Properties of spontaneous inhibitory synaptic currents in cultured rat spinal cord and medullary neurons

Abstract

1. To identify the type(s) and properties of inhibitory postsynaptic receptor(s) involved in synaptic transmission in cultured rat embryonic spinal cord and medullary neurons, we have used whole cell patch-clamp techniques to record miniature inhibitory postsynaptic currents (mIPSCs) in the presence of tetrodotoxin, DL-2-amino-5-phosphonovaleric acid, and 6-cyano-7-nitroquinoxaline-2,3-dione. 2. The mIPSCs recorded from both spinal cord and medullary neurons had skewed amplitude distributions. 3. The glycinergic antagonist strychnine and the GABAergic antagonist bicuculline each decreased both the frequency and mean peak amplitudes of mIPSCs. We conclude that both glycine and gamma-aminobutyric acid (GABA) are neurotransmitters at inhibitory synapses in our cultured cells. 4. Most (approximately 96-97%) mIPSCs decay with single-exponential time constants, and decay time distributions were consistently best fitted by the sum of four Gaussians with decay constants as follows: D1 = 5.8 +/- 0.1 (SE) ms (n = 63), D2 = 12.2 +/- 0.2 ms (n = 61), D3 = 23.2 +/- 0.4 ms (n = 54), and D4 = 44.7 +/- 1.0 ms (n = 57). We conclude that the four classes of decay times represent kinetically different inhibitory postsynaptic receptor populations. 5. Strychnine and bicuculline usually had one of two different effects on the mIPSC decay time constant distributions; either selective decreases in the frequency of mIPSCs with decay times in certain classes (i.e., the D1 class was reduced by bicuculline, the D2 class by strychnine, and the D3 and D4 classes by both antagonists) or a nonselective depression in the frequency of mIPSCs with decay times in all four classes. The particular effect observed in a given neuron was correlated with the presence or absence of ATP and guanosine 5'-triphosphate (GTP) in the patch pipette. Namely, in 71% of the antagonist applications where the pipette contained ATP and GTP, the result was a nonselective decrease in mIPSCs in all decay time constant classes. Conversely, in 54% of the antagonist applications in their absence, the result was a selective decrease in the frequency of mIPSCs in specific decay time constant classes. 6. In some experiments, mIPSCs reappeared in antagonist solution after an essentially complete block. Recovery from block in the continued presence of antagonist was never observed in the absence of ATP and GTP (8 neurons), and, at the same time, 5 of 9 neurons patched with ATP and GTP in the pipette did show recovery (56%).

Junctional versus Extrajunctional Glycine and GABAAReceptor-Mediated IPSCs in Identified Lamina I Neurons of the Adult Rat Spinal Cord

“…Even in cells with both types of mIPSCs present, these synaptic events represent separate populations. The differences in kinetics of the GlyR-versus GABA A R-mediated mIPSCs are consistent with previous findings in the dorsal horn (Takahashi and Momiyama, 1991;Takahashi et al, 1992;Baba et al, 1994;Nishi, 1993, 1995) and in medullary neurons (Lewis and Faber, 1996;Grudt and Henderson, 1998), in contrast with retinal ganglion cells in which GABA A R-mediated IPSCs have a faster decay than GlyRmediated ones (Protti et al, 1997).…”

Astrocytes regulate the developmental appearance of GABAergic and glutamatergic postsynaptic currents in cultured embryonic rat spinal neurons

“…Spontaneously occurring GABAergic transients with fast rise times and exponential decays appeared in a minority of neurons cultured on cortical astrocytes at the end of~24 h, but not until~96 h in neurons on PDL, which by that time had developed a large number of spinal astrocytes. The delayed appearance of GABAergic (and glycinergic) transients in rat spinal cord neurons cultured on PDL in serum-containing medium has been reported by Lewis & Faber (1996). In these experiments, spontaneous GABAergic transients emerged at 5 days in vitro, and formed a skewed distribution with a mode at 13± 17 pA in the presence of TTX.…”

Activity‐dependent modulation of GABAergic synapses in developing rat spinal networks in vitro

“…In embryonic interneurons monosynaptic responses can be evoked by proximal electrical stimulation in the presence of AMPA/kainate, NMDA and glycine receptor antagonists. These ePSCs were identified as GABA A receptor‐mediated events on the basis of: (a) kinetic properties, in particular the detected slow decay time values (Lewis & Faber, 1996; Gao et al ., 1998; Donato & Nistri, 2000; Galante et al . 2001); (b) bicuculline sensitivity (Galante et al .…”