Yx. Li et al., ASTROCYTES REGULATE DEVELOPMENTAL-CHANGES IN THE CHLORIDE-ION GRADIENT OF EMBRYONIC RAT VENTRAL SPINAL-CORD NEURONS IN CULTURE, Journal of physiology, 509(3), 1998, pp. 847-858
1. Embryonic rat ventral spinal cord neurons were dissociated at day 1
5 and grown on: (i) poly-D-lysine (PDL); (ii) a confluent monolayer of
type I astrocytes; or (iii) PDL in astrocyte conditioned medium (ACM)
to examine the influence of astroglia on the regulation of GABA(A) re
ceptor/Cl- channel properties. 2. Potentiometric oxonol dye recordings
of intact cells indicated that embryonic neurons were uniformly depol
arized by muscimol. The depolarizing effects disappeared in cells diss
ociated during the early postnatal period and recovered in culture for
24 h. Similar recordings using the calcium-imaging dye fura-2 AM reve
aled that GABA or muscimol triggered a sustained rise in cytosolic Ca2
+ (Ca-c(2+)) in embryonic neurons that was dependent on extracellular
Ca2+, blocked by bicuculline and nifedipine and sensitive to changes i
n extracellular chloride. The incidence and amplitude of the Ca2+ resp
onse decreased with time in vitro and was accelerated in neurons cultu
red on astrocytes compared with those on PDL. 3. Perforated patch-clam
p recordings revealed that GABA depolarized neurons in a Cl--dependent
and bicuculline-sensitive manner. Both the resting membrane potential
and the GABA equilibrium potential became more hyperpolarized with ti
me in vitro. 4. Astrocytes and ACM accelerated the transformation of G
ABAergic potential responses from depolarizing to hyperpolarizing. The
change occurred over the first 4 days in co-culture or in ACM but too
k more than 2 weeks in neurons cultured on PDL alone. 5. The intrinsic
, elementary properties of GABA, receptor/Cl- channels including open
time and unitary conductance changed independently of the presence of
astrocytes or ACM. Mean open time of the dominant kinetic component de
creased and conductance increased with time in vitro. 6. In sum, astro
cytes accelerate the developmental change in the Cl- ion gradient extr
insic to GABA, receptor/Cl- channels, which is critical for triggering
Ca2+ entry, without influencing parallel changes in the intrinsic pro
perties of the channels.