Search

Feeding behavior is comprised of both appetitive and consummatory responses to food. Appetitive responses include the motivated acquisition of food. Consummatory responses, including swallowing, are those that move the food from the mouth to the stomach. Intraoral delivery of liquid food bypasses the requirement for appetitive responses and has been used to examine consummatory responses directly in intact rats. In the present study, we administered neuropeptide Y (NPY), agouti-related protein (AGRP) and norepinephrine (NE), into the paraventricular nucleus of the hypothalamus (PVN) or into the fourth cerebral ventricle to examine their effects on the consummatory component of feeding behavior in the rat. To measure consummatory responses, milk (40% lactose free cow's milk diluted with water) was infused intraorally through a chronic cheek fistula (1 ml/min), using an alternating 5 min on -1 min off schedule, until rejection occurred. We found that both hypothalamic and fourth ventricle injections of NPY, AGRP and NE significantly increased consumption of the intraorally-delivered milk. Our results indicate that the circuitry for modulation of consummatory ingestive responses includes NE, NPY and AGRP receptors operating in both hypothalamic and hindbrain sites.

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.

The hypothalamus is jointly innervated by hindbrain and hypothalamic neuropeptide Y (NPY) cell bodies. While the specific roles of these distinct sources of innervation are not known, NPY neurotransmission within the hypothalamus appears to contribute to glucoregulatory feeding. Here we examine the involvement of hindbrain NPY neurons in glucoregulation using in situ hybridization to assess their responsiveness to glucoprivation. The hindbrain NPY innervation of the hypothalamus is derived from cell bodies that coexpress norepinephrine or epinephrine. Therefore, we quantified NPY mRNA hybridization signal in hindbrain catecholamine cell groups 90 min after subcutaneous administration of the glycolytic inhibitor 2-deoxy-d-glucose (2DG, 250 mg/kg) to male rats. Catecholamine cell groups A1, A1/C1 and C2 (that provide the major NPY innervation of the hypothalamus) showed a basal level of NPY mRNA hybridization signal that was dramatically increased by 2DG. In C1 and C3, where basal NPY mRNA expression was close to or below our detection threshold, the hybridization signal was also significantly increased by 2DG. In cell groups A2, A5, A6 and A7, neither basal nor 2DG-stimulated NPY mRNA expression was detected. Hypothalamic microinjection of the retrogradely transported catecholamine immunotoxin saporin conjugated to anti-dopamine-beta-hydroxylase destroyed hindbrain catecholamine/NPY neurons and abolished basal and 2DG-stimulated increases in NPY expression in hindbrain cell groups. The responsiveness of hindbrain NPY neurons to glucose deficit suggests that these neurons participate in glucoprivic feeding or other glucoregulatory responses.

Leptin administration into the hindbrain, and specifically the nucleus of the solitary tract, increases phosphorylated signal transducer and activator of transcription 3 (pSTAT3), a marker of leptin receptor activation, in hypothalamic nuclei known to express leptin receptors. The ventromedial nucleus of the hypothalamus (VMH) shows the greatest response, with a threefold increase in pSTAT3. This experiment tested the importance of VMH leptin receptor-expressing neurons in mediating weight loss caused by fourth ventricle (4V) leptin infusion. Male Sprague-Dawley rats received bilateral VMH 75-nL injections of 260 ng/μL of leptin-conjugated saporin (Lep-Sap) or blank-saporin (Blk-Sap). After 23 days they were fitted with 4V infusion cannulas and 1 wk later adapted to housing in a calorimeter before they were infused with 0.9 μg leptin/day for 14 days. There was no effect of VMH Lep-Sap on weight gain or glucose clearance before leptin infusion. Leptin inhibited food intake and respiratory exchange ratio in Blk-Sap but not Lep-Sap rats. Leptin had no effect on energy expenditure or brown adipose tissue temperature of either group. Inguinal and epididymal fat were significantly reduced in leptin-treated Blk-Sap rats, but the response was greatly attenuated in Lep-Sap rats. VMH pSTAT3 was increased in leptin-treated Blk-Sap but not Lep-Sap rats. These results support the concept that leptininduced weight loss results from an integrated response across different brain areas. They also support previous reports that VMH leptin receptors do not play a significant role in maintaining energy balance in basal conditions but limit weight gain during positive energy balance. [ABSTRACT FROM AUTHOR]

Previously, we investigated the role of neuropeptide Y and leptin-sensitive networks in the mediobasal hypothalamus in sleep and feeding and found profound homeostatic and circadian deficits with an intact suprachiasmatic nucleus. We propose that the arcuate nuclei (Arc) are required for the integration of homeostatic circadian systems, including temperature and activity. We tested this hypothesis using saporin toxin conjugated to leptin (Lep-SAP) injected into Arc in rats. Lep-SAP rats became obese and hyperphagic and progressed through a dynamic phase to a static phase of growth. Circadian rhythms were examined over 49 days during the static phase. Rats were maintained on a 12:12-h light-dark (LD) schedule for 13 days and, thereafter, maintained in continuous dark (DD). After the first 13 days of DD, food was restricted to 4 h/day for 10 days. We found that the activity of Lep-SAP rats was arrhythmic in DD, but that food anticipatory activity was, nevertheless, entrainable to the restricted feeding schedule, and the entrained rhythm persisted during the subsequent 3-day fast in DD. Thus, for activity, the circuitry for the light-entrainable oscillator, but not for the food-entrainable oscillator, was disabled by the Arc lesion. In contrast, temperature remained rhythmic in DD in the Lep-SAP rats and did not entrain to restricted feeding. We conclude that the leptin-sensitive network that includes the Arc is required for entrainment of activity by photic cues and entrainment of temperature by food, but is not required for entrainment of activity by food or temperature by photic cues. [ABSTRACT FROM AUTHOR]

Neural sites that interact with the suprachiasmatic nuclei (SCN) to generate rhythms of unrestricted feeding remain unknown. We used the targeted toxin, leptin conjugated to saporin (Lep-SAP), to examine the importance of leptin receptor-B (LepR-B)-expressing neurons in the arcuate nucleus (Arc) for generation of circadian feeding rhythms. Rats given Arc Lep-SAP injections were initially hyperphagic and rapidly became obese (the "dynamic phase" of weight gain). During this phase, Lep-SAP rats were arrhythmic under 12:12-h light-dark (LD) conditions, consuming 59% of their total daily intake during the daytime, compared with 36% in blank-SAP (B-SAP) controls. Lep- SAP rats were also arrhythmic in continuous dark (DD), while significant circadian feeding rhythms were detected in all B-SAP controls. Approximately 8 wk after injection, Lep-SAP rats remained obese but transitioned into a "static phase" of weight gain marked by attenuation of their hyperphagia and rate of weight gain. In this phase, Arc Lep-SAP rats exhibited circadian feeding rhythms under LD conditions, but were arrhythmic in continuous light (LL) and DD. Lep-SAP injections into the ventromedial hypothalamic nucleus did not cause hyperphagia, obesity, or arrhythmic feeding in either LD or DD. Electrolytic lesion of the SCN produced feeding arrhythmia in DD but not hyperphagia or obesity. Results suggest that both Arc Lep-SAP neurons and SCN are required for generation of feeding rhythms entrained to photic cues, while also revealing an essential role for the Arc in maintaining circadian rhythms of ad libitum feeding independent of light entrainment. [ABSTRACT FROM AUTHOR]

Glucose is required for brain energy metabolism. Decerebration, aqueduct occlusion, and cannula mapping studies have established that glucose-sensing cells capable of eliciting feeding and adrenal medullary responses to glucoprivation are localized in the hindbrain. Glucoprivation also evokes corticosterone and glucagon secretion, but the location of receptors mediating these responses is unknown. To determine whether glucoreceptive sites controlling these responses are present in the hindbrain, we administered the antiglycolytic agent, 5-D-thioglucose (5TG, 24 µg in 200 nl) into brain stem sites through implanted cannulas and examined plasma concentrations of corticosterone and glucagon. Both hindbrain and hypothalamic sites were tested. Blood was collected remotely from intra-atrial catheters at 0, 30, 60, 90, 120, 180, and 240 min after 5TG or control injection. Caudal hindbrain 5TG injections potently increased circulating corticosterone and glucagon concentrations. For corticosterone, the mean peak response (maximum concentration minus time 0 concentration) elicited at positive sites (23 of 40 sites) was 391 ng/ml (SE = 16). For glucagon, the mean peak response at positive sites (27 of 40 sites) was 46 pg/mI (SE = 6). Glucoprivically evoked glucagon secretion was abolished by the ganglionic blocker, hexamethonium, but not by adrenal denervation. Six of twenty-five hypothalamic sites were positive for corticosterone secretion, yielding plasma levels of 279 ± 23 ng/mI, but none of the hypothalamic injection sites elevated glucagon concentrations. Results demonstrate that receptor cells responsive to glucose deficit and capable of increasing corticosterone and glucagon concentrations exist within the hindbrain, thus further delineating central glucoregulatory neural circuitry. [ABSTRACT FROM AUTHOR]

To better understand the involvement of hindbrain catecholamine neurons in hypovolemia-induced secretion of AVP, we injected antidopamine β-hydroxylase saporin (DSAP) or unconjugated saporin (SAP) control solution into the hypothalamic paraventricular nucleus (PVH) of anesthetized rats to retrogradely lesion catecholamine neurons innervating magnocellular areas of the hypothalamus. Subsequently, hypotensive hypovolemia was induced by remote blood withdrawal (4.5 ml, 1 ml/min) using an intra-atrial catheter. Blood was sampled at 2, 5, 20, and 50 min after onset of blood withdrawal. The AVP response was severely impaired by DSAP. Peak responses at 50 min were 51 pg/ml in SAP control and 17 pg/ml in DSAP-lesioned rats, indicating the importance of catecholamine neurons for this response. We also measured AVP responses to osmotic challenge induced by administration of hypertonic saline (1 M, 15 ml/kg, sc) and to insulin-induced hypoglycemia. Osmotic challenge increased AVP levels, but the response was not impaired by DSAP, indicating that AVP neurons were not damaged by the DSAP injection. Insulin-induced hypoglycemia did not increase AVP levels in either DSAP- or SAP-treated rats. However, the same dose of insulin increased food intake and corticosterone secretion in SAP controls, and these responses were profoundly impaired by DSAP. Thus catecholamine neurons are required for both the AVP response to hypotensive hypovolemia and for feeding and corticosterone responses to hypoglycemia. Lack of an AVP response to insulin-induced hypoglycemia in intact rats therefore indicates that responses to hypovolemia and hypoglycemia are mediated by different catecholamine neurons under distinct sensory controls. [ABSTRACT FROM AUTHOR]

Previous work using the retrogradely transported immunotoxin, saporin (SAP) conjugated to a monoclonal antibody against dopamine-β-hydroxylase (DBH; DSAP), to selectively lesion norepinephrine (NE) and epinephrine (E) neurons projecting to the medial hypothalamus, demonstrated the essential role of these neurons for appetitive ingestive responses to glucoprivation. Here, we again utilized this lesion to assess the importance of these same neurons for the consummatory phase of glucoprivic feeding. To test consummatory responses, milk was infused intraorally through a chronic cheek fistula until rejected. Appetitive responses were tested in the same rats using pelleted food. Feeding responses to insulin-induced hypoglycemia, 2-deoxy-d-glucose (2DG)-induced blockade of glucose utilization, mercaptoacetate (MA)-induced blockade of fatty acid oxidation, 0.9% saline, and 18-h food deprivation were assessed. Unlike unconjugated SAP controls, the DSAP rats did not increase their food intake in response to glucoprivic challenges in either the pelleted food or the intraoral feeding tests. However, the DSAP rats did not differ from SAPs in their ingestive responses to food deprivation and blockade of fatty acid oxidation. The selective impairment of glucoprivic feeding responses indicates that DSAP did not impair the underlying circuitry required for either appetitive or consummatory ingestive responding but eliminated the mechanism for control of this circuitry specifically by glucoprivation. Results suggest that both appetitive and consummatory responses to glucoprivation are controlled and coordinated by multilevel terminations of the same catecholamine neurons. [Copyright &y& Elsevier]

A condition of reduced responsiveness to hypoglycemia, known as hypoglycemia-associated autonomic failure (HAAF), occurs in diabetic patients in the wake of a prior hypoglycemic episode. This condition suggests that hypoglycemia alters central glucose-sensing mechanisms. This experiment examined the effects of repeated 2-deoxy-D-glucose (2DG)-induced glucoprivation on subsequent 2DG-induced feeding and hyperglycemic responses in rats. Fos immunoreactivity (ir) in adrenal medulla and brain sites involved in these responses was also examined. Rats were injected daily for 10 days with 2DG (200 mg/kg) or saline (0.9%) or were handled. On day 11, rats were injected with 2DG (200 mg/kg). After injection, food intake was measured in one group. In another group, food was withheld, and multiple blood samples were collected for glucose determination. In a third group, food was withheld, and rats were killed after 2 h for evaluation of Fos-ir. Prior repeated glucoprivation reduced subsequent feeding and hyperglycemia responses to 2DG to baseline levels. Double-label immunohistochemistry showed that Fos-ir was reduced or abolished in catecholamine cell groups A1, A1/C1, C1, C3, and A6 and in the paraventricular nucleus of the hypothalamus and adrenal medulla. In other brain sites, 2DG-induced Fos-ir was diminished or unaffected by prior glucoprivation. Sites in which Fos-ir was abolished have been implicated previously in glucoprivic control of feeding and adrenal medullary secretion. Therefore, the present findings may identify crucial neuroanatomical sites that are altered by prior glucoprivation and that mediate some of the physiological deficits observed in HAAF. [ABSTRACT FROM AUTHOR]