The rhythmically active respiratory network in the brainstem slice of the mouse was investigated under in vitro conditions using patch clamp and microfluorometric techniques. Rhythmic respiratory activity persisted over the whole course of an experiment. Electrophysiologically recorded rhythmic activity in respiratory neurones was accompanied by oscillations in intracellular calcium, which displayed a maximal concentration of 300 nm and decayed to basal levels with a mean time constant of 1.6 ± 0.9 s. Elevations of calcium concentrations were highly correlated with the amplitude of rhythmic membrane depolarization of neurones, indicating that they were initiated by a calcium influx across the plasma membrane through voltage‐gated calcium channels. Voltage clamp protocols activating either high voltage‐activated (HVA) or both HVA and low voltage‐activated (LVA) calcium channels showed that intracellular calcium responses were mainly evoked by calcium currents through HVA channels. Somatic calcium signals depended linearly on transmembrane calcium fluxes, suggesting that calcium‐induced calcium release did not substantially contribute to the response. For calcium elevations below 1 μm, decay time constants were essentially independent of the amplitude of calcium rises, indicating that calcium extrusion was adequately approximated by a linear extrusion mechanism. Cytosolic calcium oscillations observed in neurones of the ventral respiratory group provide further evidence for rhythmic activation of calcium‐dependent conductances or second messenger systems participating in the generation and modulation of rhythmic activity in the central nervous system.

Brainstem nuclei serve a diverse array of functions in many of which ionotropic glutamate receptors are known to be involved. However, little detailed information is available on the expression of different glutamate receptor subunits in specific nuclei. We used RT-PCR in mice to analyze the glutamate receptor subunit composition of the pre-Bö tzinger complex, the hypoglossal nucleus, the nucleus of the solitary tract, and the inferior olive. Analyzing 15 receptor subunits and five variants, we found all four ␣-amino-3-hydroxy-5-methyl-4-propionic acid (AMPA) and six NMDA receptor (NR) subunits as well as three of five kainate (KA) receptors (GluR5, GluR6, and KA1) to be expressed in all nuclei. However, some distinct differences were observed: The inferior olive preferentially expresses flop variants of AMPA receptors, GluR7 is more abundant in the pre-Bö tzinger complex than in the other nuclei, and NR2C is most prominent in the nucleus of the solitary tract. In single hypoglossal motoneurons and interneurons of the pre-Bö tzinger complex investigation of GluR2 editing revealed strong expression of the GluR2-R editing variant, suggesting low Ca 2ϩ permeability of AMPA receptors. Thus, Ca 2ϩ -permeable AMPA receptors are unlikely to be the cause for the reported selective vulnerability of hypoglossal motoneurons during excitotoxic events. Key Words: ␣-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-NMDA-Kainate -Pre-Bö tzinger complexMouse -RT-PCR.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterised by selective degeneration of motoneurones. Familial ALS is an age-dependent autosomal dominant disorder in which mutations in the homodimeric enzyme Cu/Zn superoxide dismutase 1 (SOD1) is linked to the disease. An animal model for this disease is a transgenic mouse expressing the mutated human SOD1(G93A) gene. Recent electrophysiological data emphasised that the striking selective vulnerability of motoneurones might be due to their differential calcium buffering capacities. Therefore we have investigated, using immunohistochemistry, the expression of different calcium binding proteins in brainstem and spinal cord from normal and SOD1 mutated mice. Among the 13 calcium-binding proteins screened, only one, S100A6, a homodimeric calcium-binding protein able to bind four Zn(2+), appeared to be highly expressed in the SOD1 mutated mice. In brainstem, reactive astrocytes, but not motoneurones, from several regions, including nerve 12 root, were highly S100A6-positive. Hypoglossal nucleus was negative for S100A6. In dorsal root, reactive astrocytes from both white matter and anterior horn were highly reactive. If overexpression of S100A6 is specific for ALS, it will be a valuable diagnostic marker for this disease.

NMDA receptors are involved in a variety of brainstem functions. The excitatory postsynaptic NMDA currents of pre-Bö tzinger complex interneurons and hypoglossal motoneurons, which are located in the medulla oblongata, show remarkably fast deactivation kinetics of approximately 30 ms compared with NMDA receptors in other types of neurons. Because structural heterogeneity might be the basis for physiological properties, we examined the expression of six NMDA receptor subunits (NMDAR1, NR2A)2D, and NR3A) plus eight NMDR1 splice variants in pre-Bö tzinger complex, hypoglossal and, for comparison, neurons from the nucleus of the solitary tract in young rats using single cell multiplex RT-PCR. Expression of NR2A, NR2B, and NR2D was observed in all three cell types while NR3A was much more abundant in pre-Bö tzinger complex interneurons, which belong to the rhythm generator of respiratory activity. In hypoglossal neurons, the NMDAR1 splice variants NMDAR1-4a and NMDAR1-4b were found. In neurons of the nucleus of the solitary tract, instead of NMDAR1-4b, the NMDAR1-2a splice variant was detected. This differential expression of modulatory splice variants might be the molecular basis for the characteristic functional properties of NMDA receptors, as neurons expressing a special NMDAR1 splice variant at the mRNA level show fast kinetics compared with neurons lacking this splice variant. Keywords: hypoglossal nucleus, N-methyl-D-aspartate receptor kinetics, nucleus tractus solitarius, pre-Bö tzinger complex, rat, reverse transcription-polymerase chain reaction. Ionotropic glutamate receptors are found throughout the mammalian brain, where they constitute the major excitatory neurotransmitter system. In spontaneous oscillating networks like the mammalian respiratory network, excitatory glutamatergic signals are of essential importance during generation and maintenance of rhythmic activity (Pierrefiche et al. 1994;Bonham 1995). A variety of ionotropic glutamate receptors, including NMDA receptors, are expressed in respiratoryrelated neurons. The NMDA receptor subfamily comprises three different types of subunits, namely NR1, NR2 and NR3 subunits. NMDAR1 subunits show RNA splicing at three independent positions, giving rise to eight different splice variants, NMDAR1-1a to NMDAR1-4a, and NMDAR1-1b to NMDAR1-4b (Hollmann et al. 1993). These splice variants differ in their potentiation by protein kinase C (Durand et al. 1993), zinc (Hollmann et al. 1993, spermine (Zheng et al. 1994) and neurosteroids (Malayev et al. 1998;Ceccon et al. 2001). Co-expression of NMDAR1 with any of the four NR2 subunits (NR2A to NR2D) yields much larger currents than NMDAR1 alone (Ikeda et al. 1992;Kutsuwada