Characterization of Functional GABAergic Synapses Formed between Rat Hypothalamic Neurons and Pituitary Intermediate Lobe Cells in Coculture: Ca²⁺Dependence of Spontaneous IPSCs

Abstract

Rat hypothalamic neurons and endocrine cells from the intermediate lobe of the pituitary were grown in dissociated coculture. Neurons positively stained with an antibody against glutamate decarboxylase established apparent contacts with the ␣-melanocyte-stimulating hormone-positive endocrine cells. These sites of contact were intensely labeled with an antibody against the synaptic protein synapsin I and displayed ultrastructural features characteristic of synapses. Using patchclamp recordings, we have demonstrated that these contacts correspond to functional GABAergic synapses. The synaptic currents were blocked reversibly by bicuculline (5 M) and SR95531 (5 M), two competitive antagonists of the GABA A receptor. At a holding potential of Ϫ60 mV, spontaneously occurring IPSCs (s-IPSCs) had small amplitudes (10 -100 pA), whereas electrically evoked IPSCs (ee-IPSCs) had amplitudes up to 1 nA. The rise times of both types of IPSCs were fast (Յ1 msec), and their decaying phases were fitted in most cases with a single exponential function (time constant, 50 msec). The amplitude distribution of s-IPSCs did not reveal clear, equally spaced peaks and was little affected by tetrodotoxin, suggesting that most s-IPSCs were miniature IPSCs. Reduction of extracellular calcium concentration to 0.3 mM induced a marked decrease in s-IPSC frequency and revealed a single amplitude peak at 10 pA, suggesting that a single quantum of GABA activates 8 -10 GABA A channels. Thus, our preparation might be an interesting model to study different aspects of synapse formation between a central neuron and its target as well as the fundamental mechanisms of synaptic transmission at central synapses.

Correlation of Miniature Synaptic Activity and Evoked Release Probability in Cultures of Cortical Neurons

“…However, under physiological conditions, a strong [Ca 2ϩ ] i dependence of miniature synaptic activity is well documented (Matthews and Wickelgren, 1977;Marcus et al, 1992;Doze et al, 1995;Katz et al, 1995;Scanziani et al, 1995;Capogna et al, 1996aCapogna et al, , 1997Poisbeau et al, 1996;Schoppa and Westbrook, 1997;Bao et al, 1998;Li et al, 1998). Using somatic recordings, we confirmed these findings by showing significantly enhanced rates of miniature activity after the elevation of [Ca 2ϩ ] o from 0 to 5 mM (see Results).…”

“…This variability among synapses indicates that miniature release can be regulated on the level of individual synapses. It has been shown that [C a 2ϩ ] i influences the frequency of miniature activity in C NS (Minota et al, 1991;Doze et al, 1995;Scanziani et al, 1995;C apogna et al, 1996aPoisbeau et al, 1996;Schoppa and Westbrook, 1997;Bao et al, 1998;Li et al, 1998) and peripheral nervous system (PNS) (Matthews and Wickelgren, 1977;Marcus et al, 1992;Katz et al, 1995). Furthermore, downstream of C a 2ϩ influx second messenger systems such as protein kinase C (PKC) (Ghiradi et al, 1992;Parfitt and Madison, 1993;C apogna et al, 1995; C arroll et al, 1998;Stevens and Sullivan, 1998) and cAM P-dependent protein kinase A (PKA) (Chavez-Noriega and Stevens, 1994; Kondo and Marty, 1997; Chen and Regehr, 1997) have been implicated in the regulation of miniature activity.…”

Oxytocin Modulates Glutamatergic Synaptic Transmission between Cultured Neonatal Spinal Cord Dorsal Horn Neurons

“…Acquisition and analysis were performed with the pC lamp6 and Axograph 3 software (Axon Instruments). Details on the analysis of synaptic currents can be found elsewhere (Poisbeau et al, 1996).…”

Reactive Oxygen Species Mediate the Potentiating Effects of ATP on GABAergic Synaptic Transmission in the Immature Hippocampus

“…The analysis of SPSCs was performed with the Mini Analysis Program (Synaptosoft Inc., Decatur, GA). General principles and details on the analysis of synaptic currents can be found elsewhere (18). The analysis of spontaneous synaptic currents was performed on sequences with at least 100 events each.…”