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Faculty of Agriculture, University of the Ryukyus, Nishihara-cho, Okinawa 903-0213, Japan
2 Correspondence:
phone: +81-98-895-8798; fax: +81-98-895-8734; E-mail:
b986094{at}agr.u-ryukyu.ac.jp.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Key Words: Feeding Behavior • Goats • Hypothalamus
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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When the lateral hypothalamic area (LHA) in rats and goats was stimulated electrically, feed intake increased (Delgado and Anand, 1953; Larsson, 1954). Anand and Brobeck (1951) and Teitelbaum and Epstein (1962) reported that when the LHA in rats was electrically lesioned, they became anorexic. It was reported that when the ventromedial hypothalamic nucleus (VMH) was lesioned with a direct infusion of a small amount of goldthioglucose, hyperphagia and obesity was caused in mice (Smith, 1972), whereas feed intake remained unchanged in sheep (Baile et al., 1970). Wyrwicka and Dobrzecka (1960) reported that feed intake in goats was restricted when the VMH was electrically stimulated. However, Tarttelin (1976) reported that even if the VMH in sheep were electrically lesioned, there was still no effect on feed intake. The location of the feeding centers in the brain of ruminants has not yet been determined nor have the feed intake control mechanisms. In this study, it is hypothesized that changes in eating rates with feeding are centrally regulated in goats fed on crushed alfalfa hay cubes.
As part of the research to determine feeding centers in the brain of ruminants, the present research utilized the single-unit activity (SUA) recording electrode to investigate LHA neurons related to feeding behavior.
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Materials and Methods |
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Seven male Japanese Saanen goats (between 1 and 3 yr old, weighing 36.0 to 55.0 kg) were used in this experiment. The animals were maintained in individual metabolic cages that allowed for the separate collection of urine and feces. The cages were placed in a shielded room (24°C, 83% relative humidity) and the animals were fed twice a day. During the morning 2-h feeding period (1000 to 1200), the animals were fed 1.5 kg of roughly crushed alfalfa hay cubes. At 1600 each day, the animals were fed again with 200 g of commercial ground concentrate feed. Throughout the experiment, the animals were given unrestricted access to water. The alfalfa hay cubes (84.30% DM) contained, on a DM basis, 18.7% CP, 2.4% crude fat, 29.7% crude fiber, 39.7% nitrogen-free extracts (NFE), 45.9% NDF, and 36.6% ADF. The proportion of each ingredient in the ground concentrate feed was 48% maize, 24% sorghum, 1% barley, 3.5% soybean meal, 9.5% rapeseed meal, 6.0% wheat bran, 5.0% rice polishing, 0.5% molasses, 1.4% calcium carbonate, 0.5% alfalfa meal, 0.5% sodium chloride, 0.05% dicalcium phosphate, and 0.05% vitamin trace minerals premix. The concentrate feed (86.90% DM) contained, on a DM basis, 13.4% CP, 3.6% crude fat, 3.7% crude fiber, 71.0% NFE, 14.6% NDF, and 5.4% ADF. Alfalfa hay cubes were ground with a Wiley mill (type 40-525P, Ikemoto Rika Kougyou, Tokyo, Japan). The chemical components of their feeds were quantified using the procedures described by Nihon Shiryo Kyoukai (Kato, 1988).
Recording Electrode
A recording electrode was made using a platinum-iridium wire. Eight platinum-iridium wires were inserted into a stainless steel tube so as not to be bent when the electrode was introduced into the brain. The stainless steel tube was insulated except for 10 mm at the top and 1 mm at the bottom (Ono, 1985).
Placement of the Recording Electrode
The animals were anesthetized with intravenous injection of sodium pentobarbital and were fixed to a stereotaxic instrument (SN-2S, Narishige, Tokyo, Japan). Under fluothane anesthesia, the top of the animal’s head was shaved and sterilized with a 0.5% hibitene-70% ethanol solution. The skin and membrane bone were removed to expose the bregma and lambda on the skull. A 1.0-cm2 hole was then trephined on the sagital eminence with a dental drill. Using lateral X-rays for guidance, the site was chosen for vertical access to the LHA. The recording electrode was then inserted into the LHA. The tip of the inserted recording electrode was fixed with dental acrylic resin (GC Corp., Tokyo, Japan) to screws that were implanted into the skull. Finally, a protector was attached to the skull to protect the recording electrode. Animals were then allowed to recover for approximately 2 wk postsurgery. When feed intake had returned to the presurgery level, SUA recording was commenced. All surgical and experimental procedures were approved by the Animal Experimental Ethics Committee of the University of the Ryukyus and were in compliance with the Japanese code of practice for the care and use of animals for scientific purposes.
SUA Recording
One hour before the commencement of the experiment, heart rate, respiration rate, and rectal temperature were measured to confirm the animal’s health.
During the 2-h morning feeding, feed and water intake were measured every 10 and 15 min, respectively. The rate of eating was determined using a measuring scale to measure the weight of feed. Roughly 1.5 kg of crushed alfalfa hay cubes was placed in a feed box attached to a 6-kg measuring scale. The weight of the remaining feed was measured every 10 min for the duration of the 2-h feeding period. The rate of drinking was determined using a measuring scale to measure the weight of the water. Four kilograms of water was placed in a bucket and the weight of bucket with water was measured using a 12-kg measuring scale. The weight of the remaining water was measured every 15 min for the duration of the 2-h feeding period.
The SUA recording in the LHA was conducted over a 2.25-h period beginning 15 min prior to feeding commencement (0945) and continued until feeding completion (1200). Single-unit signals were inputted via a probe to the high-gain amplifier (AVH-11, Nihon Kohden, Tokyo) and were displayed on the memory oscilloscope (VC-11, Nihon Kohden, Tokyo) and recorded via a Maclab/4s (AD Instruments, NSW, Australia). Single-unit signals were recorded to an online magnetic optical (MO) disk with a Power Macintosh 4400/200 (Apple Computer Inc., Cupertino, CA) and a MO drive (MOS 350B/MA, Olympus, Nagano, Japan) connected to the recording instrument. Before each recording, the action potentials were checked to ensure their appearance. If the action potentials were not apparent, the electrode was moved toward the brain ventral and recording was conducted in a location where the action potentials were apparent.
Histology
After the completion of recording, a marking was made by passing electricity through the electrode in order to confirm the recording sites. The animals were then given a lethal dose of sodium pentobarbital and their brains were perfused via vertebral arteries with 10% formolsaline to make a brain block. After embedding with celloidine, the brain block was then cut at a 40-µm thickness and sections were made. The cell and fibers of the sections were stained with cresyl violet and luxol fast blue. With reference to the stereotaxic brain atlas of the Japanese Saanen goat, the nucleus was identified and the location of the electronic tip was confirmed.
Statistical Analyses
Regression analyses were used to calculate the regression equation of eating rates, cumulative feed intake, drinking rates, and cumulative water intake. A one-way classification and subsequent Duncan’s Multiple Range Tests were used to compare the data of firing rates from 10 min before commencement of feeding (control) and 0 to 10 min, 10 to 20 min, 20 to 30 min, and 30 to 40 min after commencement of feeding. A one-way classification and subsequent Fisher’s LSD methods were used to compare the data of spike duration between the five units and the 26 units that did not respond to feeding. For statistical analysis, GLM and REG procedures of SAS (SAS Inst., Inc., Cary, NC) were used.
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Results |
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Eating rates of roughly crushed alfalfa hay cubes rapidly decreased during the first 40 min of feeding (0 to10 min, 304.4 g/10 min; 30 to 40 min, 76.5 g/10 min; P < 0.05). A decrease in eating rates following the first 40 min of the feeding period slowed somewhat, but continued to decline (P < 0.05). The cumulative feed intake after the completion of the 2-h feeding period was 1164 ± 38 g (Figure 1a
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Drinking rates during the first 45 min increased sharply from 65.4 mL/15 min (0 to 15 min) to 374.9 mL/15 min (30 to 40 min; P < 0.05). Drinking rates were maintained at a high level between 45 to 60 min (406.3 mL/15 min) and 60 to 75 min (403.7 mL/15 min; P < 0.05). After 75 min of the feeding period had elapsed, drinking rates decreased (75 to 90 min 287.7 mL/15 min; P < 0.05). The cumulative water intake upon the conclusion of the 2-h feeding period was 2422 ± 107 mL (Figure 1b
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Single-Unit Activity Recording
In this study, 31 units were recorded, of which five responded to feeding and changed their neuronal activities. The remaining 26 units did not change their neuronal activities during the recording period.
Figure 2 shows the raw data of a recorded unit and an enlarged wave of a spike. The 31 units recorded in this study were all single units. The amplitude for the five units whose firing rates responded to feeding was 60 ± 15.8 µV, with a duration of 1.2 ± 0.12 ms. On the other hand, the amplitude for those units whose firing rates did not respond to feeding was 60 ± 25.3 µV with a duration of 1.0 ± 0.04 ms. Although there was no significant difference in the amplitude between the two groups, the duration of the latter group was significantly shorter than that of the former (P < 0.05; Table 1).
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shows the change in firing rates of the five units that responded to feeding. Although the average firing rate of unit I (amplitude, 50 µV; duration, 1.3 ms) was 1.9 ± 0.23 spikes/s 10 min prior to feeding, it increased with feeding to 3.2 ± 0.33 spikes/s in the first 10 min of feeding, and to 3.7 ± 0.23 spikes/s between 10 and 20 min of the feeding period (P < 0.05). After 20 min of the feeding period had elapsed, firing rate decreased, and following 30 min of feeding, firing had stopped completely (Table 2).
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The average firing rate of unit III (amplitude, 60 µV; duration, 1.3 ms) was 1.8 ± 0.19 spikes/s 10 min prior to feeding. It increased to 2.3 ± 0.11 spikes/s in the first 10 min of feeding, and to 2.3 ± 0.10 spikes/s between 10 and 20 min of the feeding period (P < 0.05). Following 20 min of feeding however, firing rate slightly decreased (Table 2).
The average firing rate of unit IV (amplitude, 40 µV; duration, 0.8 ms) was 2.6 ± 0.17 spikes/s 10 min prior to feeding. It increased with feeding to 4.8 ± 0.29 spikes/s in the first 10 min of feeding. Fifteen minutes after the commencement of feeding, firing rates decreased, and by the time 20 min of the feeding period had elapsed, average firing rate was significantly lower than prefeeding rates (P < 0.05; Table 2, Figure 3).
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). Histology
Figure 4 is a transverse section showing the location of the 31 units recorded in this study. It was confirmed that while the electrode used to record the 31 units in this study deviated approximately 2.2 mm toward the caudal region from the AP coordinates of the VMH, all of the units were located between the dorsal and ventral region of the LHA. The five units in which changes in firing rates with feeding were observed were all located in the dorsolateral hypothalamic area close to the fornix. On the other hand, the 26 units in which no changes were recorded were located in an area close to the fasciculus mamillothalamicus, the zona incerta, and the base of the brain.
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Discussion |
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In order to elucidate brain mechanisms controlling food intake in animals, it is necessary that the relationship between eating rates and changes in neuronal activity be investigated over a longer term during the feeding period. In the present experiment, the recording of each of the LHA neuron’s activity was continued for 2.25-h before and during feeding. The activities of the five single units recorded in the present experiment were correlated with the changes in eating rates during the 2-h feeding period. Five of the recorded units responded to feeding and changed their neuronal activities (Table 2). In all five units, firing rates increased to a level higher than that of prefeeding (P < 0.05) in response to a sharp increase in eating rates. As the animals reached a level of satiety (eating rates declined to very low levels), firing of units I and II stopped completely, whereas the firing rates of units III, IV, and V decreased. Examination of a serial histological section confirmed that the five units in which changes in firing rates with feeding were observed were all located in the dorsolateral hypothalamic area close to the fornix (Figure 4).
In goats fed twice a day, a feeding system utilized on the farm, eating rates of alfalfa hay cubes were highest 10 min after feeding commencement, but had decreased sharply by the time 40 min had elapsed (Figure 1a). From this result, it is thought that prior to feeding, directly after feeding had commenced, and at 40 min after feeding had started, the goats were in a state of hunger, at the peak of feeding, and in a state of satiety, respectively. The neurons in the LHA have intimate functional connections with afferent and efferent autonomic nerves (Barone et al., 1979; Carmona and Slangen, 1973; Misher and Brooks, 1966; Schmitt, 1973). In addition, LHA neuronal activity is influenced by metabolites and hormones contained in the blood and cerebrospinal fluid (Oomura, 1976; 1980). Sunagawa et al. (2002a) reported that both heart rate and ruminal movement greatly increased directly after feeding commenced. Sunagawa et al. (2002b) observed that the rate of parotid saliva secretion also increased directly after feeding commenced.
Internal visceral (hepatogastro-intestinal) and humoral signals, such as those from hepatic glucose receptors, or the degree of stomach distension, converge in the LHA (Niijima, 1969; Schmitt, 1973; Oomura, 1980; Ono et al., 1981). Katafuchi et al. (1985) reported that LHA neuronal activity in freely moving rats was increased through central glucoprivation produced by an intra-third-cerebroventricular injection of 2-deoxy-D-glucose. From these reports, it is thought that the increased firing rates of LHA neurons during the early stages of feeding in the present experiment were produced by internal visceral and humoral signals and were active in promoting feeding. Until now, the LHA neurons have not been found to increase their firing rates during feeding. In nonruminants such as rats, guinea pigs, monkeys, and humans, blood glucose concentration increased rapidly after feeding commenced (Steffens, 1970). Glucose is a major energy source in these animals. Frequency of spikes recorded from the satiety center neurons of cats increased, whereas the spikes recorded from the feeding center neurons decreased significantly after glucose was given intravenously (Anand et al., 1964). Oomura et al. (1969) found a glucose-receptive neuron located in the VMH neurons of rats that increased its firing rate upon systemic or electroosmotic application of glucose. Niijima (1969) found that glucose receptors existed in the liver of the guinea pig. Oomura et al. (1974) also found a glucose-sensitive neuron in the LHA neurons of rats that decreased its firing rate by glucose application. In ruminants, an endogenous substance that changes its concentration with feeding and regulates feed intake has not been found.
Volatile fatty acids (acetate, propionate, butyrate, ß-hydroxy-butyrate) contributed approximately 70% of total energy expenditure in ruminants (Annison and Armstrong, 1970). When cows and sheep were fed either grass or concentrate feed, intraruminal infusion of VFA increased in concentration and feed intake decreased (Baile and Mayer, 1969; Anil et al., 1993). Anil and Forbes (1988) reported that forage intake was reduced as a consequence of intraportal sodium propionate administration in sheep. Although VFA is a major energy substrate in goats fed alfalfa hay cubes twice a day, the VFA concentrations in ruminal fluid and blood increased slowly during feeding and peaked 2 h after feeding commenced (Sunagawa et al., 1988; 1997). In goats fed twice a day, eating rates of alfalfa hay cubes was greatest 10 min after feeding commencement, but decreased sharply by the time 40 min had elapsed (Figure 1a). Changes in the goats’ eating rates of alfalfa hay cubes in this experiment are similar to the results of experiments conducted by Prasetiyono et al. (2000) and Sunagawa et al. (2001a) on goats given alfalfa hay cubes once or twice a day in experiments. Campling and Balch (1961) and Anil et al. (1993) reported that when a balloon was inserted into the rumen to restrict the rumen capacity of cows fed on hay and silage, feed intake decreased. Hidari (1987) reported that when sheep given free access to hay and silage reached a certain level of rumen fill, they stopped feeding. On the other hand, Sunagawa et al. (2001a) intravenously infused goats fed alfalfa hay cubes twice a day with a mixed artificial saliva, a hypoosmotic artificial saliva, and an isoosmotic mannitol solution on different days. The infusions were conducted from 1 h prior to feeding and continued until after 1 h of the 2-h feeding period had elapsed. This infusion supplemented the fluid in the blood lost through accelerated salivary secretion during the early stage of dry forage feeding and increased feed intake. These results suggest that an increase in rumen fill and a decrease in circulating plasma volume accompanied by feeding are controlling factors in the regulation of hay intake. From these reports, it is thought that the decrease or disappearance of the firing rates of LHA neurons during the second feeding time in this experiment reflected the goat’s state of satiety. The LHA neurons recorded in this experiment characteristically increased their neuronal activity at high levels of feeding, but decrease them at low levels. It is therefore thought that these neurons are involved in the regulation of hay intake.
Research of the central regulation of feed intake in ruminants is still in its early stages and the location of appetite centers is still unclear. Therefore, in order to ascertain the existence of feeding centers in the LHA and the existence of satiety centers in the VMH in ruminants, more research on the various conditions (Sunagawa et al., 2000; 2001b,c) of feed intake changes is needed.
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Implications |
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Footnotes |
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Received for publication April 19, 2002. Accepted for publication September 17, 2002.
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, y = 246.015 + 11.706x - 0.035x2, r2 = 0.99, P < 0.05), and bottom (b) shows changes in rate of drinking (•, y = -51.811 + 12.312x - 0.083x2, r2 = 0.81, P < 0.05) and cumulative water intake (
: increased activities with feeding, 
: unchanged activities with feeding. AM = corpus amygdaloideum; ARC = nucleus arcuatus hypothalami; CAM = cornu ammonis; cp = pedunculus cerebri; DF = fascia dentata; fx = fornix; LHA = area lateralis hypothalamica; mt = fasciculus mamillothalamicus; ot = tractus opticus; TV = ventriculus tertius; VM = nucleus ventralis medialis thalami; ZI = zona incerta.