Rhythm generation in organotypic medullary cultures of newborn rats

Abstract

Organotypic transverse medullary slices (obex level) from six-day-old rats, cultured for two to four weeks in chemically defined medium contained rhythmically discharging neurones which were activated by CO2 and H+. The mechanisms underlying this rhythmicity and the spread of excitation and synaptic transmission within this organotypic tissue were examined by modifying the composition of the external solution. Our findings showed that (1) Exposure to tetrodotoxin (0.2 microM) or to high magnesium (6 mM) and low calcium (0.2 mM) concentrations abolished periodic activity. (2) Neither the blockade of GABAergic potentials with bicuculline methiodide (200 microM) and/or hydroxysaclofen (200 microM) nor the blockade of glycinergic potentials with strychnine hydrochloride (100 microM) abolished rhythmicity. (3) While atropine sulphate (5 microM) was ineffective in modulating periodic discharges nicotine (100 microM) - like CO2-shortened the intervals between the periodic events; hexamethonium (50-100 microM) reduced both periodic and aperiodic activity. (4) Exposure to the NMDA antagonist 2-aminophosphonovaleric acid (50 microM) suppressed periodic events only transiently. In the presence of 2-aminophosphonovaleric acid rhythmicity recovered. However, the AMPA-antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (10-50 microM), abolished periodic activity reversibly within less than 5 min. When 6-cyano-7-nitroquinoxaline-2,3-dione and nicotine were administered simultaneously periodic events persisted for up to 10 min. These findings indicate that synaptic excitatory drive is a prerequisite for the generation of rhythmic discharges of medullary neurones in this preparation. This drive may activate voltage-dependent channels or it may facilitate endogenous cellular mechanisms which initiate oscillations of intracellular calcium concentration. To test the latter possibility (5) calcium antagonists were added to the bath saline. The organic calcium antagonists verapamil and flunarizine (50-100 microM each) and the inorganic calcium antagonists cobalt (2 mM) and magnesium (6 mM) suppressed periodic activity and abolished or weakened the chemosensitivity towards CO2/acidosis. (6) Dantrolene (10 microM). an inhibitor of intracellular calcium release decreased the periodicity, while thapsigargin (2 microM) which blocks endoplasmic Ca(2+)-ATPase, transiently accelerated the occurrence of periodic events. (7) Oscillations of intracellular free calcium concentrations in Fura-2 AM-loaded cells were weakened or abolished by cobalt (2 mM). The results of (5)-(7) indicate that transmembrane calcium fluxes as well as intracellular Ca(2+)-release and -clearance mechanisms are a prerequisite for intracellular free calcium oscillations which may be important in the generation of rhythmic discharges in medullary neurones.

Physiological consequences of selective suppression of synaptic transmission in developing cerebral cortical networks in vitro: Differential effects on intrinsically generated bioelectric discharges in a living ‘model’ system for slow-wave sleep activity

“…Such transformations can also simplify the application of cross-correlation and other analog time-series techniques for quantitatively analyzing the complex interactions occurring within neuronal networks (Beggs and Plenz, 2004;Chiappalone et al, 2007;Grzywacz and Sernagor, 2000;Le Feber et al, 2007;Segev et al, 2002Segev et al, , 2004. Since spike-associated local field potentials themselves largely reflect supra-threshold membrane fluctuations (Bove et al, 1996;Elul, 1972;Grinvald et al, 2003;Kerr et al, 2005;Lopes da Silva, 1991;Purpura, 1959;Whittington et al, 2000), and since such potentials are readily measurable in organotypic explants (see Aptowicz et al, 2004;Baker et al, 1995Baker et al, , 2006Bausch et al, 2006;Plenz, 2003, 2004;Corner et al, 2002Corner et al, , 2005Crain, 1976;Mohajerani and Cherubini, 2005), field potential recordings represent yet another potentially useful approach to the tricky question of burst detection and characterization. Indeed, by virtue of the inclusion of sub-threshold synaptic activities, field potential measurements might even provide a more sensitive indicator of underlying neuronal interactions -albeit at the cost of reduced resolution at the neuronal level -than is obtainable using action potentials only.…”

“…Hyperexcitability can be induced in isolated organotypic neocortex explants by chronically suppressing all synaptic transmission by means of a high extracellular magnesium concentration (see Baker et al, 1995;Corner, 1994). Blockade of AMPA along with NMDA receptors, too, causes a well-nigh total cessation of spiking, followed upon return to control medium by exaggerated spontaneous burst discharges (Bacci et al, 2001;Corner et al, 2002;Van den Pol et al, 1996).…”

Selective inhibition of the Na + /H + exchanger type 3 activates CO 2 /H + -sensitive medullary neurones

“…Transverse medullary slices (360 µm thick) from 1-to 4-day-old rat pups were cut from 300 µm caudal to 600 µm rostral to the obex [3] and cultured on nylon grids for up to 6 weeks in R16 medium as described previously [25]. Acute hippocampal slices were prepared from adult guinea-pigs which were deeply anaesthetized with ether as described [5].…”

“…Based on these findings we suggest that the regulation of pH i is ultimately linked to central chemosensitivity. In fact, regulation of pH i in VLNcs or nucleus tractus solitarius neurones differs from that of neurones within non-chemosensitive brain areas [23,24] as they do not counteract a moderate (∆pH i <0.2 units) intracellular acidosis due to hypercapnia (pCO 2 elevated to 80-100 mmHg) or withdrawal of CO 2 /HCO 3 -amiloride, but not by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), pointing to a major contribution of Na + /H + exchange (NHE), while (Na + -dependent) Cl -/HCO 3 -antiport appears to be less involved. Previous in vivo studies show that inhibition of NHE by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) fails to activate respiration [14], and amiloride stimulates breathing frequency only at high (>1 mol/l) concentrations [30].…”

“…The activation by CO 2 /H + appears to be an inherent property of VLNcs and can be studied in vitro using long-term organotypic explants of the brainstem [3]. After weeks in culture these preparations fully retain their recognizable cytoarchitecture in a three-dimensional network composed of neurones within a glial environment with no cell debris.…”