Evidence for the presynaptic location of adenylate cyclase and the cyclic AMP-stimulated protein kinase which is bound to synaptic membranes

By comparison of activities measured with either intact or ruptured synaptosomes it was found that about half of the cerebral adenylate cyclase is presynaptic while all the membrane bound, cyclic AMP-stimulated protein kinase activity appears to be presynaptic with the cyclic AMP receptor facing inward.     

Role of cyclic nucleotides in the long-lasting histaminergic inhibition in the mollusc,Onchidium neuron

1. The effects of histamine and presynaptic nerves on changes in cyclic nucleotide levels in the Onchidium Be-1 neurons were measured under experimental conditions in which they also produced histamine-induced hyperpolarization or histaminergic inhibition (ILD).2. Stimulation by histamine or of presynaptic nerves not only generated the hyperpolarization or ILD in Be-1, but also increased cyclic AMP and GMP levels in the Be-1, and these responses were antagonized by cimetidine.3. However, the rise in these cyclic AMP and GMP levels followed that of the hyperpolarization, though the increased hyperpolarization occurred simultaneously with the elevation of the level of another nucleotide, ATP in Be-1.4. The present results suggest that cyclic AMP is responsible for a positive-feedforward enhancement, and cyclic GMP for a negative-feedforward depression of the hyperpolarization or ILD.     

Lack of the presynaptic RhoGAP protein oligophrenin1 leads to cognitive disabilities through dysregulation of the cAMP/PKA signalling pathway

Loss-of-function mutations in the gene encoding for the RhoGAP protein of oligophrenin-1 (OPHN1) lead to cognitive disabilities (CDs) in humans, yet the underlying mechanisms are not known. Here, we show that in mice constitutive lack of Ophn1 is associated with dysregulation of the cyclic adenosine monophosphate/phosphate kinase A (cAMP/PKA) signalling pathway in a brain-area-specific manner. Consistent with a key role of cAMP/PKA signalling in regulating presynaptic function and plasticity, we found that PKA-dependent presynaptic plasticity was completely abolished in affected brain regions, including hippocampus and amygdala. At the behavioural level, lack of OPHN1 resulted in hippocampus- and amygdala-related learning disabilities which could be fully rescued by the ROCK/PKA kinase inhibitor fasudil. Together, our data identify OPHN1 as a key regulator of presynaptic function and suggest that, in addition to reported postsynaptic deficits, loss of presynaptic plasticity contributes to the pathophysiology of CDs.     

Physiological roles of adenosine derivatives which are released during neurotransmission in mammalian brain.

1. Experiments using synaptosome beds suggested that ATP was released from presynaptic sites and degraded to adenosine in the synaptic cleft and that the resulting adenosine was taken up again into nerve endings where it was re-phosphorylated to ATP. 2. Adenosine derivatives in the synaptic cleft inhibited the postsynaptic potentials in olfactory cortex slices in vitro, presumably by the inhibition of Ca2+ influx into nerve endings which resulted in the reduction of transmitter release. 3. The adenosine derivatives also increased the level of cyclic AMP in the slices under the same conditions as above. 4. Although the nature of the "adenosine receptors" for both functions was remarkably similar, the increase of cyclic AMP did not mediate the inhibitory action, but the presynaptic increase of cyclic AMP induced by adenosine derivatives might mediate the facilitation observed in the olfactory cortex. 5. Possible physiological roles of extracellular adenosine derivatives in mammalian brain were classified, at different sites of action around the synapses, with different time courses and modes of action, directly or via the increase of intracellular cyclic AMP.     

Change of Cyclic AMP Level in Synaptosomes from Cerebral Cortex; Increase by Adenosine Derivatives

The endogenous level of cyclic AMP in incubated synaptosomes from cerebral cortex of guinea pigs was investigated after the addition of various agents to the incubation medium. It appeared that the synaptosomal suspension already contained exogenous adenosine. Preincubation with theophylline or with adenosine deaminase (ADase) decreased both the exogenous level of adenosine and the intrasynaptosomal level of cyclic AMP. The level of cyclic AMP was reincreased by the addition of adenosine agonists, especially 2-chloroadenosine. This increase was antagonized by deoxyadenosine and was not inhibited by dipyridamole. These results suggest that the adenosine derivatives in the synaptic cleft regulate the level of cyclic AMP in nerve terminals through adenosine receptor on the presynaptic membrane. ADP, ATP, dopamine, and histamine also stimulate the formation of cyclic AMP in the ADase-treated synaptosomes.     

A relationship between protein kinase C phosphorylation and calmodulin binding to the metabotropic glutamate receptor subtype 7.

Metabotropic glutamate receptor subtype 7 (mGluR7) is coupled to the inhibitory cyclic AMP cascade and is selectively activated by a glutamate analogue, L-2-amino-4-phosphonobutyrate. Among L-2-amino-4-phosphonobutyrate-sensitive mGluR subtypes, mGluR7 is highly concentrated at the presynaptic terminals and is thought to play an important role in modulation of glutamatergic synaptic transmission by presynaptic inhibition of glutamate release. To gain further insight into the intracellular signaling mechanisms of mGluR7, with the aid of glutathione S-transferase fusion affinity chromatography, we attempted to identify proteins that interact with the intracellular carboxyl terminus of mGluR7. Here, we report that calmodulin (CaM) directly binds to the carboxyl terminus of mGluR7 in a Ca(2+)-dependent manner. The CaM-binding domain is located immediately following the 7th transmembrane segment. We also show that the CaM-binding domain of mGluR7 is phosphorylated by protein kinase C (PKC). This phosphorylation is inhibited by the binding of Ca(2+)/CaM to the receptor. Conversely, the Ca(2+)/CaM binding is prevented by PKC phosphorylation. Collectively, these results suggest that mGluR7 serves to cross-link the cyclic AMP, Ca(2+), and PKC phosphorylation signal transduction cascades.     

Alpha adrenergic receptor mediated formation of cyclic AMP in rat spinal cord

Methoxamine and phenylephrine (PE), postsynaptic alpha adrenergic agonists stimulated the accumulation of cyclic AMP in spinal cord tissue slices. Naphazoline, oxymetazoline and clonidine, previously shown to have greater efficacy at presynaptic alpha receptors did not alter accumulation and, in fact, blocked the PE response. The PE-stimulation was completely inhibited by postsynaptic alpha antagonists, incompletely by agents which bl ock presynaptic alpha receptors, and slightly by the beta blocker propranolol. Pe-stimulated accumulation was potentiated by phosphodiesterase inhibition (RO 20-1724). In contrast to previous reports on the requirement of the copresence of adenosine for alpha receptor stimulated accumulation of cyclic AMP in neuronal tissue, the PE-stimulation in spinal cord slices was unchanged by adenosine receptor blockade (theophylline), hydrolysis of endogenous adenosine (adenosine deaminase), inhibition of adenosine deaminase (EHNA) or blockade of adenosine uptake (dipyridamole). Added adenosine increased basal accumulation and produced a marked potentiation of the PE response. From this data it is evident that, in spinal cord tissue slices, there occurs a postsynaptic alpha adrenergic receptor linked to cyclic AMP accumulation which does not require the presence of other neurohumoral agents for activation.     

Presynaptic control as a mechanism of sensory-motor integration.

In studies of central nervous system networks, it is synaptic transmission to the postsynaptic soma-dendritic membrane that has received the most attention, in particular in relation to the analysis of sensory-motor integration. Sensory transmission is gated during ongoing movements in both invertebrates and vertebrates, such that it may be depressed in one phase of a cyclic movement and facilitated in another, in order to optimize the execution of the ongoing motor task. This presynaptic modulation is not limited to sensory afferents, but also occurs in synapses of both excitatory and inhibitory premotor interneurons. The modulation can be mediated by the release of different transmitters at axo-axonal synapses, which activate different types of receptors. In addition, presynaptic sensory axons can be coupled via gap junctions, which under certain conditions may mediate a presynaptic facilitation.     

Multiple factors regulating the release of norepinephrine consequent to nerve stimulation.

Whereas extracellular calcium is absolutely required for neurotransmitter release consequent to stimulation of adrenergic and other neurons, a large number of substances are known to modify the amount of norepinephrine released per nerve impulse. In general, cyclic nucleotides, phosphodiesterase inhibitors, beta-adrenoceptor agonists, cholinergic nicotinic agonists, and angiotensin are able to enhance neurally mediated norepinephrine release, whereas alpha-adrenoreceptor agonists, cholinergic muscarinic agonists, prostaglandins of the E series, opiates, enkephalins, dopamine, and adenosine inhibit neurally mediated norepinephrine release. Although it has been proposed that cyclic AMP may enhance, and endogenous cyclic GMP may inhibit, neurotransmitter release, no consistent relationship between the effects of the several modulators of neurally mediated norepinephrine release and their effects on adenylate and guanylate cyclase is as yet apparent. The demonstration of whether such a relationship exists must await the development of techniques that will allow the measurement of cyclic nucleotide levels in the presynaptic adrenergic nerve terminal after exposure to the putative modulators of release and consequent to nerve stimulation.     

Presynaptic and Postsynaptic Effects of Neuropeptide Y in the Rat Pineal Gland

Abstract: Neuropeptide Y is colocalized with noradrena-line in sympathetic fibers innervating the rat pineal gland. In this article we present a study of the effects and mechanisms of action of neuropeptide Y on the pineal noradrenergic transmission, the main input leading to the rhythmic secretion of melatonin. At the presynaptic level, neuropeptide Y inhibits by 45%, with an EC₅₀ of 50 n M , the potassium-evoked noradrenaline release from pineal nerve endings. This neuropeptide Y inhibition occurs via the activation of pertussis toxin-sensitive G protein-coupled neuropeptide Y-Y2 receptors and is independent from, but additive to, the α₂-adrenergic inhibition of noradrenaline release. At the postsynaptic level, neuropeptide Y decreases by a maximum of 35%, with an EC₅₀ of 5 n M , the β-adrenergic induction of cyclic AMP elevation via the activation of neuropeptide Y-Y1 receptors. This moderate neuropeptide Y-induced inhibition of cyclic AMP accumulation, however, has no effect on the melatonin secretion induced by a β-adrenergic stimulation. On the contrary, in the presence of 1 m M ascorbic acid, neuropeptide Y potentiates (up to threefold) the melatonin secretion. In conclusion, this study has demonstrated that neuropeptide Y modulates the noradrenergic transmission in the rat pineal gland at both presynaptic and postsynaptic levels, using different receptor subtypes and transduction pathways.     

Intracellular cAMP involvement in the synchronized activity of noradrenaline in response to evoked release of the transmitter quanta in the frog synapses

An analogue of cyclic AMP (db-cAMP) penetrating into the frog neuromuscular junction's cell, as well as the adenylyl cyclase activator forskolin, and inhibitor of nucleotide-depending phosphodiesterase isobutilmethylxantine alter the kinetics of the quanta secretion resulting in synchronizing of the process of the transmitter release. Following a db-cAMP preliminary action, no such synchronizing of the transmitter release occurred. Action of noradrenaline on the time course of the secretion seems to be realised through activation of presynaptic beta-adrenoreceptors, augmentation of the adenylyl cyclase activity, and the rise of the intracellular cAMP.     

Presynaptic cyclic nucleotide-gated ion channels modulate neurotransmission in the mammalian olfactory bulb

Cyclic nucleotide-gated channels (CNGCs) on the dendritic cilia of olfactory receptor neurons (ORNs) are critical for sensory transduction in the olfactory system. Do CNGCs also play a role in the axons and/or nerve terminals of ORNs? We find that the cyclic nucleotides cAMP and cGMP can both facilitate and depress synaptic transmission between olfactory nerve fibers and their targets in olfactory bulb glomeruli. Cyclic nucleotides increase intracellular Ca(2+) in ORN terminals and enhance spontaneous transmitter release; at higher concentrations, cyclic nucleotides depress evoked transmission by altering olfactory nerve excitability. Cyclic nucleotides have no effect on transmission or nerve excitability, however, in mice lacking olfactory CNGCs. Taken together, our results identify a novel role for presynaptic CNGCs in modulating neurotransmission.     

Relaxation of Mytilus catch muscle by 8-bromo-cyclic GMP and related compounds

Relaxation of catch tension by 8-bromo-cyclic GMP in the ABRM of Mytilus was blocked in the presence of mersalyl and was markedly reduced after treatment of the muscle with alpha-methyldopa. In the muscle depolarized by 540 mM KCl + 5 mM EGTA solution, 8-bromo-cyclic GMP could not relax Ca-contracture. Hexylamine and phenylethylamine, which are assumed to relax the catch acting on relaxing nerve terminals, could not relax the contracture either. Serotonin and dopamine, which are known to relax the catch acting directly on the muscle fibre membrane, could relax it. In the muscle depolarized by 250 mM KCl + 5 mM EGTA solution, all of the cyclic nucleotides tested (cyclic AMP, cyclic GMP and their analogues), serotonin and dopamine relaxed Ca-contracture, but hexylamine and phenylethylamine did not relax the contracture. The possibilities of the involvement of cyclic GMP in the presynaptic and postsynaptic relaxing mechanisms in the ABRM are discussed.     

Muscarinic receptors modulating acetylcholine release from insect synaptosomes

1. Cholinergic synapses in the central nervous system of insects contain inhibitory muscarinic receptors whose stimulation by agonists leads to a diminished output of acetylcholine; antagonists, like atropine, facilitate acetylcholine release. 2. The receptors involved appear to be of the M2-subtype. Upon activation of presynaptic receptors a significant reduction of the intrasynaptosomal cyclic AMP level as well as a significantly increased membrane potential was observed. 3. The observed membrane hyperpolarization is apparently not a consequence of a lower cyclic AMP level, thus both effects may offer alternative or synergistical mechanisms for modulating transmitter release.     

Regulation of a presynaptic adenylate cyclase from bovine cerebellum by β-adrenergic receptors

Intact crude synaptosomes from bovine cerebellum contain, in addition to an externally accessible (postsynaptic) adenylate cyclase, an enzyme with its catalytic center oriented towards the inside of the synaptosome (presynaptic adenylate cyclase). This is demonstrated by the unmasking of latent adenylate cyclase activity by Triton X-100. Furthermore, intact crude synaptosomes can synthesize cyclic AMP from adenine. This synthesis takes place inside the synaptosome as the postsynaptic adenylate cyclase is inactive in the Krebs-Ringer buffer. Presynaptic adenylate cyclase activity is not influenced by depolarization, as shown by [³H]adenine pulse-labeling, but is stimulated by (?)-norepinephrine and (?)-isoproterenol. (±)-Propranolol inhibits this stimulation whereas phentolamine has no effect, suggesting the presence of a β-adrenergic receptor-coupled presynaptic adenylate cyclase.     

Macroautophagy can press a brake on presynaptic neurotransmission

The mechanistic target of rapamycin (MTOR) has been implicated in regulating synaptic plasticity and neurodegeneration, but MTOR’s role in modulating presynaptic function through autophagy is unexplored. We studied presynaptic function in ventral dopamine neurons, a system from which neurotransmitter release can be measured directly by cyclic voltammetry. We generated mutant mice that were specifically deficient for macroautophagy in dopaminergic neurons by deleting the Atg7 gene in cells that express the dopamine uptake transporter. Dopamine axonal profiles in the mutant dorsal striatum were ~one third larger in the mutant mice, released ~50% more stimulus-evoked dopamine release, and exhibited more rapid presynaptic recovery than controls. Rapamycin reduced dopamine neuron axon profile size by ~30% in control mice, but had no effect on macroautophagy deficient axons. Acute rapamycin decreased dopaminergic synaptic vesicle density by ~25% and inhibited evoked dopamine release by ~25% in control mice, but not in the Atg7 deficient mutants. Thus, both basal and induced macroautophagy can provide a brake on presynaptic activity in vivo, perhaps by regulating the turnover of synaptic vesicles, and further regulates terminal volume and the kinetics of transmitter release.     

CYCLIC AMP AND PHOSPHODIESTERASE IN SYNAPTIC VESICLES FROM MOUSE BRAIN

The isolation of synaptic vesicles from mouse brain by a modification of previously reported methodology is described. Homogeneity of the preparations was ascertained by electron microscopy. Vesicles thus isolated contained cyclic AMP (365 ± 44 pmol/mg of protein; mean ± S.E.M.) and adenosine 3′,5′-monophosphate phosphodiesterase activity (500 pmol/mg of protein per 30 min). The number of vesicles in three of the vesicle preparations was determined by a visual count of grid fields under the electron microscope. The content of cyclic AMP in the vesicles was calculated at 5000-7500 molecules/vesiclc. The presence of cyclic AMP in the synaptic vesicles from presynaptic nerve endings would support a role for this nucleotide in nerve transmission.     

Effects of Cannabinoids on Dopamine Release in the Corpus Striatum and the Nucleus Accumbens In Vitro

Cannabinoid receptors are widely distributed in the nuclei of the extrapyramidal motor and mesolimbic reward systems; their exact functions are, however, not known. The aim of the present study was to characterize the effects of cannabinoids on the electrically evoked release of endogenous dopamine in the corpus striatum and the nucleus accumbens. In rat brain slices dopamine release elicited by single electrical pulses was determined by fast cyclic voltammetry. Dopamine release was markedly inhibited by the OP2 opioid receptor agonist U-50488 and the D2/D3 dopamine receptor agonist quinpirole, indicating that our method is suitable for studying presynaptic modulation of dopamine release. In contrast, the CB1/CB2 cannabinoid receptor agonists WIN55212-2 (10(-6) M) and CP55940 (10(-6)-10(-5) M) and the CB1 cannabinoid receptor antagonist SR141716A (10(-6) M) had no effect on the electrically evoked dopamine release in the corpus striatum and the nucleus accumbens. The lack of a presynaptic effect on terminals of nigrostriatal and mesolimbic dopaminergic neurons is in accord with the anatomical distribution of cannabinoid receptors: The perikarya of these neurons in the substantia nigra and the ventral tegmental area do not synthesize mRNA, and hence protein, for CB1 and CB2 cannabinoid receptors. It is therefore unlikely that presynaptic modulation of dopamine release in the corpus striatum and the nucleus accumbens plays a role in the extrapyramidal motor and rewarding effects of cannabinoids.     

Regulation of high affinity choline uptake

High affinity uptake of choline, the rate-limiting, regulatory step for the synthesis of acetylcholine (ACh), was found to be regulated via presynaptic auto- and heteroreceptors. The transport rate was reduced by a muscarinic agonist and neuropeptides, but was significantly enhanced by octopamine. Intracellular messengers, including cyclic nucleotides, appear to modulate the transport activity, apparently by activating specific protein kinases.     

Macroautophagy can press a brake on presynaptic neurotransmission

The mechanistic target of rapamycin (MTOR) has been implicated in regulating synaptic plasticity and neurodegeneration, but MTOR’s role in modulating presynaptic function through autophagy is unexplored. We studied presynaptic function in ventral dopamine neurons, a system from which neurotransmitter release can be measured directly by cyclic voltammetry. We generated mutant mice that were specifically deficient for macroautophagy in dopaminergic neurons by deleting the Atg7 gene in cells that express the dopamine uptake transporter. Dopamine axonal profiles in the mutant dorsal striatum were ~one third larger in the mutant mice, released ~50% more stimulus-evoked dopamine release, and exhibited more rapid presynaptic recovery than controls. Rapamycin reduced dopamine neuron axon profile size by ~30% in control mice, but had no effect on macroautophagy deficient axons. Acute rapamycin decreased dopaminergic synaptic vesicle density by ~25% and inhibited evoked dopamine release by ~25% in control mice, but not in the Atg7 deficient mutants. Thus, both basal and induced macroautophagy can provide a brake on presynaptic activity in vivo, perhaps by regulating the turnover of synaptic vesicles, and further regulates terminal volume and the kinetics of transmitter release.