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Fasting Induces a Large, Leptin-Dependent Increase in the Intrinsic Action Potential Frequency of Orexigenic Arcuate Nucleus Neuropeptide Y/Agouti-Related Protein Neurons

Vollum Institute and The Center for the Study of Weight Regulation and Associated Disorders (K.A.T., R.D.C.), Oregon Health & Science University, Portland, Oregon 97239; and Department of Neuroscience (K.A.T.), Albert Einstein College of Medicine, Bronx, New York 10461

Address all correspondence and requests for reprints to: Kanji A. Takahashi, Albert Einstein College of Medicine, Rose F. Kennedy Center, Room 703, 1410 Pelham Parkway South, Bronx, New York 10461. E-mail: katakaha{at}aecom.yu.edu.

	Abstract

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The neuropeptide Y (NPY)/Agouti-related protein (AgRP) neurons of the hypothalamic arcuate nucleus are thought to promote feeding. Here, we demonstrate that feeding state in vivo, through a leptin-dependent process, induces large and persistent changes in the electrophysiological activity of these neurons as measured extracellularly in vitro. Consistent with an orexigenic role, fasting induced a 4-fold increase in the basal action potential frequency of NPY/AgRP neurons. Leptin, when injected into fasted wild-type mice, induced a dose- and time-dependent decrease in spike frequency, which approached fed levels 2–3 h post treatment. In leptin-deficient (lep^ob/lep^ob) and leptin receptor-deficient (lepr^db/lepr^db) mice, NPY/AgRP spike frequency was not significantly increased by fasting, and even in mutant mice fed ad libitum, spike frequency was at least as high as in fasted wild-type mice. All recordings included GABA_A and ionotropic glutamate receptor antagonists, suggesting that expression of this modulation is potentially intrinsic and not synaptically dependent. Recorded neurons were unambiguously identified using NPY-Sapphire transgenic mice. This is a remarkably straightforward example of a very robust in vitro electrophysiogical effect produced by a simple behavioral manipulation, food restriction.

	Introduction

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REGULATION OF FOOD intake by the neuropeptide Y (NPY)/Agouti-related protein (AgRP) neurons of the hypothalamic arcuate nucleus is an area of active investigation (1, 2, 3) (reviewed in Refs.4 and 5). When delivered centrally, NPY is noted for the voraciousness and AgRP for the extreme duration of the induced feeding (6). Chronic infusion of NPY and overexpression of AgRP both lead to obesity. Most NPY-expressing somata in the hypothalamus and all AgRP somata in the brain are located in the arcuate nucleus, where they are coexpressed (7, 8). AgRP acts as an endogenous antagonist of the anorectic effect of -MSH at melanocortin receptors (9, 10, 11). Fasting, mainly through a leptin-dependent process, induces large increases in arcuate nucleus NPY and AgRP expression, and this has been assumed to reflect modulation of the action potential activity of NPY/AgRP neurons (7, 8, 12, 13, 14). However, neuropeptide expression as a proxy measure of action potential activity is suggestive but not definitive. Because neuropeptide-containing neurons generally also release a fast neurotransmitter (15), changes in action potential activity could be important independent of changes in neuropeptide expression. In addition, as long as spike activity is not so low as to prevent peptide release, it is possible for changes in neuropeptide expression to alter peptide release without altering spike frequency.

We report direct measurement of arcuate NPY/AgRP neuronal activity in acute slices and its modulation by fasting and leptin signaling in vivo. Loose patch extracellular recording was used to measure the basal activity of visually identified neurons without artifactually influencing that activity, and recording solutions contained kynurenic acid and picrotoxin to isolate the cells from synaptic inputs via GABA_A and ionotropic glutamate receptors. A strict cell selection protocol was followed to obtain a large (20–35 cells), unbiased sample of NPY neurons widely distributed in the arcuate nucleus from every mouse in each treatment group.

	Materials and Methods

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Mice
All mice used in recordings were hemizygous for the NPY-Sapphire transgene in which expression of a Tau-Sapphire (green fluorescent protein variant) fusion protein is directed by NPY genomic elements and selectively expressed in NPY neurons (1). NPY-Tau-Sapphire (NPY-SAP) mice were a kind gift of Dr. Hongyan Liu and Dr. Jeffrey Friedman of Rockefeller University (New York, NY) and were received on a mostly C57Bl/6J background with a smaller CBA contribution (Pinto, S., personal communication). B6.V-Lep^ob/J (lep^ob/+), B6.Cg-m +/+ Lepr^db/J (lepr^db/+), and C57Bl/6J mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Subsequent breeding produced the following mice used for recording: 1) +/+; NPY-SAP Tg/+ (wild type). 2) lep^ob/lep^ob; NPY-SAP Tg/+ (lep^ob/lep^ob). 3) lepr^db/lepr^db; NPY-SAP Tg/+ (lepr^db/lepr^db). PCR-based tests of ear-snip DNA were used to genotype mice for the lep^ob (16) and lepr^db (17) mutations and for the NPY-Sapphire transgene (forward: 5'-GGGCAGAAGGTCCAGTC-3'; reverse: 5'-GGTCTTGCAGGGTGTAGTCC-3'; Liu, H., personal communication). Of the uninjected wild-type mice used in the experiments of Fig. 1, five of six mice in both fed and fasted groups were the progeny of the same matings used to produce lep^ob/lep^ob, NPY-SAP Tg/+ and lepr^db/lepr^db, NPY-SAP Tg/+ mice for recordings. The injected wild-type mice of Fig. 2 were the progeny of wild-type parents, except for five mice distributed among five injection groups that had at least one lep^ob/+ parent.

View larger version (28K): [in this window] [in a new window]   FIG. 1. Fasting for 24 h greatly increases the basal spike frequency of arcuate nucleus NPY neurons in wild-type mice. In lepob/lepob (ob/ob) and leprdb/leprdb (db/db) mice, spike frequency was high even in fed animals and was not significantly increased by fasting. A, Typical extracellular recordings of NPY/AgRP neuron spiking from fed and fasted mice. Scale bars apply to both traces. wt, Wild-type. B, Grand mean ± SEM of mean frequencies per mouse. Significantly different from fed wild-type mice (*, P < 0.001). Significantly different from fasted wild-type mice (+, P < 0.05; ++, P < 0.01). ns, Not significant. C, Relative distribution of all cells in A by spike frequency, plotted as a cumulative frequency histogram (0.1 Hz bins). In fed wild-type mice, the spike frequency of most NPY neurons was silent or very low. In fasted wild-type mice and in lepob/lepob and leprdb/leprdb mice, the entire distribution shifted to higher frequencies.

View larger version (22K): [in this window] [in a new window]   FIG. 2. Leptin injection induces a dose- and time-dependent decrease in the basal spike frequency of arcuate nucleus NPY neurons from 24-h fasted mice. Data displayed as mean spike frequencies of individual mice (filled circles, 31.1 ± 0.3 cells/mouse) and grand mean ± SEM. Mice were injected ip with leptin or vehicle 1–3 h before decapitation. In each of the vehicle-injected categories, equal numbers of mice were injected at 1 or 3 h; results were similar and thus were combined (see Materials and Methods). Statistical significance vs. vehicle (veh)-injected, fed and fasted controls is displayed. One outlier (open circle), probably the result of injection error, was not included in the analysis (see Materials and Methods). ns, Not significant.

In the experiments of Fig. 1 (uninjected), 30- to 40-d-old mice were weighed (0752–0803 PST) and decapitated (0801–0810 PST) 1 h, 45 min after lights on, and experiments using mice of the different genotypes were interleaved. In the experiments of Fig. 2 (ip injected), 30- to 48-d-old mice were weighed (0916–0929 PST) and decapitated (0930–0935 PST) 3 h, 15 min after lights on. Recombinant murine leptin (Lot AFP352C, purity 95–99% by SDS-PAGE) was obtained from A. F. Parlow (National Hormone and Peptide Program, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD) and stored at –80 C until stock solutions were made. Leptin was dissolved in sterile-filtered PBS (Invitrogen Corp., Carlsbad, CA; adjusted to pH 7.9 with NaOH) at 1 mg/1.5 ml and stored in single-use aliquots at –80 C until injection. For the 5-mg/kg dose, undiluted leptin stock was injected. For lower doses of leptin or controls, injection volume per body weight was kept constant with PBS (pH 7.9). Dose was calculated from body weight 24 h before decapitation, when fasting was started. In both the fed and fasted vehicle-injected groups, three mice were injected at 1 h before decapitation, and another three mice were injected at 3 h before decapitation. Mean spike frequencies were similar at 1 and 3 h (fed 1 h: 0.48 ± 0.02 Hz, fed 3 h: 0.38 ± 0.10 Hz, P = 0.39; fasted 1 h: 1.71 ± 0.17 Hz, fasted 3 h: 1.65 ± 0.23 Hz, P = 0.84; unpaired, two-tailed, heteroscedastic t tests), and so these groups were combined. Recordings using mice subjected to the various feeding and leptin-injection regimens were interleaved.

In the fasted, 5 mg/kg leptin, 3 h category of Fig. 2, an outlier of 2.19 Hz (indicated by an open circle) was more than 6 SDs greater than the mean of the remaining six mice (SD calculated without outlier). Because it was probably the result of experimental error (i.e. misinjection of leptin into the intestinal lumen), it was omitted from analysis. Animal protocols were approved by the Oregon Health & Science University Animal Care and Use Committee.

Electrophysiology
Recording methods were modified from those used earlier (18). Halothane-anesthetized mice were decapitated at a fixed time after lights on (1 h, 45 min—Fig. 1; 3 h, 15 min—Fig. 2). Coronal slices of hypothalamus (200 µm thick) were prepared on a vibrating slicer (VT1000S; Leica Microsystems, Wetzlar, Germany) using standard methods. After slice preparation was complete, at least 1 h was allowed for recovery before use. A slice was transferred to the recording chamber and allowed to equilibrate for 10 min. For the subsequent 40 min, moving deliberately across the arcuate nucleus, as many fluorescent neurons as possible of healthy appearance but of every brightness were subjected to loose-patch (19) extracellular recording of action potential activity. This process was repeated until 5 h, 0 min after decapitation (or earlier if all slices were used). Care was taken to achieve a seal resistance high enough to prevent action potentials from going undetected, and current clamp (I = 0) mode was used to avoid the possibility that current passed through the pipette might influence the recorded cell. Fluorescent cells on the surface of the slice with no overlying tissue were avoided because they were sometimes swollen and were more likely to have been damaged during slicing. The slice holding chamber (submersion type) and recording chamber were maintained at 30 C. Spike frequency for each cell was calculated from the first 120 sec of recording. These were combined to calculate mean spike frequency for each mouse. These means of each mouse, grouped by treatment category, were analyzed by one-way ANOVA and the Newman-Keuls posttest and presented as grand mean ± SEM. Significance level was set at 0.05 for all tests.

Through the recording period, spike frequency did not vary with either time after decapitation, time in recording chamber, or rostral-caudal position. For each mouse, each of these parameters was plotted against spike frequency, and regression lines were drawn to look for potential correlations. The slopes of these relationships, combined for each treatment group of mice, were never significantly different from zero (one sample t test vs. a theoretical value of zero, P > 0.05).

Saline used for slice preparation and maintenance contained 126.2 mM NaCl, 3.1 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 1 mM NaH₂PO₄, 26.2 mM NaHCO₃, 10 mM glucose, and 16.24 mM sucrose (320 mosm/kg, pH 7.39, when gassed with 95% O₂-5% CO₂). Saline for recording contained 133.2 mM NaCl, 3.1 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 1 mM NaH₂PO₄, 26.2 mM NaHCO₃, 10 mM glucose, and 1.05 mM NaOH (ACS reagents; Mallinckrodt Chemical, Inc., Paris, KY; and Fisher Scientific, Fair Lawn, NJ). Recording saline also contained picrotoxin (0.1 mM; Sigma Chemical Co., St. Louis, MO) and kynurenic acid (Fig. 1, 1.0 mM, Sigma; Fig. 2, 0.9 mM, Tocris Cookson Inc., Ellisville, MO). Patch pipettes were filled with recording saline. The recording chamber was perfused at approximately 2.3 ml/min and was approximately 0.7 ml in volume. To ensure consistency, all equipment in contact with solutions, including the pipette holder and recording chamber, were cleaned daily.

Sapphire-fluorescent neurons were unambiguously identified and patched using epifluorescence and IR-DIC optics (Axioskop 2 FS microscope; Carl Zeiss, GmbH, Jena, Germany). Equipment was optimized for detection of even dim Sapphire fluorescence (Sapphire GFP filter cube; Chroma Technology Corp., Rockingham, VT; and C7500–50 high sensitivity CCD camera and C2400 camera controller; Hamamatsu Photonics, Hamamatsu, Japan), and exposure to near UV excitation light was limited to reduce bleaching. Under these conditions, cells close enough to the surface to be targeted for recording were unambiguously identifiable as fluorescent or not fluorescent in fed mice (i.e. fluorescence intensity was not a limiting factor in identification of NPY/AgRP neurons). Perhaps for this reason, although fasting greatly increased the fluorescence intensity of arcuate nucleus neurons (as expected for a transgene controlled by NPY genomic elements), it seemed to not cause a large increase, if any, in the number of patchable fluorescent neurons; however, formal cell counts were not done. Because fluorescent cells were recorded without regard to intensity, we consider it unlikely that fasting-induced increases in fluorescence caused a substantial bias in the selection of cells for recording. After injecting fasted mice with leptin 1–3 h before decapitation, fluorescence intensity remained very high (perhaps due to slow degradation of the Tau-Sapphire fusion protein), eliminating selection bias as an explanation for the large effect on spike frequency.

Pipettes (1.4–1.8 M) were used to form seals of 11 M, and no cell was considered silent unless seal resistance (R_seal) was greater than 4 M. R_seal was kept low to avoid damaging the patched membrane. Over time, R_seal tends to increase as the patched membrane is drawn further into the pipette. When this increase was large, additional strain was prevented by applying a small amount of positive pressure from a short column of water attached via tubing to the pipette holder. Unlike whole-cell patch clamp and sharp microelectrode recording, loose-patch recording does not disturb cytosolic contents and intracellular processes; and neuronal activity is not affected by seal strength or current passed through the pipette. Data were acquired at 10 kHz using an Axopatch 1D amplifier (100x gain, –3 dB filter freq: 5 kHz) and Clampex 8.2 software (Axon Instruments, Union City, CA). Data were analyzed using Mini Analysis Program 5.6.28 (Synaptosoft, Decatur, GA), GraphPad Prism 3.0 (Graphpad Software, Inc., San Diego, CA), and Excel 2000 (Microsoft Corp., Bellevue, WA).

	Results

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In fed wild-type mice, the basal spike frequency of arcuate nucleus NPY neurons was very low (0.38 ± 0.06 Hz/mouse; Fig. 1, A and B). Fifty-six percent of all neurons from fed wild-type mice had spike frequencies less than 0.1 Hz (Fig. 1C). After a 24-h fast, in which body weight decreased 21.9 ± 1.1% (Supplemental Table 1, published on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org), mean spike frequency per mouse increased over 4-fold to 1.70 ± 0.13 Hz (Fig. 1B). There was a correspondingly large, rightward shift in the frequency distribution of cells from fasted mice (Fig. 1C). Because previous studies had suggested a role for leptin in mediating fasting-induced increases in neuropeptide expression (see Introduction, reviewed in Refs.5 and 14), recordings using leptin-deficient (lep^ob/lep^ob) and leptin receptor-deficient (lepr^db/lepr^db) mice were interspersed with those using wild-type mice. In fed lep^ob/lep^ob and lepr^db/lepr^db mice, NPY spike frequency was at least as high as in fasted wild-type mice. Importantly, in neither mutant strain did fasting significantly increase NPY spike frequency (Fig. 1, B and C). This strongly suggests that in order for fasting to have a large effect on spike frequency, the leptin signaling system must be intact. Alternatively, it is formally possible that developmental defects (20) in lep^ob/lep^ob and lepr^db/lepr^db mice account for these results.

These results suggest that fasting increases the spike frequency of NPY neurons by reducing leptin levels. If so, then exogenous leptin should be able to reverse this effect. Recombinant mouse leptin at 1–5 mg/kg or vehicle (PBS, pH 7.9) was injected ip 1–3 h before decapitation of fed or 24-h fasted mice. Leptin induced a dose- and time-dependent decrease in the spike frequency of NPY neurons in fasted mice (Fig. 2 and Supplemental Table 2, published on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). Leptin (5 mg/kg) induced reductions in spike frequency equivalent to 45% (1 h post injection), 71% (2 h post injection), and 90% (3 h post injection) reversals of the fasting-induced increase in controls, revealing a slow time course for this type of inhibition.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have demonstrated that fasting induces a large increase in the basal spike frequency of NPY neurons measured in an in vitro slice preparation. Mechanistically, the results from lep^ob/lep^ob and lepr^db/lepr^db mice suggest that, in order for feeding state to greatly impact spike frequency, it is necessary for leptin signaling to be intact. Our injection experiments demonstrate that increasing leptin in vivo (although probably to supraphysiological levels) is sufficient to reduce spike frequency in fasted mice. Taken together, these data suggest that deviations from energy homeostasis modulate the spike activity of arcuate NPY/AgRP neurons through changes in leptin. According to this model, homeostatic levels of leptin tonically suppress the activity of NPY/AgRP neurons, perhaps directly, given that arcuate nucleus NPY neurons have been reported to express leptin receptor (21). Fasting, by reducing circulating leptin, alleviates this inhibition and causes spike frequency to increase. Genetic ablation of leptin or leptin receptor eliminates this inhibition entirely, driving spike frequency even higher and making feeding state irrelevant. This model predicts that clamping leptin at normal fed levels would be sufficient to prevent a fasting-induced increase in spike frequency. Our data do not address whether leptin acts alone in inducing these changes in spike frequency; leptin may act indirectly through, or in concert with, additional hormones, transmitters, or other inputs. Insulin, for example, when intracerebroventricularly administered, can prevent the normal fasting-induced increase in NPY expression (22). In addition, it is possible that synaptic input in vivo is involved in inducing these changes in firing or that non-GABA_A, nonionotropic glutamate receptor-driven synaptic activity might be involved in expression of these changes in vitro.

After ip injection in mice, leptin levels have been reported to peak after a delay of 30–60 min in serum (23, 24) and 30 min in brain (23). In contrast, inhibition of spiking developed rather slowly with a 3-h time course. Also, the effects of fasting and leptin injection were very persistent, lasting unchanged for several hours in vitro, even while bathed in saline containing a superphysiological concentration (10 mM) of glucose (25). These properties of slow onset and persistence are consistent with an effect on transcription or translation. In this regard, it has been reported that leptin regulation of feeding and body weight are dependent on leptin receptor-long form activation of the STAT3 transcription factor (26), and leptin-induced transcriptional effects have been reported in NPY neurons (27, 28).

We have described a homeostatic process in which orexigenic NPY/AgRP neurons sense negative energy balance via reduced leptin signaling and respond by increasing spike frequency. This form of spike frequency regulation is persistent, surviving several hours after initiation in vivo, and is observable even in the presence of GABA_A and ionotropic glutamate receptor antagonists. In vivo, arcuate NPY neurons are doubtless also subject to numerous other forms of regulation that might not be detected by this assay, either because they are more transient in effect or because they require GABA_A or ionotropic glutamate receptor activity (2, 3, 4, 29, 30). Recently, Pinto et al. (1) described the effects of the ob mutation on GABA_A and ionotropic glutamate receptor-mediated synaptic inputs to arcuate nucleus NPY neurons. They reported that spontaneous, CNQX/AP5-sensitive excitatory postsynaptic currents in whole-cell recordings of arcuate NPY neurons were 1.5 times higher in frequency in lep^ob/lep^ob mice, suggesting that leptin can influence NPY neurons via their synaptic inputs. However, in our recordings, where the inputs recorded by Pinto et al. (1) were blocked by antagonists, intrinsic spike frequency was 6.4 times higher in fed lep^ob/lep^ob mice relative to fed wild-type mice, suggesting that leptin has a much greater, nonsynaptic effect directly on the NPY/AgRP neurons.

These methods and results will be useful in the further study of feeding and energy homeostasis. As shown here for leptin, the long-term effects of potential regulators can be reliably tested by in vivo injection and in vitro extracellular recording of spike frequency. One advantage of spike frequency recording is its sensitivity. The fasting-induced increase in spike frequency reported here, although quite large, can be caused by a change in membrane potential too small to be detected above experimental variability using a more invasive procedure such as whole-cell patch clamp. Recording spike activity extracellularly is an excellent way to quickly screen potential modulators of neuronal activity without the artifacts associated with other recording methods, but when applied to NPY/AgRP neurons, it must be remembered that in fed animals, the spike activity of most neurons will be extremely low or silent (Fig. 1B). Thus, an anorectic compound that reversed the fasting-induced increase in NPY-AgRP activity would be of great interest, but such a substance might have no measurable effect in slices from fed mice.

	Acknowledgments

 
NPY-Sapphire transgenic mice were a kind gift of Dr. Hongyan Liu and Dr. Jeffrey M. Friedman (Rockefeller University). We thank our colleagues of the Cone Laboratory for helpful discussion.

	Footnotes

 
This work was supported by National Institutes of Health Grant DK51730.

First Published Online December 9, 2004

Abbreviations: AgRP, Agouti-related protein; GABA, -aminobutyric acid; NPY, neuropeptide Y; NPY-SAP, NPY-Tau-Sapphire fluorescent protein; PST, pacific standard time; R_seal, seal resistance.

Received October 22, 2004.

Accepted for publication November 30, 2004.

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