Safety and efficacy of endoscopic spray cryotherapy for esophageal cancer.

Although surgery is traditionally the standard of care for esophageal cancer, esophagectomy carries significant morbidity. Alternative endoscopic therapies are needed for patients who are not candidates for conventional treatment. The objective of this study is to assess the safety, efficacy, and tolerability of spray cryotherapy of esophageal adenocarcinoma. This study includes patients with esophageal adenocarcinoma who had failed or were not candidates for conventional therapy enrolled retrospectively and prospectively in an open-label registry and patients in a retrospective cohort from 11 academic and community practices. Endoscopic spray cryotherapy was performed until biopsy proven local tumor eradication or until treatment was halted due to progression of disease, patient withdrawal or comorbidities. Eighty-eight patients with esophageal adenocarcinoma (median age 76, 80.7% male, mean length 5.1 cm) underwent 359 treatments (mean 4.4 per patient). Tumor stages included 39 with T1a, 25 with T1b, 9 with unspecified T1, and 15 with T2. Eighty-six patients completed treatment with complete response of intraluminal disease in 55.8%, including complete response in 76.3% for T1a, 45.8% for T1b, 66.2% for all T1, and 6.7% for T2. Mean follow-up was 18.4 months. There were no deaths or perforations related to spray cryotherapy. Strictures developed in 12 of 88 patients (13.6%) but were present before spray cryotherapy in 3 of 12. This study suggests that endoscopic spray cryotherapy is a safe, well-tolerated, and effective treatment option for early esophageal adenocarcinoma.

Safety and efficacy of endoscopic spray cryotherapy for Barrett's dysplasia: results of the National Cryospray Registry.

Retrospective series have shown the efficacy of endoscopic spray cryotherapy in eradicating high-grade dysplasia (HGD) in Barrett's esophagus (BE); however, prospective data are lacking, and efficacy for low-grade dysplasia (LGD) is unclear. The aim of this study was to assess the efficacy and safety of spray cryotherapy in patients with LGD or HGD. A multicenter, prospective open-label registry enrolled patients with dysplastic BE. Spray cryotherapy was performed every 2-3 months until there was no endoscopic evidence of BE and no histological evidence of dysplasia, followed by surveillance endoscopies up to 2 years. Primary outcome measures were complete eradication of dysplasia (CE-D) and complete eradication of all intestinal metaplasia (CE-IM). Ninety-six subjects with Barrett's dysplasia (67% HGD; 65% long-segment BE; mean length 4.5 cm) underwent 321 treatments (mean 3.3 per subject). Mean age was 67 years, 83% were male. Eighty patients (83%) completed treatment with follow-up endoscopy (mean duration 21 months). In patients with LGD, rate of CE-D was 91% (21/23) and rate of CE-IM was 61% (14/23). In HGD, CE-D rate was 81% (46/57) and CE-IM was 65% (37/57). In patients with short-segment BE (SSBE) with any dysplasia, CE-D was achieved in 97% (30/31) and CE-IM in 77% (24/31). There were no esophageal perforations or related deaths. One subject developed a stricture, which did not require dilation. One patient was hospitalized for bleeding in the setting of non-steroidal anti-inflammatory drug use. In the largest prospective cohort to date, data suggest endoscopic spray cryotherapy is a safe and effective modality for eradication of BE with LGD or HGD, particularly with SSBE.

Reflex control of inflammation by sympathetic nerves, not the vagus.

We investigated a neural reflex that controls the strength of inflammatory responses to immune challenge - the inflammatory reflex. In anaesthetized rats challenged with intravenous lipopolysaccharide (LPS, 60 µg kg(-1)), we found strong increases in plasma levels of the key inflammatory mediator tumour necrosis factor α (TNFα) 90 min later. Those levels were unaffected by previous bilateral cervical vagotomy, but were enhanced approximately 5-fold if the greater splanchnic sympathetic nerves had been cut. Sham surgery had no effect, and plasma corticosterone levels were unaffected by nerve sections, so could not explain this result. Electrophysiological recordings demonstrated that efferent neural activity in the splanchnic nerve and its splenic branch was strongly increased by LPS treatment. Splenic nerve activity was dependent on inputs from the splanchnic nerves: vagotomy had no effect on the activity in either nerve. Together, these data demonstrate that immune challenge with this dose of LPS activates a neural reflex that is powerful enough to cause an 80% suppression of the acute systemic inflammatory response. The efferent arm of this reflex is in the splanchnic sympathetic nerves, not the vagi as previously proposed. As with other physiological responses to immune challenge, the afferent pathway is presumptively humoral: the present data show that vagal afferents play no measurable part. Because inflammation sits at the gateway to immune responses, this reflex could play an important role in immune function as well as inflammatory diseases.

Adaptive appetites for salted and unsalted food in rats: differential effects of sodium depletion, DOCA, and dehydration.

Most ingested sodium is contained in food. The aim was to investigate whether sodium depletion, dehydration, or DOCA alters intakes of salted and unsalted foods by rats given choices of two foods: salted (0.2-0.5% Na) and unsalted food containing either similar or different other dietary components. Diuretic-induced (furosemide or acetazolamide, two treatments on successive days) sodium depletion always caused pronounced falls in intake of unsalted food within 24 h, continuing at least another 2 days (e.g., 20.9 ± 1.6 pretreatment to 14.8 ± 1.2, 10.6 ± 1.5, and 14.3 ± 1.3 g/day for 3 days of depletion). Intake and preference for salted food increased after 24-72 h (e.g., 6.5 ± 1.2 pretreatment to 7.1 ± 1.1, 16.4 ± 2.3, and 17.0 ± 1.5 g/day at 1, 2, and 3 days of depletion). Valsartan (10 mg/day) blocked the increased intake of salted food but not the reduced intake of unsalted food. DOCA (2 mg/day) caused equivalent increase and decrease in intakes of salted and unsalted food, respectively. Water-deprived rats reduced intake (e.g., 14.2 ± 3.1 to 3.2 ± 2.0 g/day) of and preference for salted food (e.g., 56 ± 13% to 21 ± 11%) after 2 days of dehydration but did not consistently reduce intake of unsalted food. Total food ingested/day fell in both sodium-depleted and dehydrated rats. Rats regulate intakes of different foods to balance sodium needs, osmoregulatory homeostasis, and energy requirements. Reduced appetite for unsalted food may be a homeostatic response to sodium depletion, which together with subsequent generation of appetite for salted food, drives animals to ingest sodium-containing food, thereby restoring sodium balance.

Neural regulation of inflammation: no neural connection from the vagus to splenic sympathetic neurons.

The 'inflammatory reflex' acts through efferent neural connections from the central nervous system to lymphoid organs, particularly the spleen, that suppress the production of inflammatory cytokines. Stimulation of the efferent vagus has been shown to suppress inflammation in a manner dependent on the spleen and splenic nerves. The vagus does not innervate the spleen, so a synaptic connection from vagal preganglionic neurons to splenic sympathetic postganglionic neurons was suggested. We tested this idea in rats. In a preparatory operation, the anterograde tracer DiI was injected bilaterally into the dorsal motor nucleus of vagus and the retrograde tracer Fast Blue was injected into the spleen. On histological analysis 7-9 weeks later, 883 neurons were retrogradely labelled from the spleen with Fast Blue as follows: 89% in the suprarenal ganglia (65% left, 24% right); 11% in the left coeliac ganglion; but none in the right coeliac or either of the superior mesenteric ganglia. Vagal terminals anterogradely labelled with DiI were common in the coeliac but sparse in the suprarenal ganglia, and confocal analysis revealed no putative synaptic connection with any Fast Blue-labelled cell in either ganglion. Electrophysiological experiments in anaesthetized rats revealed no effect of vagal efferent stimulation on splenic nerve activity or on that of 15 single splenic-projecting neurons recorded in the suprarenal ganglion. Together, these findings indicate that vagal efferent neurons in the rat neither synapse with splenic sympathetic neurons nor drive their ongoing activity.

Cortical activation and lamina terminalis functional connectivity during thirst and drinking in humans.

The pattern of regional brain activation in humans during thirst associated with dehydration, increased blood osmolality, and decreased blood volume is not known. Furthermore, there is little information available about associations between activation in osmoreceptive brain regions such as the organum vasculosum of the lamina terminalis and the brain regions implicated in thirst and its satiation in humans. With the objective of investigating the neuroanatomical correlates of dehydration and activation in the ventral lamina terminalis, this study involved exercise-induced sweating in 15 people and measures of regional cerebral blood flow (rCBF) using a functional magnetic resonance imaging technique called pulsed arterial spin labeling. Regional brain activations during dehydration, thirst, and postdrinking were consistent with the network previously identified during systemic hypertonic infusions, thus providing further evidence that the network is involved in monitoring body fluid and the experience of thirst. rCBF measurements in the ventral lamina terminalis were correlated with whole brain rCBF measures to identify regions that correlated with the osmoreceptive region. Regions implicated in the experience of thirst were identified including cingulate cortex, prefrontal cortex, striatum, parahippocampus, and cerebellum. Furthermore, the correlation of rCBF between the ventral lamina terminalis and the cingulate cortex and insula was different for the states of thirst and recent drinking, suggesting that functional connectivity of the ventral lamina terminalis is a dynamic process influenced by hydration status and ingestive behavior.

Hyperdipsia in sheep bearing lesions in the medial septal nucleus.

Previous experiments in rodents showed that ablation of the septal brain region caused hyperdipsia. We investigated which part of the septal region needs ablation to produce hyperdipsia in sheep, and whether increased drinking was a primary hyperdipsia. Following ablation of the medial septal region (n = 5), but not parts of the lateral septal region (n = 4), daily water intake increased from ~2.5-5 L/day up to 10 L/day for up to 3 months post-lesion. In hyperdipsic sheep, plasma osmolality increased on the first day post-lesion and body weight fell, suggesting that initial hyperdipsia was secondary to fluid loss. However hyperosmolality was not sustained long-term and plasma hypo-osmolality persisted from 0.5 to 3 months post-lesion. Acute dipsogenic responses to intravenous hypertonic saline, intravenous or intracerebroventricular angiotensin II, water deprivation for 2 days, or feeding over 5 h were not potentiated by medial septal lesions, showing that the rapid pre-systemic inhibitory influences that cause satiation of thirst upon the act of drinking were intact. However, hyperdipsic sheep continued to ingest water when hyponatremic (plasma [Na] was 127-132 mmol/l) and plasma osmolality was 262-268 mosmol/kg due to retention of ingested fluid resulting from intravenous infusion of vasopressin administered to maintain a basal blood level of antidiuretic hormone. The results show that septal lesion-induced hyperdipsia is not due to disruption of acute pre-systemic influences associated with drinking water that initiates rapid satiation of thirst. Rather, inhibitory influences of hyponatremia, hypo-osmolality or hypervolemia on drinking appear to be disrupted by medial septal lesions.

Specific control of sympathetic nerve activity to the mammalian heart and kidney.

There is a large body of evidence indicating that sympathetic nerves to individual organs are specifically controlled, but only few studies have compared the control of cardiac sympathetic nerve activity (CSNA) with activity in other sympathetic nerves. In this review, changes in sympathetic activity to the heart and kidneys are described during increases in brain [Na+] and in heart failure (HF). In conscious sheep, increases in brain [Na+] increased CSNA and arterial pressure and, conversely, decreased renal sympathetic nerve activity (RSNA), promoting urinary sodium loss. These organ-specific effects are mediated via a neural pathway that includes an angiotensinergic synapse, the lamina terminalis and the paraventricular nucleus of the hypothalamus. There is also evidence of differential control of SNA in HF. In normal sheep, the resting burst incidence of CSNA was much lower than that of RSNA, whereas in HF they increased to similar, almost maximal levels in both nerves. Arterial baroreflex control of both these nerves was unchanged in HF, but the response of CSNA to changes in blood volume was almost absent. These data indicate that in HF the lower arterial pressure leads to reduced baroreflex inhibition of SNA, which, together with the lack of an inhibitory response to the increased volume and cardiac pressures, would contribute to the sympathoexcitation observed. These studies demonstrate differences in the control of CSNA and RSNA, enabling selective actions on the heart and kidney to restore fluid and electrolyte homeostasis in the case of elevated brain [Na+] and to increase cardiac output in HF.

Sodium appetite in sheep induced by cerebral ventricular infusion of angiotensin: comparison with sodium deficiency.

Intraventricular administration of supraphysiological amounts of renin, nerve growth factor preparation, or angiotensin II greatly increased the consumption of water and hypertonic sodium bicarbonate solution by sheep. These effects were antagonized by intraventricular administration of drugs that prevent the formation of angiotensin II or block its receptors. The fact that these angiotensin-blocking drugs did not change the sodium intake of sodium-deficient sheep challenges the idea that central angiotensin action is involved in sodium appetite due to a deficiency.

The role of primordial emotions in the evolutionary origin of consciousness.

Primordial emotions are the subjective element of the instincts which are the genetically programmed behaviour patterns which contrive homeostasis. They include thirst, hunger for air, hunger for food, pain and hunger for specific minerals etc. There are two constituents of a primordial emotion--the specific sensation which when severe may be imperious, and the compelling intention for gratification by a consummatory act. They may dominate the stream of consciousness, and can have plenipotentiary power over behaviour. It is hypothesized that early in animal evolution complex reflex mechanisms in the basal brain subserving homeostatic responses, in concert with elements of the reticular activating system subserving arousal, melded functionally with regions embodied in the progressive rostral development of the telencephalon. This included the emergent limbic and paralimbic areas, and the insula. This phylogenetically ancient organization subserved the origin of consciousness as the primordial emotion, which signalled that the organisms existence was immediately threatened. Neuroimaging confirms major activations in regions of the basal brain during primordial emotions in humans. The behaviour of decorticate humans and animals is discussed in relation to the possible existence of primitive awareness. Neuroimaging of the primordial emotions reveals that rapid gratification of intention by a consummatory act such as ingestion causes precipitate decline of both the initiating sensation and the intention. There is contemporaneous rapid disappearance of particular regions of brain activation which suggests they may be part of the jointly sufficient and severally necessary activations and deactivations which correlate with consciousness [Crick, F. & Koch, C. (2003). A framework for consciousness. NatureNeuroscience,6, 119-126].

Effect of central urotensin II on heart rate, blood pressure and brain Fos immunoreactivity in conscious rats.

Central administration of urotensin II (UII) increases heart rate (HR), cardiac contractility, and plasma levels of epinephrine and glucose. To investigate the mechanisms causing these responses we examined the effects of i.c.v. administration of rat UII (10 microg) on the sympatho-adrenal and pituitary-adrenal axes in conscious rats, and we mapped the brain sites activated by UII by immunohistochemically detecting Fos expression. In six conscious rats i.c.v. UII, but not vehicle, increased HR significantly 60-90 min after treatment and increased plasma glucose at 60 and 90 min, both indicators of increased epinephrine release. Plasma corticosterone levels were significantly elevated 90 min after i.c.v. UII. Conscious rats, given i.c.v. UII (n=12) and killed after 100 or 160 min, showed increased Fos-immunoreactivity (Fos-IR) in the nucleus of the solitary tract and the central nucleus of the amygdala (CeA) at both time points, compared with vehicle (n=11). In UII-treated rats, Fos-IR in the paraventricular nucleus of the hypothalamus (PVN) was significantly elevated at 160 min, but not 100 min, compared with vehicle. There were no increases in Fos-IR in the rostral ventrolateral medulla or the A5 cell group, areas associated with sympathetic outflow to the adrenal gland. In summary, i.c.v. UII increased HR and plasma glucose and corticosterone in conscious rats. UII increased Fos-IR in the CeA and PVN, but over a longer time course in the latter. These findings indicate that UII acts on specific brain nuclei to stimulate the hypothalamo-pituitary-adrenal axis and to stimulate adrenal sympathetic nerve activity.

Central osmoregulatory influences on thermoregulation.

1. Many mammals maintain a constant core body temperature in the face of a heat load by using evaporative cooling responses, such as sweating, panting and spreading of saliva. These cooling mechanisms incur a body fluid deficit if the fluid lost as sweat, saliva or respiratory moisture is not replaced by the ingestion of water; body fluid hypertonicity and hypovolaemia result. 2. Evidence in several mammals shows that, as they become dehydrated, evaporative cooling mechanisms such as sweating and panting are inhibited so that further fluid loss from the body is reduced. As a result, core temperature in the dehydrated animal is maintained at a higher than normal level. 3. Increasing the osmotic pressure of plasma has an inhibitory effect on panting and sweating in mammals. It has been proposed that osmoreceptors mediate these inhibitory influences of plasma hypertonicity on sweating and panting. 4. The suppression of panting in dehydrated sheep is mediated by cerebral osmoreceptors that are probably located in the lamina terminalis. We speculate that osmoreceptors in the lamina terminalis may also influence thermoregulatory sweating. 5. When dehydrated animals drink water, sweating and panting resume rapidly before water has been absorbed from the gut. It is likely that the act of drinking initiates a reflex that can override the osmoreceptor inhibition of panting, resulting in core temperature falling back quickly to a normal level.

Amylin induces natriuresis by a central angiotensin-dependent mechanism.

This study provides evidence that amylin acts centrally to increase sodium excretion in the sheep. Amylin was infused at 8 mg/h into a carotid artery (IC), via a lateral ventricle (ICV), intravenously (IV) or intra-renally (IR) into conscious sheep (n=5 per group). Renal sodium excretion increased by at least 3-fold after 1 h of amylin infusion by ICV (66+/-14 to 367+/-35 mmol/min) and IC (78+/-14 to 244+/-22 mmol/min) routes of administration. Amylin infusion IV caused a 1.5-fold increase in sodium excretion while IR infusion did not have a significant effect. The natriuretic effect of ICV infused amylin was blocked by pre-treatment with the angiotensin AT1 receptor antagonist, losartan (1 mg/h). No changes in blood pressure or heart rate were recorded at this dose of amylin by any route of administration. Plasma renin concentration increased (1.32+/-0.22 to 2.55+/-0.73 pmol/Ang I/h; P<0.05) following IR infusion of amylin, and remained unchanged when amylin was infused by the other routes of administration. We conclude that amylin causes changes in sodium excretion in sheep through a central, angiotensin-dependent pathway and that amylin may increase renin secretion by a direct effect on the kidney.

The brain as an endocrine target for Peptide hormones.

Unlike circulating steroid hormones, which have a relatively unhindered passage into the central nervous system, blood-borne peptides are usually restricted by the blood-brain barrier. Some circulating peptides, such as angiotensin II, atrial natriuretic peptide and relaxin, influence central neural pathways subserving cardiovascular and body fluid homeostasis by acting on neurons in the subfornical organ, organum vasculosum of the lamina terminalis and area postrema, all of which lack a blood-brain barrier. There are some circulating peptides such as insulin and leptin that are transported from the bloodstream across cerebral blood vessel walls into sites in the hypothalamus that have appropriate neural connections to influence food intake and sympathetic control of brown fat.

The brain angiotensin system: insights from mapping its components.

Mapping of components of the angiotensin (Ang) system in the brain suggests that it serves multiple central roles, including regulation of fluid and electrolyte balance, central autonomic control, and pituitary hormone release.

The median preoptic nucleus: front and centre for the regulation of body fluid, sodium, temperature, sleep and cardiovascular homeostasis.

Located in the midline anterior wall of the third cerebral ventricle (i.e. the lamina terminalis), the median preoptic nucleus (MnPO) receives a unique set of afferent neural inputs from fore-, mid- and hindbrain. These afferent connections enable it to receive neural signals related to several important aspects of homeostasis. Included in these afferent projections are (i) neural inputs from two adjacent circumventricular organs, the subfornical organ and organum vasculosum laminae terminalis, that respond to hypertonicity, circulating angiotensin II or other humoural factors, (ii) signals from cutaneous warm and cold receptors that are relayed to MnPO, respectively, via different subnuclei in the lateral parabrachial nucleus and (iii) input from the medulla associated with baroreceptor and vagal afferents. These afferent signals reach appropriate neurones within the MnPO that enable relevant neural outputs, both excitatory and inhibitory, to be activated or inhibited. The efferent neural pathways that proceed from the MnPO terminate on (i) neuroendocrine cells in the hypothalamic supraoptic and paraventricular nuclei to regulate vasopressin release, while polysynaptic pathways from MnPO to cortical sites may drive thirst and water intake, (ii) thermoregulatory pathways to the dorsomedial hypothalamic nucleus and medullary raphé to regulate shivering, brown adipose tissue and skin vasoconstriction, (iii) parvocellular neurones in the hypothalamic paraventricular nucleus that drive autonomic pathways influencing cardiovascular function. As well, (iv) other efferent pathways from the MnPO to sites in the ventrolateral pre-optic nucleus, perifornical region of the lateral hypothalamic area and midbrain influence sleep mechanisms.

Identification of CNS neurons with polysynaptic connections to both the sympathetic and parasympathetic innervation of the submandibular gland.

Coordinated modulation of sympathetic and parasympathetic nervous activity is required for physiological regulation of tissue function. Anatomically, whilst the peripheral sympathetic and parasympathetic pathways are separate, the distribution of premotor neurons in higher brain regions often overlaps. This co-distribution would enable coordinated regulation and might suggest individual premotor neurons could project to both sympathetic and parasympathetic outflows. To investigate this one submandibular gland was sympathectomized. One of two isogenic strains of the pseudorabies virus, expressing different fluorophores, was injected into the cut sympathetic nerve and the other into the submandibular gland. Independent labeling of the peripheral sympathetic and parasympathetic pathways was observed. Dual-labeled neurons were observed in many CNS regions known to be involved in regulating salivary function. We propose these observations highlight a common pattern of organization of the CNS, providing the anatomical framework for the fine control of organ function required for homeostatic regulation and the coordination of organ responses to enable complex behaviors.

The brain renin-angiotensin system: location and physiological roles.

Angiotensinogen, the precursor molecule for angiotensins I, II and III, and the enzymes renin, angiotensin-converting enzyme (ACE), and aminopeptidases A and N may all be synthesised within the brain. Angiotensin (Ang) AT(1), AT(2) and AT(4) receptors are also plentiful in the brain. AT(1) receptors are found in several brain regions, such as the hypothalamic paraventricular and supraoptic nuclei, the lamina terminalis, lateral parabrachial nucleus, ventrolateral medulla and nucleus of the solitary tract (NTS), which are known to have roles in the regulation of the cardiovascular system and/or body fluid and electrolyte balance. Immunohistochemical and neuropharmacological studies suggest that angiotensinergic neural pathways utilise Ang II and/or Ang III as a neurotransmitter or neuromodulator in the aforementioned brain regions. Angiotensinogen is synthesised predominantly in astrocytes, but the processes by which Ang II is generated or incorporated in neurons for utilisation as a neurotransmitter is unknown. Centrally administered AT(1) receptor antagonists or angiotensinogen antisense oligonucleotides inhibit sympathetic activity and reduce arterial blood pressure in certain physiological or pathophysiological conditions, as well as disrupting water drinking and sodium appetite, vasopressin secretion, sodium excretion, renin release and thermoregulation. The AT(4) receptor is identical to insulin-regulated aminopeptidase (IRAP) and plays a role in memory mechanisms. In conclusion, angiotensinergic neural pathways and angiotensin peptides are important in neural function and may have important homeostatic roles, particularly related to cardiovascular function, osmoregulation and thermoregulation.

Water drinking in rats resulting from intravenous relaxin and its modification by other dipsogenic factors.

The purpose of the study was to determine whether iv infusion of relaxin would acutely stimulate water drinking in rats and, if it did, whether such drinking is affected by other dipsogenic stimuli or is blocked by centrally administered losartan. iv infusions of human gene 2 relaxin at doses of 25, 40, 55, or 80 microg/kg x h for 1 h induced dose-dependent water drinking in both male and female rats within 15-30 min of commencement of infusions. iv infusion of a nondipsogenic dose of angiotensin II (0.5 microg/h), combined with relaxin (40 microg/kg x h), almost tripled the relaxin-induced water intake. iv infusion of hypertonic (1 M) NaCl did not potentiate relaxin-induced drinking. Intracerebroventricular injection of the angiotensin AT1 antagonist losartan (10 microg) reduced water drinking induced by iv infusion of relaxin. The water drinking induced by iv infusion of relaxin in the rat suggests that blood-borne relaxin may be a dipsogenic hormone. Potentiation of this relaxin-induced drinking by moderate levels of circulating angiotensin II is additional evidence in support of this view. The results also indicate that a central angiotensinergic neural pathway, utilizing AT1 receptors, subserves relaxin-induced drinking.

Localization and characterization of insulin receptors in rat brain and pituitary gland using in vitro autoradiography and computerized densitometry.

In order to identify likely sites of action in insulin in rat brain we have used the technique of in vitro autoradiography and computerized densitometry to map, characterize, and quantify its receptors in coronal and sagittal sections. A discrete and characteristic distribution of insulin receptor binding was demonstrated, with specific binding representing 92% of total binding. Displacement and specificity competition curves in olfactory bulb are typical for authentic insulin receptors, and computer analysis indicates a single class of binding site with a dissociation constant (Kd) 0.48 nM for choroid plexus and 0.44 nM for olfactory bulb external plexiform layer. Insulin receptor density is maximum in the choroid plexus, and high in the external plexiform layer of olfactory bulb. Structures of the limbic system and hypothalamus reveal moderate to high insulin receptor density, particularly the lateral septum, amygdala, subiculum, hippocampal CA1 region, mammillary body, and arcuate nucleus. Moderate insulin receptor density occurs in regions of cerebral cortex and cerebellum, and moderate to low binding occurs in discrete brainstem and midbrain structures. Insulin binding in the pituitary gland is greatest in the anterior lobe, with clear distinction from intermediate and posterior lobes. The circumventricular organs and the thalamus show low insulin binding. We conclude that insulin receptors are widespread throughout rat brain, with concentration in regions concerned with olfaction, appetite, and autonomic functions. The distribution is distinct from other neuropeptides and not related to either vascularity or cell density. A common feature of regions rich in insulin receptors is that they contain dendritic fields receiving rich synaptic input. Whether insulin plays a specific neurotransmitter or metabolic role in these sites remains unclear, but these studies have provided detailed information on potential sites of action of insulin in the brain, and will allow further studies to examine insulin receptor function in specific brain regions.