Feeding specific glutamate surge in the rat lateral hypothalamus revealed by low-flow push–pull perfusion

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Abstract

Substantial evidence implicates the lateral hypothalamus (LH) in the control of ingestive behavior and previous studies have found that glutamate release within the LH increases during meals. It is not known, however, whether this effect is selective for feeding, or whether similar changes are also seen during drinking. In this work, we examined this question using low-flow push–pull perfusion which allows sampling from small tissue volumes. Presentation of highly palatable solid or liquid foods to food-deprived rats resulted in an immediate increase in glutamate output of more than 200% over baseline. The response was maximal immediately after food presentation. In contrast, significant changes in glutamate output were not seen when water was presented to water-deprived animals, despite the occurrence of vigorous drinking. These findings confirm reports of feeding related glutamate release in the LH and demonstrate that this effect is specific to feeding, rather than being a general concomitant of all ingestive behaviors. The push–pull technique described here may allow the relevant region of the LH to be identified with greater precision than other methods.

Introduction

Obesity is a major health problem, contributing to disorders such as diabetes, heart disease, hypertension and cancer (Angelopoulos et al., 2005, Dhillo, 2007, King et al., 2007, Morton et al., 2006, Wilding, 2002) and which is associated with increased mortality (Adams et al., 2006, Conway and Lip, 2004). The prevention and treatment of obesity, and other disorders of food intake, clearly requires a better understanding of the neural mechanisms underlying ingestive behavior than is currently available. A great deal of evidence indicates that the lateral hypothalamic area (LH) plays an important role in the control of food intake. Classical studies indicated that electrolytic lesions of the LH lead to long lasting reductions in food intake and body weight and, conversely, electrical stimulation of this region was shown to induce feeding. Although these results could have been due to damage to fibers of passage, more recent work has confirmed that disturbances in feeding behavior can be produced by axon sparing lesions of the LH and by injections of a variety of neurotransmitters into the LH. In particular, Stanley and his coworkers have shown that injections of the excitatory amino acid glutamate into the lateral hypothalamus are able to induce robust feeding (Stanley et al., 1993, Stanley et al., 1996). Similar effects can be observed following injections of glutamate agonists (Duva et al., 2002) and mapping studies have shown the central, tuberal, portion of the LH is more sensitive to this effect than are a number of adjacent regions (Stanley et al., 1993). Conversely, repeated local injections of NMDA type glutamate receptor antagonists are able to reduce food intake and body weight (Stanley et al., 1996). The possibility that glutamate in the LH is involved in the normal, physiological, control of feeding is further by studies which have used in vivo microdialysis to demonstrate that glutamate release in the tuberal LH is dramatically increased at the onset of feeding (Rada et al., 1997, Rada et al., 2003). In contrast, glutamate output from the nucleus accumbens shell has been shown to decrease in association with feeding (Rada et al., 1997), demonstrating that the response observed in the LH is not present globally throughout the brain.

The LH is clearly involved in the control of feeding, but a substantial body of work using lesioning, electrical stimulation, and imaging techniques indicates that it also exerts a pronounced influence on water intake. It is striking, therefore, that intra-LH injections of glutamate agonists have no effect on water intake, in marked contrast to their robust effects on feeding (Duva et al., 2002, Stanley et al., 1993). These results raise the possibility that glutamate in the tuberal LH is specifically involved in the control of food, but not water, intake. In evaluating this possibility, it would be interesting to know whether drinking, like feeding, is associated with increased LH glutamate output, but such information is not available at this time.

In the current study, we examined the relation between ingestive behavior and LH glutamate release using the method of low-flow push–pull perfusion (LFPP) combined with capillary electrophoresis (CE) to monitor glutamate output. This technique allows sampling from much smaller tissue volumes that would be possible using other techniques, such as in vivo microdialysis (Kottegoda et al., 2002, Stanley et al., 1993), and has been previously used to measure the extracellular content of a number of neuroactive compounds (Cellar et al., 2005, Gao et al., 2004, Gao et al., 2007, Kottegoda et al., 2002, Kottegoda et al., 2007, Thongkhao-on et al., 2004, Zhao et al., 2003, Zhao et al., 2004). In this work, we have examined feeding behavior during LFPP sampling to determine extracellular glutamate levels in the perifornical region of the LH. In particular, we examined glutamate output during the consumption of solid food and during the drinking of water. In addition, to determine whether differences observed between these to conditions might reflect the occurrence of chewing versus licking behavior, we examined glutamate output in animals consuming Ensure®, a highly palatable liquid diet. A brief version of some of these results has previously been presented in abstract form (Thongkhao-on et al., 2006).

Section snippets

Subjects

Adult, male Sprague–Dawley rats weighing from 290 to 450 g at the time of surgery were used. These rats were bred in the Psychology Department of the University of Illinois at Chicago, and were descended from rats obtained from Charles River. They were individually housed in a temperature controlled room on a 12 h:12 h light:dark cycle (lights on at 06:00) and allowed free access to standard Purina rat chow pellets and tap water, except as noted below. All experiments were performed under a

Experiment 1. Glutamate output during intake of solid food or water

Histological examination indicated that the tips of the push–pull probes were located within the tuberal portion of the LH in all subjects. An example of a typical probe location is shown in Fig. 2.

All subjects given food or water began to eat or drink within 1 min of presentation of the ingestates. Most subjects ate or drank continuously for several minutes and then stopped, and sometimes began subsequent bouts of ingestion at later times. Most subjects either ignored the Nylabones, or

Discussion

We demonstrate here for the first time that low-flow push–pull perfusion combined with CE-LIF analysis can be used to monitor extracellular glutamate levels in the brain of awake, freely behaving animals. As we have discussed elsewhere (Kottegoda et al., 2002) it is likely that this method allows for sampling from substantially more restricted volumes than would be possible using conventional in vivo microdialysis. In agreement with previous studies (Rada et al., 2003) which used in vivo

Acknowledgement

This work was supported by NIH Grants MH067971 (S.A.S.) and DA20802 (D.W.).

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