Synapsin I and calcium/calmodulin-dependent protein kinase II were pressure-injected into the preterminal digit of the squid giant synapse to test directly the possible regulation of neurotransmitter release by these substances. Neurotransmitter release was determined by measuring the amplitude, rate of rise, and latency of the postsynaptic potential generated in response to presynaptic depolarizing steps under voltage clamp conditions. Injection of dephosphosynapsin I decreased the amplitude and rate of rise of the postsynaptic potential, whereas injection of either phosphosynapsin I or heat-treated dephosphosynapsin I was without effect. Conversely, injection of calcium/calmodulin-dependent protein kinase II, which phosphorylates synapsin I on site II, increased the rate of rise and amplitude and decreased the latency of the postsynaptic potential. The effects of these proteins were observed without any detectable change in the initial phase of the presynaptic calcium current. A synapsin I-like protein and calcium/calmodulin-dependent protein kinase II were demonstrated by biochemical and immunochemical techniques to be present in squid nervous tissue. The data support the hypothesis that synapsin I regulates the availability of synaptic vesicles for release; we propose that calcium entry into the nerve terminal activates calcium/calmodulin-dependent protein kinase II, which phosphorylates synapsin I on site II, dissociating it from the vesicles and thereby removing a constraint in the release process.The coupling between presynaptic membrane depolarization and transmitter release in chemical synaptic transmission has been the subject of a large number of studies over several decades (cf. ref. 1). It is clear that this depolarization causes the opening of voltage-sensitive calcium channels, entry of calcium into the nerve terminal, and calcium-dependent release of neurotransmitter (cf. refs. 1 and 2). The present study represents a step towards elucidating the molecular mechanism(s) by which the increase in intracellular calcium regulates transmitter availability or release.Synapsin I is a neuron-specific phosphoprotein localized to presynaptic terminals, where it is associated with synaptic vesicles (3)(4)(5)(6) (3-6, 16, 17). In the present study the possible roles of synapsin I and of calmodulin kinase II in synaptic transmission were examined by injecting these substances into the presynaptic terminal of the squid giant synapse and measuring the effect on calcium entry and neurotransmitter release. For this purpose, well-established electrophysiological methods for studying the squid giant synapse were used (18-21). METHODS Intracellular Injection and Electrophysiological Recording.The experiments were performed on Loligo pealii at the Marine Biological Laboratory (Woods Hole, MA), and the techniques for the electrophysiological portion of the work were similar to those described previously (19). The stellate ganglion was held in a three-compartment chamber that was superfused with Tris-buffered a...

SUMMMARY1. Presynaptic or simultaneous pre-and postsynaptic voltage-clamp protocols w^ere implemented in the squid giant synapse in order to determine the magnitude and time course of the presynaptic calcium current (ICa) and its relation to transmitter release before and after presynaptic injection of proteins. These included several forms of synapsin I, calcium-calmodulin-dependent protein kinase II (CaM kinase II) and avidin.2. The quantities and location of these proteins were monitored by fluorescence video-enhanced microscopy during the electrophysiological measurements.3. Presynaptic injection of dephosphorylated synapsin I inhibited synaptic transmission with a time course consistent with diffusion of the protein through the terminal and action at the active release zone. A mathematical model relating the diffusion of synapsin I into the terminal with transmitter release was developed to aid in the interpretation of these results.4. Synapsin I inhibition of transmitter release was reversible. 5. The action of synapsin I was highly specific, as phosphorylation of the tail region only or head and tail regions prevented synapsin I from inhibiting release.6. Injections of heat-treated synapsin I or of avidin, a protein with a size and isoelectric point similar to those of synapsin I, had no effect on transmitter release.7. CaM kinase II injected presynaptically was found to facilitate transmitter release. This facilitation, which could be as large as 700 % of the control response, was related to the level of penetration of the enzyme along the length of the preterminal. A mathematical model of this facilitation indicates a reasonable fit between the distribution of CaM kinase II within the terminal and the degree of facilitation.8. The overall shape of the postsynaptic response was not modified by either synapsin I or CaM kinase II injection.9. The data suggest that, in addition to releasing transmitter, calcium also penetrates the presynaptic cytosol and activates CaM kinase II. When activated, CaM kinase II phosphorylates synapsin I, which reduces its binding to vesicles MS 8704 PHY 436 R. LLINAS AND OTHERS and/or cytoskeletal structures, enabling more vesicles to be released during a presynaptic depolarization. The amplitude of the postsynaptic response will then be both directly and indirectly regulated by depolarization-induced Ca21 influx. This model provides a molecular mechanism for synaptic potentiation.

Calcium/calmodulin-dependent protein kinase II (CaM kinase II) is a prominent enzyme in mammalian brain capable of phosphorylating a variety of substrate proteins. In the present investigation, the subcellular and regional distribution of CaM kinase II has been studied by light and electron microscopic immunocytochemistry using an antibody that recognizes the Mr 50,000 and 60,000/58,000 subunits of the enzyme. Light microscopy demonstrates strong immunoreactivity in neuronal somata and dendrites and weak immunoreactivity in axons. Electron microscopy, in addition to confirming light microscopic observations, reveals moderate immunoreactivity in spines and weak immunoreactivity ii nerve terminals. An accumulation of immunoreaction product is also present on postsynaptic densities. The presence of CaM kinase II in diverse structures throughout the neuron supports the view that this enzyme may be involved in mediating a variety of calcium-dependent physiological processes. CaM kinase II immunoreactivity is present in neurons throughout the brain, but a marked regional variation in the strength of the immunoreactivity exists. Overall, there is a gradient of staining intensity with the strongest immunoreactivity in the telencephalon and the weakest in the myelencephalon. The most heavily labeled regions of the telencephalon are the hippocampal formation, lateral septum, cortical regions, neostriatum, and amygdaloid complex.

Synapsin I, a neuron-specific, synaptic vesicle-associated phosphoprotein, is thought to play an important role in synaptic vesicle function. Recent microinjection studies have shown that synapsin I inhibits neurotransmitter release at the squid giant synapse and that the inhibitory effect is abolished by phosphorylation of the synapsin I molecule (Llinas et al., 1985). We have considered the possibility that synapsin I might modulate release by regulating the ability of synaptic vesicles to move to, or fuse with, the plasma membrane. Since it is not yet possible to examine these mechanisms in the intact nerve terminal, we have used video-enhanced microscopy to study synaptic vesicle mobility in axoplasm extruded from the squid giant axon. We report here that the dephosphorylated form of synapsin I inhibits organelle movement along microtubules within the interior of extruded axoplasm and that phosphorylation of synapsin I on sites 2 and 3 by calcium/calmodulin-dependent protein kinase II removes this inhibitory effect. Phosphorylation of synapsin I on site 1 by the catalytic subunit of cAMP-dependent protein kinase only partially reduces the inhibitory effect. In contrast to the inhibition of movement along microtubules seen within the interior of the axoplasm, movement along isolated microtubules protruding from the edges of the axoplasm is unaffected by dephospho-synapsin I, despite the fact that the synapsin I concentration is higher there. Thus, synapsin I does not appear to inhibit the fast axonal transport mechanism itself. Rather, these results are consistent with the possibility that dephospho-synapsin I acts by a crosslinking mechanism involving some component(s) of the cytoskeleton, such as F-actin, to create a dense network that restricts organelle movement. The relevance of the present observations to regulation of neurotransmitter release is discussed.

An afterdischarge in the bag cell neurons of Aplysia was previously shown to be associated with calcium entry into these cells and with changes in the phosphorylation state of at least two bag cell proteins (BC-I and BC-II). We have now investigated the role of calcium plus calmodulin (Cal CaM) in the control of phosphorylation of Aplysia nervous system proteins, including those of the bag cell neurons.In cell-free preparations of Aplysia CNS, we demonstrated Ca/CaM-stimulated protein phosphorylation that could be inhibited by the calmodulin-blocking drugs R24571, trifluoperazine, chlorpromazine, and W7. A number of substrate proteins for Ca/CaM-dependent protein phosphorylation with M, values from 17,000 to 310,000 were consistently observed in homogenates of the Aplysia CNS. In the bag cells, we found that a major substrate for Ca/CaM-dependent protein phosphorylation was the bag cell-specific, M, = 21,000 protein (BC-II). BC-I (M, = 33,000), on the other hand, appeared not to be a substrate for a Ca/CaM-dependent protein kinase.We found that there are a minimum of two Ca/CaM-dependent protein kinases in the Aplysia nervous system. These enzymes were distinguished on the basis of their subcellular distribution and their ability to phosphorylate distinct sites on synapsin I, an exogenous neuronal protein from vertebrates. Phosphorylation by one of these kinases (calmodulin kinase I) was on a site recovered in an M, = 10,000 proteolytic fragment of synapsin I, and phosphorylation by the other (calmodulin kinase II) was on a site recovered in an M, = 30,000 fragment. The predominant enzyme in the Aplysia CNS, as in the mammalian nervous system, was calmodulin kinase II.In addition, we compared an M, = 51,000 Aplysia substrate for Ca/CaM-dependent phosphorylation with the M, = 50,000 to 51,000 subunit of mammalian calmodulin kinase II. Both proteins showed immunoreactivity with monoclonal antibodies raised against rat calmodulin kinase II, both bound calmodulin, and phosphorylation of both proteins followed by partial proteolysis with Staphylococcus aureus protease V8 led to similar phosphopeptide maps. Our results indicate that the major form of Ca/CaM-dependent protein kinase in Aplysia CNS is homologous to mammalian calmodulin kinase II. The findings also raise the possibility that calcium/ calmodulin-dependent phosphorylation may mediate some of the long-lasting effects of intracellular calcium entry during an afterdischarge of the bag cell neurons.Calcium plays a dual role within nerve cells. It carries electrical current through voltage-gated channels in the membrane, and it also acts as a second messenger within