The ß-Hydroxybutyrate-GPR109A Receptor Regulates Fasting-induced Plasticity in the Mouse Adrenal Medulla.

During fasting, increased sympathoadrenal activity leads to epinephrine release and multiple forms of plasticity within the adrenal medulla including an increase in the strength of the preganglionic → chromaffin cell synapse and elevated levels of agouti-related peptide (AgRP), a peptidergic cotransmitter in chromaffin cells. Although these changes contribute to the sympathetic response, how fasting evokes this plasticity is not known. Here we report these effects involve activation of GPR109A (HCAR2). The endogenous agonist of this G protein-coupled receptor is ß-hydroxybutyrate, a ketone body whose levels rise during fasting. In wild-type animals, 24-hour fasting increased AgRP-ir in adrenal chromaffin cells but this effect was absent in GPR109A knockout mice. GPR109A agonists increased AgRP-ir in isolated chromaffin cells through a GPR109A- and pertussis toxin-sensitive pathway. Incubation of adrenal slices in nicotinic acid, a GPR109A agonist, mimicked the fasting-induced increase in the strength of the preganglionic → chromaffin cell synapse. Finally, reverse transcription polymerase chain reaction experiments confirmed the mouse adrenal medulla contains GPR109A messenger RNA. These results are consistent with the activation of a GPR109A signaling pathway located within the adrenal gland. Because fasting evokes epinephrine release, which stimulates lipolysis and the production of ß-hydroxybutyrate, our results indicate that chromaffin cells are components of an autonomic-adipose-hepatic feedback circuit. Coupling a change in adrenal physiology to a metabolite whose levels rise during fasting is presumably an efficient way to coordinate the homeostatic response to food deprivation.

Recurrent hypoglycemia inhibits the counterregulatory response by suppressing adrenal activity.

Hypoglycemia activates the counterregulatory response (CRR), a neural-endocrine reflex that restores euglycemia. Although effective if occasionally activated, repeated induction of the CRR leads to a decline in responsiveness and prolonged exposure to hypoglycemia. The mechanism underlying this impairment is not known. We found that the reduction in epinephrine release that characterizes a suppressed CRR involves a long-lasting form of sympatho-adrenal synaptic plasticity. Using optogenetically evoked catecholamine release, we show that recurrent hypoglycemia reduced the secretory capacity of mouse adrenal chromaffin cells. Single activation of the CRR increased the adrenal levels of tyrosine hydroxylase (TH), the rate-limiting enzyme for catecholamine synthesis, but this was prevented by repeated activation. In contrast, the level of neuropeptide Y (NPY), an adrenal cotransmitter, remained elevated after recurrent hypoglycemia. Inhibition of NPY or Y1 signaling, either transgenically or pharmacologically, prevented the attenuation of both TH expression and epinephrine release. These results indicate that impairment of the CRR involves suppressed activity at the adrenal level. Interfering with the peripheral NPY-dependent negative feedback loop may provide a way to avoid the pathophysiological consequences of recurrent hypoglycemia which are common in the diabetic state.

AgRP-Expressing Adrenal Chromaffin Cells Are Involved in the Sympathetic Response to Fasting.

Fasting evokes a homeostatic response that maintains circulating levels of energy-rich metabolites and increases the drive to eat. Centrally, this reflex activates a small population of hypothalamic neurons that are characterized by the expression of AgRP, a neuropeptide with an extremely restricted distribution. Apart from the hypothalamus, the only other site with substantial expression is the adrenal gland, but there is disagreement about which cells synthesize AgRP. Using immunohistochemistry, flow cytometry, and reverse transcription-polymerase chain reaction, we show AgRP is present in the mouse adrenal medulla and is expressed by neuroendocrine chromaffin cells that also synthesize the catecholamines and neuropeptide Y. Short-term fasting led to an increase in adrenal AgRP expression. Because AgRP can act as an antagonist at MC3/4 receptors, we tested whether melanotan II, an MC3/4 receptor agonist, could regulate pre- and postsynaptic signaling within the adrenal medulla. Melanotan II decreased the paired-pulse ratio of evoked synaptic currents recorded in chromaffin cells; this effect was blocked by exogenous AgRP. In contrast, neither melanotan II nor AgRP altered the optogenetically evoked release of catecholamines from isolated chromaffin cells. These results are consistent with the idea that AgRP regulates the strength of the sympathetic input by modulation of presynaptic MC3/4 receptors located on preganglionic neurons. We conclude that a small population of neuroendocrine cells in the adrenal medulla, and the arcuate nucleus of the hypothalamus, express AgRP and neuropeptide Y and are functionally involved in the systemic response to fasting.

Fasting induces a form of autonomic synaptic plasticity that prevents hypoglycemia.

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.

Neuropeptide y gates a stress-induced, long-lasting plasticity in the sympathetic nervous system.

Acute stress evokes the fight-or-flight reflex, which via release of the catecholamine hormones affects the function of every major organ. Although the reflex is transient, it has lasting consequences that produce an exaggerated response when stress is reexperienced. How this change is encoded is not known. We investigated whether the reflex affects the adrenal component of the sympathetic nervous system, a major branch of the stress response. Mice were briefly exposed to the cold-water forced swim test (FST) which evoked an increase in circulating catecholamines. Although this hormonal response was transient, the FST led to a long-lasting increase in the catecholamine secretory capacity measured amperometrically from chromaffin cells and in the expression of tyrosine hydroxylase. A variety of approaches indicate that these changes are regulated postsynaptically by neuropeptide Y (NPY), an adrenal cotransmitter. Using immunohistochemistry, RT-PCR, and NPY(GFP) BAC mice, we find that NPY is synthesized by all chromaffin cells. Stress failed to increase secretory capacity in NPY knock-out mice. Genetic or pharmacological interference with NPY and Y1 (but not Y2 or Y5) receptor signaling attenuated the stress-induced change in tyrosine hydroxylase expression. These results indicate that, under basal conditions, adrenal signaling is tonically inhibited by NPY, but stress overrides this autocrine negative feedback loop. Because acute stress leads to a lasting increase in secretory capacity in vivo but does not alter sympathetic tone, these postsynaptic changes appear to be an adaptive response. We conclude that the sympathetic limb of the stress response exhibits an activity-dependent form of long-lasting plasticity.

Stress-induced changes in adrenal neuropeptide Y expression are regulated by a negative feedback loop.

Neuropeptide Y is a co-transmitter that is synthesized by chromaffin cells in the adrenal medulla. During the fight-or-flight response these cells release NPY in addition to epinephrine and norepinephrine. Following the stress-induced reflex, the levels of NPY are increased as part of a homeostatic response that modulates catecholaminergic signaling. Here, we examined the control of NPY expression in mice after brief exposure to the cold water forced swim test. This treatment led to a shift in NPY expression between two populations of chromaffin cells that reversed over the course of 1 week. When NPY(GFP) BAC transgenic animals were exposed to stress, there was an increase in cytoplasmic, non-secretable GFP, indicating that stress increased NPY promoter activity. In vivo blockage of Y2 (but not Y1 or Y5) receptors increased basal adrenal NPY expression and so modulated the effects of stress. We conclude that release of NPY mediates a negative feedback loop that inhibits its own expression. Thus, the levels of NPY are determined by a balance between the potentiating effects of stress and the tonic inhibitory actions of Y2 receptors. This may be an efficient way to ensure the levels of this modulator do not decline following intense sympathetic activity.

Cell type-dependent trafficking of neuropeptide Y-containing dense core granules in CNS neurons.

Neuropeptide transmitters are synthesized throughout the CNS and play important modulatory roles. After synthesis in the neuronal cell body, it is generally assumed that peptides are transported to nonspecialized sites of release. However, apart from a few cases, this scenario has not been thoroughly examined. Using wild-type and NPY(GFP) transgenic mice, we have studied the subcellular distribution of neuropeptide Y (NPY), a prototypical and broadly expressed neuropeptide. NPY puncta were found in the dendrites and axons of hippocampal GABAergic interneurons in situ. In contrast in hypothalamic GABAergic interneurons, NPY was restricted to the axon. Surprisingly this differential trafficking was preserved when the neurons were maintained in vitro. When hippocampal and hypothalamic neurons were transfected with NPY-Venus, the distribution of the fluorescent puncta replicated the cell type-specific distribution of endogenous neuropeptide Y. The NPY puncta in the axons of hippocampal and hypothalamic neurons colocalized with the sites of classical transmitter release (identified by staining for synapsin and the vesicular GABAergic transporter, VGAT). In hippocampal neurons, most of the postsynaptic NPY puncta were clustered opposite synapsin-containing varicosities. When neurons were stained for a second neuropeptide, agouti-related protein, immunoreactivity was found in the axon and dendrites of hippocampal neurons but only in the axons of hypothalamic neurons, thus mimicking the polarized distribution of NPY. These results indicate that the trafficking of neuropeptide-containing dense core granules is markedly cell type specific and is not determined entirely by the characteristics of the particular peptide per se.

Pancreatic beta cells synthesize neuropeptide Y and can rapidly release peptide co-transmitters.

BACKGROUND: In addition to polypeptide hormones, pancreatic endocrine cells synthesize a variety of bioactive molecules including classical transmitters and neuropeptides. While these co-transmitters are thought to play a role in regulating hormone release little is known about how their secretion is regulated. Here I investigate the synthesis and release of neuropeptide Y from pancreatic beta cells. METHODOLOGY/PRINCIPAL FINDINGS: NPY appears to be an authentic co-transmitter in neonatal, but not adult, beta cells because (1) early in mouse post-natal development, many beta cells are NPY-immunoreactive whereas no staining is observed in beta cells from NPY knockout mice; (2) GFP-expressing islet cells from an NPY(GFP) transgenic mouse are insulin-ir; (3) single cell RT-PCR experiments confirm that the NPY(GFP) cells contain insulin mRNA, a marker of beta cells. The NPY-immunoreactivity previously reported in alpha and delta cells is therefore likely to be due to the presence of NPY-related peptides. INS-1 cells, a beta cell line, are also NPY-ir and contain NPY mRNA. Using the FMRFamide tagging technique, NPY secretion was monitored from INS-1 beta cells with high temporal resolution. Peptide release was evoked by brief depolarizations and was potentiated by activators of adenylate cyclase and protein kinase A. Following a transient depolarization, NPY-containing dense core granules fused with the cell membrane and discharged their contents within a few milliseconds. CONCLUSIONS: These results indicate that after birth, NPY expression in pancreatic islets is restricted to neonatal beta cells. The presence of NPY suggests that peptide co-transmitters could mediate rapid paracrine or autocrine signaling within the endocrine pancreas. The FMRFamide tagging technique may be useful in studying the release of other putative islet co-transmitters in real time.

Trafficking and fusion of neuropeptide Y-containing dense-core granules in astrocytes.

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 peptide-containing 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.

A common single nucleotide polymorphism alters the synthesis and secretion of neuropeptide Y.

A single nucleotide polymorphism (SNP) in the neuropeptide Y gene has been associated with elevated serum lipid levels and cardiovascular disease. The polymorphism (T1128C) changes the seventh amino acid in the prohormone from leucine to proline. It has been speculated this alters neuropeptide Y (NPY) synthesis, trafficking, or secretion. We tested this hypothesis by expressing the mutant and wild-type prohormones in CNS neurons and endocrine cells. Synthesis and trafficking were followed using immunocytochemistry and fluorescent protein-tagged fusion constructs. Mutant prohormone was synthesized and entered the regulated secretory pathway. When expressed in endocrine cells, wild-type and mutant proteins were found in the same large dense core granules. However, the T1128C polymorphism altered the degree of copackaging, and, on average, individual granules contained more mutant prohormone. This was not attributable to codon bias but to the change in prohormone sequence. Global prohormone targeting was normal, because in hippocampal neurons, the polarized distribution of the mutant prohormone was indistinguishable from the wild-type. When secretion was measured from chromaffin cells, brief depolarizations triggered peptide secretion, confirming the entry of the mutant prohormone into the regulated secretory pathway. However, cells that expressed the mutant protein had increased levels of peptide secretion. We conclude that the T1128C polymorphism alters the packaging and secretion of NPY. In contrast to SNPs in other prohormones, we could not find a phenotype until the prohormone was tracked at the single granule level. These results are consistent with studies showing the T1128C polymorphism has pleiotropic effects.

Cytomegalovirus induces interferon-stimulated gene expression and is attenuated by interferon in the developing brain.

Cytomegalovirus (CMV) is considered the most common infectious agent causing permanent neurological dysfunction in the developing brain. We have previously shown that CMV infects developing brain cells more easily than it infects mature brain cells and that this preference is independent of the host B- and T-cell responses. In the present study, we examined the innate antiviral defenses against mouse (m) and human (h) CMVs in developing and mature brain and brain cells. mCMV infection induced interferon (IFN)-stimulated gene expression by 10- to 100-fold in both glia- and neuron-enriched cultures. Treatment of primary brain cultures with IFN-alpha, -beta, and -gamma or a synthetic RNA, poly(I:C), reduced the number of mCMV-infected cells, both in older cells and in fresh cultures from embryonic mouse brains. When a viral dose that killed almost all unprotected cells was used, IFN-protected cells had a natural appearance, and when they were tested with whole-cell patch clamp recording, they appeared physiologically normal with typical resting membrane potentials and action potentials. mCMV infection increased expression of representative IFN-stimulated genes (IFIT3, OAS, LMP2, TGTP, and USP18) in both neonatal and adult brains to similarly large degrees. The robust upregulation of gene expression in the neonatal brain was associated with a much higher degree of viral replication at this stage of development. In contrast to the case for downstream gene induction, CMV upregulated IFN-alpha/beta expression to a greater degree in the adult brain than in the neonatal brain. Similar to the case with cultured brain cells, IFN treatment of the developing brain in vivo depressed mCMV replication. In parallel work with cultured primary human brain cells, IFN and poly(I:C) treatment reduced hCMV infection and prevented virus-mediated cell death. These results suggest that coupling IFN administration with current treatments may reduce CMV infections in the developing brain.

Near simultaneous release of classical and peptide cotransmitters from chromaffin cells.

Adrenal chromaffin cells are an important part of the neuroendocrine system and under stressful conditions release catecholamines into the blood, thus regulating many physiological processes. In addition to the catecholamines, chromaffin cells also synthesize a range of peptides, including neuropeptide Y. Although the catecholamines and peptides are both contained within dense core granules, whether they are copackaged is less clear. Here, I investigate whether a single dense core granule can be loaded with both types of transmitter molecules. Using amperometry and FMRFamide tagging, I simultaneously measure the secretion of the catecholamines and a neuropeptide from mouse chromaffin cells in vitro. I find that fusion of a single dense core granule releases both types of transmitters into the extracellular space. Significant amounts of peptide escape from a fusing granule in 1-2 ms: almost as rapidly as the catecholamines. This suggests that the kinetics of peptide secretion might not be as sluggish as sometimes thought.

Regulation of the timing of MNTB neurons by short-term and long-term modulation of potassium channels.

The firing patterns of neurons in central auditory pathways encode specific features of sound stimuli, such as frequency, intensity and localization in space. The generation of the appropriate pattern depends, to a major extent, on the properties of the voltage-dependent potassium channels in these neurons. The mammalian auditory pathways that compute the direction of a sound source are located in the brainstem and include the connection from bushy cells in the anteroventral cochlear nucleus (AVCN) to the principal neurons of the medial nucleus of the trapezoid body (MNTB). To preserve the fidelity of timing of action potentials that is required for sound localization, these neurons express several types of potassium channels, including the Kv3 and Kv1 families of voltage-dependent channels and the Slick and Slack sodium-dependent channels. These channels determine the pattern of action potentials and the amount of neurotransmitter released during repeated stimulation. The amplitude of currents carried by one of these channels, the Kv3.1b channel, is regulated in the short term by protein phosphorylation, and in the long term, by changes in gene expression, such that the intrinsic excitability of the neurons is constantly being regulated by the ambient auditory environment.

FMRFamide tagging--how an ionotropic receptor can be used to measure peptide secretion.

In this chapter, we describe a technique, FMRFamide tagging, that in principle can be used to measure the release of any sequenced neuropeptide. The method relies upon the addition of an "electrophysiologically active" tag to the prohormone that encodes the neuropeptide of interest. Secretion of the electrophysiological tag (and thus the peptide of interest) is detected by activation of the ionotropic "tag receptor." Both the tagged prohormone and the tag receptor are expressed in the cell type under investigation. Since the tag and the neuropeptide of interest are on the same prohormone they are co-secreted and thus secretion of the tag reflects the co-secretion of the neuropeptide of interest. This method can be used to detect neuropeptide secretion on a millisecond timescale.

Measuring peptide secretion using the FMRFamide tagging technique.

Peptides are transmitters produced by a wide variety of neurons and neuroendocrine cells. They mediate a remarkable range of physiological processes. To better understand the roles played by peptides, a number of methods have been developed that can monitor their secretion. Although each has particular strengths, they cannot rapidly detect the secretion of chemically defined peptides. However, a recently developed approach termed "FMRFamide-tagging" may be useful in this regard. A genetically encoded electrophysiological tag is attached to the peptide prohormone of interest. The "tagged" prohormone together with an ionotropic receptor that binds the tag are expressed in the cell type under investigation. Secretion of the tag (and the co-secreted peptide of interest) are revealed by rapid inward membrane currents that are due to the activation of the tag receptor. In this manner, peptide secretion can be followed on a millisecond time scale. This protocol gives the details of the approach and its potential application to a range of cell types.