expressing cells in a dose-dependent fashion and in the absence of APC, indicating that TGF-beta1 can inhibit Th1 development by acting directly on the CD4(+) T cell. During priming, TGF-beta1 strongly inhibited the expression of both T-bet (T box expressed in T cells) and Stat4. We evaluated the importance of these two molecules in the suppression of IFN-gamma expression at the two phases of Th1 responses. Enforced expression of T-bet by retrovirus prevented TGF-beta1's inhibition of Th1 development, but did not prevent TGF-beta1's inhibition of IFN-gamma expression at priming. Conversely, enforced expression of Stat4 partly prevented TGF-beta1's inhibition of IFN-gamma expression during priming, but did not prevent TGF-beta1's inhibition of Th1 development. These data show that TGF-beta1 uses distinct mechanisms to inhibit IFN-gamma expression in CD4(+) T cells at priming and at recall.