K+-stimulated neuropeptide Y release into the paraventricular nucleus and relation to feeding behavior in free-moving rats

Neuropeptides (1993) 24,307-3 12 0 LollgrIlan GroupUK Ltd 1993

K+-stimulated Neuropeptide Y Release into the Paraventricular Nucleus and Relation to Feeding Behavior in Free-moving Rats A. STRICKER-KRONGRAD, G. BARBANEL”, B. BECK, A. BURLET, J. P. NICOLAS and C. BURLET INSERM LJ.308, MBcanismes de Rdgulation du Comportement Alimentaire-38, rue Lionnois 54000 Nancy, France and “Laboratoire de Neurobiologie Endocrinologique, URA 1797 CNRS 34000 Montpellier, France (Reprint requests to AS-K)

Abstract-Neuropeptide Y (NPY) strongly stimulates food intake when it is injected in the central nervous system and especially in the hypothalamus. The major site of NPY synthesis in the hypothalamus is the arcuate nucleus which projects to the paraventricular nucleus. These two nuclei form the arcuate-paraventricular axis, a local circuit in the control of food intake. It was demonstrated that neuropeptide Y concentration in the paraventricular nucleus can be modified by ingestive or metabolic factors. Actually, these modifications cannot be associated with the existence of a release of neuropeptide Y in this nucleus. That is why we used pushpull perfusion during the light phase in freely-behaving rats with food and water available. Perfusates were collected with standard artificial cerebrospinal fluid (CSF) as medium and hyperosmotic CSF obtained by addition of potassium chloride (55 mM). Hyperosmotic perfusion was repeated a second time for some animals. Results clearly demonstrated that neuropeptide Y is released into the paraventricular nucleus during normal perfusion with a mean value of 35.5 f 1.5 pgAube. The potassium perfusion produced an increase in the release of neuropeptide Y (peak at 71.4 +I 7.1 pg/tube; p e 0.011, and this phenomenon was reproduced with the second potassium stimulation (peak at 47.7 f 2.3 vs pg/tube; p c 0.05). Neuropeptide Y release returned to normal values after or between stimulations. Behavioral analysis showed that these stimulations were associated with an increase in food intake. Neuropeptide Y is therefore physiologically released into the paraventricular nucleus of the hypothalamus. This release is associated with ingestive behavior and might be induced through voltage-dependent channels sensible to the high depolarisation associated with potassium excess in the extracellular fluid. Introduction Neuropeptide Y (NPY), a member of the pancreatic polypeptide family, is widely distributed in the brain Date received 19 June 1992 Date accepted 4 February 1993

of mammals10J3 and was proposed as a putative neurotransmitter.2 The major site of NPY synthesis in the hypothalamus is the arcuate nucleus.* NPY neurons of the arcuate nucleus projects to the ventromedian, dorsomedian and paraventricular (PVN) nuclei to form a non-catecholaminergic NPY net-

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work.’ The latter nucleus is also the site of NPYergic fibers originating in the brainstem.17 NPY strongly stimulates food intake when it is injected in brain ventricles11~21~22 or more specifically in the PVN.18JgRecently, the role of the brainstem NPYergic innervation of the PVN was shown as minor in the modulation of spontaneous feeding behavior, l6 and the arcuate-PVN axis was proposed as a local circuit in the control of food intake regulation.7*g Some recent studies showed that NPY concentrations in the PVN are modulated by internal or external factors. In the rat, neonatal treatment with monosodium glutamate,’ ingestion of a carbohydrate-rich diet* or refeeding6 are associated with lower NPY concentrations. On the other hand, augmentations were noted during starvation6 in a food choice situation2o and in the obese hyperphagic Zucker rat.5 NPY therefore plays an important role in the regulation of the feeding behavior. However, this conclusion is only based on the site specific effects of NPY injections and on the modifications of NPY concentrations measured in micropunched PVN areas and not on a dynamic release of NPY. Actually, the push-pull perfusion in the brain is the only adequate method for harvesting extracellular compounds of higher molecular weight’* like neuropeptides. This method can be associated with direct stimulation with high-K+ to demonstrate the role of these molecules as neuromodulators.15 In this study, we therefore measured NPY contents in perfusates of the PVN by the use of pushpull perfusion under standard or hyperosmotic stimulation in freely-behaving rats and tried to relate them to behavioral modifications.

Materials

and methods

Animals Male Long-Evans rats weighing 250-300 g bred in our laboratory were given food and water ad libitum. They were individually housed in plastic cages in a temperature-controlled room (24 f 1’C) with a 12/l 2 h automatic light-dark cycle with light on at 7.00 a.m. Push-pull technique in freely-moving

animals

After 7 days of habituation to these conditions, they were anesthetized with chlorhydrate ketamine

(Ketalar, Parke-Davis, France; 150 mg/kg body weight). They were stereotaxically implanted with a push-pull cannula (external cannula 0.5 mm, internal cannula 0.1 mm, 0.25 mm protusion)4 aimed in the right paraventricular nucleus (antero-posteriority: -1.5 mm; lateral@: -0.5 mm from bregma; depth: -7.45 mm from cranial skull). After 7 days of recovery, they were perfused in their own cage during the middle of the light phase (between 11 .OO h and 17.00 h) at a rate of 13 pl/mn4 30-minute samples were collected during 6 hours into polypropylene tubes containing 10 pl aprotinin (Iniprol, Laboratoires Choay, France) and kept on ice. Tubes were lyophilized and stored at -40°C before assay for NPY. Animals were divided into 4 groups. The first group was placed in the conditions of push-pull perfusion with food and water available, but not perfused (behavioral controls; n = 3). The animals of the second group (perfusion controls; n = 5) were perfused under standard free-moving conditions in their own cage with artificial cerebrospinal fluid (aCSF; NaCl: 124 mM; KCl: 3.3 mM; m2Po4: 1.24 mM; MgS04: 1.3 n&I; CaC122.5 mM; NaHC03: 26 mM; glucose 10 mM and pyrogen-free bidistilled water). In two other groups (experimental groups), samples were collected with hyperosmotic aCSF (55 mM KCl) as perfusion medium. In the first of these experimental groups, KC1 dissolved in aCSF was perfused once during 30 min (n = 6) whereas in the second experimental group, the first KC1 perfusion was followed by a second one 90 min later (n = 3). Food and water were available to all rats. Animals behavior was recorded during all the perfusion time by one of the experimentators. Each of the mutually non-exclusive following behaviors was recorded in duration: moving, sleeping (light period), resting and eating. After perfusion, the animals were immediately sacrificed and brains were removed for histological checking. Animals with misplacement of cannula, tissue damage, or with abnormal behavior (as compared with behavioral control) during standard perfusion were discarded of the study. Radioimmunoassay Immunoreactive NPY was measured by a radioimmunoassay developed in our laboratory.5 Briefly,

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EFFECTS OF K+-STIMULATED NPY RELEASE ON FEEDING BEHAVIOR IN RATS

standard (porcine NPY - SIGMA, La Verpilliere, France) or lyophilized unknown push-pull samples were reconstituted with assay buffer :0.04 M phosphate buffer pH 7.4 containing bovine serum albumin (fraction V, Sigma Chemicals, La Verpilliere, aprotinin (4000 IU/ml, IniprolR, France), Laboratoires Choay, Paris, France) and sodium azide (Merck, Darmstadt, Germany). 100 ~1 of antiserum diluted in assay buffer and 100 ~1 of standard or unknown sample were preincubated for 24 h at 4°C. Then, 100 ul of L251-labelledneuropeptide Y 2000 activity: (Amersham IM 170; specific Ci/mmol; Les Ulis, France) were added and incubated for a further 24 h. Bound and free fraction were separated by the addition of 500 p.1of a solution of 2% charcoal (Norit A, Kodak, Rochester, NY, USA) and 0.2% dextran (T70, Pharmacia, Uppsala, Sweden) in assay buffer. Bound fraction was measured in a gamma counter coupled to a microcomputer (MDA 3 12 system, Kontron, Velizy, France) for the plotting of the standard curve and the calculation of the results. Assay sensitivity was 12 pg/tube, and intra-assay and inter-assay variation coefficients were 4.7 and 13.2%, respectively. Non

specific binding was not affected by aCSF or KC1 (6.8% (KC1 in aCSF) and 7.2% (aCSF) vs 7.1% (assay buffer)) and maximal binding range was 55-60%. Statistical analysis Multiple comparisons were made with KruskalWallis non-parametric test followed by MannWithney non-parametric U intergroup comparisons. For each group analysis at different times of perfusion were made with paired Wilcoxon non-parametric T test. Behavioral data were compared with unpaired Mann-Withney non-parametric U test. Only probability values less than 5% were considered significant.

Results NPY in standard and K+-supplemented perfusions Immunoreactive NPY was above the detection limit of were at least twice as great The average content ofNPY

present in all samples the assay. All samples as the detection limit. was 35.5 + 1.5 pg/tube

NPY (pghube)

180

210

210

270

TIME mn

Neuropeptide Y (NFV) content in 30 - minutes samples of push-pull perfusion of the paraventricular nucleus during the light phase (mean f S.E.M. in p&k.) following one perfusion of KCl. O-O: rats perksed with artiUaJ cerebrospinal fluid (n=5); 0-O: rats perfused with 55 mM KC1 dissolved in artificial cerebrospinal fluid between 120 to 150 min (n = 6). For statistics, see text.

Ftg. 1

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0; 0

30

KCI

30

30

120

KCI

130

I I

130

210

2.0

270

300

0

TIME mn

Neuropeptide Y (NF’Y) content in 30 - minutes samples of push-pull perfusion of the paraventricular nucleus during the light phase (mean f S.E.M. in pgtube) following two perfusions of KCl. O-O: rats perfused with artificial cerebrospinal fluid (n = 5); 0-O: rats perfused with 55 mM KC1 dissolved in artificial cerebrospinal fluid between 120 to 150 min and between 210 to 240 mm (n = 3). For statistics, see text.

Fig. 2

DURATION

(mn)

RESTING

SLEEPING

EATING

BEHAVIORS Fig. 3

Duration in minutes (mn) of behavioral responses in behavioral control rats (no perfusion; open bars; n = 3) and in rats perfused with artiticial cerebrospinal fluid (hatched bars; n = 5) or KC1 (black bars; n = 9). * p < 0.05 and ** p < 0.01 between normal and hyperosmotic perfusion.

EFFECTS OF K--STIMULATED

NPY RELEASE ON FEEDING BEHAVIOR IN RATS

in the standard condition and before hyperosmotic perfusion. This was not different of the NPY content after the hyperosmotic stimulation (35.5 f 1.5 vs 35.2 f 1.8 pgtube; ns). No major variations around the average level of NPY were noted in the standard condition. As shown in Figure 1, KC1 stimulation produced an increase of NPY content when compared to basal prestimulation values (71.4 f 7.1 vs 36.4 + 2.1 pgltube; p < 0.01) and the poststimulation values (42.6 + 3.7 pg/tube; p < 0.01) of the same animals or when compared to standard perfusates sampled atthesametime(26.3f4.3pg/tube;p
During a standard perfusion with aCSF, the animal spent about half of its time to sleep, and only small feeding episodes were observed. The hyperosmotic stimulation with KC1 induced augmentations in time spent to rest and to eat. Diminutions were noted in time spent to move and to sleep (Fig. 3). The following pattern ofbehavioral response was observed. As perfusion started, the animal stayed immobile during approximately 15-20 min. During this time, long periods of teeth grinding and rapid breathing were observed. After this episode, the rat was hyperactive during a very short period (l-3 min). Then, the animal became much more quiet and started to eat. Eating persisted during at least 10 min.

311

Discussion This study clearly demonstrates that NPY is released into the paraventricular nucleus of the hypothalamus in extracellular fluid in basal conditions and is the first to demonstrate that this release can be stimulated by potassium. In all the animals, normal release was relatively identical with time of perfusion. Stimulation with 55 mM KC1 produced an increase in NPY release, and after the stimulation, NPY release returned to normal values showing that standard release was not disturbed. This was clearly demonstrated by the presence of the response of the NPY release during the second stimulation, even if this response did not reach the level of the first one. These facts indicated that the physiological integrity of paraventricular cellular elements was maintained during push-pull perfusion at this flow rate. K’stimulations showed that NPY might be released by voltage-dependent channels sensible to the high depolarisation associated with potassium excess in the extracellular fluid. The smaller NPY release after the second KC1 stimulation might be related to a decrease of the NPY pool induced by the first stimulation. It might also be related to the establishment of counterregulatory mechanisms induced by food intake. Indeed, we observed during this experiment frequent feeding episodes after the beginning of the first KC1 stimulation. We have previously shown that ingestion of food after a period of 48 h of fast induces a decrease in NPY concentrations in the arcuate nucleus 6 h after refeeding.6 Moreover, in more physiological conditions, the high NPY levels measured in the paraventricular nucleus at the end of the light period (1 h before dark) decrease after 1 h of darkness when the rats have taken their first meals.14 In the same way, increase of central NPY concentrations through acute or chronic injections stimulates food intake11J8Jg,21Jz and NPY concentrations in the paraventricular nucleus are modulated by ingestive or metabolic factors.i,5J,20 All these effects of exogenous and endogenous NPY modifications in the paraventricular nucleus added to those ofNPY release in normal and hyperosmotic conditions indicate that NPY release in the paraventricular nucleus is really involved in the regulation of ingestive behavior.

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Acknowledgement Thispaper has been presented under abstract form in the 9th annual conference of the French Association for the Study of Obesity (AFERO; Paris). It was supported by the MRE grant 92.G.0341. During the submission of these results to the AFERO, a paper of Kalra’s group (PNAS 88: 10931-10935, 1991) has confirmed that NPY release varies with the feeding state.

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