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1. The electrophysiological properties of inspiratory neurons were studied in a rhythmically active thick-slice preparation of the newborn mouse brain stem maintained in vitro. Whole cell patch recordings were performed from 60 inspiratory neurons within the rostral ventrolateral part of the slice with the aim of extending the classification of inspiratory neurons to include analysis of active membrane properties. 2. The slice generated a regular rhythmic motor output recorded as burst of action potentials on a XII nerve root with a peak to peak time of 11.5 +/- 3.4 s and a duration of 483 +/- 54 ms (means +/- SD, n = 50). Based on the electroresponsive properties and membrane potential trajectories throughout the respiratory cycle, three types of inspiratory neurons could be distinguished. 3. Type-1 neurons were spiking in the interval between the inspiratory potentials (n = 9) or silent with a resting membrane potential of -48.6 +/- 10.1 mV and an input resistance of 306 +/- 130 M omega (n = 15). The spike activity between the inspiratory potentials was burst-like with spikes riding on top of an underlying depolarization (n = 11) or regular with no evidence of bursting (n = 12). Hyperpolarization of the neurons below threshold for spike initiation did not reveal any underlying phasic synaptic activity, that could explain the bursting behavior. 4. Type-1 neurons showed delayed excitation after hyperpolarizing square current pulses or when the neurons were depolarized from a hyperpolarized level. This membrane behavior resembles the response seen in other CNS neurons expressing an IA. The response to 1-s long depolarizing pulses with a large current strength showed signs of activation of an active depolarizing membrane response leading to a transient reduction in the spike amplitude. The relationship between the membrane potential and the amplitude of square current pulses (Vm-I) showed a small upward rectification below -70 mV, and spike adaptation throughout a 1-s pulse had a largely linear time course. 5. Type-1 neurons depolarized and started to fire spikes 398 +/- 102 ms (n = 20) before the upstroke of the integrated XII nerve discharge. The inspiratory potential was followed by fast hyperpolarization, a short fast-repolarizing phase (1,040 +/- 102 ms, n = 5) and a longer slow-repolarizing phase (lasting until the next inspiratory discharge). 6. Type-2 neurons were spiking in the interval between the inspiratory potentials with no evidence of bursting behavior and had an input resistance of 296 +/- 212 M omega (n = 26). The response to hyperpolarizing pulses revealed an initial sag and postinhibitory rebound depolarization. This membrane behavior resembles the response seen in other CNS neurons expressing an Ih. The Vm-I relationship was linear at depolarized potentials and showed a marked upward rectification below -60 mV. Spike trains elicited by 1-s long pulses showed a pronounced early and late adaptation. 7. Type-2 neurons depolarized and started to fire spikes 171 +/- 87 ms (n = 23) before the upstroke...

Previous pharmacological studies have indicated the possible existence of functional interactions between -, ␦-and -opioid receptors in the CNS. We have investigated this issue using a genetic approach. Here we describe in vitro and in vivo functional activity of ␦-and -opioid receptors in mice lacking the -opioid receptor (MOR). Measurements of agonist-induced [ ] enkephalin exhibited similar potency to inhibit smooth muscle contraction in both wild-type and MOR Ϫ/Ϫ mice. ␦-Analgesia induced by deltorphin II was slightly diminished in mutant mice, when the tail flick test was used. Deltorphin II strongly reduced the respiratory frequency in wild-type mice but not in MOR Ϫ/Ϫ mice. Analgesic and respiratory responses produced by the selective -agonist U-50,488H were unchanged in MOR-deficient mice. In conclusion, the preservation of ␦-and -receptor signaling properties in mice lacking -receptors provides no evidence for opioid receptor cross-talk at the cellular level. Intact antinociceptive and respiratory responses to the -agonist further suggest that the -receptor mainly acts independently from the -receptor in vivo. Reduced ␦-analgesia and the absence of ␦-respiratory depression in MOR-deficient mice together indicate that functional interactions may take place between -receptors and central ␦-receptors in specific neuronal pathways.

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.

SUMMARY1. Synaptic transmission and neuronal morphology were studied in the nucleus tractus solitarius and in the dorsal vagal motor nucleus (solitary complex), in coronal brain-stem slices of rat or cat, superfused in vitro.2. Electrical stimulation of afferent fibres of the solitary tract evoked two different types of post-synaptic response recorded intracellularly in different solitary complex neurones. Labelling with horseradish peroxidase showed that these two sorts of orthodromically evoked responses were correlated with different post-synaptic neuronal morphologies.3. The majority of recorded neurones (n = 93) showed a prolonged reduction in excitability following the initial solitary-tract-evoked excitatory post-synaptic potential (e.p.s.p.). A smaller number of neurones (n = 53) showed a prolonged increase in excitability following solitary tract stimulation. In no case did the solitary tract stimulation induce a burst of action potentials at high frequency.4. The time-to-peak and the half-width of the initial solitary-tract-evoked e.p.s.p. were shorter in neurones with prolonged increased excitability than in those with prolonged reduced excitability. In neurones with prolonged reduced excitability, this e.p.s.p. was followed by a hyperpolarization lasting 60-100 ms. The latency of this inhibitory post-synaptic potential (i.p.s.p.) was 3-5 ms longer than that of the initial e.p.s.p. and its reversal potential was 10 mV more negative than the reversal potential of the response measured following application of y-aminobutyric acid or glycine. In neurones with prolonged increased excitability, at a membrane potential of -40 to -50 mV, the initial solitary tract e.p.s.p. was followed by a prolonged depolarization lasting 100-400 ms.5. Background synaptic activity was high in neurones with prolonged increased excitability, consisting of unitary e.p.s.p.s with an amplitude of more than 0-8 mV. This activity was increased for a period of 300-800 ms following solitary tract stimulation. Spontaneous excitatory potentials of more than 0-5 mV were not seen in neurones with prolonged reduced excitability. In these neurones, after intracellular injection of choride ions, reversed unitary i

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1. Activation of N-methyl-D-aspartate (NMDA) glutamate receptors in the brainstem network of respiratory neurones is required to terminate inspiration in the absence of lung afferents, but it is not required in the inspiratory motor act of lung inflation. In the present study we examined the involvement of non-NMDA ionotropic glutamate receptors in these two mechanisms in the adult mammal. 2. Adult cats were either decerebrated or anaesthetized with sodium pentobarbitone, paralysed and ventilated. Inspiratory motor output was recorded from the phrenic nerve and central respiratory activity from neurones in the bulbar ventral respiratory group. 3. In decerebrate vagotomized cats, ionophoretic application of 2,3-dihydroxy-6-nitro-7-sulphamoylbenzo(F)quinoxaline (NBQX) onto single respiratory neurones decreased their spontaneous discharge rate and abolished the excitatory effect of exogenously applied (IRS) a-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) but not NMDA. 4. In these animals, intravenous infusion (12 mg kg') of the non-NMDA receptor blockers GYKI 52466 (1-(4-aminophenyl)-4-methyl-7,8-methylene-dioxy-5-H-2,3-benzodiazepine) or NBQX: (1) decreased (in 10/15 cats) or abolished (in 5/15 cats) the inspiratory-related discharge of the phrenic nerve; (2) did not prolong the inspiratory phase; (3) reduced or abolished the spontaneous discharge of respiratory neurones; and (4) profoundly decreased the excitatory effects of AMPA but not NMDA ionophoresed onto these neurones. When both the phrenic nerve and the recorded respiratory neurone were silenced, neuronal excitation by ionophoretic application of NMDA first revealed a subthreshold respiratory modulation without lengthening of the inspiratory phase, then respiratory modulation became undetectable.5. Additional blockade of NMDA receptors by a small dose (0-15 mg kg') of dizocilpine (MK-801), abolished the phrenic nerve activity which persisted after NBQX (apnoea), but the discharge or the subthreshold modulation of the bulbar respiratory neurones showed a lengthening of the inspiratory phase (apneusis).

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