NPY derived from AGRP neurons controls feeding via Y1 and energy expenditure and food foraging behaviour via Y2 signalling.

OBJECTIVE: Aguti-related protein (AGRP) neurons in the arcuate nucleus of the hypothalamus (ARC), which co-express neuropeptide Y (NPY), are key regulators of feeding and energy homeostasis. However, the precise role NPY has within these neurons and the specific pathways that it control are still unclear. In this article, we aimed to determine what aspects of feeding behaviour and energy homeostasis are controlled by NPY originating from AGRP neurons and which Y-receptor pathways are utilised to fulfil this function. METHODS: Novel conditional Agrpcre/+;Npylox/lox knockout mice were generated and comprehensively phenotyped, both under standard chow as well as high-fat-diet conditions. Designer receptor exclusively activated by designer drugs (DREADD) technology was used to assess the altered responses on feeding and energy homeostasis control in the absence of NPY in these neurons. Rescue experiments utilising Npy1r- and Npy2r-selective NPY ligands were performed to assess which component of the energy homeostasis control is dependent by which specific Y-receptor pathway. RESULTS: We show that the specific deletion of Npy only in AGRP neurons leads to a paradoxical mild obese phenotype associated with reduced locomotion and energy expenditure and increased feeding and Respiratory Quotient (RQ) that remain elevated under a positive energy balance. The activation of Npy-deficient AGRP neurons via DREADD's is still able to drive feeding, yet with a delayed onset. Additionally, Clozapine-N-oxide (CNO) treatment reduces locomotion without impacting on energy expenditure. Rescue experiments re-introducing Npy1r- and Npy2r-selective NPY ligands revealed that the increased feeding and RQ are mostly driven by Npy1r, whereas energy expenditure and locomotion are controlled by Npy2r signalling. CONCLUSION: Together, these results demonstrate that NPY originating from AGRP neurons is not only critical to initiate but also for continuously driving feeding, and we for the first time identify which Y-receptor controls which pathway.

Both NPY-Expressing and CART-Expressing Neurons Increase Energy Expenditure and Trabecular Bone Mass in Response to AP1 Antagonism, But Have Opposite Effects on Bone Resorption.

Energy metabolism and bone homeostasis share several neuronal regulatory pathways. Within the ventral hypothalamus (VHT), the orexigenic neurons co-express Agouti-related peptide (AgRP) and neuropeptide Y (NPY) and the anorexigenic neurons co-express, α-melanocyte stimulating hormone derived from proopiomelanocortin (POMC), and cocaine and amphetamine-regulated transcript (CART). These neurons regulate both processes, yet their relative contribution is unknown. Previously, using genetically targeted activator protein (AP1) alterations as a tool, we showed in adult mice that AgRP or POMC neurons are capable of inducing whole-body energy catabolism and bone accrual, with different effects on bone resorption. Here, we investigated whether co-residing neurons exert similar regulatory effects. We show that AP1 antagonists targeted to NPY-producing or CART-producing neurons in adult mice stimulate energy expenditure, reduce body weight gain and adiposity and promote trabecular bone formation and mass, yet again via different effects on bone resorption, as measured by serum level of carboxy-terminal collagen type I crosslinks (CTX). In addition, AP1 antagonists promote neurite expansion, increasing neurite number, length, and surface area in primary hypothalamic neuronal cultures. Overall, our data demonstrate that the orexigenic NPY and anorexigenic CART neurons both have the capacity to stimulate energy burning state and increase bone mass. © 2020 American Society for Bone and Mineral Research.

Endocannabinoid and nitric oxide systems of the hypothalamic paraventricular nucleus mediate effects of NPY on energy expenditure.

OBJECTIVE: Neuropeptide Y (NPY) is one of the most potent orexigenic peptides. The hypothalamic paraventricular nucleus (PVN) is a major locus where NPY exerts its effects on energy homeostasis. We investigated how NPY exerts its effect within the PVN. METHODS: Patch clamp electrophysiology and Ca2+ imaging were used to understand the involvement of Ca2+ signaling and retrograde transmitter systems in the mediation of NPY induced effects in the PVN. Immuno-electron microscopy were performed to elucidate the subcellular localization of the elements of nitric oxide (NO) system in the parvocellular PVN. In vivo metabolic profiling was performed to understand the role of the endocannabinoid and NO systems of the PVN in the mediation of NPY induced changes of energy homeostasis. RESULTS: We demonstrated that NPY inhibits synaptic inputs of parvocellular neurons in the PVN by activating endocannabinoid and NO retrograde transmitter systems via mobilization of Ca2+ from the endoplasmic reticulum, suggesting that NPY gates the synaptic inputs of parvocellular neurons in the PVN to prevent the influence of non-feeding-related inputs. While intraPVN administered NPY regulates food intake and locomotor activity via NO signaling, the endocannabinoid system of the PVN selectively mediates NPY-induced decrease in energy expenditure. CONCLUSION: Thus, within the PVN, NPY stimulates the release of endocannabinoids and NO via Ca2+-influx from the endoplasmic reticulum. Both transmitter systems appear to have unique roles in the mediation of the NPY-induced regulation of energy homeostasis, suggesting that NPY regulates food intake, energy expenditure, and locomotor activity through different neuronal networks of this nucleus.

Organization of the Orexin/Hypocretin System in the Brain of Holostean Fishes: Assessment of Possible Relationships with Monoamines and Neuropeptide Y.

Holosteans form a small group of actinopterygian fishes considered the sister group of teleosts. Despite this proximity to the biggest group of vertebrates, relatively few studies have been conducted to investigate the organization of the central nervous system of this group of fishes. In this study, the neuroanatomical distribution of orexin/hypocretin-like immunoreactive (OX-ir) cell bodies and fibers was analyzed in the brain of 3 representative species of the 2 orders of extant holosteans, the spotted gar Lepisosteus oculatus, the Florida gar Lepisosteus platyrhincus, and the bowfin Amia calva. Antibodies against orexin-A (OXA) and orexin-B (OXB) were used, which labeled the same cells and fibers throughout the brain. In addition, double immunohistofluorescence was performed for the simultaneous detection of OXA and OXB with tyrosine hydroxylase, serotonin, and neuropeptide Y (NPY), in an attempt to localize the orexinergic structures precisely and study the possible interactions between these neuroactive substances. The pattern of distribution of OX-ir cells in the 3 species was largely similar, showing labeled cells in the preoptic area (POA), and the tuberal and retrotuberal hypothalamic regions, with only subtle differences between species in the density of labeled cells. OX-ir fibers were found in all main brain subdivisions of the 3 species, mostly in the ventral subpallial areas, POA, hypothalamus, posterior tubercle, thalamus, and mesencephalic tectum. Different densities of orexinergic fibers were observed in relation to catecholaminergic and serotoninergic cell groups, as well as an absence of colocalization between orexins and NPY in the same hypothalamic neurons. The comparison of these results with those obtained in other vertebrates highlights a constant pattern of distribution of this system of neurotransmission among different groups of actinopterygian fishes, especially in teleosts. Conserved features shared by all vertebrates studied were also observed, such as the presence of OX-ir cells in the basal hypothalamus, reflecting the preserved functions of these neuropeptides over the course of evolution.

A Hypothalamic Phosphatase Switch Coordinates Energy Expenditure with Feeding.

Beige adipocytes can interconvert between white and brown-like states and switch between energy storage versus expenditure. Here we report that beige adipocyte plasticity is important for feeding-associated changes in energy expenditure and is coordinated by the hypothalamus and the phosphatase TCPTP. A fasting-induced and glucocorticoid-mediated induction of TCPTP, inhibited insulin signaling in AgRP/NPY neurons, repressed the browning of white fat and decreased energy expenditure. Conversely feeding reduced hypothalamic TCPTP, to increase AgRP/NPY neuronal insulin signaling, white adipose tissue browning and energy expenditure. The feeding-induced repression of hypothalamic TCPTP was defective in obesity. Mice lacking TCPTP in AgRP/NPY neurons were resistant to diet-induced obesity and had increased beige fat activity and energy expenditure. The deletion of hypothalamic TCPTP in obesity restored feeding-induced browning and increased energy expenditure to promote weight loss. Our studies define a hypothalamic switch that coordinates energy expenditure with feeding for the maintenance of energy balance.

Unaltered Hypothalamic Metabolic Gene Expression in Kiss1r Knockout Mice Despite Obesity and Reduced Energy Expenditure.

Kisspeptin controls reproduction by stimulating gonadotrophin-releasing hormone neurones via its receptor Kiss1r. Kiss1r is also expressed other brain areas and in peripheral tissues, suggesting additional nonreproductive roles. We recently determined that Kiss1r knockout (KO) mice develop an obese and diabetic phenotype. In the present study, we investigated whether Kiss1r KOs develop this metabolic phenotype as a result of alterations in the expression of metabolic genes involved in the appetite regulating system of the hypothalamus, including neuropeptide Y (Npy) and pro-opiomelanocortin (Pomc), as well as leptin receptor (Lepr), ghrelin receptor (Ghsr), and melanocortin receptors 3 and 4 (Mc3r, Mc4r). Body weights, leptin levels and hypothalamic gene expression were measured in both gonad-intact and gonadectomised (GNX) mice at 8 and 20 weeks of age that had received either normal chow or a high-fat diet. We detected significant increases in Pomc expression in gonad-intact Kiss1r KO mice at 8 and 20 weeks, although there were no alterations in the other metabolic-related genes. However, the Pomc increases appeared to reflect genotype differences in circulating sex steroids, because GNX wild-type and Kiss1r KO mice exhibited similar Pomc levels, along with similar Npy levels. The altered Pomc gene expression in gonad-intact Kiss1r KO mice is consistent with previous reports of reduced food intake in these mice and may serve to increase the anorexigenic drive, perhaps compensating for the obese state. However, the surprising overall lack of changes in any of the hypothalamic metabolic genes in GNX KO mice suggests that the aetiology of obesity in the absence of kisspeptin signalling may reflect peripheral rather than central metabolic impairments.

Insulin ameliorating endotoxaemia-induced muscle wasting is associated with the alteration of hypothalamic neuropeptides and inflammation in rats.

OBJECTIVES: Septic patients always develop muscle wasting, which delays the rehabilitation and contributes to the increased complications and mortality. Previous studies have implied the crucial role of central inflammation and neuropeptides in the energy balance and muscle metabolism. Insulin has been confirmed to attenuate muscle degradation and inhibit inflammation. We tested the hypothesis whether insulin ameliorating muscle wasting was associated with modulating hypothalamic inflammation and neuropeptides. DESIGN AND SUBJECTS: Thirty-two adult male Sprague-Dawley rats were in intraperitoneally injected with lipopolysaccharide (LPS) (5 mg/kg) or saline, followed by subcutaneous injection of insulin (5 IU/kg) or saline. Twenty-four hours after injection, skeletal muscle and hypothalamus tissues were harvested. Muscle wasting was measured by the mRNA expression of two E3 ubiquitin ligases, muscle ring finger 1 (MuRF-1) and muscle atrophy F-box (MAFbx), as well as 3-methylhistidine (3-MH) and tyrosine release. Hypothalamic inflammatory markers and neuropeptides expression were also measured in four groups. RESULTS: LPS injection led to significant increase in hypothalamic inflammation as well as muscle wasting. Also, increased hypothalamic neuropeptides, proopiomelanocortin (POMC), cocaine and amphetamine-related transcript (CART) and neuropeptides Y (NPY) and decreased agouti-related protein (AgRP) were observed. Insulin treatment ameliorated endotoxaemia-induced muscle wasting and hypothalamic inflammation, and attenuated the alteration of neuropeptides, POMC, CART and AgRP. CONCLUSION: Hypothalamic inflammation and neuropeptides are involved in the endotoxaemia-induced muscle wasting. Insulin treatment can reduce muscle wasting, which is associated with reduced hypothalamic inflammation and alteration of hypothalamic neuropeptides.

Assessment of a fully active class A G protein-coupled receptor isolated from in vitro folding.

We provide a protocol for the preparation of fully active Y2 G protein-coupled receptors (GPCRs). Although a valuable target for pharmaceutical research, information about the structure and dynamics of these molecules remains limited due to the difficulty in obtaining sufficient amounts of homogeneous and fully active receptors for in vitro studies. Recombinant expression of GPCRs as inclusion bodies provides the highest protein yields at lowest costs. But this strategy can only successfully be applied if the subsequent in vitro folding results in a high yield of active receptors and if this fraction can be isolated from the nonactive receptors in a homogeneous form. Here, we followed that strategy to provide large quantities of the human neuropeptide Y receptor type 2 and determined the folding yield before and after ligand affinity chromatography using a radioligand binding assay. Directly after folding, we achieved a proportion of ~25% active receptor. This value could be increased to ~96% using ligand affinity chromatography. Thus, a very homogeneous sample of the Y2 receptor could be prepared that exhibited a K(D) value of 0.1 ± 0.05 nM for the binding of polypeptide Y, which represents one of the natural ligands of the Y2 receptor.

Neuropeptide Y and agouti-related peptide mediate complementary functions of hyperphagia and reduced energy expenditure in leptin receptor deficiency.

Neuropeptide Y (NPY) and agouti-related peptide (AGRP) can produce hyperphagia, reduce energy expenditure, and promote triglyceride deposition in adipose depots. As these two neuropeptides are coexpressed within the hypothalamic arcuate nucleus and mediate a major portion of the obesity caused by leptin signaling deficiency, we sought to determine whether the two neuropeptides mediated identical or complementary actions. Because of separate neuropeptide receptors and signal transduction mechanisms, there is a possibility of distinct encoding systems for the feeding and energy expenditure aspects of leptin-regulated metabolism. We have genetically added NPY deficiency and/or AGRP deficiency to LEPR deficiency isolated to AGRP cells. Our results indicate that the obesity of LEPR deficiency in AGRP/NPY neurons can produce obesity with either AGRP or NPY alone with AGRP producing hyperphagia while NPY promotes reduced energy expenditure. The absence of both NPY and AGRP prevents the development of obesity attributable to isolated LEPR deficiency in AGRP/NPY neurons. Operant behavioral testing indicated that there were no alterations in the reward for a food pellet from the AGRP-specific LEPR deficiency.

C-type natriuretic peptide as a new regulator of food intake and energy expenditure.

The physiological implication of C-type natriuretic peptide (CNP) including energy metabolism has not been elucidated, because of markedly short stature in CNP-null mice. In the present study we analyzed food intake and energy expenditure of CNP-null mice with chondrocyte-targeted CNP expression (CNP-Tg/Nppc(-/-) mice), in which marked skeletal dysplasia was rescued, to investigate the significance of CNP under minimal influences of skeletal phenotypes. In CNP-Tg/Nppc(-/-) mice, body weight and body fat ratio were reduced by 24% and 32%, respectively, at 20 wk of age, and decreases of blood glucose levels during insulin tolerance tests were 2-fold exaggerated at 17 wk of age, as compared with CNP-Tg/Nppc(+/+) mice. Urinary noradrenalin excretion of CNP-Tg/Nppc(-/-) mice was greater than that of CNP-Tg/Nppc(+/+) mice by 28%. In CNP-Tg/Nppc(-/-) mice, rectal temperature at 1600 h was higher by 1.1 C, and uncoupling protein-1 mRNA expression in the brown adipose tissue was 2-fold increased, which was canceled by propranolol administration, as compared with CNP-Tg/Nppc(+/+) mice. Oxygen consumption was significantly increased in CNP-Tg/Nppc(-/-) mice compared with that in CNP-Tg/Nppc(+/+) mice. Food intake of CNP-Tg/Nppc(-/-) mice upon ad libitum feeding and refeeding after 48 h starvation were reduced by 21% and 61%, respectively, as compared with CNP-Tg/Nppc(+/+) mice. This study unveiled a new aspect of CNP as a molecule regulating food intake and energy expenditure. Further analyses on precise mechanisms of CNP actions would lead to the better understanding of the significance of the CNP/guanylyl cyclase-B system in food intake and energy expenditure.

Global life-long health benefits of repression of hypothalamic NPY system by central leptin gene therapy.

A minute-to-minute crosstalk between the hypothalamic neuropeptide Y (NPY) network and the hormone leptin is essential for energy homeostasis. Leptin insufficiency i.e. lack of leptin restraint due to genetic and environmental factors on the hypothalamic NPY system confers obesity, a cluster of metabolic afflictions and shorter lifespan. A state-of-the-art gene transfer technology using recombinant adeno-associated viral vector to overcome hypothalamic leptin insufficiency was employed in rodent models of obesity, metabolic syndrome and shorter lifespan. Our findings show that life-long tonic repression of NPY system with a stable increase in leptin availability in the hypothalamus prevented the age-related and high fat-diet-induced obesity, hyperinsulinemia and diabetes and extended lifespan. Additional health benefits include increased energy expenditure and normalization of neuroendocrine control on reproduction, and promotion of brain and bone growth. We propose that central leptin gene therapy or novel long-acting leptin mimetics should be tested clinically to decelerate the worldwide epidemic of obesity, diabetes and shortened lifespan.

Antiobesity effects of the beta-cell hormone amylin in diet-induced obese rats: effects on food intake, body weight, composition, energy expenditure, and gene expression.

Effects of amylin and pair feeding (PF) on body weight and metabolic parameters were characterized in diet-induced obesity-prone rats. Peripherally administered rat amylin (300 microg/kg.d, 22d) reduced food intake and slowed weight gain: approximately 10% (P<0.05), similar to PF. Fat loss was 3-fold greater in amylin-treated rats vs. PF (P<0.05). Whereas PF decreased lean tissue (P<0.05 vs. vehicle controls; VEH), amylin did not. During wk 1, amylin and PF reduced 24-h respiratory quotient (mean+/-se, 0.82+/-0.0, 0.81+/-0.0, respectively; P<0.05) similar to VEH (0.84+/-0.01). Energy expenditure (EE mean+/-se) tended to be reduced by PF (5.67+/-0.1 kcal/h.kg) and maintained by amylin (5.86+/-0.1 kcal/h.kg) relative to VEH (5.77+/-0.0 kcal/h.kg). By wk 3, respiratory quotient no longer differed; however, EE increased with amylin treatment (5.74+/-0.09 kcal/.kg; P<0.05) relative to VEH (5.49+/-0.06) and PF (5.38+/-0.07 kcal/h.kg). Differences in EE, attributed to differences in lean mass, argued against specific amylin-induced thermogenesis. Weight loss in amylin and pair-fed rats was accompanied by similar increases arcuate neuropeptide Y mRNA (P<0.05). Amylin treatment, but not PF, increased proopiomelanocortin mRNA levels (P<0.05 vs. VEH). In a rodent model of obesity, amylin reduced body weight and body fat, with relative preservation of lean tissue, through anorexigenic and specific metabolic effects.

Acute intracerebroventricular administration of either carboxyl-terminal or amino-terminal fragments of agouti-related peptide produces a long-term decrease in energy expenditure in rats.

We investigated if agouti-related peptide (AgRP), an endogenous antagonist of melanocortin receptors (MC3-R and MC4-R), effects energy expenditure in rats. Fragments of the carboxyl-terminal, AgRP (83-132), and the amino-terminals, AgRP (25-51) and AgRP (54-82), were administered intracerebroventricularly (ICV). Food intake, body weight and fat weight changes were measured 5 and/or 24 h after a single ICV injection of the fragments. Oxygen consumption and colonic temperature were measured as indices of energy expenditure, during 3 and 24 h after the ICV injections, respectively. An oral glucose tolerance test was performed 24 h after ICV AgRP (83-132) injection. Binding experiments were performed in HEK-293 cells that over-expressed human MC4-R. AgRP (83-132), but not AgRP (25-51) nor AgRP (54-82), induced a potent and long-lasting increase in the cumulative food intake. Both the carboxyl-terminal and amino-terminal AgRP fragments significantly decreased oxygen consumption and colonic temperature. Despite the absence of hyperphagia and cross-reactivities with MC4-R, AgRP (25-51) and AgRP (54-82) significantly increased body weight and epididymal/mesenteric fat weight. AgRP (83-132) did not affect glucose and insulin responses to the oral glucose tolerance test. AgRP causes a potent and long-lasting decrease in energy expenditure; an effect that is exhibited by carboxyl-terminal fragments and amino-terminal fragments that lack antagonist activity at the MC receptors. This suggests that the amino-terminal region of AgRP plays a regulatory role in energy metabolism.

Stereological assessment of vulnerability of immunocytochemically identified striatal and hippocampal neurons after global cerebral ischemia in rats.

Detailed quantitative analysis of the vulnerability of different hippocampal and striatal neurons to global forebrain ischemia has not previously been performed. Here we have studied the survival of immunocytochemically identified neurons using an unbiased stereological method in rats subjected to global forebrain ischemia for 30 min and sacrificed 48 h, 1 week or 4 weeks thereafter. Within the hippocampal formation, there was extensive, progressive loss of CA1 pyramidal neurons and dentate hilar neuropeptide Y (NPY)-positive interneurons. In contrast, no reduction of the number of CA3 and CA4 pyramidal neurons or hilar parvalbumin-positive interneurons was detected. In the dorsolateral striatum, the insult caused a major loss of projection neurons immunoreactive to dopamine- and adenosine 3':5'-monophosphate-regulated phosphoprotein with a molecular weight of 32 kilodalton (DARPP-32). The number of parvalbumin-positive striatal interneurons was significantly reduced, while NPY-positive interneurons were resistant. All striatal cholinergic interneurons survived the ischemic insult. At 48 h following the ischemia, the cholinergic interneurons within the lesioned striatum transiently expressed the p75 neurotrophin receptor (p75(NTR)), as shown by double-label immunocytochemistry. Furthermore, there was a significant increase in the number of choline acetyltransferase (ChAT)- and TrkA-immunoreactive interneurons at 4 weeks after the insult. Injections with the cell mitotic division marker BrdU provided no evidence that the elevated cholinergic cell number was due to neurogenesis. Probably, the higher number of ChAT- and TrkA-positive interneurons reflected increased intracellular levels of the corresponding proteins leading to more cells detectable with immunocytochemistry. This study gives the first quantitative description of the vulnerability of defined hippocampal and striatal neurons after global forebrain ischemia. The ischemia-induced increases of p75(NTR), TrkA and ChAT in cholinergic striatal interneurons at various time points after the insult suggest that neurotrophin signaling might be important for the survival and function of these cells in the post-ischemic phase.

Activation of the NPY Y5 receptor regulates both feeding and energy expenditure.

Intracerebroventricular (ICV) administration of neuropeptide Y (NPY) has been shown to decrease energy expenditure, induce hypothermia, and stimulate food intake. Recent evidence has suggested that the Y5 receptor may be a significant mediator of NPY-stimulated feeding. The present study attempts to further characterize the role of NPY Y5-receptor subtypes in feeding and energy expenditure regulation. Satiated Long-Evans rats with temperature transponders implanted in the interscapular brown adipose tissue (BAT) displayed a dose-dependent decrease in BAT temperature and an increase in food intake after ICV infusion of NPY. Similar effects were induced by ICV administration of peptide analogs of NPY that activate the Y5 receptor, but not by analogs that activate Y1, Y2, or Y4 receptors. Furthermore, ICV infusion of the Y5 selective agonist D-[Trp(32)]-NPY significantly reduced oxygen consumption and energy expenditure of rats as measured by indirect calorimetry. These data suggest that the NPY Y5-receptor subtype not only mediates the feeding response of NPY but also contributes to brown fat temperature and energy expenditure regulation.

A quantitative assessment of innervation in the conduction system of the calf heart.

BACKGROUND: The aim of the present investigation was to determine the relative distribution of autonomic and sensory nerves in the cardiac conduction tissues of calves. METHODS: A quantitative immunohistochemical and histochemical technique was adopted. RESULTS: Immunoreactivity to the general neuronal marker protein gene product 9.5 (PGP 9.5) demonstrated that all regions of the conduction system possessed a higher relative density of total nerves when compared with the surrounding myocardial tissues. Unlike myocardial innervation, the conduction system did not display an atrial-to-ventricular gradient in nerve density. PGP 9.5-immunoreactive nerve trunks and varicose nerve fibres were more numerous in the transitional atrioventricular node and the penetrating atrioventricular bundle than in either the sinus node, compact atrioventricular node, or bundle branches. The Purkinje network of the ventricular conduction tissues possessed a rich supply of PGP 9.5-immunoreactive nerve trunks and varicose nerve fibres. Acetylcholinesterase (AChE)-positive nerves were the main subtype identified in the sinus and atrioventricular nodes and in the ventricular conduction tissues, representing 50-80% of the area occupied by PGP 9.5-immunoreactive nerves. The compact atrioventricular node possessed AChE-positive and tyrosine hydroxylase (TH)-immunoreactive nerves in similar proportions (45%), although, in general, TH-immunoreactive nerves had a lower relative nerve density than AChE-positive nerves. Neuropeptide Y (NPY)-immunoreactive nerves represented the main peptide-containing subpopulation and occurred throughout the conduction system, displaying a similar pattern of distribution and relative density to those demonstrating TH immunoreactivity. Nerve fibres immunoreactive for somatostatin, vasoactive intestinal polypeptide, substance P, and calcitonin gene-related peptide formed relatively minor subpopulations. CONCLUSIONS: The general innervation of the bovine conduction tissues exhibits significant regional variation. Throughout all regions of the conduction system, AChE-positive nerve represented the dominant subtype when compared with TH- and NPY-immunoreactive nerves. The distribution and relative density of nerve subtypes in the tissues of the bovine conduction system are similar to those observed in man, whereas differences were observed in other regions, such as the atrioventricular bundle and bundle branches. This finding must be considered by those making interspecies comparisons.

Interactions between NPY and its receptor: assessment using ant-NPY antibodies.

The present data show that monoclonal antibodies (NPY02, NPY03, NPY04, NPY05) directed against 4 distinct epitopes on NPY may have different actions on NPY binding and NPY-induced cellular responses. NPY02 and NPY05 recognize the 11-24 and 32-36 amidated form of NPY, respectively. These 2 antibodies block the binding of NPY to its receptor as well as the NPY-induced inhibition of cAMP accumulation caused in SK-N-MC cells by forskolin. NPY02 and NPY05 have also an inhibitory action on NPY-induced contraction of rabbit femoral arteries. NPY03 and NPY04 are directed against the 27-34 and 1-12 part of NPY, respectively. NPY03 and NPY04 inhibit the binding of NPY only at very high concentrations and have a weak effect on cAMP response to NPY. NPY02 and NPY05 might provide useful tools to study the effect of NPY in cellular systems and organ preparations.

A quantitative assessment of somatostatin-like and neuropeptide Y-like immunostained cells in the frontal and temporal cortex of patients with Alzheimer's disease.

Immunocytochemical studies utilizing radioimmunoassay and morphological techniques have provided conflicting evidence for the involvement of somatostatin and neuropeptide Y in Alzheimer's disease (AD). However, previous investigators have not considered the effects of cortical atrophy in AD tissue on their findings. This study reports the numbers of somatostatin-like (SLI) and neuropeptide Y-like immunoreactive (NPYLI) neuronal perikarya and the length of SLI and NPYLI immunoreactive fibres, with appropriate corrections for atrophy in 6 control and 6 AD cases. There were significantly fewer SLI neurones in AD in layers II + III combined from the temporal cortex, and fewer NPYLI neurones in layers V + VI in both frontal and temporal cortices. Using a randomized method to quantify immunostained fibre length in the neuropil, an analysis of variance revealed no significant differences in the mean SLI or NPYLI fibre length per cortical strip between control and AD groups in frontal or temporal cortex. However, using a second measure of fibre length by tracing the fibres attached to consecutive immunostained perikarya, there were significant reductions in the AD brains in the mean fibre length per cell in layers V + VI for SLI in the temporal cortex, and for NPYLI in the frontal cortex. This reduction in fibre length per individual cell was presumably masked by the large variation in the fibre length found between cases using the randomized approach. It was concluded that in order to evaluate the involvement of these neuropeptides in AD from any measurements of concentration, it is essential to include some compensation for the extent of cortical atrophy that occurs with the disease.