Although transmission of signals along nerves is primarily electrical, interneuronal communication is mainly chemical. Neurotransmitters, such as noradrenaline and acetyl choline, are released by the terminals of one nerve and stimulate the receptors on one or several other neurones, which respond by an increase or decrease of firing rate. There is more scope for both physiological and pharmacological mechanisms to work at the synapse than along the nerve fibre and, in recent years, chemical stimulation of the brain has been used as the major experimental technique. The development of pharmacological antagonists to neural transmitters has also provided useful tools for the investigation of brain function.
Methods of introducing drugs into the brain include injection via cannulae implanted in the lateral ventricle, micro-injection via fine cannulae implanted or advanced through guides into brain tissue and by elecrophoretic introduction.
Invariably, drugs must be injected in amounts greater than the amount normally present in brain tissue in order to cause a significant response, and the question arises as to whether results can be considered to be ‘physiological’. Despite such methodological problems, it seems likely that substances in the extracellular fluid
Central Nervous System and the Special Senses 95
0 200 400 600 800
Food Salt Water Control
Mean change in firing rate (%)
Fig. 5.1. Response in firing rate of neurones in the zona incertaof sheep to the sight of food, water and salt when the animals were food-deprived for 12 h (solid bars) or sodium-deprived for 4–6 days (open bars) (from Kendrick, 1992).
of the brain are involved with the physiological control of feeding. Martin et al.
(1973) transferred 1 ml of cerebrospinal fluid (CSF) from fasted sheep into the lateral ventricle of recipients and found a fivefold increase in food intake during the following 15 min; CSF from satiated sheep had no effect.
Anaesthetics
By preventing neural transmission through affected parts of the brain, local anaesthetics are expected to have similar effects on feeding to lesions in the same areas. In general, this has proved to be the case.
Pentobarbital (a local anaesthetic) injected into the third ventricle or basomedial hypothalamus of chickens caused intake to increase from 14 to 32 g during the 1 h after injection, even though the birds were almost asleep;
there was no effect when the injections were made into the posterior lateral hypothalamus (Snapiret al., 1973). A dose of 6.5 mg of sodium pentobarbital given into the LHA depresses intake in hungry pigs, while in the VMH or cerebroventricles it stimulates intake in satiated animals.
Cerebroventricular injections of pentobarbital stimulate feeding in calves, goats and sheep. Detailed studies with sheep, in which a range of barbiturates with different durations of action were compared, showed that long-acting ones were more effective than those with a short action in inducing feeding when injected into the third ventricle (Seoaneet al., 1973). The effects of barbiturates were similar in a hot environment and at normal temperatures, and with high roughage and high concentrate rations. Micro-injection of barbiturates into the medial hypothalamus also stimulates feeding in sheep.
Adrenergic agents
Substances such as anaesthetics are, of course, unnatural, and manipulation of the concentrations of substances found naturally in the brain, and their analogues and antagonists, have formed the basis of most recent work.
Since the original work with rats there has been ample confirmation of a major place for adrenergic transmitters in the control of intake, and numerous experiments have demonstrated that noradrenaline given into the medial hypo- thalamus stimulates intake in several species, including farm animals. This effect can be prevented by prior injection of the appropriate blocker, but blockers given alone do not stimulate feeding. Work originally concentrated on the VMH and LHA and the paraventricular nucleus (PVN) as being the most sensitive sites mediating the actions of adrenergic agonists on eating. It seems likely that the PVN and its ␣-adrenergic receptors are important in the control of eating in response to deprivation and, possibly, to post-ingestional satiety signals.
Brain turnover of noradrenaline is increased by glucoprivation, a situation that can be reversed by providing substrates, either by ingestion or infusion, which could be used by the brain. This is evidence for a physiological role for noradrenaline in the control of feeding.
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Poultry
Cerebroventricular injections of adrenaline or noradrenaline increase food intake in broiler-type chickens, while dopamine has no effect. However, the injection of adrenaline had no effect in birds of a layer strain. A differential response was also noted to cerebroventricular injections of 5-HT which, in broilers, depressed food intake in fully fed birds but not in 24-h fasted birds, while in layers 5-HT decreased food intake in both fully fed and 24-h fasted birds.
Further studies (Denbowet al., 1986), with lines of broilers selected for fast and slow growth, showed that methoxamine (an ␣-adrenergic agonist) increased feeding in the fast- but not in the slow-growing strain, while 5-HT had no effect in fully fed birds but did depress intake in 24-h fasted birds of both lines. Genetic selection for growth has been demonstrated to alter the meal pattern as well as to alter the feeding response to intubations of amino acids and cerebroventricular injections of biogenic amines. Further studies are required to explore the mechanisms of these differences between breeds and strains.
Injection of 6-hydroxy-dopamine, which depletes noradrenaline, into the cerebroventricles or hypothalamus not only increases food intake but also causes obesity and testicular atrophy in geese.
Fenfluramine is a well-known appetite suppressant in mammals and, when incorporated in food, it induced a dose-dependent reduction in food intake by broiler chickens (Hocking and Bernard, 1993).
Pigs
In the pig, noradrenaline injected into the hypothalamus stimulates feeding while isoproterenol (a synthetic -adrenergic agonist) causes temporary anorexia, blocked by propranolol (a synthetic -adrenergic antagonist). The volume injected was large, however, so these results tell us little about the exact site of action.
Cattle
Lateral ventricular injection of noradrenaline in cattle stimulated voluntary intake over a very narrow range of doses (50–200 nmol), whereas isoproterenol was effective over a broad range (500–2000 nmol).
Sheep
Baile and his colleagues were, for several years, particularly active in this field (see Baile, 1975). They found that intraventricular injection of noradrenaline at doses of 542 nmol induced hyperphagia for some 30 min after injection. The effect was blocked by prior injection of ␣-adrenergic blockers, and it was later confirmed that intrahypothalamic injections had similar effects. At very low doses (4–16 nmol), the -adrenergic agonist isoproterenol stimulated intake for about 1 h and this was blocked by - but not by ␣-adrenergic antagonists. This
suggests the existence of two classes of adrenergic receptors and adrenaline, which has both ␣- and -adrenergic activity, stimulated intake at two dose ranges, presumably corresponding to optimum stimulation of the two types of receptor.
One major drawback to ventricular injection is that the site of action cannot be determined because CSF secreted in the lateral ventricles transports the injected material through several parts of the brain. More precise targeting can be achieved by implanting guides through the cranium, which are directed towards certain loci in the brain. Using sheep prepared in this way it was shown that noradrenaline (240 nmol) and isoproterenol (8 nmol) had similar effects on feeding when given into hypothalamic tissue as they did when given intraventricularly. Again, effects of each were blocked by the appropriate blocking agent and the dose–response curve showed an optimum range with depression at high doses.
A brain locus sensitive to noradrenaline was not usually responsive to isoproterenol, and prostaglandins (PG) injected at loci sensitive to noradrenaline had different effects on feeding than at loci sensitive to isoproterenol. Doses of 14 or 28 nmol of PGE1 depressed food intake when injected at loci that showed feeding in response to noradrenaline. A PG antagonist, polyphloretin phosphate, not only blocked the effects of subsequent PG injection but by itself significantly increased intake. Whether this means that PGE1 normally inhibits feeding is not clear. At sites that responded to isoproterenol, PGE1 significantly stimulated intake. This subject is complex and will not be properly understood until the full range of the pharmacological and metabolic effects of these substances is known.
Serotonin (5-hydroxytryptamine, 5-HT)
In the mammalian central nervous system, 5-HT systems occupy a strategic anatomical location, projecting to, and passing through, the hypothalamus.
Experimental manipulations of 5-HT metabolism can, under certain conditions, produce marked changes in food intake, food preferences and body weight, most obviously the suppression of food intake by experimental treatments that directly or indirectly activate 5-HT receptors. More controversial is the role of 5- HT in diet selection (see Chapter 7). Serotonin binds to several distinct receptor types in peripheral tissues but, while the significance of these receptor types in the CNS is uncertain, there is evidence linking them with effects on eating (see Forbes and Blundell, 1989).
In chickens of an egg-laying strain, 33–100 g 5-HT injected intraventricularly depressed intake, while in broilers it had this effect only if the birds were relatively satiated.
Intravenous injection of the serotonin agonist 8-OH-DPAT significantly increased food intake in the 45 min following injection in non-deprived pigs, while after 4 h of deprivation it had no effect on food intake (Baldwin et al., 1995). After deprivation, pigs are likely to eat so quickly that it will be difficult to demonstrate effects of experimental treatments unless they are severe.
In sheep, 5-HT has smaller effects on feeding than noradrenaline and at fewer sites, the responsive ones being concentrated in the area of the anterior commissure.
Cholinergic agents
Acetyl choline is a neural transmitter found in significant amounts in the hypothalamus. In the rat, ventricular or hypothalamic injection of carbachol, a slowly metabolized cholinergic agonist, causes an increase in water intake but a consistent and large depression in food intake.
Carbachol injected into the hypothalamus of sheep at doses as low as 7 nmol stimulated feeding for 30–60 min and when atropine, a cholinergic antagonist, was injected a few minutes before a 28 nmol dose of carbachol, the hyperphagia was prevented (Forbes and Baile, 1974; Fig. 5.2). The adrenergic antagonists phentolamine and LB46 did not attenuate the carbachol effect, showing that it was acting on cholinergic receptors in the hypothalamus rather than causing non-specific stimulation. However, atropine alone had no effect on intake, which implies that endogenous acetyl choline is not involved in feeding control.
It had been shown that significantly increased feeding followed the injection of 50 nmol of carbachol into the lateral ventricle. However, larger amounts of carbachol (56–436 nmol) given into the lateral ventricle of sheep almost totally
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0 50 100 150 200 250
0 30 60 90 120
Time after injection (min)
Cumulative food intake (g)
Carb Carb/LB 46 Carb/phent
Atr CSF Art/carb
Fig. 5.2. Food intake by sheep 30, 60 and 120 min after intrahypothalamic injection of: (i) synthetic cerebrospinal fluid (control, CSF); (ii) carbachol (28 nmol, Carb); (iii) atropine (28 nmol, Atr); (iv) carbachol preceded by atropine (Atr/carb); (v) carbachol preceded by phentolamine (120 nmol, Carb/phent); or (vi) carbachol preceded by LB46 (120 nmol, Carb/LB46) (Forbes and Baile, 1974).
prevented feeding for 1 h, accompanied by significantly elevated plasma levels of growth hormone (Driver et al., 1979). Experimental differences that might be responsible for this discrepancy between the two sets of results have not been identified.
It is unlikely that the carbachol-induced stimulation of growth hormone secretion directly caused the reduction in food intake because intrajugular growth hormone infusion to mimic peaks of this type had no effect on feeding behaviour in sheep (P.M. Driver and J.M. Forbes, unpublished results). It is equally unlikely that the low food intake following carbachol injection would give such a rapid response in growth hormone secretion; feeding and growth hormone were probably affected independently by carbachol.
Gamma-amino butyric acid (GABA)
Intracerebroventricular (ICV) injection of several GABA agents induced hyperphagia in layer-type chicks (Bungo et al., 2003). However, in broiler chicks two of the agents (muscimol and nipecotic acid) had similar effects while baclofen depressed feeding. These results suggest that there are some differences in central GABAergic systems between these strains of chicks, but that GABAergic systems have an important role in the regulation of food intake in neonatal chicks.
Muscimol, the GABA-A receptor agonist, increased operant responding for food in pigs after injection into the lateral ventricles, an effect that was completely abolished by simultaneous administration of bicuculine, a GABA antagonist (Baldwinet al., 1990b). GABA itself at 800 and 1600 nmol increased intake, and this was also abolished by bicuculine.
It has also been found that muscimol given intraventricularly stimulated feeding in sheep, and this was prevented by the GABA antagonist, ␥-vinyl GABA; GABA itself had little effect on feeding, however (Seoane et al., 1988).
Cholecystokinin (CCK)
CCK was first implicated in the control of feeding following experiments involving peripheral injections (see Chapter 4). CCK is found in the brain and reduces food intake when injected intraventricularly. The possibility that CCK is a neurotransmitter involved in a physiological control is discussed by Baile et al.(1986).
Poultry
ICV injection of chickens with 0.1 or 0.4 g CCK-8/kg (CCK octapeptide, CCK-8, is the active moiety of CCK) had no effect on gizzard or duodenal motility but caused drowsiness in some birds and reduced intakes by 20 and 31%, respectively, during the following 30 min (Savory and Gentle, 1984). Dibutyryl
cyclic GMP, a CCK receptor blocker, stimulated intake, supporting a physiological role for CCK. The mean concentrations of CCK-8 in different parts of the brain did not differ between fed birds and those fasted for 24 h, however, but no measurements of turnover have been reported.
Four-week-old broilers injected intracerebroventricularly with 100 and 150 ng of CCK-8 reduced intake, respectively, for 60 and 105 min. The fact that smaller amounts of CCK are required to depress intake by a given amount when injected into the brain, compared with peripheral injection, has been used to support the concept that brain CCK receptors are more important than gut CCK receptors in the control of food intake. However, brain injections are made into a much smaller volume than peripheral injections, making comparisons of efficacy very difficult.
Pigs
Doses of CCK-8 thought to be within the physiological range based on concentrations of CCK found in human CSF were injected into the lateral ventricles of prepubertal pigs. There was a dose-dependent reduction in food intake, cessation of feeding appearing to be similar to normal satiety, with no effect on drinking (Parrott and Baldwin, 1981). Greater doses injected intra- venously were without effect, strongly suggesting that the effect on feeding was on central mechanisms. However it has been suggested that exogenous CCK (given intravenously) causes a general malaise in the pig and thus depresses food intake non-specifically (see Chapter 4).
Sheep
Infusions of CCK-8 into the lateral ventricles of sheep at 0.01 pmol/min significantly depressed intake and ruminal motility during a 3-h period and were much more effective than a single injection (Della-Fera and Baile, 1980).
There was no effect on water intake or body temperature, and increasing the time of fasting before the infusion reduced the magnitude of the effect. This experiment offers no proof of a physiological role for CCK in the central nervous control of feeding. However, continuous ventricular infusion of dibutyryl cyclic GMP (a CCK antagonist) led to a large increase in food intake in the sheep (Della-Fera et al., 1981) suggesting that endogenous CCK in the brain is a natural satiety factor.
Bombesin
This brain peptide is as effective as CCK in inhibiting feeding when given into the lateral ventricle of sheep, but in rats there is considerable evidence that it induces abnormal behaviour. Injected intracerebroventricularly at doses of 1.25–5.00 g, bombesin depressed food intake in the 17-h food-deprived young pig (Parrott and Baldwin, 1982), but the behaviour induced was not
Central Nervous System and the Special Senses 101
typical of normal satiety as the pigs appeared uncomfortable and occasionally vomited. In water-deprived pigs, bombesin also inhibited drinking showing that it, like intravenously administered CCK, was non-specific in its behavioural effects.
Opioid peptides
Many peptides of the opiate family are synthesized in the brain and the existence of brain receptors for these peptides suggests that they have a physiological role. Amongst other effects the opiates have been found to stimu- late feeding in rats and sheep after cerebroventricular injection. Endogenous opioids might play a role in the regulation of food intake as the opioid antagonist naloxone decreases feeding in rats and in a variety of other species ranging from the slug to the wolf. It is likely that more than one opioid receptor and more than one brain site are involved in the opioid modulation of feeding.
Resistance to the inhibitory effects of naloxone on feeding occurs when animals are eating large amounts of food per kg of body mass and/or have a high basal blood glucose concentration. This strongly suggests that, although opioids play an important role in initiating food-seeking behaviours, they are not the only food drive system. Much evidence has accumulated suggesting that stress-induced eating is driven by activation of the opioid system. Similarly, the hyperphagia associated with diabetes mellitus appears to have an opioid component.
Poultry
A stable analogue of met-enkephalin, D-ala2-methionine enkephalinamide, stimulated food intake by pullets for 30 min after 2 and 8 g/kg were given into the cerebroventricles, but had no effect when given intravenously at 15 and 60 g/kg (Savory et al., 1989). Naloxone had no effect after either cerebroventricular (50 and 200 g/kg) or intravenous (1 and 4 mg/kg) injection.
Nalmefene, a more potent and long-lasting opioid antagonist than naloxone, inhibited feeding in a dose-related manner at doses from 0.2 to 1.6 mg. It appears that central release of endogenous opioids may reinforce feeding in birds. Further evidence of opioid release during feeding in the chicken comes from the observation that the reduction in tonic pain during feeding is reversed by naloxone (Wylie and Gentle, 1998).
Pigs
There was no effect on feeding of naloxone injected into the lateral ventricles at doses of 0.4 or 0.8 mg, 10 min after access to food was given following an overnight fast. This was attributed to the fact that the pigs were young and had been subjected to a fast, and were therefore very highly motivated to feed.
An injection of 200 g of dynorphin (a natural endorphin) given into the lateral ventricles resulted in a meal within a few minutes (Baldwinet al., 1990a).
Leumorphin and ␣-neo-endorphin also elicited feeding but ␣-neo-endorphin did not. Administration after the start of a meal taken after 4 h of deprivation increased the size of that meal; 400 g of naloxone significantly reduced intake after 4 h of deprivation and abolished the effects of dynorphin. It appears that dynorphin and related endogenous opioids might be involved in the regulation of food intake in pigs.
Feeding seems to induce an opioid-based analgesia, as the latency of tail- flick to a painful stimulus was longer after feeding than before, and this was abolished by naloxone. However, sows with marked behavioural stereotypies had shorter tail-flick latencies after feeding.
Sheep
Opioid levels are elevated in the brain of sheep after a 4-h fast, at which time the animal would be expected to be hungry, and intracerebroventricular injections of several opioids stimulate feeding. Baile et al. (1987), reviewing their work on involvement of opioids in feeding control in sheep, concluded that more specific agonists are needed before a full elucidation is possible.
Naloxone given intravenously prevents the feeding stimulated by cerebro- ventricular injection of enkephalamide (Buenoet al., 1983). Many times more analogue were required to generate a similar effect on feeding when it was given intravenously compared with injection into the lateral ventricles, implying that the major site of action is the CNS.
Neuropeptide Y and peptide YY
The pancreatic polypeptide family consists of pancreatic polypeptide, neuropeptide Y (NPY) and peptide YY (PYY). Very low doses increase feeding in rats when injected directly into the paraventricular nucleus (PVN), the magnitude of the increased food intake following NPY injections being much greater than that seen following central administration of opioid peptides or of noradrenaline. PYY has been shown to be an even more potent stimulator of feeding in rats than is NPY, and when PYY is administered every 6 h for 48 h it causes massive hyperphagia (80.5 g/day compared with 31.1 g in the control group) and stomach distension, attesting to the fact that central factors can override the physiological satiety signals from the periphery.
Poultry
Central (lateral ventricle) administration of NPY or PYY significantly stimulates feeding in chicks, possibly as a result of stimulation of insulin secretion, and NPY immunoreactivity has been found around the area of the PVN in chickens (Keunzel and McMurtry, 1988).
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