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.

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 15, 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 ofB15 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 stiniulation 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.Over the last decade, it has become clear that many neurons contain multiple transmitters, often one or more peptide transmitters with a single conventional transmitter (1, 2). Knowledge of the regulation of release of coexisting transmitters is crucial to our understanding of the physiological roles of each transmitter and the interactions between them (3-5). The metabolism of conventional transmitters and peptide transmitters differs significantly. Conventional transmitters are synthesized enzymatically at synaptic terminals and, once released, usually have high-affinity uptake systems. Thus, there are cellular mechanisms for maintaining homeostatic levels of conventional transmitters. In contrast, peptide transmitters are cleaved from precursors in the neuronal cell body and transported to remote terminals and there is little evidence for efficient reuptake of most peptides. Consequently, during sustained activation, either the rates of peptide release must be very low compared to total content in the terminals or the peptide content must decline.One class of peptides that have been commonly found to coexist with other transmitters in Aplysia neurons are the small cardioactive peptides (SCPs) (6-8). The SCPs are represented by two peptides, SCPA and SCPB, which are 11 and 9 amino acids in length, respectively. These peptides have similar se...

It is becoming clear that astrocytes are active participants in synaptic functioning and exhibit properties, such as the secretion of classical transmitters, previously thought to be exclusively neuronal. Whether these similarities extend to the release of neuropeptides, the other major class of transmitters, is less clear. Here we show that cortical astrocytes can synthesize both native and foreign neuropeptides and can secrete them in a stimulation-dependent manner. Reverse transcription-PCR and mass spectrometry indicate that cortical astrocytes contain neuropeptide Y (NPY), a widespread neuronal transmitter. Immunocytochemical studies reveal NPY-immunoreactive (IR) puncta that colocalize with markers of the regulated secretory pathway. These NPY-IR puncta are distinct from the synaptic-like vesicles that contain classical transmitters, and the two types of organelles are differentially distributed. After activation of metabotropic glutamate receptors and the release of calcium from intracellular stores, the NPY-IR puncta fuse with the cell membrane, and the peptidecontaining dense cores are displayed. To determine whether peptide secretion subsequently occurred, exocytosis was monitored from astrocytes expressing NPY-red fluorescent protein (RFP). In live cells, after activation of glutamate receptors, the intensity of the NPY-RFP-labeled puncta declined in a step-like manner indicating a regulated release of the granular contents. Because NPY is a widespread and potent regulator of synaptic transmission, these results suggest that astrocytes could play a role in the peptidergic modulation of synaptic signaling in the CNS.

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During fasting, activation of the counter-regulatory response (CRR) prevents hypoglycemia. A major effector arm is the autonomic nervous system that controls epinephrine release from adrenal chromaffin cells and, consequently, hepatic glucose production. However, whether modulation of autonomic function determines the relative strength of the CRR, and thus the ability to withstand food deprivation and maintain euglycemia, is not known. Here we show that fasting leads to altered transmission at the preganglionic → chromaffin cell synapse. The dominant effect is a presynaptic, long-lasting increase in synaptic strength. Using genetic and pharmacological approaches we show this plasticity requires neuropeptide Y, an adrenal cotransmitter and the activation of adrenal Y5 receptors. Loss of neuropeptide Y prevents a fasting-induced increase in epinephrine release and results in hypoglycemia in vivo. These findings connect plasticity within the sympathetic nervous system to a physiological output and indicate the strength of the final synapse in this descending pathway plays a decisive role in maintaining euglycemia.hypoglycemia | autonomic nervous system | synaptic plasticity | adrenal | chromaffin cells F ailure to avoid hypoglycemia can lead to dysphoria, ventricular arrhythmia, and even sudden death (1). That these effects are rare and observed only in response to prolonged fasting or severe insulin-induced hypoglycemia is because of the remarkable effectiveness of the counter-regulatory response (CRR). This sensory-motor homeostatic feedback loop detects a fall in blood glucose through central and peripheral receptors and initiates a neuronal, endocrine, and behavioral response that restores euglycemia (2). One of the principal effector arms of the CRR is the sympatho-adrenal branch of the autonomic nervous system. Hypoglycemia elevates sympathetic activity, increasing hepatic glucose production and the release of gluconeogenic substrates while suppressing insulin and potentiating glucagon secretion (3, 4).During fasting, these autonomic actions are mediated by epinephrine, which enters the systemic circulation after release from adrenal neuroendocrine chromaffin cells and by norepinephrine, secreted directly onto target tissues from postganglionic sympathetic neurons. The importance of sympathetic activity, and in particular circulating epinephrine in the CRR, is illustrated by the poor recovery from insulin-induced hypoglycemia when the release of this hormone is suppressed during hypoglycemia-associated autonomic failure (5, 6), and by recent work showing that deletion of melanocortin 4 receptors from preganglionic sympathetic neurons leads to elevated levels of blood glucose (7).Given the involvement of the sympathetic nervous system in the CRR, modulation of autonomic activity is thus likely to alter the ability to respond to a hypoglycemic challenge. However, unlike in the CNS, where long-lasting changes in synaptic strength are known to be associated with functional consequences (8-10), whether the output ...