Basomedial Hypothalamic Injections of Neuropeptide Y Conjugated to Saporin Selectively Disrupt Hypothalamic Controls of Food Intake

Abstract

Neuropeptide Y (NPY) conjugated to saporin (NPY-SAP), a ribosomal inactivating toxin, is a newly developed compound designed to selectively target and lesion NPY receptor-expressing cells. We injected NPY-SAP into the basomedial hypothalamus (BMH), just dorsal to the arcuate nucleus (ARC), to investigate its neurotoxicity and to determine whether ARC NPY neurons are required for glucoprivic feeding. We found that NPY-SAP profoundly reduced NPY Y1 receptor and alpha MSH immunoreactivity, as well as NPY, Agouti gene-related protein (AGRP), and cocaine and amphetamine-related transcript mRNA expression in the BMH. NPY-SAP lesions were localized to the injection site with no evidence of retrograde transport by hindbrain NPY neurons with BMH terminals. These lesions impaired responses to intracerebroventricular (icv) leptin (5 microg/5 microl x d) and ghrelin (2 microg/5 microl), which are thought to alter feeding primarily by actions on ARC NPY/AGRP and proopiomelanocortin/cocaine and amphetamine-related transcript neurons. However, the hypothesis that NPY/AGRP neurons are required downstream mediators of glucoprivic feeding was not supported. Although NPY/AGRP neurons were destroyed by NPY-SAP, the lesion did not impair either the feeding or the hyperglycemic response to 2-deoxy-D-glucose-induced blockade of glycolysis use. Similarly, responses to glucagon-like peptide-1 (GLP-1, 5 microg/3 microl icv), NPY (5 microg/3 microl icv), cholecystokinin octapeptide (4 microg/kg ip), and beta-mercaptoacetate (68 mg/kg ip) were not altered by the NPY-SAP lesion. Thus, NPY-SAP destroyed NPY receptor-expressing neurons in the ARC and selectively disrupted controls of feeding dependent on those neurons but did not disrupt peptidergic or metabolic controls dependent upon circuitry outside the BMH.

Circadian integration of sleep-wake and feeding requires NPY receptor-expressing neurons in the mediobasal hypothalamus

“…All known rat NPY receptors, except the NPY-Y4 subtype, are internalizing receptors (56,57,94). Accordingly, we have shown previously that NPY-SAP binds to NPY receptors with greater affinity than NPY itself (5). We have also shown previously and in the present study that known NPY receptor-expressing neurons are profoundly reduced in number by NPY-SAP injections directed at the Arc.…”

“…In this experiment, we investigated the interactions of sleepwake and feeding rhythms in rats given bilateral injections of a targeted toxin, neuropeptide Y (NPY) conjugated to saporin (SAP), a ribosomal disaggregating agent (5,35). This conjugate (NPY-SAP) targets NPY receptor-expressing neurons for destruction by binding to internalizing NPY receptors.…”

Leptin-sensitive neurons in the arcuate nuclei contribute to endogenous feeding rhythms

“…For experiments in which immunohistochemistry (IHC) was used for lesion analysis, rats were killed at the end of experimentation by deep isoflurane anesthesia. After perfusion and fixation in 4% formalin in PBS, brains were sectioned coronally into serial sets (30 or 40 m thickness) and stained using standard avidin-biotin-peroxidase or immunofluorescent IHC technique (13,52). The following primary antibodies were used: goat anti-AGRP (1:1,000; Neuromics), sheep anti-alpha-melanocyte stimulating hormone (␣-MSH) (1:10,000; Millipore), rabbit antisteroidogenic factor-1 (SF-1) was (1:1,000; Millipore), rabbit anti-arginine vasopressin (AVP) (1:15,000; Millipore), and goat anti-SAP (1:1,000; Advanced Targeting Systems) antibodies.…”

“…Leptin's actions are mediated by its effect on the leptin receptor B, LepR-B (28), which is densely expressed in the Arc on various cell types of known importance for food intake (6). Arc area lesions block major effects of exogenous leptin on food intake and body weight (13,15,21,23). Furthermore, LepR-B has been implicated in the circadian control of feeding, as both ob/ob mice that lack leptin (99) and Zucker fa/fa rats that lack functional leptin receptors (96) have disrupted day-night feeding patterns (3,27,29,36,58,82).…”

Effects of hindbrain melanin-concentrating hormone and neuropeptide Y administration on licking for water, saccharin, and sucrose solutions.

“…36, 68). For example, hypothalamic NPY systems are not essential for the mediation of exogenous NPY-induced feeding, as NPY-related lesions of the ARC, paraventricular nucleus, or basal hypothalamus fail to prevent either hyperphagic responses to ICV NPY (2,17,52,54) or the attainment of normal levels of food intake, body weight, and adiposity in adulthood (13,38).…”