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.

The pre-Bötzinger Complex (preBötC) inspiratory centre remains active in isolated brainstem-spinal cords and brainstem slices. The extent to which findings in these models depend on their dimensions or superfusate [K + ] and [Ca 2+ ] (both of which determine neuronal excitability) is not clear. We report here that inspiratory-related rhythms in newborn rat slices and brainstem-spinal cords with defined boundaries were basically similar in physiological Ca 2+ (1.2 mM) and K + (3 mM). Hypoglossal nerve rhythm was 1 : 1-coupled to preBötC activity in slices and to cervical nerve bursts in en bloc preparations lacking the facial motonucleus (VII). Hypoglossal rhythm was depressed in brainstems containing (portions of) VII, while pre/postinspiratory lumbar nerve bursting was present only in preparations with > 79% VII. preBötC-related slice rhythms were inhibited in 1.5 mM Ca 2+ solution, whereas their longevity and burst rate were substantially augmented in 1 mM Ca 2+ . Ca 2+ depression of slice rhythms was antagonized by raising superfusate K + to 8-10 mM. This strong extracellular Ca 2+ /K + antagonism of inspiratory (motor) rhythms was also revealed in brainstem-spinal cords without VII, while the inhibition was progressively attenuated with increasing amount of rostral tissue. We hypothesize that depression of hypoglossal rhythm and decreased Ca 2+ sensitivity of preBötC rhythm are probably not related to an increased content of rostral respiratory structures, but rather to larger brainstem dimensions resulting in interstitial gradients for neuromodulator(s) and K + , respectively. We discuss whether block of pre/postinspiratory activity in preparations with < 79% VII is due to impairment of the pathway from preinspiratory interneurons to abdominal muscles

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