The pre-Bötzinger complex (PBC) inspiratory center remains active in a transverse brainstem slice. Such slices are studied at high (8 -10 mM) superfusate [K ϩ ], which could attenuate the sensitivity of the PBC to neuromodulators such as opiates. Findings may also be confounded because slice boundaries, drug injection sites, or location of rhythmogenic interneurons are rarely verified histologically. Thus, we first generated PBC slices with defined boundaries using novel "on-line histology" based on our finding that rostrocaudal extensions of brainstem respiratory marker nuclei are constant in newborn rats between postnatal days 0 -4. At physiological superfusate ] generate rhythm with a high sensitivity to neuromodulators for extended time periods, whereas spontaneous "in vitro apnea" is an important tool to study the interaction of signaling pathways that modulate rhythm. Our approaches and findings provide the basis for a pharmacological and structure-function analysis of the isolated respiratory center in a histologically well defined substrate at physiological [K ϩ ].

The discovery of the rhythmogenic pre-Bötzinger complex (preBötC) inspiratory network, which remains active in a transverse brainstem slice, greatly increased the understanding of neural respiratory control. However, basic questions remain unanswered such as (1) What are the necessary and sufficient slice boundaries for a functional preBötC? (2) Is the minimal preBötC capable of reconfiguring between inspiratory-related patterns (e.g., fictive eupnea and sighs)? (3) How is preBötC activity affected by surrounding structures? Using newborn rat slices with systematically varied dimensions in physiological [K ϩ ] (3 mM), we found that a 175 m thickness is sufficient for generating inspiratory-related rhythms. In 700-m-thick slices with unilaterally exposed preBötC, a kernel Ͻ100 m thick, centered 0.5 mm caudal to the facial nucleus, is necessary for rhythm generation. Slices containing this kernel plus caudal structures produced eupneic bursts of regular amplitude, whereas this kernel plus rostral tissue generated sighs, intermingled with eupneic bursts of variable amplitude ("eupnea-sigh pattern"). After spontaneous arrest of rhythm, substance-P or neurokinin-1 (NK1) receptor agonist induced the eupnea-sigh burst pattern in Ն250-m-thick slices, whereas thyrotropin-releasing hormone or phosphodiesterase-4 blockers evoked the eupnea burst pattern. Endogenous rhythm was depressed by NK1 receptor antagonism. Multineuronal Ca 2ϩ imaging revealed that preBötC neurons reconfigure between eupnea and eupnea-sigh burst patterns. We hypothesize a (gradient-like) spatiochemical organization of regions adjacent to the preBötC, such that a small preBötC inspiratory-related oscillator generates eupnea under the dominant influence of caudal structures or thyrotropin-releasing hormone-like transmitters but eupnea-sigh activity when the influence of rostral structures or substance-P-like transmitters predominates.

CX546 effectively reverses opioid- and barbiturate-induced respiratory depression without reversing the analgesic response. These studies suggest that ampakines may be useful in preventing or reversing opioid-induced respiratory depression and identify the potential of ampakines for alleviating other forms of respiratory depression including sedative use and sleep apnea.

ATP released during hypoxia from the ventrolateral medulla activates purinergic receptors (P2Rs) to attenuate the secondary hypoxic depression of breathing by a mechanism that likely involves a P2Y 1 R-mediated excitation of preBötzinger complex (preBötC) inspiratory rhythm-generating networks. In this study, we used rhythmically active in vitro preparations from embryonic and postnatal rats and ATP microinjection into the rostral ventral respiratory group (rVRG)/preBötC to reveal that these networks are sensitive to ATP when rhythm emerges at embryonic day 17 (E17). The peak frequency elicited by ATP at E19 and postnatally was the same (ϳ45 bursts/min), but relative sensitivity was threefold greater at E19, reflecting a lower baseline frequency (5.6 Ϯ 0.9 vs 19.0 Ϯ 1.3 bursts/min). Combining microinjection techniques with ATP biosensors revealed that ATP concentration in the rVRG/preBötC falls rapidly as a result of active processes and closely correlates with inspiratory frequency. A phosphate assay established that preBötC-containing tissue punches degrade ATP at rates that increase perinatally. Thus, the agonist profile [ATP/ADP/adenosine (ADO)] produced after ATP release in the rVRG/preBötC will change perinatally. Electrophysiology further established that the ATP metabolite ADP is excitatory and that, in fetal but not postnatal animals, ADO at A 1 receptors exerts a tonic depressive action on rhythm, whereas A 1 antagonists extend the excitatory action of ATP on inspiratory rhythm. These data demonstrate that ATP is a potent excitatory modulator of the rVRG/preBötC inspiratory network from the time it becomes active and that ATP actions are determined by a dynamic interaction between the actions of ATP at P2 receptors, ectonucleotidases that degrade ATP, and ATP metabolites on P2Y and P1 receptors.

Although there is considerable evidence implicating a role for CD43 (leukosialin) in leukocyte cell–cell interactions, its precise function remains uncertain. Using CD43-deficient mice (CD43−/−) and intravital microscopy to directly visualize leukocyte interactions in vivo, we investigated the role of CD43 in leukocyte–endothelial cell interactions within the cremasteric microcirculation under flow conditions. Our studies demonstrated significantly enhanced leukocyte rolling and adhesion after chemotactic stimuli in CD43−/− mice compared with wild type mice. Using an in vitro flow chamber, we established that the enhanced rolling interactions of CD43−/− leukocytes, primarily neutrophils, were also observed using immobilized E-selectin as a substrate, suggesting that passive processes related to steric hindrance or charge repulsion were likely mechanisms. Despite increased adhesion and rolling interactions by CD43−/− leukocytes, we uncovered a previously unrecognized impairment of CD43−/− leukocytes to infiltrate tissues. Oyster glycogen–induced neutrophil and monocyte infiltration into the peritoneum was significantly reduced in CD43−/− mice. In response to platelet activating factor, CD43−/− leukocytes were impaired in their ability to emigrate out of the vasculature. These results suggest that leukocyte CD43 has a dual function in leukocyte–endothelial interactions. In addition to its role as a passive nonspecific functional barrier, CD43 also facilitates emigration of leukocytes into tissues.