It is well established that GABAA-mediated postsynaptic potentials are excitatory in many brain regions during embryonic and early postnatal life. The pre-Bötzinger complex (PBC) in the brainstem is an essential component of the respiratory rhythm-generating network, where GABAA-mediated inhibition plays a critical role in generating a stable respiratory rhythm in adult animals. In the present study, using the perforated patch technique, we investigated the maturation of GABAA receptor-mediated effects on rhythmically active PBC neurons and on the motor output in slice preparations from P0-15 neonatal mice. The reversal potential of GABAA receptor-mediated current (EGABA-A) switched from depolarizing to hyperpolarizing within the first postnatal week. EGABA-A was -13.7 +/- 9.8 mV at P0, then it changed to -44.8 +/- 7.0 mV at P2 and -71.5 +/- 6.8 mV at P4. Perfusion of bicarbonate-free saline has no detectable influence on EGABA-A, indicating that a lack of Cl- extrusion during P0-3 is mainly responsible for early GABAA-ergic excitation. At the network level, blockade of GABAA receptors with bicuculline did not significantly change the frequency of rhythmic bursts recorded from hypoglossal nerve roots before P3, whereas it increased the coefficient of variation. After P3, bicuculline increased burst frequency with little effect on the coefficient of variation. Thus, chloride-mediated inhibition, which appears in PBC neurons after P3, coincides with the appearance of GABAA-mediated modulation of the respiratory rhythm. GABAA receptor-activated inhibition may therefore be necessary for frequency modulation in the respiratory network beginning on the fourth postnatal day in the mouse brainstem.

Rhythm generation in mature respiratory networks is influenced strongly by synaptic inhibition. In early neonates, GABAA‐receptor‐ and glycine‐receptor‐mediated inhibition is not present, thus the question arises as to whether GABAB‐receptor‐mediated inhibition plays an important role. Using brainstem slices of neonatal mice (postnatal day, P0‐P15), we analysed the role of GABAB‐mediated modulation of GABA and glycine synaptic transmission in the respiratory network. Blockade of GABA uptake by nipecotic acid (0.25–2 mm) reduced the respiratory frequency. This reduction was prevented by the selective GABAB receptor antagonist CGP55845A (CGP) alone at P0‐P3, but by bicuculline as well as CGP at P7‐P15. Blockade of GABAB receptors by CGP increased the respiratory frequency at P0‐P3, whereas it caused a reduction of frequency in older animals. The effect of CGP on respiratory frequency was diminished in the presence of bicuculline and strychnine in older but not in younger animals. The relative contribution of GABAB‐receptor‐mediated pre‐ and postsynaptic modulation was examined by analysing the effect of GABAB receptors on spontaneous and miniature IPSCs. In younger animals (P0‐P3), the GABAB receptor agonist baclofen had no detectable effect on IPSC frequency, but caused a significant decrease in the amplitude. In older animals (P7‐P15), baclofen decreased both the frequency and amplitude of spontaneous and miniature IPSCs. These results demonstrate that GABAB‐receptor‐mediated postsynaptic modulation plays an important role in the respiratory network from P0 on. GABAB‐receptor‐mediated presynaptic modulation develops with a longer postnatal latency, and becomes predominant within the first postnatal week.

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GABA(B) receptors play a critical neuromodulatory role in the central nervous system. It has been suggested that both the functional role and the cellular distribution of GABA(B) receptors in the neuronal network change during post-natal maturation. In the present study, the cellular and subcellular distribution patterns of the GABA(B) R1a/b receptors have been analysed in different brain regions of the mouse using immunocytochemistry with isoform-specific antisera. GABA(B) R1-immunoreactivity (IR) was present from the first post-natal day (P0) on in most regions of the brain. Neurones exhibited diffuse GABA(B) R1-IR labelling throughout somata and larger proximal dendrites as well as some fine neuronal processes. After P5, distinct punctuated staining was apparent. The number of such GABA(B) IR granules per cell increased with age in a sigmoidal manner from P5 to P60. Electron microscopy revealed GABA(B) IR as clusters of small clear vesicles of 30-50 nm diameter within the cytoplasm and close to the cell membrane at extrasynaptic locations, as well as at pre-synaptic and post-synaptic specialisations. The increase in GABA(B) R1-IR punctuate staining during brain maturation points to increasing functional participation and heterogeneity of GABA(B) receptors as the complexity of the central nervous system expands with growth and development.

It has previously been shown that cephalic, segmental, and caudal ganglia from the medicinal leech show differences in their protein composition. Here we studied whether the neuronal reorganization that occurs in cultured segmental ganglia from the medicinal leech is accompanied by detectable changes in the protein expression pattern. Using silver‐stained two‐dimensional gels we showed that after 5 and 12 days in culture changes in the protein patterns can be detected in isolated ganglia. The changes observed in the two‐dimensional gels occurred concomitantly with a sprouting of serotoninergic neurites and a decreased transmitter content of dopaminergic neurites as shown by using the glyoxylic acid condensation reaction. In addition, we present evidence that Retzius cells, which can be identified by their characteristic morphology and action potential waveform, exhibit biochemically unique properties with respect to their protein expression pattern. © 2000 John Wiley & Sons, Inc. J Neurobiol 44: 320–332, 2000