Mitochondrial membrane potential (DeltaPsi) and Ca(2+)-induced differentiation in HaCaT keratinocytes.

We have used the human calcium- and temperature-dependent (HaCaT) keratinocyte cell line to elucidate mechanisms of switching from a proliferating to a differentiating state. When grown in low calcium medium (<0.1 mM) HaCaT cells proliferate. However, an increase in the calcium concentration of the culture medium, [Ca(2+)](0), induces growth arrest and the cells start to differentiate. Numerous studies have already shown that the increase in [Ca(2+)](0) results in acute and sustained increases in intracellular calcium concentration, [Ca(2+)](i). We find that the Ca(2+)-induced cell differentiation of HaCaT cells is also accompanied by a significant decrease in mitochondrial membrane potential, DeltaPsi. By combining patch-clamp electrophysiological recordings and microspectrofluorimetric measurements of DeltaPsi on single cells, we show that the increase in [Ca(2+)](i) led to DeltaPsi depolarization. In addition, we report that tetraethylammonium (TEA), a blocker of plasma membrane K(+) channels, which is known to inhibit cell proliferation, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a blocker of plasma membrane Cl(-) channels, also affect DeltaPsi. Both these agents stimulate HaCaT cell differentiation. These data therefore strongly suggest a direct causal relationship between depolarization of DeltaPsi and the inhibition of proliferation and induction of differentiation in HaCaT keratinocytes.

Forelimb locomotor generators and quadrupedal locomotion in the neonatal rat.

The spinal localization of the forelimb locomotor generators and their interactions with other spinal segments were investigated on in vitro brainstem-spinal cord preparations of new-born rats. Superfusion of the cervicothoracic cord (C1-T4) with high K+/low Mg2+ artificial cerebrospinal fluid (aCSF) evoked rhythmic motor root activity that was limited to low cervical (C7, C8) and high thoracic (T1) spinal levels. This activity consisted of synchronous, homolateral bursts and a typical alternating bilateral pattern. Rhythmic activity with similar locomotor-like characteristics could be induced with either serotonin (5-HT, 5 microm), N-methyl-d-aspartate (NMDA, 5 microm), kainate (10 microm) or a "cocktail" of 5-HT (5 microm) and NMDA (5 microm). During 5-HT/NMDA perfusion of the cervicothoracic cord, induced bursting was no longer restricted to C7-T1 levels, but also occurred at cervical C3-C5 levels and with C5-C8 homolateral alternation. Spinal transections between C6 and C7 cervical segments did not abolish rhythmic activity in C7-T1, but suppressed locomotor-like rhythmicity at C3-C5 levels. Reduced regions comprising the C7-C8 or C8-T1 segments maintained rhythmicity. Superfusion of the whole cord with 5-HT/NMDA induced ventral root bursting with similar frequencies at all recorded segments (cervical, thoracic and lumbar). After isolation, the T3-T10 cord was unable to sustain any rhythmic activity while cervical and lumbar segmental levels continued to burst, albeit at different frequencies. We also found that the faster caudal and the slower rostral locomotor generators interact to produce coordinated locomotor-like activity in all segments of the intact spinal cord. In conclusion, C7-T1 spinal levels display a strong motor rhythmogenic ability; with the lumbar generators, they contribute to coordinated rhythmic activity along the entire spinal cord of a quadrupedal locomoting mammal.

alpha1-adrenergic receptor-induced slow rhythmicity in nonrespiratory cervical motoneurons of neonatal rat spinal cord.

Previous studies have reported that the alpha1-adrenergic system can activate spinal rhythm generators belonging to the central respiratory network. In order to analyse alpha1-adrenergic effects on both cranial and spinal motoneuronal activity, phenylephrine (1-800 microM) was applied to in vitro preparations of neonatal rat brainstem-spinal cord. High concentration of phenylephrine superfusion exerted multiple effects on spinal cervical outputs (C2-C6), consisting of a lengthening of respiratory period and an increase in inspiratory burst duration. Furthermore, in 55% of cases a slow motor rhythm recorded from the same spinal outputs was superimposed on the inspiratory activity. However, this phenylephrine-induced slow motor rhythm generated at the spinal level was observed neither in inspiratory cranial nerves (glossopharyngeal, vagal and hypoglossal outputs) nor in phrenic nerves. Whole-cell patch-clamp recordings were carried out on cervical motoneurons (C4-C5), to determine first which motoneurons were involved in this slow rhythm, and secondly the cellular events underlying direct phenylephrine effects on motoneurons. In all types of motoneurons (inspiratory and nonrespiratory) phenylephrine induced a prolonged depolarization with an increase in neuronal excitability. However, only nonrespiratory motoneurons showed additional rhythmic membrane depolarizations (with spiking) occurring in phase with the slow motor rhythm recorded from the ventral root. Furthermore the tonic depolarization produced in all motoneurons results from an inward current [which persists in the presence of tetrodotoxin (TTX)] associated with a decrease in neuron input conductance, with a reversal potential varying as a Nernstian function of extracellular K+ concentration. Our results indicate that the alpha1-adrenoceptor activation: (i) affects both the central respiratory command (i.e. respiratory period and inspiratory burst duration) and spinal inspiratory outputs; (ii) induces slow spinal motor rhythmicity, which is unlikely to be related to the respiratory system; and (iii), increases motoneuronal excitability, probably through a decrease in postsynaptic leak K+ conductance.

Development of lumbar rhythmic networks: from embryonic to neonate locomotor-like patterns in the mouse.

Different aspects of spinal locomotor organization have been studied in the mouse during embryonic and neonatal development using in vitro preparations of isolated lumbosacral cords. The first consideration was the embryonic development of an alternating bilateral pattern. From embryonic day (E) 12, perfusion of serotonin could induce relatively synchronous lumbar bursts across the cord. Bilateral activity became progressively alternate at E15 due to the appearance of glycinergic inhibitory interactions (revealed by strychnine application). Strictly alternating patterns were expressed at E18 and were maintained after birth. In a second step, we investigated cellular properties involved in lumbar rhythmogenesis in postnatal day 0-2 preparations which displayed spontaneous locomotor-like activity. Perfusion of receptor antagonists showed the co-operative involvement of N-methyl-D-aspartate (NMDA)- and non-NMDA-receptors for excitatory amino acids-mediated operation of locomotor networks. In a final step we investigated the localization of locomotor networks within the lumbar cord. Data obtained from preparations exhibiting spontaneous or Mg2+-free induced bursts revealed that the networks are present throughout the lumbar cord and that rhythmogenesis is distributed throughout all segmental levels.