Neurotransmitters are stored in synaptic vesicles, where they have been assumed to be in free solution. Here we report that in Torpedo synaptic vesicles, only 5% of the total acetylcholine (ACh) or ATP content is free, and that the rest is adsorbed to an intravesicular proteoglycan matrix. This matrix, which controls ACh and ATP release by an ion-exchange mechanism, behaves like a smart gel. That is, it releases neurotransmitter and changes its volume when challenged with small ionic concentration change. Immunodetection analysis revealed that the synaptic vesicle proteoglycan SV2 is the core of the intravesicular matrix and is responsible for immobilization and release of ACh and ATP. We suggest that in the early steps of vesicle fusion, this internal matrix regulates the availability of free diffusible ACh and ATP, and thus serves to modulate the quantity of transmitter released.

A method was developed to transplant assembled nicotinic acetylcholine receptors (AcChoRs) and Cl1 channels from the electric organ of Torpedo to the membrane of Xenopus oocytes. Membrane vesicles from Torpedo electroplaques were injected into the oocytes and,within a few hours, the oocyte membrane acquired AcChoRs and Cl-channels.The mechanism of exprpssion of these receptors and channels is very different from that which follows the injection of mRNA, since the 4ppearance of receptors after membrane injection does not require'de novo protein synthesis or Nglycosylation. This, and other controls, indicate that the foreign receptor-bearing membranes fuse with the oocyte membrane and cause the appearance of functional receptors and channels. All this makes the Xenopus oocyte an even more powerful tool for studies of the structure and function of membrane proteins.Xenopus oocytes are a useful system to study the structure and function of many neurotransmitter receptors, ion channels, and other membrane proteins. In the first place, Xenopus oocytes and their enveloping follicular cells, which are electrically coupled to the oocyte, are endowed with a variety of native neurotransmitter and hormone receptors and voltageoperated channels (1-4). Moreover, after injection of appropriate mRNAs, Xenopus oocytes will express a large variety of foreign receptors and channels.' The oocytes translate the heterologous mRNA, process the products, and incorporate them into their plasma membrane where they form functional receptors and channels (5).The present experiments were done to see if the oocyte membrane could be made to acquire foreign receptors, already assembled in other cell membranes, without injecting the nucleic acids. If this could be achieved, the oocytes would become even more useful for studies of many receptors, channels, and signaling events. Our experiments, carried out early in 1990, showed that acetylcholine receptors (AcChoRs) and Cl-channels from Torpedo can be transplanted directly from the Torpedo electroplaques to the oocyte membrane. Some of the results have been presented in brief (6). MATERIALS AND METHODSTo determine whether foreign membranes carrying neurotransmitter receptors could be incorporated directly into the oocyte plasma membrane, Xenopus oocytes were injected with membranes isolated from the electric organ of Torpedo, because the electroplaque cells are very rich in AcChoRs and C1 channels (7-11). Membrane Preparation. Membranes were prepared as described (10, 11) with slight modifications. About 300 g of frozen electric organ from Torpedo californica was thawed in 1-2 liters of a hypoosmotic solution (1 mM EDTA, 10. mM Hepes buffer, pH 7.2). This solution was discarded and freshThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.solution was added to make 1 liter. The thawed, or sometimes fresh, tissue was homogenized first ...

The regulation of neostriatal cholinergic function by tachykinins (TKs) has been studied by measuring endogenous ACh released from rat neostriatal slices. Septide (SEP; a highly selective substance P analog), neurokinin A (NKA), and neurokinin B (NKB) elicited endogenous ACh release in a concentration-dependent manner. The rank order in potency was the following: NKB (EC50 approximately 0.5 nM) greater than NKA (EC50 approximately 7 nM) greater than SEP (EC50 approximately 12 nM). Spantide (SPA) was less effective (39% inhibition) than [D-Arg6, D- Trp7,9, N-Methyl-Phe8]-substance P fragment 6–11 (53% inhibition) at antagonizing ACh release evoked by SEP and NKA. Smaller doses of the antagonists inhibited the effects of SEP compared to NKA, and the effects of NKB could only be antagonized by SPA. These findings suggest the involvement of the three neurokinin (NK) receptors in ACh release evoked by TKs with the following rank order: NK3 greater than NK2 greater than NK1. 6-Hydroxydopamine lesions of nigrostriatal neurons and tetrodotoxin (TTX) intoxication of striatal tissue revealed two different patterns of regulation of cholinergic function by TKs. On the one hand, SEP and NKA evoked ACh release, independently of the nigrostriatal dopaminergic system, by acting on NK1 and NK2 receptors that are probably localized on the somatodendritic field of cholinergic neurons receiving substance P terminals. On the other hand, dopaminergic terminals seem to regulate NKB neurons that modulate cholinergic neurons, because NKB-evoked ACh release decreased by 24% in the denervated striata. In addition, TTX partially blocked (50%) ACh release evoked by NKB, suggesting that NKB acts on NK3 receptors at both the nerve terminals and the somatodendritic field of cholinergic neurons.