Selective clustering of glutamate and gamma-aminobutyric acid receptors opposite terminals releasing the corresponding neurotransmitters.

Abstract

Several immunocytochemical and physiological studies have demonstrated a concentration of neurotransmitter receptors at postsynaptic sites on neurons, but an overall picture of receptor distribution has not emerged. In particular, it has not been clear whether receptor clusters are selectively localized opposite terminals that release the corresponding neurotransmitter. By using antibodies against the excitatory glutamate receptor subunit GluRl and the inhibitory type A yaminobutyric acid (GABA) receptor P2/3 subunits, we show that these different receptor types cluster at distinct postsynaptic sites on cultured rat hippocampal neurons. The GABAA receptor .32/3 subunits clustered on cell bodies and dendritic shafts opposite GABAergic terminals, whereas GluRl clustered maiuly on dendritic spines and was associated with glutamatergic synapses. Chronic blockade of evoked transmitter release did not block receptor clustering at postsynaptic sites. These results suggest that complex mechanisms involving nerve terminal-specific signals are requlired to allow different postsynaptic receptor types to cluster opposite only appropriate presynaptic terminals.At the neuromuscular junction, postsynaptic receptors are precisely localized opposite presynaptic terminals (1). There is now considerable evidence that neurotransmitter receptors are also clustered at postsynaptic sites on neurons, albeit in many cases less tightly than at the neuromuscular junction. By immunoelectron microscopy, Triller et al. (2) (21, 22). We found that glutamate receptor subunits GluRl and GluR2/3 cluster at a subset of postsynaptic sites on cultured hippocampal neurons, which is suggestive of an element of specificity in receptor clustering (13). These results are consistent with the possibility that receptors selectively cluster opposite appropriate presynaptic terminals, but direct evidence has been lacking.To resolve this question, we have performed a series of double-label immunolocalization experiments to compare the distributions of different types of postsynaptic receptors in individual cells and to relate the localization of postsynaptic receptors with that of different types of presynaptic terminals. We present here direct evidence that the glutamate receptor subunit GluRl and the GABAA receptor 82/3 subunits (GABAARP82/3) selectively cluster opposite terminals releasing the corresponding neurotransmitters and, furthermore, that postsynaptic receptor clustering occurs in the absence of evoked transmitter release. MATERIALS AND METHODSHippocampal cells were prepared from 18-day rat embryos by dissociation with trypsin, plated on poly(L-lysine)-coated glass coverslips at 2400 cells per cm2, and maintained in serum-free medium above an astroglial monolayer (23, 24).Cytosine arabinoside (5 ,AM) was added 3 days after plating to inhibit the proliferation ofnonneuronal cells. For inhibition of synaptic activity, tetrodotoxin (TTX; 1 ,uM) was added to the culture medium on day 0 when the coverslips were placed over glial monolayers and ...

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