BDNF and AMPA receptors in the cNTS modulate the hyperglycemic reflex after local carotid body NaCN stimulation.

The application of sodium cyanide (NaCN) to the carotid body receptors (CBR) (CBR stimulation) induces rapid blood hyperglycemia and an increase in brain glucose retention. The commissural nucleus tractus solitarius (cNTS) is an essential relay nucleus in this hyperglycemic reflex; it receives glutamatergic afferents (that also release brain derived neurotrophic factor, BDNF) from the nodose-petrosal ganglia that relays CBR information. Previous work showed that AMPA in NTS blocks hyperglycemia and brain glucose retention after CBR stimulation. In contrast, BDNF, which attenuates glutamatergic AMPA currents in NTS, enhances these glycemic responses. Here we investigated the combined effects of BDNF and AMPA (and their antagonists) in NTS on the glycemic responses to CBR stimulation. Microinjections of BDNF plus AMPA into the cNTS before CBR stimulation in anesthetized rats, induced blood hyperglycemia and an increase in brain arteriovenous (a-v) of blood glucose concentration difference, which we infer is due to increased brain glucose retention. By contrast, the microinjection of the TrkB antagonist K252a plus AMPA abolished the glycemic responses to CBR stimulation similar to what is observed after AMPA pretreatments. In BDNF plus AMPA microinjections preceding CBR stimulation, the number of c-fos immunoreactive cNTS neurons increased. In contrast, in the rats microinjected with K252a plus AMPA in NTS, before CBR stimulation, c-fos expression in cNTS decreased. The expression of AMPA receptors GluR2/3 did not change in any of the studied groups. These results indicate that BDNF in cNTS plays a key role in the modulation of the hyperglycemic reflex initiated by CBR stimulation.

Glutamatergic Receptor Activation in the Commisural Nucleus Tractus Solitarii (cNTS) Mediates Brain Glucose Retention (BGR) Response to Anoxic Carotid Chemoreceptor (CChr) Stimulation in Rats.

Glutamate, released from central terminals of glossopharyngeal nerve, is a major excitatory neurotransmitter of commissural nucleus tractus solitarii (cNTS) afferent terminals, and brain derived neurotrophic factor (BDNF) has been shown to attenuate glutamatergic AMPA currents in NTS neurons. To test the hypothesis that AMPA contributes to glucose regulation in vivo modulating the hyperglycemic reflex with brain glucose retention (BGR), we microinjected AMPA and NBQX (AMPA antagonist) into the cNTS before carotid chemoreceptor stimulation in anesthetized normal Wistar rats, while hyperglycemic reflex an brain glucose retention (BGR) were analyzed. To investigate the underlying mechanisms, GluR2/3 receptor and c-Fos protein expressions in cNTS neurons were determined. We showed that AMPA in the cNTS before CChr stimulation inhibited BGR observed in aCSF group. In contrast, NBQX in similar conditions, did not modify the effects on glucose variables observed in aCSF control group. These experiments suggest that glutamatergic pathways, via AMPA receptors, in the cNTS may play a role in glucose homeostasis.

Nitric oxide in the hypothalamus-pituitary axis mediates increases in brain glucose retention induced by carotid chemoreceptor stimulation with cyanide in rats.

Neuronal nitric oxide synthase (nNOS), which catalyzes the generation of nitric oxide (NO), is expressed by neuron subpopulations in the CNS. Nitric oxide is involved in neurotransmission and central glucose homeostasis. Our prior studies have shown that carotid body receptors participate in brain glucose regulation in vivo, and suggest the presence of a NO tonic mechanism in the solitary tract nucleus (STn). However, the role of NO within STn in glucose control remains unknown. In this study, we explored the potential regulatory role of NO on brain glucose retention induced by carotid body chemoreceptor anoxic stimulation with sodium cyanide (NaCN) which inhibits oxidative metabolism. Intracisternal infusions of nitroxidergic drugs before carotid chemoreceptor stimulation in anesthetized rats, elicited changes in nitrite concentration in plasma and hypothalamus-pituitary (H-P) tissue, as well as in gene expression of neuronal and inducible isoforms (nNOS and iNOS) in H-P tissue. The changes observed in above variables modified brain glucose retention in an opposite direction. When the NO donor, sodium nitroprusside (SNP), was given before carotid stimulation, nitrite concentration in plasma and H-P tissue, and gene expression of nNOS and iNOS in H-P tissue increased, whereas brain glucose retention decreased. In contrast, when the NOS inhibitor, Nomega-nitro-L-arginine methyl ester (L-NAME) was infused immediately before carotid chemoreceptor stimulation, nitrite levels and nNOS expression decreased in plasma and H-P tissue, whereas brain glucose retention increased. Anoxic stimulation by itself induced an increase in the expression of both genes studied. All these results indicate that de novo expression of the nNOS gene in H-P tissue may be critically involved in central glucose changes observed after anoxic carotid chemoreceptor stimulation in conjunction with NO.

Nitric oxide in the solitary tract nucleus (STn) modulates glucose homeostasis and FOS-ir expression after carotid chemoreceptor stimulation.

We evaluate in rats the role of NO in the solitary tract nucleus (STn) after an anoxic stimulus to carotid body chemoreceptor cells (CChrc) with cyanide (NaCN), on the hyperglycemic reflex with glucose retention by the brain (BGR) and FOS expression (FOS-ir) in the STn. The results suggest that nitroxidergic pathways in the STn may play an important role in glucose homeostasis. A NO donor such as sodium nitroprusside (NPS) in the STn before CChrc stimulation increased arterial glucose level and significantly decreased BGR. NPS also induced a higher FOS-ir expression in STn neurons when compared to neurons in control rats that only received artificial cerebrospinal fluid (aCSF) before CChrc stimulation. In contrast, a selective NOS inhibitor such as Nomega-nitro-L-arginine methyl ester (L-NAME) in the STn before CChrc stimulation resulted in an increase of both, systemic glucose and BGR above control values. In this case, the number of FOS-ir positive neurons in the STn decreased when compared to control or to NPS experiments. FOS-ir expression in brainstem cells suggests that CChrc stimulation activates nitroxidergic pathways in the STn to regulate peripheral and central glucose homeostasis. The study of these functionally defined cells will be important to understand brain glucose homeostasis.

Arginine-vasopressin in nucleus of the tractus solitarius induces hyperglycemia and brain glucose retention.

Hypothalamic arginine-vasopressin (AVP) plays an important role both as a neurotransmitter and hormone in the regulation of blood glucose and feeding behavior. AVP-containing axons from the parvocellular subdivision of paraventricular nucleus of the hypothalamus terminate in the nucleus of the tractus solitarius (NTS), but the function of this projection is not known. Interestingly, the NTS also receives afferent information from the carotid body and other peripheral receptors involved in glucose homeostasis. We have previously reported that stimulation of the carotid body receptors initiates a hyperglycemic reflex and increases brain glucose retention. Here we show that direct administration of micro-doses of AVP into the NTS of anesthetized or awake rats rapidly increased the levels of blood glucose concentration and brain arterio-venous (A-V) glucose difference. This effect was not observed when the same doses of AVP were injected in the brainstem outside NTS. Arginine-vasopressin antagonist microinjections alone produced a small but significant reduction in brain A-V glucose. Pre-administered VP1-receptor antagonist [beta-mercapto-beta,beta-cyclopentamethylene-propionyl(1),O-Me-Tyr(2),Arg(8)]vasopressin blocked the effects of AVP. These results indicate that AVP acting on its receptors locally within the NTS participates in glucose homeostasis, increasing both blood glucose concentration and brain A-V glucose differences. Hypothalamic AVP may facilitate hyperglycemic responses initiated by peripheral signals processed at the level of the NTS.

Influence of the pituitary gland on the immune response in young rats.

The response of hypophysectomized (HYPOX) and sham-operated (S-HYPOX) female and male Wistar young rats (8 weeks old) to antigenic stimulation was compared. Humoral antigenic responses against hemocyanin were measured by ELISA. [3H]thymidine incorporation into cultured spleen cells was used to determine proliferative response to concanavalin A (ConA) or antigenic stimulation. Anti-hemocyanin serum titers in the HYPOX animals was about half of that observed in control S-HYPOX rats. Similarly, the cellular proliferative response was significantly decreased in HYPOX animals when compared to S-HYPOX rats; the blastogenic response to hemocyanin in UC rats (which did not receive the antigen injection) was close to zero. S-HYPOX control rats responded to direct ConA stimulation as UC controls. Body weight and the weight of pituitary target organs (adrenal, thyroid, ovary and testes) was about 1/4 of that of controls. Hypophysectomy also resulted in a striking reduction in spleen weight. These results indicate that the pituitary gland is involved in cellular and humoral immune regulation in young rats.

Changes in blood glucose concentration in the carotid body-sinus modify brain glucose retention.

To test whether blood glucose concentration in the carotid body-sinus may influence the amount of glucose retained by the brain, the isolated carotid sinus was perfused with glucose-rich blood or glucose-poor blood from a second animal. The circulation of the right carotid body-sinus was temporarily isolated in rat A, and perfused with blood coming from rat B. Blood glucose in rat B was modified by injections of glucose or insulin. Changes in glucose retention by the brain were measured in rat A. When the isolated carotid body-sinus in rat A was perfused with hyperglycemic blood (16.7 mM), brain glucose retention in rat A decreased significantly from 0.14 +/- 0.02 mumol/g/min (t = 0) to 0.08 +/- 0.01 mumol/g/min at 4 min after the beginning of perfusion. In contrast, the perfusion of the isolated carotid body-sinus of rat A with hypoglycemic blood (2.7 mM) from rat B, had the opposite effect. Brain glucose retention in rat A increased (0.23 +/- 0.03 mumol/g/min) at t = 4 min in comparison to control values (0.13 +/- 0.01 mumol/g/min). Chemoreceptor activity was also manipulated by the injection of cyanide (NaCN) in rat B, under these conditions, brain glucose retention in rat A increased from 0.13 +/- 0.01 mumol/g/min to 0.28 +/- 0.03 mumol/g/min between 4 to 8 min after the beginning of perfusion. These results indicate that chemosensory activity within the carotid body-sinus, superfused in vivo with different glucose concentrations, modify glucose retention by the brain.

Technical note: removal of the unfragmented pituitary gland (hypophysectomy) in the rat.

A technique to excise the pituitary gland (hypophysis) in rats is described. The basisphenoid bone is reached from the ventral neck and is perforated to expose the pituitary gland and its stalk. An aspirator allows the removal of the hypophysis and the stalk, including pars tuberalis, in one piece. The advantages of this new technique include: 1) immediate verification of the entirety of hypophysectomy; 2) broad operating field which exposes the pituitary stalk up to the hypothalamus; 3) the use of tracheal intubation and artificial respiration to improve postoperative recovery and to allow expanded operation field even during prolonged surgery. Pre- and postoperative care are described. The mean survival rate after this type of operation was 79% in rats weighing 50 to 130 g and 90% in rats larger than 130 g.

Carotid sinus receptors participate in glucose homeostasis.

This paper describes (a) the influence of glucose on carotid chemoreceptor activity, and (b) the participation of carotid receptors in glucose homeostasis. After eliminating the carotid body baroreceptors in anesthetized cats, the injection of glucose to the vascularly isolated carotid sinus region reduced by 20% the electrical activity of carotid body chemoreceptors and increased their threshold to hypoxia. Mannitol in the same concentration did not change the chemoreceptor activity. A decrease in baroreceptor activity elicited by carotid occlusion, or carotid chemoreceptor stimulation with 50 micrograms/kg cyanide (NaCN), produced an immediate increase in the output of hepatic glucose, raising the hepatic venous-arterial glucose difference above basal levels. Bilateral adrenalectomy eliminated these reflex responses. Cyanide injected in the same conditions caused a sharp increase in glucose retention by the brain. In control experiments, after sectioning the carotid nerve, NaCN injections were ineffective. However, electrical stimulation of the central stump of carotid nerve elicited reflex effects similar to those obtained with NaCN stimulation.

Effects of intracisternal glucose or insulin injections on glucose homeostasis in cat.

The injection of glucose (100 mg) into the cisterna magna of intact anesthetized cats elicited immediate glycosuria and natriuresis without significant changes in blood glucose concentration. Immunoreactive insulin (IRI) increased 140% in plasma, and Na+ concentration decreased in cerebrospinal fluid (CSF). After kidney denervation there was a significant decrease in glucose and Na+ concentrations in urine. Control injections with mannitol did not elicit changes in the studied parameters. Abdominal vagotomy abolished the rise in IRI levels and the decrease in Na+ concentration in CSF. Vagotomy or adrenalectomy also attenuated the glycosuria and the rise in urine Na+ concentration. The intracisternal injection of insulin (0.5 U/kg) caused first, a decrease in glucose concentration in CSF and afterwards a longer latency in plasma. Again, these responses were significantly attenuated when insulin was administered in vagotomized cats. These experiments indicate that the nervous system, through the vagi, adrenal glands, and kidneys, plays an important role in glucose homeostasis after increasing glucose or insulin levels in the CSF above physiologic concentrations. The results obtained with a denervated kidney confirm the participation of nervous system in the effector mechanism that brings the sugar and Na+ into the urine. Evidence is presented for an interrelationship between glucose and Na+ concentrations in blood, urine, and CSF.

Histological study of the operative zone in dogs with parotid gland grafts in the sella turcica.

No remnants of adenohypophyseal tissue were found in 83% of the 70 dogs studied; in 17% of the animals remnants amounting to from 1 to 3% of normal hypophyseal tissue were found. These traces showed significant histological and cytological changes, and were most frequently found located on the floor of the sella turcica at some distance from their usual site. It is doubtful whether these residua have any functional significance, since the hypophysectomized dogs with such remnants had a survival time that corresponded to that of dogs with total hypophysectomy (6 months). The microscopic structure of the fragment of transplanted parotid (salivary gland) presents rich vascularization, changes in cellular distribution and a loss of the excretory duct. A better histological aspect was observed in the transplanted cells of the dogs with longer survival time. An important correlation exists between functional behavior of the operated animals and the histological state of the transplanted parotid tissue.

Histological study of the operative zone in dogs with parotid gland grafts in the sella turcica

No remnants of adenohypophyseal tissue were found in 83

of the 70 dogs studied; in 17

of the animals remnants amounting to from 1 to 3

of normal hypophyseal tissue were found. These traces showed significant histological and cytological changes, and were most frequently found located on the floor of the sella turcica at some distance from their usual site. It is doubtful whether these residua have any functional significance, since the hypophysectomized dogs with such remnants had a survival time that corresponded to that of dogs with total hypophysectomy (6 months). The microscopic structure of the fragment of transplanted parotid (salivary gland) presents rich vascularization, changes in cellular distribution and a loss of the excretory duct. A better histological aspect was observed in the transplanted cells of the dogs with longer survival time. An important correlation exists between functional behavior of the operated animals and the histological state of the transplanted parotid tissue.

Histological study of the operative zone in dogs with parotid gland grafts in the sella turcica.

No remnants of adenohypophyseal tissue were found in 83

of the 70 dogs studied; in 17

of the animals remnants amounting to from 1 to 3

of normal hypophyseal tissue were found. These traces showed significant histological and cytological changes, and were most frequently found located on the floor of the sella turcica at some distance from their usual site. It is doubtful whether these residua have any functional significance, since the hypophysectomized dogs with such remnants had a survival time that corresponded to that of dogs with total hypophysectomy (6 months). The microscopic structure of the fragment of transplanted parotid (salivary gland) presents rich vascularization, changes in cellular distribution and a loss of the excretory duct. A better histological aspect was observed in the transplanted cells of the dogs with longer survival time. An important correlation exists between functional behavior of the operated animals and the histological state of the transplanted parotid tissue.