Effect of paraventricular injection of neuropeptide Y on milk and water intake of preweanling rats

Neuropeplides (1993) 24,177-l 82 0 Longman Group UK Ltd 1993

Effect of Paraventricular Injection of Neuropeptide on Milk and Water Intake of Preweanling Rats C. A. CAPUANO*,

S. F. LElBOWlTZt

Y

and G. A. BARRSS

*Department of Psychology, Fairleigh Dickinson University, Teaneck, NJ 07666 USA; tThe Rockefeller University, New York, NY 10021 USA; *Department of Psychology, Hunter College, CUNV, New York, NY 10021 USA and SDivision of Developmental Psychobiology, Department of Psychiatry, New York State Psychiatric Institute, Columbia University, College of Physicians and Surgeons, New York, NY 10032 USA (Reprint requests to CAC)

Abstract-This study investigated the effect of paraventricular nucleus (PVN) injection of neuropeptide Y (NPY) on milk and water intake of 2- and 15-day-old sated rats. On the day prior to testing, rat pups were removed from their mothers and implanted with a cannula directed unilaterally at the PVN. On the following day, each pup was implanted with an intra-oral cannula for oral infusion of milk or water that could be swallowed or rejected. Following a l-hour period of satiation, each pup received a PVN injection of saline or a single dose of NPY (23.5-235.0 pmol). Milk or water intake was then assessed in a l-hour test period. Injection of NPY into the PVN enhanced milk and water intake equally at 2 days of age. At 15 days, NPY produced a significantly greater enhancement of milk than water intake. These findings, which are similar to those observed previously with PVN injections of norepinephrine (NE), suggest that: (1) NPY receptors in the PVN, like o?2-noradrenergic receptors, are functional very early in the postnatal development of the rat; (2) NPY, in addition to its orexigenic effect, produces a small but significant dipsogenic effect; and (3) NPY may function cooperatively with NE in the PVN to stimulate feeding and drinking beginning at a very early age.

Introduction Neuropeptide Y (NPY), a 36-amino acid member of the pancreatic polypeptide family, is known to play a role in the control of feeding behavior. Investigations in rats have shown that NPY is heavDate received 4 December 1992 Date accepted 14 December 1992 Correspondence to: C. A. Capuano, Department of Psychology, Fairleigh Dickinson University, 1000 River Road, Teaneck, NJ 07666 USA

ily concentrated in the hypothalamus, especially in the paraventricular nucleus (PVN), and is one of few neuropeptides that stimulates food intake.5**5J9 Injections of relatively low doses of NPY into the third ventricle or PVN of adult rats produces a feeding response which is more potent than that produced by any other neurochemical agent studied to date.L5J8-20In fact, a single PVN injection of NPY has been shown to induce sated adult rats to eat an average day’s worth of food in just a 4 h period.20 Similar to NPY, the classical neurotransmitter

177

178 norepinephrine (NE), working through u2-noradrenergic receptors, has been shown to stimulate food intake following injection into the PVN.12 Evidence suggests that, while NPY and NE can clearly act independently of one another, they have similar neuroendocrine and behavioral actions and very likely function in a cooperative fashion to support ingestive behavior. L3~14 These neurochemicals, which coexist in a subset of neurons ascending from the brainstem to the PVN,7JoJ6J7both produce a small increase in drinking behavior in association with a feeding response. I9 Moreover, they both increase appetite specifically for carbohydrate and become most active at the beginning of the natural feeding cycle.14 In light of these findings revealing a close association between NE and NPY, and given the results of a recent developmental study reporting the early functional onset of the NE-PVN-a*-noradrenergic receptor system,4 the present study was undertaken to investigate the ontogeny of NPY’s orexigenic effect in the PVN. Additionally, given that NPY has been shown to stimulate water intake in adult rats following injection into the third ventricle or PVN,15J8Jgthe ontogeny ofNPY’s dipsogenic effect was similarly investigated. Unilateral cannulas were used to administer NPY directly into the PVN of preweanling rats. Milk and water intake were assessed using an ‘independent feeding’ procedure* which has been used in previous developmental studies of this kind.3*4+6

Method Subjects Male and female offspring of Long-Evans hooded rats were used as subjects. All animals were bred and reared in the laboratory animal facility. The facility was kept at a constant temperature (22°C) and maintained on a 12/l 2 h light/dark cycle with lights on at 7:00 a.m. Mothers were checked for litters at 8:00 a.m. and 6:00 p.m. each day, and rat pups found at either of these times were designated 0 days of age. All litters were culled, without regard to gender, to a minimum of 5 and maximum of 10 pups 3 days after birth. Litters with fewer than 5 pups were not used. Other than culling, litters were left undisturbed until the time of surgery, with the exception

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of providing food and water and changing the bedding material twice each week. Surgery/Post-surgery On the day prior to testing, pups from a single litter, at either 1 or 14 days of age, were removed from their mother, weighed and stereotaxically implanted, under deep methoxyfluorane (Metofane) anesthesia, with a chronic indwelling 26-gauge stainless-steel guide cannula (Plastic Products Co.) that terminated 1.O mm dorsal to the targeted injection site in the PVN. The guide cannula, with a removable stylet, was anchored to the skull with successive applications of a thin layer of grip cement (Dentsply International) and a non-irritating, selfcuring acrylic (Silverman’s) that was used to construct a supporting crown. The stereotaxic instrument (David Kopf Instruments, Model 1430) was equipped with a custom-fitted infant stereotaxic accessory.g The stereotaxic coordinates for the targeted PVN site were taken from a stereotaxic atlas of the infant rat hypothalamus** and, in pilot studies, were carefully readjusted for each age following histological examination. Methods for surgery and histological examination and confirmation are detailed elsewhere.2 The stereotaxic coordinates used for each age are shown in Table. Following surgery, each pup was placed in a small, clear plastic container (10 x 12.2 x 12.7 cm). Each container was then placed in an incubator that was maintained at nest temperature (32-34°C). All pups remained in the incubator until the time of testing. To minimize the level of food deprivation, pups were given access to a small spoonful of commercially available plain, low-fat yogurt that was placed on the floor of each container. Table Stereotaxic Coordinates for PVN Injections in 2and 15-Day-Old Pups

Injection site PVN

Age (days) 2 15

Stereotaxic Coordinates (mm) M-L D-V A-P -1.2 -1.5

-0.4 -0.5

-4.9 -6.9

PVN = paraventricular nucleus. All coordinates are relative to bregma: A-P, anterior-posterior; M-L, medial-lateral; D-V, dorsal-ventral (to the surface of the skull). Note: Pups were tested at one of the two ages cited in the table. Cannulas were implanted on the day prior to testing.

EFFECT

OF PVN INJECTION

OF NPY

179

ON RATS

Testing On the day following surgery, each pup was removed fi-om the incubator and transferred to one of a similar set of containers with the same type of bedding material (pine shavings) found within their home cages. These containers were then placed in a water bath that maintained the ambient temperature within the containers at 32-34°C throughout each individual test period. At this time, an intra-oral cannula was implanted in the anterior portion of the mouth of each pup.* The free end of the cannula was then attached through a larger piece of polyethylene tubing (PE 50, Clay Adams) to a 5 or 10 ml syringe that was placed in a dual infusion/withdrawal pump (Harvard Apparatus Co., Model 600-910). The pump infused milk or water continuously at rates of 0.66 and 3.30 ml per h for 2- and 15-day-olds, respectively. In each case, the rate of infusion was approximately equal to 10% of the pup’s body weight per h, which exceeded intake by a moderate amount. Milk or water that was not ingested simply spilled out of the mouths of the pups. The milk diet used for all tests was commercially available Halfand-Half (milk and cream), which has certain similarities to rat’s milk.* After voiding the pups’ bladders by gently stroking their anogenital area with a moist Q-tip swab, their urogenital and anal openings were sealed with a drop of non-irritating, self-curing acrylic (Silverman’s) to minimize weight loss through excretion. The pups were then weighed. As reported in an earlier study,6 pups taken directly from their mothers can ingest large volumes of milk when it is offered via intra-oral cannulas. Therefore, all pups in the present study were sated for a 1.5-hour period prior to injection in order to prevent large spontaneous intake from obscuring the effect of the neurochemical agent tested. Each pup was sated with the diet (milk or water) for which they were to be tested. Following the period of satiation, each pup was reweighed and injected intracerebrally, without anesthesia, with a single dose (23.5, 70.5 or 235.0 pmol; 0. l-l .O pg) of NPY (Peninsula Laboratories, Belmont, CA, USA) or an equivalent volume of the vehicle (0.85% bacteriostatic saline). While 2-dayold pups tested for milk intake were administered one oftwo doses ofNPY (23.5 or 70.5 pmol), 2-dayold pups tested for water intake were given only one

dose of NPY (70.5 pmol) the most effective dose in 2-day-olds tested for milk intake. All 15-day-old pups, tested for milk or water intake, were given 235.0 pmol of NPY, a dose used in similar tests of food and water intake in adult rats.19 The injection volume was 0.1 fl, and injections were made directly into the PVN through a 33-gauge injector cannula (Plastic Products Co.) that extended 1.Omm beyond the tip of the guide cannula. Immediately following injections, the infusion pump was started, and a continuous milk or water infusion was delivered for a 1-hour test period. Following each test period, pups were gently dried and weighed. Body weight gain was used as a measure of milk or water ingested, since it has been shown that weight gain is a valid measure of intake in suckling rats.” Increases in body weights of pups administered NPY were compared to that of their littermate given the vehicle. Histology Following each test, all pups received an intracerebra1 injection of 0.1 pl of India ink to aid histological verification of injection sites. All pups were then perfused intracardially, under deep anesthesia, with isotonic saline followed by 10% buffered formalin. Brains were excised, frozen, cut in 50 pm coronal sections, and stained with Cresyl violet. The injection sites were determined with the aid of a stereotaxic atlasz2 Statistics A total of 63 rat pups from 28 different litters were tested in this study. Litters were regarded as experimental units for each test and analysis. A litter was tested at one age, for milk or water intake only. Different pups within a single litter were randomly assigned to vehicle or drug conditions at the time they were removed from their mother on the day prior to testing. Statistical analyses were performed on weight gain expressed in grams and as a percentage of postsatiation weight. A 3-way (factorial) analysis of variance (age x diet x dose) was used to evaluate the data for each of the two measures of weight gain. Post hoc comparisons of drug to control conditions were made using Dunnett’s test. The lowest dose for milk intake at 2 days of age (23.5 pmol) was excluded from the above analyses,

180 but was compared to baseline using Student’s t-test. This one exception was permitted because this additional dose was tested at 2 days of age for milk intake, but not for water intake.

Results

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although spread of drug into the ventricle could not be ruled out. Only a few PVN cannulas (3163) terminated immediately lateral (less than 0.1 mm) to the nucleus. Due to the close proximity of these three injections to the PVN and because there was spread of ink into the PVN for each of them, these data were included among the data reported.

Histology The locations of all injection sites are shown in Figure 1 on a plate reproduced from an atlas of the developing rat brain.** Nearly all of the cannulas targeted for the PVN (60/63) terminated within the dorsal (wing) portion of the nucleus. Moreover, there was no indication of spread of India ink into the third ventricle for any of the paraventricular injections,

Fig. 1 Histological verification of injection sites as confirmed by-the spread ofindia ink. Nearly all of the cannulas targeted for the PVN (60/63) terminated within the dorsal (wing) portion of the nucleus (0): A few PVN cannulas (3/63) tern&ted immediately lateral (less than 0.1 mm) to the nucleus (0). AH = anterior nucleus of the hypothalamus; F = fomix; PVN = paraventricular nucleus; V = third ventricle; Vh4 = ventromedial nucleus of the hypothalamus.

Weight gain Figure 2 shows weight gain from milk and water ingestion in 2- and 15day-old sated pups following injection ofNPY into the PVN. Relative to baseline, NPY stimulated milk and water intake, as measured by weight gain in grams or as a percentage of postsatiation weight, at both ages. 3-way factorial analyses of weight gain (age x diet x dose) indicated significant main effects of age, diet and dose for both measures (ps < 0.001; age for percent, p < 0.05). The 2-way interactions of age-by-diet, age-by-dose and diet-by-dose were also significant for both measures (ps < 0.001; except age-by-dose for percent, p < 0.05). The 3-way interaction of age-by-diet-by-dose was significant for both measures as well (ps < 0.001). Dunnett’s post hoc comparisons revealed significant differences (ps < 0.01) between drug and control conditions for both measures, for milk and water intake, at both 2 and 15 days of age. Using Student’s t-test, comparison ofthe lowest dose (23.5 pmol) to baseline for milk intake at 2 days of age revealed a significant difference (p < 0.01) for both measures. Inspection of the data in Figure 1 indicates that, at 2 days of age, similar doses of NPY stimulated milk and water intake equally. At 15 days, however, NPY produced a significantly greater stimulation of milk than water intake at similar doses. As reported above, a significant age-by-diet-dose interaction resulted for both measures. Despite this significant three-way interaction, a non-significant age-by-dose interaction resulted for weight gain analyzed as percent post-satiation weight. Careful examination of percent weight gain from water intake at 2 and 15 days of age indicates a slight increase for control animals over age, but a slight decrease for drugged animals over age. While percent weight gain for 15-day-old control pups was slightly greater than that for 2-day-old control pups, percent weight gain for 15-day-old drugged pups

EFFECT OF PVN INJECTION

181

OF NPY ON RATS

MILK

WATER

2 Days

6

15 Days

2 Days

I *

1Ii-l*

Vehicle

10.0 100.0 Vehicle 235.0 log scale NEUROPEPTIDE

Vehicle 70.5

Vehicle 235.0

Y (pmoles)

Fig. 2 Effect of NPY on milk and water intake of sated Z- and 15day-old pups as a function of dose in the PVN. The bottom panel represents weight gain in grams and the top panel represents a conversion of weight gain to a percentage of post-satiation weight. The error bars are one standard emor of the mean. * = p < 0.01 relative to vehicle by Dunnett’s test, or by Student’s t-test.

was slightly less than that for 2-day-old drugged pups. This minor directional difference more than likely accounts for the lack of a significant age-bydose interaction for weight gain analyzed as percent post-satiation weight.

Discussion The results of this study show that, like NE,4 injection of NPY into the PVN stimulates feeding (milk or nutrient intake) beginning very early in the postnatal development of the rat. Also similar to NE,4 NPY stimulates milk and water intake equally at 2 days of age, yet produces a greater stimulation of milk than water intake at 15 days of age. Therefore, like NE,4 NPY’s orexigenic effect becomes more pronounced as development proceeds. This finding is consistent with the developmental literature in suggesting that preweanling rats do not discriminate

or differentiate between milk and water until about 8 days of age, whether drugged or non-drugged,6 and also with the adult literature in suggesting that NPY produces a greater orexigenic than dipsogenic effect.L5,18.19 The early onset in development of NPY’s effect on feeding further suggests that NPY receptors in the PVN, like a2noradrenergic receptors,4 are functionally mature very early in life. This inference is consistent with radioimmunoassay’ and immunohistochemicalz3 analyses of the early ontogeny of the NPY system in the rat brain. These analyses have indicated that NPY is present in the brainstem and diencephalon, including the PVN, in embryos of 14 days postconception and that concentrations ofNPY show a rapid rise in these regions immediateiy after birth. Furthermore, the finding that NPY appears early in the development of the embryonic rat brain, particularly in caudal regions, was said to have some striking similarities to the developmental pattern of

182 the catecholamine systems.* In fact, areas rich in NPY immunostaining included the monoaminergic regions of the brain stem from embryonic Day 13, especially the lateral reticular nucleus and the medullary reticular formation.23 Collectively, these findings support the adult literaturelg in suggesting that NPY and NE act similarly in the PVN to enhance food and water intake in rats. Moreover, these findings strengthen the view that NPY and NE function cooperatively in the PVN,i3J4perhaps beginning very early in life, to control nutrient intake.

Acknowledgements The work described inthis manuscript was supported by Fairleigh Dickinson University laboratory funds to C. A. Capuano, by USPHS research grant MH 43422 to S. F. Leibowitz and by CUNY Research Foundation grant 667240 to G. A. Barr. This study was performed in following the standards established by the Animal Welfare Act, as cited in USDA and AAALAC guides for the Care and Use of Laboratory Animals.

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