Activation of NPY type 5 receptors induces a long-lasting increase in spontaneous GABA release from cerebellar inhibitory interneurons.

Neuropeptide Y (NPY), a widely distributed neuropeptide in the central nervous system, can transiently suppress inhibitory synaptic transmission and alter membrane excitability via Y2 and Y1 receptors (Y2rs and Y1rs), respectively. Although many GABAergic neurons express Y5rs, the functional role of these receptors in inhibitory neurons is not known. Here, we investigated whether activation of Y5rs can modulate inhibitory transmission in cerebellar slices. Unexpectedly, application of NPY triggered a long-lasting increase in the frequency of miniature inhibitory postsynaptic currents in stellate cells. NPY also induced a sustained increase in spontaneous GABA release in cultured cerebellar neurons. When cerebellar cultures were examined for Y5r immunoreactivity, the staining colocalized with that of VGAT, a presynaptic marker for GABAergic cells, suggesting that Y5rs are located in the presynaptic terminals of inhibitory neurons. RT-PCR experiments confirmed the presence of Y5r mRNA in the cerebellum. The NPY-induced potentiation of GABA release was blocked by Y5r antagonists and mimicked by application of a selective peptide agonist for Y5r. Thus Y5r activation is necessary and sufficient to trigger an increase in GABA release. Finally, the potentiation of inhibitory transmission could not be reversed by a Y5r antagonist once it was initiated, consistent with the development of a long-term potentiation. These results indicate that activation of presynaptic Y5rs induces a sustained increase in spontaneous GABA release from inhibitory neurons in contrast to the transient suppression of inhibitory transmission that is characteristic of Y1r and Y2r activation. Our findings thus reveal a novel role of presynaptic Y5rs in inhibitory interneurons in regulating GABA release and suggest that these receptors could play a role in shaping neuronal network activity in the cerebellum.

Targeted attenuation of electrical activity in Drosophila using a genetically modified K(+) channel.

We describe here a general technique for the graded inhibition of cellular excitability in vivo. Inhibition is accomplished by expressing a genetically modified Shaker K(+) channel (termed the EKO channel) in targeted cells. Unlike native K(+) channels, the EKO channel strongly shunts depolarizing current: activating at potentials near E(K) and not inactivating. Selective targeting of the channel to neurons, muscles, and photoreceptors in Drosophila using the Gal4-UAS system results in physiological and behavioral effects consistent with attenuated excitability in the targeted cells, often with loss of neuronal function at higher transgene dosages. By permitting the incremental reduction of electrical activity, the EKO technique can be used to address a wide range of questions regarding neuronal function.

Presynaptic target of Ca2+ action on neuropeptide and acetylcholine release in Aplysia californica.

1. When buccal neuron B2 of Aplysia californica is co-cultured with sensory neurons (SNs), slow peptidergic synapses are formed. When B2 is co-cultured with neurons B3 or B6, fast cholinergic synapses are formed. 2. Patch pipettes were used to voltage clamp pre- and postsynaptic neurons and to load the caged Ca2+ chelator o-nitrophenyl EGTA (NPE) and the Ca2+ indicator BTC into presynaptic neurons. The relationships between presynaptic [Ca2+]i and postsynaptic responses were compared between peptidergic and cholinergic synapses formed by cell B2. 3. Using variable intensity flashes, Ca2+ stoichiometries of peptide and acetylcholine (ACh) release were approximately 2 and 3, respectively. The difference did not reach statistical significance. 4. ACh quanta summate linearly postsynaptically. We also found a linear dose-response curve for peptide action, indicating a linear relationship between submaximal peptide concentration and response of the SN. 5. The minimum intracellular calcium concentrations ([Ca2+]i) for triggering peptidergic and cholinergic transmission were estimated to be about 5 and 10 microM, respectively. 6. By comparing normal postsynaptic responses to those evoked by photolysis of NPE, we estimate [Ca2+]i at the release trigger site elicited by a single action potential (AP) to be at least 10 microM for peptidergic synapses and probably higher for cholinergic synapses. 7. Cholinergic release is brief (half-width approximately 200 ms), even in response to a prolonged rise in [Ca2+]i, while some peptidergic release appears to persist for as long as [Ca2+]i remains elevated (for up to 10 s). This may reflect differences in sizes of reserve pools, or in replenishment rates of immediately releasable pools of vesicles. 8. Electron microscopy revealed that most synaptic contacts had at least one morphologically docked dense core vesicle that presumably contained peptide; these were often located within conventional active zones. 9. Both cholinergic and peptidergic vesicles are docked within active zones, but cholinergic vesicles may be located closer to Ca2+ channels than are peptidergic vesicles.

A novel technique that measures peptide secretion on a millisecond timescale reveals rapid changes in release.

Neuropeptides are ubiquitous transmitters that have been implicated in a wide variety of physiological and pathological conditions, and it is important to understand the processes that control their secretion. We have developed a technique that measures neuropeptide secretion with high temporal resolution. This method involves placing an electrophysiological "tag" in a neuropeptide prohormone. The tagged prohormone is subsequently expressed together with an ionotropic receptor that binds the tag. Because the neuropeptide of interest and the tag enter the same population of dense core granules, neuropeptide secretion gives rise to fast, synaptic-like currents. Using this method, we show that peptide secretion can be modulated on a millisecond time scale. This technique could be readily adapted to measure the secretion of any neuropeptide.

Heterologous expression of the Kv3.1 potassium channel eliminates spike broadening and the induction of a depolarizing afterpotential in the peptidergic bag cell neurons.

The bag cell neurons of Aplysia are a cluster of cells that control egg laying behavior. After brief synaptic stimulation, they depolarize and fire spontaneously for up to 30 min. During the first few seconds of this afterdischarge, the action potentials of the bag cell neurons undergo pronounced broadening. Single bag cell neurons in culture also show spike broadening in response to repeated depolarizations. This broadening is frequency-dependent and associated with the induction of a depolarizing afterpotential lasting minutes. In some neurons the depolarizing afterpotential is sufficient to trigger spontaneous firing. To test the possibility that spike broadening during stimulation is required to trigger the depolarizing afterpotential, we eliminated frequency-dependent broadening by heterologous expression of the Kv3.1 potassium channel. This channel has rapid activation and deactivation kinetics and no use-dependent inactivation. Expression of Kv3.1 prevented spike broadening and also eliminated the depolarizing afterpotential. Measurements of the integral of calcium current during voltage commands, which simulated the action potentials of the control neurons and those expressing Kv3.1, indicate that spike broadening produces up to a fivefold increase in calcium entry. Manipulations that limit calcium entry during action potentials or chelation of intracellular calcium using BAPTA AM prevented the induction of the depolarizing afterpotential. We conclude that spike broadening is essential for the induction of the depolarizing afterpotential probably by regulating calcium influx and suggest that one of the physiological roles of spike broadening may be to regulate long-term changes in neuronal excitability.

Expression of a foreign G-protein coupled receptor modulates the excitability of the peptidergic bag cell neurons of Aplysia.

Brief afferent stimulation of the bag cell neurons can trigger a sustained period of spontaneous firing (the 'afterdischarge'). Pharmacological activation of a number of second messenger pathways has previously been shown to partially replicate this behavior. One strategy to identify additional pathways may be to heterologously express a variety of G-protein coupled receptors in bag cell neurons, then test whether receptor activation can induce an afterdischarge. We find that expression of the 1alpha metabotropic glutamate receptor in single bag cell neurons mimics some features of the afterdischarge. This approach is thus feasible and may allow the reconstruction of different components of afterdischarge in the absence of the afferent input.

The secretion of classical and peptide cotransmitters from a single presynaptic neuron involves a synaptobrevin-like molecule.

It is not yet understood how the molecular mechanisms controlling the release of neuropeptides differ from those controlling the release of classical transmitters, mainly because there are few peptidergic synapses in which the environment at the presynaptic release sites can be manipulated. Using Aplysia californica neuron B2, which synthesizes both peptide and classical transmitters, we have established two synaptic types. When B2 is cocultured with a sensory neuron, a peptidergic synapse is formed. In contrast, when B2 is cocultured with neuron B6, a classical synapse is formed. In contrast to a common assumption, single action potentials can release both types of transmitters. The secretion of peptide and classical transmitters by B2 is inhibited by the presynaptic injection of tetanus toxin, but not by an inactive mutant. Thus a synaptobrevin-like molecule is involved in the secretion of these two types of transmitters.

Differential regulation of the release of the same peptide transmitters from individual identified motor neurons in culture.

Aplysia motor neurons B1, B2, and B15 synthesize the small cardioactive peptides A and B (SCPs). In previous studies using semi-intact preparations we have demonstrated that the SCPs are released from B15. Significant peptide release only occurred when B15 was stimulated at a high frequency or at lower frequencies with a long burst duration. In the behaving animal B15 fires in patterns expected to release the SCPs. In contrast, in the behaving animal, neurons B1 and B2 fire at much lower frequencies. We therefore examined whether similar aspects of the stimulation pattern governed the release of the SCPs from B1 and B2. To monitor peptide release, all three neurons were individually cultured and newly synthesized peptides labeled with 35S-methionine. Release of labeled SCPs was detected by HPLC of extracts of superfusates. By keeping spike number constant and varying the stimulation pattern, the release of the SCPs from B1 and B2 was found to be pattern insensitive. That is, regardless of the stimulation paradigm, each action potential released a similar amount of peptide. By severing the primary neurite, peptide release was found to occur mainly from the regenerated neurites and most likely from the intensely immunoreactive varicosites. Calcium- and stimulation-dependent release of the SCPs and a third neuropeptide termed buccalin A from motor neuron B15 was also observed in culture. The release of these peptides from B15 was found to be pattern sensitive as was observed in the semi-intact preparations.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of neuromuscular transmission by conventional and peptide transmitters released from excitatory and inhibitory motor neurons in Aplysia.

The anterior portion of intrinsic buccal muscle (I3a) is innervated by two excitatory motor neurons, B3 and B38, and the newly identified inhibitory motor neuron, B47. We show that B47 is cholinergic while B3 and B38 are not. B3 and B38 have previously been shown to express the neuropeptides FMRFamide and the small cardioactive peptides (SCPs) A and B, respectively. We present evidence here that B47 synthesizes the neuropeptide myomodulin A (Mma). When placed in culture, B3, B38, and B47 continued to synthesize their respective peptides. These peptides were released in a stimulation- and Ca(2+)-dependent manner, suggesting that they are transmitters in these neurons. By using B3-evoked excitatory junction potentials (EJPs) and muscle contractions as assays, we next examined the modulatory effects of superfusion of peptides and stimulation of motor neurons B38 and B47. Superfusing the muscle with low concentrations of the SCPs, FMRFamide, or Mma enhanced B3-evoked EJPs and contractions. Stimulation of B47 simultaneously with B3 reduced the amplitude of B3-evoked contractions. However, when either B47 or B38 was stimulated in extended bursts designed to release their peptide transmitters, subsequent B3-evoked EJPs and contractions were enhanced. We believe that this modulation is due at least in part to the release of peptides from the terminals of B38 and B47. The SCPs potently increase cAMP levels in I3a muscle fibers. Likewise, stimulation of B38 in extended bursts increased cAMP levels in the muscle. This provides independent evidence that the SCPs are released from B38 terminals in the muscle. Therefore, we have described a neuromuscular preparation amenable to the study of both excitatory and inhibitory motor neurons that utilize a variety of conventional and peptide transmitters. Our results suggest that these motor neurons can function in two states. When stimulated in single brief bursts, they primarily release conventional transmitters. When stimulated in a series of prolonged bursts, they release both conventional transmitters and peptide cotransmitters. These dual states are most pronounced in the case of B47, which, depending on the stimulation paradigm, can act selectively to inhibit or enhance the effects of a second motor neuron innervating the same muscle.

Functional roles of peptide cotransmitters at neuromuscular synapses in Aplysia.

Neuromuscular synapses in Aplysia have been used as model systems to study peptidergic cotransmission. Here we describe neuromuscular preparations in which it has been possible to investigate the physiological consequences of peptide transmitter release in detail. In the first preparation, the release of peptide cotransmitters from identified motor neuron B15 has been shown to be sensitive to the pattern of stimulation. High frequencies and long burst durations evoke peptide release that modulates muscle contractions in a manner similar to that produced by exogenous cotransmitter. By contrast, the release of the same peptide transmitters from motor neuron B1 show little dependence on pattern. We conclude that there are no stimulation patterns that are prerequisites for peptide release. Peptide cotransmitter release from motor neuron B47 has also been studied. B47, depending on the stimulation pattern, uses either ACh, which acts as a conventional inhibitory transmitter, or ACh plus neuropeptides, which act as excitatory modulatory cotransmitters. Thus, neuropeptide cotransmitters have the capability to greatly increase synaptic plasticity at neuromuscular synapses.

Modulation of peptide release from single identified Aplysia neurons in culture.

Aplysia neurons B1 and B2 contain large amounts of the neuropeptides SCPA and SCPB. When grown in culture, individual B1 and B2 cells incorporate 35S-methionine into the SCPs, which can be released in a stimulus- and calcium-dependent fashion (Lloyd et al., 1986). We now show that single cells can be stimulated in a manner to evoke release of the SCPs that declines only slightly with repeated stimulation. This has allowed us to examine the ability of several physiologically relevant agonists to modulate the stimulus-evoked release of the SCPs. Bath application of either FMRFamide or 5-HT resulted in a significant decrease in the amount of SCPs released by intracellular stimulation of B1 or B2. The action of 5-HT was dose dependent with an inhibition of release of approximately 70% at a concentration of 100 microM. SCPA did not significantly affect release. The bath application of several compounds that are expected to elevate intracellular levels of cAMP were also found to depress release. To investigate the possibility that the agonists inhibited the release of the SCPs via a hyperpolarization of membrane potential (and perhaps a loss of spikes in the neurites), we examined the actions of 5-HT, FMRFamide, and SCPA on several electrophysiological parameters intended to monitor the level of cell excitability. Surprisingly, even though 5-HT depressed the release of the SCPs from both cells, it depolarized and increased the excitability of B1, and hyperpolarized and decreased the excitability of B2. Furthermore, in contrast to the effects seen in culture, 5-HT depolarized both B1 and B2 in situ.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuropeptide cotransmitters released from an identified cholinergic motor neuron modulate neuromuscular efficacy in Aplysia.

Intrinsic buccal muscle 5 (I5) in Aplysia is innervated by 2 motor neurons (termed B15 and B16). In addition to the classical transmitter ACh, B15 also contains the 2 neuropeptides SCPA and SCPB. In a previous study, we demonstrated that the SCPs were released from the terminals of B15 in the I5 muscle and that this release was sufficient to raise cAMP levels in I5 muscle fibers. Significant peptide release occurred only when B15 was stimulated at high frequency or at lower frequencies with a relatively long burst duration (Whim and Lloyd, 1989). In the present article, we examine the possibility that the SCPs released from B15 modulate I5 muscle contractions produced by stimulation of the second motor neuron, B16. Application of exogenous SCPs to I5 muscles increased the amplitude and relaxation rate of B16-evoked contractions. Stimulation of B15 using paradigms that have been shown previously to cause release of the SCPs resulted in a long-lasting increase in the amplitude and relaxation rate of muscle contractions evoked by B16. This modulation is unlikely to be due to the B15-induced muscle contractions themselves, because modulation of B16-evoked contraction amplitude and relaxation rate was observed when the contractions were blocked transiently by a cholinergic antagonist during B15 stimulation. Conversely, stimulation of B15 at frequencies that produce no measurable release of the SCPs did not elicit significant modulation of B16-evoked contractions. The minimum B15 stimulation frequency required to elevate muscle cAMP levels or to modulate B16-evoked contractions was found to be within the physiological range at which B15 fires during feeding. Therefore, the mechanism underlying the modulation of B16-evoked contractions by B15 is likely to involve the release of the SCPs from B15 terminals in the I5 muscle. With respect to behavior, this modulation of muscle contractions would be most likely to occur during food-induced arousal when both motor neurons fire at high frequency with brief interburst intervals.

Frequency-dependent release of peptide cotransmitters from identified cholinergic motor neurons in Aplysia.

We have investigated the release of two peptide cotransmitters from the terminals of a cholinergic motor neuron in Aplysia. Identified motor neuron B15 synthesizes the two small cardioactive peptides (SCP) A and B in addition to acetylcholine. A symmetrical pair of B15 neurons innervate symmetrical buccal muscles, termed I5, which are involved in generating biting movements. The amplitude of I5 contractions is enhanced by the SCPs. Intracellular stimulation of one B15 produces depletion of the SCPs from the stimulated muscle as compared to the unstimulated control muscle. Significant depletion requires either high-frequency stimulation or prolonged bursts at lower frequencies. A second cholinergic motor neuron, B16, also innervates I5 but does not synthesize the SCPs. Stimulation of B16 produced no depletion of the SCPs. Exogenous SCPs potently increase cAMP levels in the muscle. If depletion is a reflection of release, it should be possible to demonstrate an effect of B15 stimulation on muscle cAMP levels. Indeed, stimulation of B15 did elevate cAMP levels in I5. Stimulation of B16 had no effect on cAMP levels. Increases in cAMP were observed only when B15 was stimulated in a manner that would produce significantly facilitated acetylcholine release. This facilitation could be produced by increased stimulation frequency, longer burst durations, or shorter interburst intervals. However, B15 is capable of producing cholinergically mediated contractions with stimulation parameters that would not cause release of the SCPs. Thus, B15 appears to function as a purely cholinergic motor neuron when firing slowly, and as a cholinergic/peptidergic neuron when firing rapidly.