Neuromedin B and gastrin-releasing peptide excite arcuate nucleus neuropeptide Y neurons in a novel transgenic mouse expressing strong Renilla green fluorescent protein in NPY neurons

Abstract

Neuropeptide Y (NPY) is one of the most widespread neuropeptides in the brain. Transgenic mice were generated that expressed bright Renilla green fluorescent protein (GFP) in most or all of the known NPY cells in the brain, which otherwise were not identifiable. GFP expression in NPY cells was confirmed with immunocytochemistry and single-cell reverse transcription-PCR. NPY neurons in the hypothalamic arcuate nucleus play an important role in energy homeostasis and endocrine control. Whole-cell patch clamp recording was used to study identified arcuate NPY cells. Primary agents that regulate energy balance include melanocortin receptor agonists, AgRP, and cannabinoids; none of these substances substantially influenced electrical properties of NPY neurons. In striking contrast, neuropeptides of the bombesin family, including gastrin-releasing peptide and neuromedin B, which are found in axons in the mediobasal hypothalamus and may also be released from the gut to signal the brain, showed strong direct excitatory actions at nanomolar levels on the NPY neurons, stronger than the actions of ghrelin and hypocretin/orexin. Bombesin-related peptides reduced input resistance and depolarized the membrane potential. The depolarization was attenuated by several factors: substitution of choline for sodium, extracellular Ni(2+), inclusion of BAPTA in the pipette, KB-R7943, and SKF96365. Reduced extracellular calcium enhanced the current, which reversed around -20 mV. Together, these data suggest two mechanisms, activation of nonselective cation channels and the sodium/calcium exchanger. Since both NPY and POMC neurons, which we also studied, are similarly directly excited by bombesin-like peptides, the peptides may function to initiate broad activation, rather than the cell-type selective activation or inhibition reported for many other compounds that modulate energy homeostasis.

Figure 1.

GFP is expressed selectively in NPY neurons. A, Micrographs of coronal sections from the arcuate nucleus (Arc; A–C). GFP expression in the NPY-Renilla GFP mice is green, and immunostaining for NPY is red. C shows the merged image; green GFP- positive cells also show red NPY immunofluorescence. D–F, In contrast, immunoreactive α-MSH (red) is not colocalized in green neurons expressing GFP. Scale bars: A, 15 μm; D, 20 μm. G, Single-cell RT-PCR was used to test for NPY mRNA expression in GFP- positive cells from the arcuate nucleus and GFP-negative neurons from the ventromedial nucleus (VMH). All GFP-expressing neurons also had NPY mRNA message. In contrast, GFP-negative cells harvested from the VMH showed no NPY mRNA, but were positive for β-actin used as a control. A final lane used water as an additional control and was negative, as expected. H, In additional experiments, three GFP-positive arcuate neurons from the NPY-GFP mouse (lanes 1–3) were compared with GFP-negative neurons from the arcuate nucleus from the same mouse (lanes 4–7) and GFP-positive cells from a different transgenic mouse in which the POMC neurons expressed GFP (lanes 8–13). A final lane served as a water control. Only the three GFP-positive neurons from the NPY-GFP mouse expressed NPY mRNA, whereas the other control cells were all negative. I, Four GFP-positive cells from the arcuate nucleus were tested for NPY and AgRP. All four showed mRNA coding for NPY and AgRP, further substantiating the view that the green cells were the NPY neurons.

Figure 2.

Confirmation of NPY expression in GFP-expressing neurons. A, Cortical neuron immunostained with NPY antiserum and labeled red with Alexa 594. B, The same cell expresses strong GFP. Scale bar, 8 μm. C, Four cortical neurons (arrows) express GFP. D, The same cells are also stained with NPY antisera, and show both the GFP and red immunolabeling. Scale bar, 12 μm. E, In the hippocampal dentate gyrus, a number of neurons show GFP expression. Three are indicated by arrows. Scale bar, 15 μm. F, The same cells show red immunolabeling. G, A neuron in the lateral hypothalamus (LH) is green with GFP expression, and also shows red immunostaining. Scale bar, 9 μm.

Figure 3.

Fluorescence micrographs of coronal sections from brains of Renilla GFP mice showing distribution of fluorescence. A–J, Sections shown are from olfactory bulb (A), nucleus accumbens (B), septum (C), amygdala (D), caudate–putamen (E), hippocampus and overlying somatosensory cortex (F), thalamus (G), paraventricular hypothalamic nucleus (H), arcuate nucleus (I), and dorsomedial hypothalamic nucleus (J). Scale bars: A, B, D–F, 100 μm; C, G–J, 50 μm. For abbreviations, see Figure 4.

Figure 4.

A–E, Micrographs of NPY-rGFP in intergeniculate leaflet (A), inferior colliculus (B), adrenergic C1 cell group in ventral medulla oblongata (C), nucleus of the solitary tract (D), and the lateral caudal medulla oblongata (E). Scale bars: B, D, 100 μm; A, C, E, 50 μm. I-VI, Layers of the cerebral cortex; 12N, hypoglossal nucleus; aca, anterior commissure anterior part; Acb, nucleus accumbens; AON, anterior olfactory nucleus; AOB, accessory olfactory bulb; AP, area postrema; Aq, cerebral aqueduct; Arc, arcuate nucleus; BAC, bed nucleus of the anterior commissure; BLA, basolateral amygdaloid nucleus anterior part; BLV, basolateral amygdaloid nucleus ventral part; BSTMPI, bed nucleus of the stria terminalis medial division posterointermediate part; cc, central canal; Ce, central amygdaloid nucleus; cic, commissure of the inferior colliculus; CIC, central nucleus of the inferior colliculus; CPu, caudate–putamen; DLG, dorsolateral geniculate nucleus; DMH, dorsomedial hypothalamic nucleus; DMX, dorsal motor nucleus of the vagus nerve; ECIC, external cortex of the inferior colliculus; EPl, external plexiform layer of the olfactory bulb; Gl, glomerular layer of the olfactory bulb; GP, globus pallidus; GrDG, granular layer of the dentate gyrus; GrO, granular cell layer of the olfactory bulb; ic, internal capsule; icp, inferior cerebellar peduncle; IGL; intergeniculate leaflet; LMol, lacunosum moleculare layer of the hippocampus; LO, lateral orbital cortex; LPGi, lateral paragigantocellular nucleus; LV, lateral ventricle; ME, median eminence; Mol, molecular layer of the dentate gyrus; Or, oriens layer of the hippocampus; PAG, periaqueductal gray; PVH, paraventricular hypothalamic nucleus; Pyr, pyramidal cell layer of the hippocampus; Rad, stratum radiatum of the hippocampus; Rt, reticular thalamic nucleus; RVL, rostroventrolateral reticular nucleus; SolM, nucleus of the solitary tract medial part; Sp5C, spinal trigeminal nucleus caudal part; V3, third ventricle; VEn, ventral endopiriform nucleus; VLG, ventrolateral geniculate nucleus; VPL, ventral posterolateral thalamic nucleus; VPM, ventral posteromedial thalamic nucleus; vsc, ventral spinocerebellar tract.

Figure 5.

Schematic illustration depicting the distribution of fluorescent cell bodies–forebrain (filled red circles) in Renilla GFP mice. Scale bar: A, 1 mm. A–H, Anatomical plates taken from Paxinos and Franklin (2001). NPY-GFP cell bodies are found throughout the brain. The left side of each brain section shows the relative density of NPY-GFP cells of this transgenic mouse, and the right shows abbreviations for different brain regions.

Figure 6.

Schematic illustration depicting the distribution of fluorescent cell bodies–forebrain (filled red circles) in Renilla GFP mice. A–D, Anatomical plates taken from Paxinos and Franklin (2001). NPY-GFP cell bodies are found throughout the brain. The left side of each brain section shows the relative density of NPY-GFP cells of this transgenic mouse, and the right shows abbreviations for different brain regions.

Figure 7.

GFP axons and presynaptic dendrites. A, GFP-expressing axons are particularly dense in the hypothalamic paraventricular nucleus. B, The same PVN section immunostained red for NPY. Scale bar, 40 μm. C, In the corpus callosum, GFP-positive axons are seen. D, In the same section examined after immunostaining, red immunoreactive axons are seen, but in lower density than seen with GFP. Scale bar, 7 μm. E, In the olfactory bulb, a subset of granule cells in the granule cell layer (GCL) send dendrites up into the external plexiform layer (EPL). Less GFP is seen in the glomerular layer (GL), but GFP is strong in the olfactory nerve layer (ONL). Scale bar, 20 μm. F, Using a longer exposure, these presynaptic dendrites terminate in the EPL, and have numerous presynaptic boutons. Long arrow shows the same region on both E and F. G, This scanning laser confocal micrograph shows strong GFP labeling in cortical axons, but not in the large pyramidal cells. Scale bar, 8 μm.

Figure 8.

Membrane properties of arcuate nucleus NPY neurons. A1, Spontaneous burst firing of a GFP-expressing NPY neuron at resting membrane potential (−52.5 mV). A2, Cell showing spontaneous irregular firing at rest (−65.9 mV). B1, Voltage responses of an NPY neuron to 700-ms-step current injections of 20 pA. B2, A cell failed to fire continuously after the 700 ms current injection of 40 pA. C1, Voltage traces evoked by a step current injection from –100 to 0 pA. C2, Mean current–voltage relationship of 20 GFP neurons. C3, Traces show an LTS evoked when the cell recovered from a hyperpolarization to −95 mV; the LTS persisted in TTX, but was abolished by NiCl₂ 200 μm. D, Postsynaptic currents (upward: inhibitory, downward: excitatory) recorded at a holding potential at –35 mV with KMeSO4 pipette solution, which were blocked by AP5 (50 μm), CNQX (10 μm) and BIC (30 μm).

Figure 9.

GRP/neuromedin B/bombesin excite NPY neurons. A–C, Bombesin (250 nm, A) and the related mammalian peptides neuromedin B (NMB, 250 nm, B), and gastrin-releasing peptide (GRP, 250 nm, C) increase spike frequency in three different NPY neurons. D, Bar graph shows the mean effect of bombesin, NMB, and GRP. For comparison purposes, we also include the magnitude of the response to ghrelin and orexin on the spike frequency of NPY neurons. Bombesin and NMB generate the biggest response of the five peptides tested, all used at 250 nm. Numbers above bars indicate the number of cells that were excited by the peptide over the total cells tested. E, Bar graph shows the mean depolarization of the membrane potential evoked by the five neuropeptides. F, Bar graph shows the mean depolarization of the membrane potential evoked by the NMB at different concentrations, including 1, 10, 100, 1000, and 5000 nm. G, Bar graph shows the mean depolarization of the membrane potential evoked by GRP at a concentration of 1, 10, 100,1000, and 5000 nm. The number of cells is shown in parentheses. Error bars indicate SEM. The asterisk indicates statistical significance. H, RT-PCR experiment showing arcuate nucleus (ARC), whole hypothalamus (HYP), hippocampus (HIP), and cortex (CTX) with equal amounts of initial template. Both NMB-R (also called BB1) and GRP-R (also called BB2) are expressed in the arcuate nucleus. I, Using single cells harvested with a patch pipette, in lane 1 5 NPY cells were pooled, and in lane 2, 10 NPY cells were pooled to increase the amount of template. NPY cells showed NMB-R expression, as tested by nested RT-PCR.

Figure 10.

Direct effect of bombesin on NPY neurons. A, In the presence of TTX, bombesin depolarizes the membrane potential of an NPY neuron. B, In a different NP4 cell, in Ca²⁺-free ACSF, membrane depolarization by bombesin is increased. C, When extracellular sodium is replaced by choline, membrane depolarization by bombesin is reduced. D, Bombesin decreases the voltage response of NPY neurons after hyperpolarizing current steps (steps shown below response). E, Graph shows the current–voltage relationship in the absence (white circle) and presence (filled circle) of bombesin. Bombesin evoked a decrease in the whole-cell input resistance (slope). F, Bombesin induced an inward current at voltages between −100 and −20 mV (left graph). The bombesin-induced current shows a reversal potential at ∼−20 mV (right graph). G, Mean membrane depolarization by bombesin in different conditions. The number of cells tested is in parentheses.

Figure 11.

Bombesin modulates synaptic input. A, Bombesin (250 nm) decreases sIPSC frequency. B, Mean effect of bombesin on the frequency or amplitude of sIPSCs. Bombesin decreases both the frequency and amplitude of sIPSCs. C, Bombesin shows little effect on mIPSC frequency in the presence of 0.5 μm TTX. D, Mean effect of bombesin on mIPSCs. Bombesin has no significant effect on the frequency or amplitude of mIPSC. The number of cells is shown in parentheses. Error bars indicate SEM. The asterisk (*) indicates statistical significance; p < 0.05.

Figure 12.

Bombesin and NMB excite POMC neurons. A, Bombesin (250 nm) increases spike frequency in a POMC neuron. B, NMB (250 nm) also increases spike frequency. C, In the presence of TTX, bombesin (250 nm) depolarizes the POMC cell, suggesting a direct effect.