Most physiological effects of 1 receptor ligands are sensitive to pertussis toxin, suggesting a coupling with cell membrane-bound G proteins. However, the cloning of the 1 receptor has allowed the identification of an intracellular protein anchored on the endoplasmic reticulum. Here, we show, using the isolated adult guinea pig brainstem preparation, that activation of the 1 receptor results in its translocation from the cytosol to the vicinity of the cell membrane and induces a robust and rapid decrease in hypoglossal activity, which is mediated by phospholipase C. The subsequent activation of protein kinase C ␤ 1 and ␤ 2 isoforms and the phosphorylation of a protein of the same molecular weight as the cloned 1 receptor lead to a desensitization of the 1 motor response. Our results indicate that the intracellular 1 receptor regulates several components implicated in plasma membrane-bound signal transduction. This might be an example of a mechanism by which an intracellular receptor modulates metabotropic responses.

Vocalization in young mice is an innate response to isolation or mechanical stimulation. Neuronal circuits that control vocalization and breathing overlap and rely on motor neurons that innervate laryngeal and expiratory muscles, but the brain center that coordinates these motor neurons has not been identified. Here, we show that the hindbrain nucleus tractus solitarius (NTS) is essential for vocalization in mice. By generating genetically modified newborn mice that specifically lack excitatory NTS neurons, we show that they are both mute and unable to produce the expiratory drive required for vocalization. Furthermore, the muteness of these newborns results in maternal neglect. We also show that neurons of the NTS directly connect to and entrain the activity of spinal (L1) and nucleus ambiguus motor pools located at positions where expiratory and laryngeal motor neurons reside. These motor neurons control expiratory pressure and laryngeal tension, respectively, thereby establishing the essential biomechanical parameters used for vocalization. In summary, our work demonstrates that the NTS is an obligatory component of the neuronal circuitry that transforms breaths into calls.V ocalization is the primary mechanism used by many vertebrate species for communication (1). Whereas adult mice call during courtship, mating, and territorial disputes, newborn mice use vocalization to communicate with their mothers (2, 3). Newborn mice, when isolated, produce ultrasonic calls (USCs) that elicit search and retrieval behavior by their mothers. Thus, vocalizations of newborn mice represent an innate behavior that is thought to rely on a genetically determined circuit. Such innate vocalizations are reminiscent of nonverbal utterances of humans like laughing, crying, sighing, and moaning.The central circuits that control vocalization have been widely studied in adult vertebrates, where they overlap in their executive components with respiratory circuits (4). Forebrain pathways that control the frequency and sequence of ultrasounds in mice are not essential for innate vocalization (5, 6); rather, it is the periaqueductal gray in the midbrain that modulates the activity of motor neurons in the hindbrain and spinal cord to implement calls and modulate breathing (7,8). Calls are shaped through a biomechanical process that involves variations in subglottal air pressure and laryngeal muscle tension (9, 10). Expiration is an important determinant of subglottal air pressure (11), suggesting that expiratory muscle activity and laryngeal tension are highly coordinated during vocalization. However, because expiratory and laryngeal motor neurons are located at markedly different axial levels of the nervous system, in the spinal cord (T11-L1 levels, expiratory) and hindbrain (nucleus ambiguus, laryngeal), how the activities of these motor pools are coordinated is unclear (12, 13). More importantly, the identity and location of functionally important premotor neurons for vocalization are little known.Using mouse genetics to investigate the ...

Intracellular calcium concentration ([Ca 2ϩ ] i ) plays a major role in neuronal excitability, especially that triggered by the N-methyl-D-aspartate (NMDA)-sensitive glutamatergic receptor. We have previously shown that 1 receptor agonists potentiate NMDA receptor-mediated neuronal activity in the hippocampus and recruit Ca 2ϩ -dependent second messenger cascades (e.g., protein kinase C; PKC) in brainstem motor structures. The present study therefore assessed whether the potentiating action of 1 agonists on the NMDA response observed in the hippocampus involves the regulation of [Ca 2ϩ ] i and PKC. For this purpose, [Ca 2ϩ ] i changes after NMDA receptor activation were monitored in primary cultures of embryonic rat hippocampal pyramidal neurons using microspectrofluorometry of the Ca 2ϩ -sensitive indicator Fura-2/acetoxymethyl ester in the presence of 1 agonists and PKC inhibitors. We show that successive activations of the 1 receptor by 1-min pulses of (ϩ)-benzomorphans or (ϩ)-N-cyclopropylmethyl-N-methyl-1,4-diphenyl-1-ethyl-but-3-en-1-ylamine hydrochloride (JO-1784) concommitantly with glutamate time dependently potentiated before inconstantly inhibiting the NMDA receptor-mediated increase of [Ca 2ϩ ] i , whereas 1,3-di-o-tolyl-guanidine, a mixed 1 / 2 agonist, did not significantly modify the glutamate response. Both potentiation and inhibition were prevented by the selective 1 antagonist N, N-dipropyl-2-[4-methoxy-3-(211phe-nylethoxy) phenyl]-ethylamine monohydrochloride (NE-100). Furthermore, only (ϩ)-benzomorphans could induce [Ca 2ϩ ] i influx by themselves after a brief pulse of glutamate. A pretreatment with the conventional PKC inhibitor 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo [2,3-a] pyrrolo [3,4-c] carbazole (Gö -6976) prevented the potentiating effect of (ϩ)-benzomorphans on the glutamate response. Our results provide further support for a general mechanism for the intracellular 1 receptor to regulate Ca 2ϩ -dependent signal transduction and protein phosphorylation.

Nucleus tractus solitarius (NTS) is the integrative sensory relay of autonomic functions in the brainstem. To explore the nonneuronal cellular basis of central chemosensitivity during the first 24 hr of ventilatory acclimatization to hypoxia (VHA), we have investigated glial activation markers in the NTS. Conscious mice (C57/BL6) were placed in a hermetic hypoxia chamber containing a plethysmograph to record ventilation. After 4 days of habituation to the normoxic environment, mice were subjected to physiological hypoxia (10% O2 ) for 1, 6, or 24 hr. To dissociate interactions between microglia and astrocytes, another group received daily minocycline, a microglia activation blocker. By immunochemical localization of astrocytes (GFAP), activated microglia (Cd11b), and total microglia (Iba-1), we identified an oxygen-sensing glial layer in the NTS, in which astrocytes are first activated after 1-6 hr of hypoxia, followed by microglia after 6-24 hr of hypoxia. Minocycline administration suppressed microglial activation and decreased astrocyte activation at 6 hr and VHA at 24 hr of hypoxia. These results suggest that astrocytes contribute to the neuronal response during the first hour of hypoxia, whereas microglial cells, via cross-talk with astrocytes, are involved in the VHA during the first 24 hr of acclimatization.