Thu T. Dinh

Immunotoxic destruction of distinct catecholamine subgroups produces selective impairment of glucoregulatory responses and neuronal activation.

The toxin‐antibody complex anti‐dβh‐saporin (DSAP) selectively destroys dβh‐containing catecholamine neurons. To test the role of specific catecholamine neurons in glucoregulatory feeding and adrenal medullary secretion, we injected DSAP, unconjugated saporin (SAP), or saline bilaterally into the paraventricular nucleus of the hypothalamus (PVH) or spinal cord (T2–T4) and subsequently tested rats for 2‐deoxy‐D‐glucose (2DG)‐induced feeding and blood glucose responses. Injections of DSAP into the PVH abolished 2DG‐induced feeding, but not hyperglycemia. 2DG‐induced Fos expression was profoundly reduced or abolished in the PVH, but not in the adrenal medulla. The PVH DSAP injections caused a nearly complete loss of tyrosine hydroxylase immunoreactive (TH‐ir) neurons in the area of A1/C1 overlap and severe reduction of A2, C2, C3 (primarily the periventricular portion), and A6 cell groups. Spinal cord DSAP blocked 2DG‐induced hyperglycemia but not feeding. 2DG‐induced Fos‐ir was abolished in the adrenal medulla but not in the PVH. Spinal cord DSAP caused a nearly complete loss of TH‐ir in cell groups A5, A7, subcoeruleus, and retrofacial C1 and a partial destruction of C3 (primarily the ventral portion) and A6. Saline and SAP control injections did not cause deficits in 2DG‐induced feeding, hyperglycemia, or Fos expression and did not damage catecholamine neurons. DSAP eliminated dβh immunoreactivity but did not cause significant nonspecific damage at injection sites. The results demonstrate that hindbrain catecholamine neurons are essential components of the circuitry for glucoprivic control of feeding and adrenal medullary secretion and indicate that these responses are mediated by different subpopulations of catecholamine neurons. J. Comp. Neurol. 432:197–216, 2001.

Localization of hindbrain glucoreceptive sites controlling food intake and blood glucose.

Feeding and blood glucose responses to local injection of nanoliter volumes of 5-thio-D-glucose (5TG), a potent antimetabolic glucose analogue, were studied at 142 hindbrain and 61 hypothalamic cannula sites. A site was considered positive if 5TG elicited at least 1.5 g more food intake or a hyperglycemic response at least 25 mg/dl greater than the respective responses elicited by vehicle injection in the same rat. Of 61 hypothalamic cannula sites tested, none were positive for blood glucose and only one was positive for feeding. Increasing the 5TG dose to 48 ug did not produce additional positive results at hypothalamic sites. In contrast, 66 hindbrain sites were positive for feeding and 49 were positive for blood glucose, with 33 of these being positive for both responses. The distribution of positive sites for feeding and hyperglycemia overlapped almost completely. Positive sites were concentrated in two distinct zones: one in the ventrolateral and one in the dorsomedial medulla. In both locations, the glucoreceptive areas extended approximately from the level of the area postrema (AP) to the pontomedullary junction. Glucoreceptive zones were co-distributed with epinephrine cell groups C1-C3, suggesting that epinephrine neurons may be important components of the neural circuitry for glucoregulation. Localization of glucoreceptive sites will facilitate positive identification of glucoreceptor cells and the direct analysis of the neural mechanisms through which they influence food intake and metabolic responses.

Subgroups of hindbrain catecholamine neurons are selectively activated by 2-deoxy-d-glucose induced metabolic challenge

Glucose is a major fuel for body energy metabolism and an essential metabolic fuel for the brain. Consequently, glucose deficit (glucoprivation) elicits a variety of physiological and behavioral responses crucial for survival. Previous work indicates an important role for brain catecholamine neurons in mediation of responses to glucoprivation. This experiment was conducted to identify the specific catecholamine neurons that are activated by glucoprivation. Activation of hindbrain catecholamine neurons by the antimetabolic glucose analogue, 2-deoxy-D-glucose (2DG; 50, 100, 200 or 400 mg/kg, s.c.) was evaluated using double label immunohistochemistry. Fos protein was used as the marker for neuronal activation and the enzymes tyrosine hydroxylase (TH) and phenethanolamine-N-methyl transferase (PNMT) were used as the markers for norepinephrine (NE) and epinephrine (E) neurons. 2-Deoxy-D-glucose (200 and 400 mg/kg) produced selective activation of distinct hindbrain catecholamine cell groups. In the ventrolateral medulla, doubly labeled neurons were concentrated in the area of A1/C1 and were predominantly adrenergic in phenotype. In the dorsal medulla, doubly labeled neurons were limited to C2 and C3 cell groups. In the pons, some A6 neurons were Fos-positive. Neurons in rostral C1, ventral C3, A2, A5 and A7 did not express Fos-ir in response to 2DG. Our results identify specific subpopulations of catecholamine neurons that are selectively activated by 2DG. Previously demonstrated connections of these subpopulations are consistent with their participation in the feeding and hyperglycemic response to glucoprivation. Finally, the predominant and seemingly preferential activation of epinephrine neurons suggests that they may play a unique role in the brains response to glucose deficit.

2-Mercaptoacetate and 2-deoxy-D-glucose induce Fos-like immunoreactivity in rat brain.

2-Deoxy-D-glucose (2-DG) and 2-mercaptoacetate (MA) are antimetabolic drugs that selectively antagonize glucose and fatty acid utilization, respectively, and stimulate feeding. Fos immunohistochemistry was employed to identify brain neurons activated by these drugs and to assess the role of the vagus nerve in the drug effects. Remote intravenous infusions of both MA and 2-DG induced Fos-like immunoreactivity (Fos-li) in specific brain sites, but the pattern was different for the two drugs. Mercaptoacetate induced Fos-li in the nucleus of the solitary tract (NTS), the central subnucleus of the lateral parabrachial nucleus (1PBN), the central nucleus of the amygdala (CNA, lateral part) and the dorsal motor nucleus of the vagus (DMV). Induction of Fos-li in the brain by MA was totally abolished by vagotomy. 2-Deoxy-D-glucose also induced Fos-li in the NTS, CNA (lateral part) and DMV, as well as in the external 1PBN subnucleus, locus coeruleus, paraventricular and supraoptic hypothalamic nuclei, and in scattered cells throughout the diencephalon. Induction of Fos-li by 2-DG was not blocked by vagotomy. Results suggest that 2-DGs effects on Fos-li are mediated by a direct central action, whereas MAs effects are mediated by peripheral sensory neurons. Thus, availability of glucose and fatty acids influences the activity of specific brain sites by different neural mechanisms. The correlation of Fos-immunoreactive sites with sites where lesions have been shown to cause deficits in MA- and 2-DG-induced feeding indicates that c-fos expression defines in part the central pathways involved in the metabolic control of feeding.(ABSTRACT TRUNCATED AT 250 WORDS)

Hindbrain catecholamine neurons control multiple glucoregulatory responses.

Reduced brain glucose availability evokes an integrated constellation of responses that protect and restore the brains glucose supply. These include increased food intake, adrenal medullary secretion, corticosterone secretion and suppression of estrous cycles. Our research has focused on mechanisms and neural circuitry underlying these systemic glucoregulatory responses. Using microinjection techniques, we found that localized glucoprivation of hindbrain but not hypothalamic sites, elicited key glucoregulatory responses, indicating that glucoreceptor cells controlling these responses are located in the hindbrain. Selective destruction of hindbrain catecholamine neurons using the retrogradely transported immunotoxin, anti-dopamine beta-hydroxylase conjugated to saporin (DSAP), revealed that spinally-projecting epinephrine (E) or norepinephrine (NE) neurons are required for the adrenal medullary response to glucoprivation, while E/NE neurons with hypothalamic projections are required for feeding, corticosterone and reproductive responses. We also found that E/NE neurons are required for both consummatory and appetitive phases of glucoprivic feeding, suggesting that multilevel collateral projections of these neurons coordinate various components of the behavioral response. Epinephrine or NE neurons co-expressing neuropeptide Y (NPY) may be the neuronal phenotype required for glucoprivic feeding: they increase NPY mRNA expression in response to glucoprivation and are nearly eliminated by DSAP injections that abolish glucoprivic feeding. In contrast, lesion of arcuate nucleus NPY neurons, using the toxin, NPY-saporin, does not impair glucoprivic feeding or hyperglycemic responses. Thus, hindbrain E/NE neurons orchestrate multiple concurrent glucoregulatory responses. Specific catecholamine phenotypes may mediate the individual components of the overall response. Glucoreceptive control of these neurons resides within the hindbrain.

Induction of Fos-like immunoreactivity (Fos-li) and stimulation of feeding by 2, 5-anhydro-d-mannitol (2, 5-AM) require the vagus nerve

The antimetabolic fructose analogue, 2,5-anhydro-D-mannitol (2,5-AM), stimulates feeding. Selective hepatic branch vagotomy has been shown to block feeding induced by low 2,5-AM doses. However, hepatic vagal fibers are not the sole mediators of 2,5-AM-induced feeding, since hepatic branch vagotomy does not impair feeding induced by higher doses of 2,5-AM. To further evaluate the role of the vagus in the response to 2,5-AM, we examined the effect of total subdiaphragmatic vagotomy on feeding induced by a high 2,5-AM dose (500 mg/kg). In addition, we assessed the ability of 2,5-AM (300 and 500 mg/kg) to induce Fos-like immunoreactivity (Fos-li) in the brain in sham-operated (SHAM), hepatic branch vagotomized (HBV) and total subdiaphragmatic vagotomized (TSDV) rats. Both doses of 2,5-AM, but not control solutions, induced Fos-li in the area postrema (AP), nucleus of the solitary tract (NTS) and lateral parabrachial nucleus (1PBN). Very weak immunoreactivity was present in the central nucleus of the amygdala and none was observed in the locus coeruleus or paraventricular nucleus of the hypothalamus. The effect of the lower 2,5-AM dose on Fos-li was blocked by HBV. The high dose effect was blocked by TSDV but not by HBV. Feeding induced by the high dose of 2,5-AM was also blocked by TSDV. Results are consistent with the hypothesis that stimulation of feeding by 2,5-AM is dependent on the vagus nerve. Hepatic branch fibers may have the lowest threshold for activation, but fibers in other vagal branches independently mediate induction of c-fos and stimulate food intake at higher doses of the analogue.

Progressive postnatal dilation of brain ventricles in spontaneously hypertensive rats.

Cross-sectional areas of the forebrain ventricles were measured from coronal sections in spontaneously hypertensive rats (SHRs) 4, 8, 12, 16, 21 and 56 weeks of age and in age-matched Wistar--Kyoto (WKY) and Sprague--Dawley (SD) normotensive rats. Progressive ventricular dilation and associated attrition of brain tissue was observed in SHRs of both sexes after 4 weeks of age, and was present in animals obtained from two different suppliers. In some SHRs, ventricle size was increased to 270% of control. Hence, it seems likely that some systemic and behavioral signs which are concomitant with hypertension in the SHR may be attributable to hydrocephalus and its neuropathological correlates.

Minireview: The Value of Looking Backward: The Essential Role of the Hindbrain in Counterregulatory Responses to Glucose Deficit

This review focuses on evidence indicating a key role for the hindbrain in mobilizing behavioral, autonomic and endocrine counterregulatory responses to acute and profound glucose deficit, and identifies hindbrain norepinephrine (NE) and epinephrine (E) neurons as essential mediators of some of these responses. It has become clear that hindbrain NE/E neurons are functionally diverse. However, considerable progress has been made in identifying the particular NE/E neurons important for particular glucoregulatory responses. Although it is not yet known whether NE/E neurons are directly activated by glucose deficit, compelling evidence indicates that if they are not, the primary glucoreceptor cells must be located in the immediate vicinity these neurons. Hindbrain studies identifying cellular markers associated with glucose-sensing functions in other brain regions are discussed, as are studies examining the relationship of these markers to counterregulatory responses of NE/E neurons. Further investigations to identify glucose-sensing cells (neurons, ependymocytes, or glia) controlling counterregulatory responses are crucial, as are studies to determine the specific functions of glucose-sensing cells throughout the brain. Likewise, examination of the roles (if any) of hindbrain counterregulatory systems in managing glucose homeostasis under basal, nonglucoprivic conditions will also be important for a full understanding of energy homeostasis. Nevertheless, the accumulated evidence demonstrates that hindbrain glucose sensors and NE/E neurons are essential players in triggering counterregulatory responses to emergencies of glucose deficit.

Simultaneous Silencing of Npy and Dbh Expression in Hindbrain A1/C1 Catecholamine Cells Suppresses Glucoprivic Feeding

Previous data have strongly implicated hindbrain catecholamine/neuropeptide Y (NPY) coexpressing neurons as key mediators of the glucoprivic feeding response. Catecholamine/NPY cell bodies are concentrated in the A1 and caudal C1 cell cluster (A1/C1) in the ventrolateral medulla, a region highly sensitive to glucoprivic challenge. To further investigate the importance of this catecholamine subpopulation in glucoregulation, we used small interfering RNA (siRNA) technology to produce a targeted gene knockdown of NPY and dopamine-β-hydroxylase (DBH), a catecholamine biosynthetic enzyme. Unilateral injection of NPY siRNA and DBH siRNA (0.02 nmol each) both significantly inhibited expression of the targeted genes up to 2 d, as revealed by real-time PCR, and reduced protein expression up to 8 d, as revealed by immunohistochemistry, compared with the control nontargeting siRNA (ntRNA) side. Subsequently, targeted siRNA or control ntRNA was injected bilaterally into A1/C1 and responses to 2-deoxy-d-glucose (2DG; 200 mg/kg)-induced glucoprivation were tested 3–7 d later. Silencing of either Npy or Dbh alone did not reduce glucoprivic feeding or hyperglycemic responses, compared with responses of ntRNA-injected controls. In contrast, simultaneous silencing of both Npy and Dbh reduced 2DG-induced feeding by 61%. Neither the hyperglycemic response to 2DG nor feeding elicited by mercaptoacetate (68 mg/kg)-induced blockade of fatty acid oxidation (“lipoprivic feeding”) was reduced by simultaneous silencing of these two genes. These results suggest that catecholamines and NPY act conjointly to control glucoprivic feeding and that the crucial NPY/catecholamine coexpressing neurons are concentrated in the A1/C1 cell group.

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

Sleep and feeding rhythms are highly coordinated across the circadian cycle, but the brain sites responsible for this coordination are unknown. We examined the role of neuropeptide Y (NPY) receptor-expressing neurons in the mediobasal hypothalamus (MBH) in this process by injecting the targeted toxin, NPY-saporin (NPY-SAP), into the arcuate nucleus (Arc). NPY-SAP-lesioned rats were initially hyperphagic, became obese, exhibited sustained disruption of circadian feeding patterns, and had abnormal circadian distribution of sleep-wake patterns. Total amounts of rapid eye movement sleep (REMS) and non-REMS (NREMS) were not altered by NPY-SAP lesions, but a peak amount of REMS was permanently displaced to the dark period, and circadian variation in NREMS was eliminated. The phase reversal of REMS to the dark period by the lesion suggests that REMS timing is independently linked to the function of MBH NPY receptor-expressing neurons and is not dependent on NREMS pattern, which was altered but not phase reversed by the lesion. Sleep-wake patterns were altered in controls by restricting feeding to the light period, but were not altered in NPY-SAP rats by restricting feeding to either the light or dark period, indicating that disturbed sleep-wake patterns in lesioned rats were not secondary to changes in food intake. Sleep abnormalities persisted even after hyperphagia abated during the static phase of the lesion. Results suggest that the MBH is required for the essential task of integrating sleep-wake and feeding rhythms, a function that allows animals to accommodate changeable patterns of food availability. NPY receptor-expressing neurons are key components of this integrative function.