Hindbrain Catecholamine Neurons Research Articles

Repeated Pharmacogenetic Catecholamine Neuron Activation in the Ventrolateral Medulla Attenuates Subsequent Glucoregulatory Responses.

Hindbrain catecholamine (CA) neurons are essential for elicitation of protective counterregulatory responses (CRRs) to glucose deficit, including increased feeding and elevation of circulating corticosterone, epinephrine, and glucose. Severe or repeated antecedent glucoprivation results in attenuation of these CRRs and failure to correct glucose deficit, constituting a potentially lethal condition known as hypoglycemia-associated autonomic failure (HAAF) that may occur in patients with diabetes on insulin therapy. Recently, we demonstrated that selective pharmacogenetic activation of CA neuron subpopulations in the ventrolateral medulla during normoglycemia elicits these CRRs in a site-specific manner. In the present experiment, we examined the effect of repeated pharmacogenetic activation of CA neurons in the A1/C1 cell group on subsequent elicitation of feeding, corticosterone secretion, and respiratory quotient. We found that this prior treatment attenuated these responses to subsequent pharmacogenetic stimulation, similar to attenuation of these CRRs following repeated antecedent glucoprivation. This suggests that functional impairment of A1/C1 CA neurons resulting from antecedent glucoprivation may account, at least in part, for impairment of specific CRRs critical for restoration of normoglycemia in response to glucose deficit. Thus, a pharmacogenetic approach to selective activation of key neural circuits could provide a means of identifying neuropathogenic mechanisms contributing to HAAF.

Thrombin action on astrocytes in the hindbrain of the rat disrupts glycemic and respiratory control.

Severe trauma can produce a postinjury “metabolic self-destruction” characterized by catabolic metabolism and hyperglycemia. The severity of the hyperglycemia is highly correlated with posttrauma morbidity and mortality. Although no mechanism has been posited to connect severe trauma with a loss of autonomic control over metabolism, traumatic injury causes other failures of autonomic function, notably, gastric stasis and ulceration (“Cushing’s ulcer”), which has been connected with the generation of thrombin. Our previous studies established that proteinase-activated receptors (PAR1; “thrombin receptors”) located on astrocytes in the autonomically critical nucleus of the solitary tract (NST) can modulate gastric control circuit neurons to cause gastric stasis. Hindbrain astrocytes have also been implicated as important detectors of low glucose or glucose utilization. When activated, these astrocytes communicate with hindbrain catecholamine neurons that, in turn, trigger counterregulatory responses (CRR). There may be a convergence between the effects of thrombin to derange hindbrain gastrointestinal control and the hindbrain circuitry that initiates CRR to increase glycemia in reaction to critical hypoglycemia. Our results suggest that thrombin acts within the NST to increase glycemia through an astrocyte-dependent mechanism. Blockade of purinergic gliotransmission pathways interrupted the effect of thrombin to increase glycemia. Our studies also revealed that thrombin, acting in the NST, produced a rapid, dramatic, and potentially lethal suppression of respiratory rhythm that was also a function of purinergic gliotransmission. These results suggest that the critical connection between traumatic injury and a general collapse of autonomic regulation involves thrombin action on astrocytes.

Glucoprivic sensitivity of hindbrain catecholamine neurons is astrocyte‐dependent

Hypoglycemia poses a threat to survival. Endocrine, metabolic, and behavioral counter‐regulatory responses (CRRs) act to correct blood glucose deficit. These responses require sensor, integrator, and effector elements to restore glucose homeostasis. Groups of hindbrain catecholamine (CA) neurons are essential for implementation of different CRRs which include activation of descending sympathetic pathways controlling epinephrine and glucagon release and ascending pathways controlling feeding behavior and glucocorticoids (3, 4). Hindbrain astrocytes respond to low glucose (1, 2, 5). Therefore, we postulated that CA neurons are activated by astrocytes during glucose deficit. To directly test the proposition that CA neuron responses to glucoprivic challenges are astrocyte dependent, we utilized male and female transgenic mice in which the calcium reporter construct (GCaMP5) was expressed selectively in tyrosine hydroxylase neurons (TH‐GCaMP5; TH+). We conducted live‐cell calcium imaging studies on tissue slices containing the nucleus of the solitary tract (NST) and the ventrolateral medulla (VLM); loci of glucoregulatory circuitry (4). Results show that hindbrain TH+ neurons are robustly activated by a glucoprivic challenge and that this response is dependent on functional astrocytes. Pretreatment of hindbrain slices with fluorocitrate (an astrocytic metabolic suppressor) abolished TH+ neuronal responses to glucoprivation, but not to glutamate stimulation. Pharmacologic results suggest that the astrocytic connection with TH+ neurons is purinergic via P2 receptors. Parallel imaging studies on hindbrain slices of NST from wild type C57BL/6J mice (astrocytes and neurons were prelabeled with a calcium reporter dye and an astrocytic vital dye) show that both cell types are activated by glucoprivation but astrocytes responded nearly a minute sooner than neurons. Pretreatment of these hindbrain slices with fluorocitrate eliminated both astrocytic and neuronal responses to glucoprivation; pretreatment with P2 antagonists only abolished neuronal responses to glucoprivation without interruption of astrocyte responses. These results support earlier work suggesting that the primary detection of glucoprivic signals by the hindbrain is mediated by astrocytes which utilize purinergic gliotransmission to activate critical effector neurons.Support or Funding InformationNIH NIDDK 108765, U54 GM 104940, ADA‐1‐15‐JF‐37,This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Catecholaminergic projections into an interconnected forebrain network control the sensitivity of male rats to diet-induced obesity

Hindbrain catecholamine neurons convey gut-derived metabolic signals to an interconnected neuronal network in the hypothalamus and adjacent forebrain. These neurons are critical for short-term glycemic control, glucocorticoid and glucoprivic feeding responses, and glucagon-like peptide 1 (GLP-1) signaling. Here we investigate whether these pathways also contribute to long-term energy homeostasis by controlling obesogenic sensitivity to a high-fat/high-sucrose choice (HFSC) diet. We ablated hindbrain-originating catecholaminergic projections by injecting anti-dopamine-β-hydroxylase-conjugated saporin (DSAP) into the paraventricular nucleus of the hypothalamus (PVH) of male rats fed a chow diet for up to 12 wk or a HFSC diet for 8 wk. We measured the effects of DSAP lesions on food choices; visceral adiposity; plasma glucose, insulin, and leptin; and indicators of long-term ACTH and corticosterone secretion. We also determined lesion effects on the number of carbohydrate or fat calories required to increase visceral fat. Finally, we examined corticotropin-releasing hormone levels in the PVH and arcuate nucleus expression of neuropeptide Y ( Npy), agouti-related peptide ( Agrp), and proopiomelanocortin ( Pomc). DSAP-injected chow-fed rats slowly increase visceral adiposity but quickly develop mild insulin resistance and elevated blood glucose. DSAP-injected HFSC-fed rats, however, dramatically increase food intake, body weight, and visceral adiposity beyond the level in control HFSC-fed rats. These changes are concomitant with 1) a reduction in the number of carbohydrate calories required to generate visceral fat, 2) abnormal Npy, Agrp, and Pomc expression, and 3) aberrant control of insulin secretion and glucocorticoid negative feedback. Long-term metabolic adaptations to high-carbohydrate diets, therefore, require intact forebrain catecholamine projections. Without them, animals cannot alter forebrain mechanisms to restrain increased visceral adiposity.

Limiting glucocorticoid secretion increases the anorexigenic property of Exendin-4.

ObjectiveGlucagon-like peptide-1 (GLP-1) analogs are attractive options for the treatment of type II diabetes and obesity because of their incretin and anorexigenic effects. Peripheral administration of the GLP-1R agonist Exendin-4 (Ex-4) also increases glucocorticoid secretion in rodents and humans, but whether the released glucocorticoids interact with Ex-4's anorexigenic effect remains unclear.MethodsTo test this, we used two experimental approaches that suppress corticosterone secretion and then assessed Ex-4 effects on eating in adult male rats. First, we combined acute and chronic low dose dexamethasone treatment with Ex-4. Second, we ablated hindbrain catecholamine neurons projecting to the hypothalamus with anti-dopamine-β-hydroxylase-saporin (DSAP) to block Ex-4-induced corticosterone secretion.ResultsCombining dexamethasone and Ex-4 produced a larger acute anorexigenic effect than Ex-4 alone. Likewise, chronic dexamethasone and Ex-4 co-treatment produced a synergistic effect on eating and greater body weight loss in diet-induced obese rats than Ex-4 alone. DSAP lesions not only blunted Ex-4's ability to increase corticosterone secretion, but potentiated the anorexigenic effect of Ex-4, indicating that Ex-4-dependent corticosterone secretion opposes Ex-4's actions. Consistent with the enhancement of Ex-4's anorexigenic effect, DSAP lesion altered Ex-4-dependent changes in neuropeptide Y, preproglucagon, and corticotropin releasing hormone gene expression involved in glucocorticoid feedback.ConclusionsOur findings demonstrate that limiting glucocorticoid secretion and actions with low dose dexamethasone or DSAP lesion increases Ex-4's ability to reduce food intake and body weight. Novel glucocorticoid receptor based mechanisms, therefore, may help enhance GLP-1-based obesity therapies.

Orexin-A enhances feeding in male rats by activating hindbrain catecholamine neurons.

Both lateral hypothalamic orexinergic neurons and hindbrain catecholaminergic neurons contribute to control of feeding behavior. Orexin fibers and terminals are present in close proximity to hindbrain catecholaminergic neurons, and fourth ventricular (4V) orexin injections that increase food intake also increase c-Fos expression in hindbrain catecholamine neurons, suggesting that orexin neurons may stimulate feeding by activating catecholamine neurons. Here we examine that hypothesis in more detail. We found that 4V injection of orexin-A (0.5 nmol/rat) produced widespread activation of c-Fos in hindbrain catecholamine cell groups. In the A1 and C1 cell groups in the ventrolateral medulla, where most c-Fos-positive neurons were also dopamine β hydroxylase (DBH) positive, direct injections of a lower dose (67 pmol/200 nl) of orexin-A also increased food intake in intact rats. Then, with the use of the retrogradely transported immunotoxin, anti-DBH conjugated to saporin (DSAP), which targets and destroys DBH-expressing catecholamine neurons, we examined the hypothesis that catecholamine neurons are required for orexin-induced feeding. Rats given paraventricular hypothalamic injections of DSAP, or unconjugated saporin (SAP) as control, were implanted with 4V or lateral ventricular (LV) cannulas and tested for feeding in response to ventricular injection of orexin-A (0.5 nmol/rat). Both LV and 4V orexin-A stimulated feeding in SAP controls, but DSAP abolished these responses. These results reveal for the first time that catecholamine neurons are required for feeding induced by injection of orexin-A into either LV or 4V.

Hindbrain Catecholamine Neurons Activate Orexin Neurons During Systemic Glucoprivation in Male Rats.

Hindbrain catecholamine neurons are required for elicitation of feeding responses to glucose deficit, but the forebrain circuitry required for these responses is incompletely understood. Here we examined interactions of catecholamine and orexin neurons in eliciting glucoprivic feeding. Orexin neurons, located in the perifornical lateral hypothalamus (PeFLH), are heavily innervated by hindbrain catecholamine neurons, stimulate food intake, and increase arousal and behavioral activation. Orexin neurons may therefore contribute importantly to appetitive responses, such as food seeking, during glucoprivation. Retrograde tracing results showed that nearly all innervation of the PeFLH from the hindbrain originated from catecholamine neurons and some raphe nuclei. Results also suggested that many catecholamine neurons project collaterally to the PeFLH and paraventricular hypothalamic nucleus. Systemic administration of the antiglycolytic agent, 2-deoxy-D-glucose, increased food intake and c-Fos expression in orexin neurons. Both responses were eliminated by a lesion of catecholamine neurons innervating orexin neurons using the retrogradely transported immunotoxin, anti-dopamine-β-hydroxylase saporin, which is specifically internalized by dopamine-β-hydroxylase-expressing catecholamine neurons. Using designer receptors exclusively activated by designer drugs in transgenic rats expressing Cre recombinase under the control of tyrosine hydroxylase promoter, catecholamine neurons in cell groups A1 and C1 of the ventrolateral medulla were activated selectively by peripheral injection of clozapine-N-oxide. Clozapine-N-oxide injection increased food intake and c-Fos expression in PeFLH orexin neurons as well as in paraventricular hypothalamic nucleus neurons. In summary, catecholamine neurons are required for the activation of orexin neurons during glucoprivation. Activation of orexin neurons may contribute to appetitive responses required for glucoprivic feeding.

The rate of fall of blood glucose determines the necessity of forebrain-projecting catecholaminergic neurons for male rat sympathoadrenal responses.

Different onset rates of insulin-induced hypoglycemia use distinct glucosensors to activate sympathoadrenal counterregulatory responses (CRRs). Glucosensory elements in the portal-mesenteric veins are dispensable with faster rates when brain elements predominate, but are essential for responses to the slower-onset hypoglycemia that is common with insulin therapy. Whether a similar rate-associated divergence exists within more expansive brain networks is unknown. Hindbrain catecholamine neurons distribute glycemia-related information throughout the forebrain. We tested in male rats whether catecholaminergic neurons that project to the medial and ventromedial hypothalamus are required for sympathoadrenal CRRs to rapid- and slow-onset hypoglycemia and whether these neurons are differentially engaged as onset rates change. Using a catecholamine-specific neurotoxin and hyperinsulinemic-hypoglycemic clamps, we found that sympathoadrenal CRRs to slow- but not rapid-onset hypoglycemia require hypothalamus-projecting catecholaminergic neurons, the majority of which originate in the ventrolateral medulla. As determined with Fos, these neurons are differentially activated by the two onset rates. We conclude that 1) catecholaminergic projections to the hypothalamus provide essential information for activating sympathoadrenal CRRs to slow- but not rapid-onset hypoglycemia, 2) hypoglycemia onset rates have a major impact on the hypothalamic mechanisms that enable sympathoadrenal CRRs, and 3) hypoglycemia-related sensory information activates hindbrain catecholaminergic neurons in a rate-dependent manner.

Stimulation of feeding by three different glucose-sensing mechanisms requires hindbrain catecholamine neurons

Previous work has shown that hindbrain catecholamine neurons are required components of the brain's glucoregulatory circuitry. However, the mechanisms and circuitry underlying their glucoregulatory functions are poorly understood. Here we examined three drugs, glucosamine (GcA), phloridzin (Phl) and 5-thio-d-glucose (5TG), that stimulate food intake but interfere in different ways with cellular glucose utilization or transport. We examined feeding and blood glucose responses to each drug in male rats previously injected into the hypothalamic paraventricular nucleus with anti-dopamine-β-hydroxylase conjugated to saporin (DSAP), a retrogradely transported immunotoxin that selectively lesions noradrenergic and adrenergic neurons, or with unconjugated saporin (SAP) control. Our major findings were 1) that GcA, Phl, and 5TG all stimulated feeding in SAP controls whether injected into the lateral or fourth ventricle (LV or 4V), 2) that each drug's potency was similar for both LV and 4V injections, 3) that neither LV or 4V injection of these drugs evoked feeding in DSAP-lesioned rats, and 4) that only 5TG, which blocks glycolysis, stimulated a blood glucose response. The antagonist of the MEK/ERK signaling cascade, U0126, attenuated GcA-induced feeding, but not Phl- or 5TG-induced feeding. Thus GcA, Phl, and 5TG, although differing in mechanism and possibly activating different neural populations, stimulate feeding in a catecholamine-dependent manner. Although results do not exclude the possibility that catecholamine neurons possess glucose-sensing mechanisms responsive to all of these agents, currently available evidence favors the possibility that the feeding effects result from convergent neural circuits in which catecholamine neurons are a required component.

Hindbrain Catecholamine Neurons Control Rapid Switching of Metabolic Substrate Use during Glucoprivation in Male Rats

Using the retrogradely transported immunotoxin, antidopamine β-hydroxylase-saporin (DSAP), we showed previously that hindbrain catecholamine neurons innervating corticotropin-releasing hormone neurons in the paraventricular nucleus of the hypothalamus are required for glucoprivation-induced corticosterone secretion. Here, we examine the metabolic consequences of the DSAP lesion in male rats using indirect calorimetry. Rats injected into the paraventricular nucleus of the hypothalamus with DSAP or saporin (SAP) control did not differ in energy expenditure or locomotor activity under any test condition. However, DSAP rats had a persistently higher respiratory exchange ratio (RER) than SAPs under basal conditions. Systemic 2-deoxy-D-glucose did not alter RER in DSAP rats but rapidly decreased RER in SAP controls, indicating that this DSAP lesion impairs the ability to switch rapidly from carbohydrate to fat metabolism in response to glucoprivic challenge. In SAP controls, 2-deoxy-D-glucose-induced decrease in RER was abolished by adrenalectomy but not adrenal denervation. Furthermore, dexamethasone, a synthetic glucocorticoid, decreased RER in both SAP and DSAP rats. Thus, rapid switching of metabolic substrate use during glucoprivation appears to be due to impairment of the catecholamine-mediated increase in corticosterone secretion. Sustained elevation of basal RER in DSAP rats indicates that catecholamine neurons also influence metabolic functions that conserve glucose under basal conditions.

2. Hypoglycemia Detection.

Insulin-induced hypoglycemia is a life-threatening iatrogenic phenomenon that activates multiple neural and humoral corrective systems. When blood glucose levels begin to fall below 80 mg/dL, a sequential series of events occurs. Insulin secretion ceases; secretion of glucagon, epinephrine (E), corticosterone, and growth hormone is initiated, and neurogenic systems trigger food intake. All of these responses are recruited when glucose decreases to approximately 60 mg/dL. Together, these corrective responses (counter-regulatory responses or CRR) rapidly restore euglycemia. Unfortunately, however, the magnitude of the CRR decreases with recurring hypoglycemic episodes, leading to a syndrome known as hypoglycemia-associated autonomic failure (HAAF). During HAAF, glucose levels can fall dangerously low without eliciting the behavioral signs that usually accompany hypoglycemia. HAAF is the major limiting factor of the intensive insulin therapy needed to control the deleterious effects of diabetic hyperglycemia (1, 2). In order to understand and develop therapies for HAAF, it is critical to understand the mechanisms underlying hypoglycemia detection and initiation of the CRR. For many decades, it has been clear that there are glucose sensors located throughout the body and the central nervous system (CNS) (3–8). The goal of this chapter is to describe glucose sensing by central and peripheral glucose sensors and evidence that supports their roles in hypoglycemia detection and HAAF. The authors will focus on 3 glucose-sensing systems that contribute importantly to glucose counterregulation: the ventrome-dial hypothalamic nucleus (VMN) (9, 10), the hindbrain catecholamine neurons (11), and the portal-mesenteric vein (PMV) (12).

Participation of Hindbrain AMP-Activated Protein Kinase in Glucoprivic Feeding

OBJECTIVETo examine the role of AMP-activated protein kinase (AMPK) in the control of glucoprivic feeding by hindbrain catecholamine neurons.RESEARCH DESIGN AND METHODSMicropunched hindbrain samples were collected from control and 2-deoxy-d-glucose (2DG)-injected rats for Western blot analysis of phosphorylated (activated) AMPK (pAMPK). Samples also were collected from 2DG-injected rats pretreated with anti-dopamine-β-hydroxylase conjugated to saporin to lesion hindbrain catecholamine neurons. In a second experiment, rats were given a fourth-ventricle injection of compound C (CC) or 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR), an inhibitor and activator of AMPK, to identify a role for AMPK in hindbrain neurons required for elicitation of 2DG-induced feeding.RESULTSSystemic 2DG stimulated food intake in controls but not in catecholamine-lesioned rats. In controls, but not catecholamine-lesioned rats, 2DG also increased phosphorylated Thr172 at AMPKα subunits (pAMPKα) in hindbrain micropunches containing catecholaminergic cell groups A1 through the middle region of C1 (A1–C1m). Increased pAMPKα was not observed in the adjacent noncatecholaminergic ventromedial medulla or in the A2–C2 catecholamine cell groups in the dorsal hindbrain. Fourth-ventricle injection of CC attenuated 2DG-induced feeding during the first 2 h of the test, and AICAR alone increased food intake only during the first 60 min of the 4-h test.CONCLUSIONSResults indicate that AMPK in catecholaminergic A1–C1m neurons is activated by glucoprivation. Therefore, AMPK may contribute to the glucose-sensing mechanism by which these neurons detect and signal a glucose deficit in the service of systemic glucoregulation.

Hindbrain Catecholamine Neurons Modulate the Growth Hormone But Not the Feeding Response to Ghrelin

The gastrointestinal peptide, ghrelin, elicits feeding and secretion when administered systemically or centrally. Previous studies have suggested that hypothalamic projections of hindbrain catecholamine neurons are involved in both of these actions of ghrelin. The purpose of this study was to further assess the role of hindbrain catecholamine neurons in ghrelin-induced feeding and GH secretion and to determine the anatomical distribution of the catecholamine neurons involved. We lesioned noradrenergic and adrenergic neurons that innervate the medial hypothalamus by microinjecting the retrogradely transported immunotoxin, saporin (SAP) conjugated to antidopamine-beta-hydroxylase (DSAP) into the paraventricular nucleus of the hypothalamus. Controls were injected with unconjugated SAP. We found that the DSAP lesion did not impair the feeding response to central or peripheral ghrelin administration, indicating that these neurons are not required for ghrelin's orexigenic effect. However, the GH response to ghrelin was prolonged significantly in DSAP-lesioned rats. We also found that expression of Fos, an indicator of neuronal activation, was significantly enhanced over baseline levels in A1, A1/C1, C1, and A5 cell groups after ghrelin treatment and in A1, A1/C1, and A5 cell groups after GH treatment. The similar pattern of Fos expression in catecholamine cell groups after GH and ghrelin and the prolonged GH secretion in response to ghrelin in DSAP rats together suggest that activation of hindbrain catecholamine neurons by ghrelin or GH could be a component of a negative feedback response controlling GH levels.

Hindbrain catecholamine neurons contribute to the growth hormone but not the feeding response to ghrelin

Hindbrain catecholamine neurons contribute to the growth hormone but not the feeding response to ghrelin

Hindbrain catecholamine neurons contribute to control of daily food intake during chronic leptin treatment.

Chronic leptin treatment sufficient to cause profound reduction of food intake and body weight does not suppress glucoprivic feeding, a control of feeding that requires hindbrain catecholamine neurons. Therefore, we speculated that hindbrain catecholamine neurons may function during leptin treatment to avert brain glucose deficit. If so, elimination of these neurons would potentiate reduction of food intake during chronic leptin treatment. To test this hypothesis, we microinjected the immunotoxin, anti-dopamine beta-hydroxylase saporin (DSAP) or IgG-saporin control (SAP), into the PVH to retrogradely destroy catecholamine neurons with projections to the hypothalamus. This DSAP lesion selectively abolishes the glucoprivic control of feeding, but does not cause anorexia or loss of body weight during ad libitum access to food. Beginning 2 weeks after injections, we administered leptin into the lateral cerebroventricle daily and measured food (standard chow) intake, body weight and plasma metabolic fuels. We found that leptin treatment caused clear changes in plasma NEFA, glycerol and triglyceride levels, but these did not differ between DSAP and SAP rats. However, DSAP lesions significantly reduced food intake during leptin treatment and potentiated leptin-induced weight loss. DSAP-lesioned rats also had lower plasma glucose levels than SAP rats beginning on day 8 of leptin treatment. Results reveal a contribution of hindbrain catecholamine neurons to control of feeding and energy homeostasis during chronic leptin. Catecholamine neurons and/or the glucoprivic control may play a more prominent role in control of daily food intake in the absence of body fat stores.

Hindbrain catecholamine neurons control multiple glucoregulatory responses

Reduced brain glucose availability evokes an integrated constellation of responses that protect and restore the brain's 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.

Hypotensive hypovolemia and hypoglycemia activate different hindbrain catecholamine neurons with projections to the hypothalamus

To better understand the involvement of hindbrain catecholamine neurons in hypovolemia-induced secretion of AVP, we injected antidopamine beta-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.

Hindbrain catecholamine neurons mediate consummatory responses to glucoprivation

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.

Immunotoxic destruction of distinct catecholaminergic neuron populations disrupts the reproductive response to glucoprivation in female rats.

We tested the hypothesis that hindbrain catecholamine (norepinephrine or epinephrine) neurons, in addition to their essential role in glucoprivic feeding, are responsible for suppressing estrous cycles during chronic glucoprivation. Normally cycling female rats were given bilateral injections of the retrogradely transported ribosomal toxin, saporin, conjugated to monoclonal dopamine beta-hydroxylase antibody (DSAP) into the paraventricular nucleus (PVN) of the hypothalamus to selectively destroy norepinephrine and epinephrine neurons projecting to the PVN. Controls were injected with unconjugated saporin. After recovery, we assessed the lesion effects on estrous cyclicity under basal conditions and found that DSAP did not alter estrous cycle length. Subsequently, we examined effects of chronic 2-deoxy-d-glucose-induced glucoprivation on cycle length. After two normal 4- to 5-d cycles, rats were injected with 2-deoxy-d-glucose (200 mg/kg every 6 h for 72 h) beginning 24 h after detection of estrus. Chronic glucoprivation increased cycle length in seven of eight unconjugated saporin rats but in only one of eight DSAP rats. Immunohistochemical results confirmed loss of dopamine beta-hydroxylase immunoreactivity in PVN. Thus, hindbrain catecholamine neurons with projections to the PVN are required for inhibition of reproductive function during chronic glucose deficit but are not required for normal estrous cyclicity when metabolic fuels are in abundance.

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

The toxin-antibody complex anti-d(beta)h-saporin (DSAP) selectively destroys d(beta)h-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 d(beta)h 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.