High temporal resolution gastric emptying breath tests in mice.

BACKGROUND: Gastric emptying is a complex physiological process regulating the division of a meal into smaller partitions for the small intestine. Disrupted gastric emptying contributes to digestive disease, yet current measures may not reflect different mechanisms by which the process can be altered. METHODS: We have developed high temporal resolution solid and liquid gastric emptying breath tests in mice using [13 C]-octanoic acid and off axis- integrated cavity output spectroscopy (OA-ICOS). Stretched gamma variate and 2-component stretched gamma variate models fit measured breath excretion data. KEY RESULTS: These assays detect acceleration and delay using pharmacological (7.5 mg/kg atropine) or physiological (nutrients, cold exposure stress, diabetes) manipulations and remain stable over time. High temporal resolution resolved complex excretion curves with 2 components, which was more prevalent in mice with delayed gastric emptying following streptozotocin-induced diabetes. There were differences in the gastric emptying of Balb/c vs C57Bl6 mice, with slower gastric emptying and a greater occurrence of two-phase gastric emptying curves in the latter strain. Gastric emptying of C57Bl6 could be accelerated by halving the meal size, but with no effect on the occurrence of two-phase gastric emptying curves. A greater proportion of two-phase gastric emptying was induced in Balb/c mice with the administration of PYY (8-80 nmol) 60 min following meal ingestion. CONCLUSIONS AND INFERENCES: Collectively, these results demonstrate the utility of high temporal resolution gastric emptying assays. Two-phase gastric emptying is more prevalent than previously reported, likely involves intestinal feedback, but contributes little to the overall rate of gastric emptying.

Epigenetics and chromatin dynamics: a review and a paradigm for functional disorders.

BACKGROUND: Motility and functional gastrointestinal disorders have high prevalence in the community, cause significant morbidity, and represent a major health care burden. Despite major advances in our understanding of the cellular and molecular basis of gastrointestinal neuromuscular functions, many of these diseases still defy mechanistic explanations. The biopsychosocial model underlying the current classification of functional gastrointestinal disorders recognizes and integrates the pathogenetic role of genetic, environmental, and psychosocial factors but has not been associated with specific molecular mechanisms. PURPOSE: Here, we propose that this integrative function is encoded in the chromatin, composed of the DNA and associated histone and non-histone proteins and non-coding RNA. By establishing epigenetically heritable 'molecular memories' of past stimuli including environmental challenges, the chromatin determines an individual's responses to future insults and translates them into high-order outputs such as symptoms and illness behavior. Thus, surveying epigenetic signatures throughout the genome of affected cells in individual patients may make it possible to better understand and ultimately control the phenomena described by the biopsychosocial model. In this review, we provide a high-level but comprehensive description of the concepts and mechanisms underlying epigenetics and chromatin dynamics, describe the mechanisms whereby the environment can alter the epigenome and identify aspects of functional gastrointestinal and motility disorders where epigenetic mechanisms are most likely to play important roles.

Platelet-derived growth factor receptor α (PDGFRα)-expressing "fibroblast-like cells" in diabetic and idiopathic gastroparesis of humans.

BACKGROUND: Emerging evidence suggests that "fibroblast-like cells" (FLC) may play a role in the regulation of gastrointestinal (GI) motor function. FLC are ultrastructurally distinct from other interstitial cells, including interstitial cells of Cajal (ICC), and express small-conductance Ca(2+) -activated K(+) channels (SK3). In mice, platelet-derived growth factor receptor α (PDGFRα) antibody has also been shown to label FLC. The aims of this study were to determine the morphology and distribution of PDGFRα-immunoreactive (ir) FLC in human gastric muscle and to determine if FLC are altered in gastroparesis, where ICC are reduced. METHODS: Full thickness gastric body biopsies from five healthy subjects, 10 diabetic, and 10 idiopathic gastroparesis patients were immunolabeled using SK3 and PDGFRα staining for FLC and Kit staining for ICC. Intramuscular FLC and ICC were quantified. KEY RESULTS: Intramuscular PDGFRα-ir cells had slender cell bodies and long, thin processes and were more abundant in the longitudinal compared with the circular muscle. In the region of myenteric plexus, FLC had smaller, rounder cell bodies with 3-4 processes and formed networks, often around ganglia. All SK3-ir cell structures showed complete overlap with PDGFRα-ir. FLC were in close proximity to ICC, but their cell bodies did not overlap. No differences were seen in the distribution, morphology, or overall numbers of FLC in gastroparesis patients. CONCLUSIONS & INFERENCES: In conclusion, PDGFRα identifies FLC in human gastric smooth muscle. FLC were not altered in distribution or overall numbers in gastroparesis. Additional studies are required to determine their role in human GI function.

Generalized neuromuscular hypoplasia, reduced smooth muscle myosin and altered gut motility in the klotho model of premature aging.

BACKGROUND: Gastrointestinal symptoms, particularly constipation, increase with aging, but their underlying mechanisms are poorly understood due to lack of experimental models. Previously we established the progeric klotho mouse as a model of aging-associated anorexia and gastric dysmotility. We also detected reduced fecal output in these animals; therefore, the aim of this study was to investigate in vivo function and cellular make-up of the small intestinal and colonic neuromuscular apparatus. METHODS: Klotho expression was studied by RT-PCR and immunohistochemistry. Motility was assessed by dye transit and bead expulsion. Smooth muscle and neuron-specific gene expression was studied by Western immunoblotting. Interstitial cells of Cajal (ICC) and precursors were analyzed by flow cytometry, confocal microscopy, and three-dimensional reconstruction. HuC/D(+) myenteric neurons were enumerated by fluorescent microscopy. KEY RESULTS: Klotho protein was detected in neurons, smooth muscle cells, and some ICC classes. Small intestinal transit was slower but whole-gut transit of klotho mice was accelerated due to faster colonic transit and shorter intestinal lengths, apparent only after weaning. Fecal water content remained normal despite reduced output. Smooth muscle myosin expression was reduced. ICC, ICC precursors, as well as nitrergic and cholinergic neurons maintained their normal proportions in the shorter intestines. CONCLUSIONS & INFERENCES: Progeric klotho mice express less contractile proteins and develop generalized intestinal neuromuscular hypoplasia mainly arising from stunted postweaning growth. As reduced fecal output in these mice occurs in the presence of accelerated colonic and whole-gut transit, it likely reflects reduced food intake rather than intestinal dysmotility.

Immunoreactivity for Ano1 detects depletion of Kit-positive interstitial cells of Cajal in patients with slow transit constipation.

BACKGROUND: Depletion of interstitial cells of Cajal (ICC) is associated with several gastrointestinal (GI) motility disorders. Changes in ICC networks are usually detected by immunolabeling for the receptor tyrosine kinase Kit. Ano1 (DOG1 or TMEM16A) was recently described as a marker of ICC in GI tract. Our aim was to determine whether Ano1 immunoreactivity can be used as a reliable marker for ICC in tissues from patients with motility disorders. METHODS: Four tissues from patients with normal ICC numbers and four tissues from patients with slow transit constipation and loss of Kit-positive ICC were studied. Interstitial cells of Cajal were detected by double labeling using antisera to Kit and Ano1. KEY RESULTS: Both the processes and cell bodies of ICC in tissue from controls and slow transit constipation were immunoreactive for Ano1. There was a near complete overlap between Kit and Ano1 immunoreactivity. Tissues from patients with slow transit constipation contained significantly fewer Ano1-positive ICC than control tissues. The numbers of ICC identified by Ano1 and Kit immunoreactivity were nearly identical across the range of ICC numbers from an average of 1.64 to 7.05 cells per field and correlated with an R(2) value of 0.99. CONCLUSIONS & INFERENCES: Ano1 is a reliable and sensitive marker for detecting changes in ICC networks in humans. Labeling with antibodies selective for Ano1 reproducibly detects depletion of Kit-positive ICC in tissues from patients with slow transit constipation.

Changes in interstitial cells of cajal with age in the human stomach and colon.

BACKGROUND: Aging produces inevitable changes in the function of most organs including the gastrointestinal tract. Together with enteric nerves and smooth muscle cells, interstitial cells of Cajal (ICC) play a key role in the control of gastrointestinal motility, yet little is known about the effect of aging on ICC. The aim of this study was to determine the effect of aging on ICC number and volume in the human stomach and colon. METHODS: Gastric and colonic tissues from patients aged 25-70 and 36-92 years old, respectively, and with no co-existent motility disorders were immunolabeled with an anti-Kit antibody and ICC were counted in the circular muscle and myenteric regions. Network volumes were measured using 3D reconstructions of confocal stacks. The effects of aging were determined by testing for linear trends using regression analysis. KEY RESULTS: In both stomach and colon, the number of ICC bodies and volume significantly decreased with age at a rate of 13% per decade. ICC size was only affected in the myenteric plexus in the colon. The changes associated with age were not differentially affected by sex or colonic region. CONCLUSIONS & INFERENCES: The number and volume of ICC networks in the normal human stomach and colon decline with age. This decrease in ICC likely reduces the functional capacity of the gastrointestinal motor apparatus, may contribute to changes in gastrointestinal motility with aging and may influence intestinal responses to insults such as disease, operative interventions and medications in older patients. Tissue specimens must be carefully age-matched when studying ICC in disease.

Gastroparesis and functional dyspepsia: excerpts from the AGA/ANMS meeting.

BACKGROUND: Despite the relatively high prevalence of gastroparesis and functional dyspepsia, the aetiology and pathophysiology of these disorders remain incompletely understood. Similarly, the diagnostic and treatment options for these two disorders are relatively limited despite recent advances in our understanding of both disorders. PURPOSE: This manuscript reviews the advances in the understanding of the epidemiology, pathophysiology, diagnosis, and treatment of gastroparesis and functional dyspepsia as discussed at a recent conference sponsored by the American Gastroenterological Association (AGA) and the American Neurogastroenterology and Motility Society (ANMS). Particular focus is placed on discussing unmet needs and areas for future research.

Cellular pathogenesis of diabetic gastroenteropathy.

Gastroenteropathy manifesting in upper gastrointestinal symptoms, delayed gastric emptying, constipation, diarrhea and fecal incontinence occurs frequently in patients with diabetes mellitus and represents a significant health care burden. Current treatments are largely symptomatic and ineffective. Better understanding of the cellular and molecular pathogenesis of these disorders is required for the development of more effective therapies. Recent advances in our understanding of the inherent, high-level complexities of the control systems that execute and regulate gastrointestinal motility, together with the utilization of new experimental models and sophisticated physiological, morphological and molecular techniques have lead to the realization that diabetic gastroenteropathies cannot be ascribed to any singular defect or dysfunction. In fact, these disorders are multifactorial and involve a spectrum of metabolic and dystrophic changes that can potentially affect all key components of motor control including the systemic autonomic and enteric nervous systems, interstitial cells of Cajal and smooth muscle cells. Candidate pathomechanisms are also varied and include imbalance between pro- and anti-oxidative factors, altered trophic stimuli to mature cells and their progenitors, and, possibly, autoimmune factors. The goal of this paper is to review the cellular changes underlying diabetic gastroenteropathies and their potential causes, with particular focus on functional interactions between various cell types. It is proposed that diabetic gastroenteropathies should be considered a form of gastrointestinal neuromuscular dystrophy rather than a "functional" disorder. Future research should identify ways to block cytotoxic factors, support the regeneration of damaged cells and translate the experimental findings into new treatment modalities.

Review article: gastric electrical stimulation for gastroparesis--physiological foundations, technical aspects and clinical implications.

BACKGROUND: Application of electrical stimulation to the gut, primarily the stomach, has rapidly advanced in the last two decades, from mostly animal studies to the clinical arena. Most studies focused on the use of electrical stimulation for gastroparesis, the only approved indication for such intervention. AIM: To review the physiological basis of gastric electrical activity and the technical aspects and clinical outcome of gastric electrical stimulation (GES) for gastroparesis. METHODS: PubMed search from 1966 to 2009, using gastroparesis and GES as search terms. Areas in focus were systematically reviewed. RESULTS: The literature consists of open-label studies, mostly from single centres, published in the last decade. Improvement in symptoms, quality of life and nutritional status was reported by most studies. Physiologically, stimulation parameters approved in clinical practice do not regulate gastric slow wave activity and have inconsistent effect on gastric emptying. The mechanism of action of GES is not fully known, but data support modulation of gastric biomechanical activity and afferent neural mechanisms. CONCLUSIONS: Gastric electrical stimulation is a helpful intervention in recalcitrant gastroparesis. Controlled studies and better understanding of mechanisms of action of electrical stimulation are needed to evaluate further the clinical utility of this intervention and to exploit its therapeutic potential better.

Interstitial cells of Cajal in diabetic gastroenteropathy.

Gastroenteropathy causes considerable morbidity in patients with diabetes mellitus and represents a major healthcare burden. Current treatments are largely symptomatic and frequently ineffective. Development of new therapeutic options is hampered by poor understanding of the underlying pathomechanisms. Experimental studies and sparse human data indicate that diabetic gastroenteropathy is multifactorial and involves not only parasympathetic and sympathetic autonomic nerves, but also enteric neurons, smooth muscle cells and interstitial cells of Cajal (ICC). ICC are mesenchymal cells that occur throughout the muscular coat of the gastrointestinal tract and provide functions critical for normal gastrointestinal motility including generation and propagation of electrical slow waves and mediation of bidirectional communication between the autonomic nervous system and smooth muscle cells. Through these functions, and in concert with other cell types of the gastrointestinal muscles, ICC support basic gastrointestinal functions such as digestion, absorption and waste removal. Loss or dysfunction of ICC in various dysmotilities and their animal models has been shown to lead to gastric dysrhythmias, gastroparesis, slow intestinal transit, impaired neuroeffector mechanisms and altered visceral afferent signalling that are considered hallmarks of diabetic gastroenteropathy. These findings and an increasing body of evidence indicating disruptions of ICC networks in diabetes suggest that the loss of ICC in this disorder is probably of functional significance and could even be a major pathogenetic factor. Future research should focus on the identification of the molecular and cellular mechanisms underlying ICC loss in diabetes and the translation of the experimental findings into treatments.

Template switching within exons 3 and 4 of KV11.1 (HERG) gives rise to a 5' truncated cDNA.

K(V)11.1 (HERG) channels contribute to membrane potential in a number of excitable cell types. We cloned a variant of K(V)11.1 from human jejunum containing a 171 bp deletion spanning exons 3 and 4. Expression of a full-length cDNA clone containing this deletion gave rise to protein that trafficked to the cell membrane and generated robust currents. The deletion occurred in a G/C-rich region and identical sequence elements of UGGUGG were located at the deletion boundaries. In recent studies these features have been implicated to cause deletions via template switching during cDNA synthesis. To examine this possibility we compared cDNAs from human brain, heart, and jejunum synthesized at lower (42 degrees C) and higher temperatures (70 degrees C). The 171 bp deletion was absent at the higher temperature. Our results suggest that the sequence and secondary structure of mRNA in the G/C rich region leads to template switching producing a cDNA product with a 171 bp deletion.

Ionic conductances involved in generation and propagation of electrical slow waves in phasic gastrointestinal muscles.

Considerable work has led many to conclude that interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. These cells form electrically coupled networks within the pacemaker regions of the GI tract, and ICC are electrically coupled to smooth muscle cells. ICC express unique ion channels that periodically produce inward (pacemaker) currents. Recent work has suggested that the inward current is produced by a calcium (Ca2+)-regulated, nonselective cation conductance. Channels responsible for this conductance oscillate in open probability in response to the periodic drop in intracellular Ca2+ concentration during the slow wave cycle. Pacemaker activity generates slow waves that are propagated actively through ICC networks. Depolarization coordinates the pacemaker activity through the ICC network by activating a dihydropyridine-resistant Ca2+ conductance. Entry of small amounts of Ca2+ into ICC entrains spontaneous pacemaker activity and produces cell-to-cell propagation of slow waves. This review discusses the mechanisms and conductances involved in generation and propagation of electrical slow waves in ICC.

Remodeling of networks of interstitial cells of Cajal in a murine model of diabetic gastroparesis.

Patients with long-standing diabetes commonly suffer from gastric neuromuscular dysfunction (gastropathy) causing symptoms ranging from postprandial bloating to recurrent vomiting. Autonomic neuropathy is generally believed to be responsible for diabetic gastropathy and the underlying impairments in gastric emptying (gastroparesis) and receptive relaxation, but the specific mechanisms have not been elucidated. Recently, it has been recognized that interstitial cells of Cajal generate electrical pacemaker activity and mediate motor neurotransmission in the stomach. Loss or defects in interstitial cells could contribute to the development of diabetic gastroparesis. Gastric motility was characterized in spontaneously diabetic NOD/LtJ mice by measuring gastric emptying and by monitoring spontaneous and induced electrical activity in circular smooth muscle cells. Interstitial cells of Cajal were studied by Kit immunofluorescence and transmission electron microscopy. Diabetic mice developed delayed gastric emptying, impaired electrical pacemaking, and reduced motor neurotransmission. Interstitial cells of Cajal were greatly reduced in the distal stomach, and the normally close associations between these cells and enteric nerve terminals were infrequent. Our observations suggest that damage to interstitial cells of Cajal may play a key role in the pathogenesis of diabetic gastropathy.

Pacemaking in interstitial cells of Cajal depends upon calcium handling by endoplasmic reticulum and mitochondria.

Pacemaker cells, known as interstitial cells of Cajal (ICC), generate electrical rhythmicity in the gastrointestinal tract. Pacemaker currents in ICC result from the activation of a voltage-independent, non-selective cation conductance, but the timing mechanism responsible for periodic activation of the pacemaker current is unknown. Previous studies suggest that pacemaking in ICC is dependent upon metabolic activity 1y1yand1 Ca2+ release from intracellular stores. We tested the hypothesis that mitochondrial Ca2+ handling may underlie the dependence of gastrointestinal pacemaking on oxidative metabolism. Pacemaker currents occurred spontaneously in cultured ICC and were associated with mitochondrial Ca2+ transients. Inhibition of the electrochemical gradient across the inner mitochondrial membrane blocked Ca2+ uptake and pacemaker currents in cultured ICC and blocked slow wave activity in intact gastrointestinal muscles from mouse, dog and guinea-pig. Pacemaker currents and rhythmic mitochondrial Ca2+ uptake in ICC were also blocked by inhibitors of IP3-dependent release of Ca2+ from the endoplasmic reticulum and by inhibitors of endoplasmic reticulum Ca2+ reuptake. Our data suggest that integrated Ca2+ handling by endoplasmic reticulum and mitochondria is a prerequisite of electrical pacemaking in the gastrointestinal tract.

Development and plasticity of interstitial cells of Cajal.

Interstitial cells of Cajal (ICC) are the pacemakers in gastrointestinal (GI) muscles, and these cells also mediate or transduce inputs from the enteric nervous system. Different classes of ICC are involved in pacemaking and neurotransmission. ICC express specific ionic conductances that make them unique in their ability to generate and propagate slow waves in GI muscles or transduce neural inputs. Much of what we know about the function of ICC comes from developmental studies that were made possible by the discoveries that ICC express c-kit and proper development of ICC depends upon signalling via the Kit receptor pathway. Manipulating Kit signalling with reagents to block the receptor or downstream signalling pathways or by using mutant mice in which Kit or its ligand, stem cell factor, are defective has allowed novel studies into the specific functions of the different classes of ICC in several regions of the GI tract. Kit is also a surface antigen that can be used to conveniently label ICC in GI muscles. Immunohistochemical studies using Kit antibodies have expanded our knowledge about the ICC phenotype, the structure of ICC networks, the interactions of ICC with other cells in the gut wall, and the loss of ICC in some clinical disorders. Preparations made devoid of ICC have also allowed analysis of the consequences of losing specific classes of ICC on GI motility. This review describes recent advances in our knowledge about the development and plasticity of ICC and how developmental studies have contributed to our understanding of the functions of ICC. We have reviewed the clinical literature and discussed how loss or defects in ICC affect GI motor function.

Development of interstitial cells of Cajal and pacemaking in mice lacking enteric nerves.

BACKGROUND & AIMS: Development of interstitial cells of Cajal (ICC) requires signaling via Kit receptors. Kit is activated by stem cell factor (SCF), but the source of SCF in the bowel wall is unclear and controversy exists about whether enteric neurons express the SCF required for ICC development. METHODS: Glial cell line-derived neurotrophic factor (GDNF) knockout mice, which lack enteric neurons throughout most of the gut, were used to determine whether neurons are necessary for ICC development. ICC distributions were determined with Kit immunofluorescence, and function of ICC was determined by intracellular electrical recording. RESULTS: ICC were normally distributed throughout the gastrointestinal tracts of GDNF-/- mice. Intracellular recordings from aganglionic gastrointestinal muscles showed normal slow wave activity at birth in the stomach and small intestine. Slow waves developed normally in aganglionic segments of small bowel placed into organ culture at birth. Quantitative polymerase chain reaction showed similar expression of SCF in the muscles of animals with and without enteric neurons. Expression of SCF was demonstrated in isolated intestinal smooth muscle cells. CONCLUSIONS: These data suggest that enteric neurons are not required for the development of functional ICC. The circular smooth muscle layer, which develops before ICC, may be the source of SCF required for ICC development.

Interstitial cells of cajal generate electrical slow waves in the murine stomach.

1. The gastric corpus and antrum contain interstitial cells of Cajal (ICC) within the tunica muscularis. We tested the hypothesis that ICC are involved in the generation and regeneration of electrical slow waves. 2. Normal, postnatal development of slow wave activity was characterized in tissues freshly removed from animals between birth and day 50 (D50). Slow wave amplitude and frequency increased during this period. Networks of myenteric ICC (IC-MY) were present in gastric muscles at birth and did not change significantly in appearance during the period of study as imaged by confocal immunofluorescence microscopy. 3. IC-MY networks were maintained and electrical rhythmicity developed in organ culture in a manner similar to normal postnatal development. Electrical activity was maintained for at least 48 days in culture. 4. Addition of a neutralizing antibody (ACK2) for the receptor tyrosine kinase, Kit, to the culture media caused progressive loss of Kit-immunoreactive cells. Loss of Kit-immunoreactive cells was associated with loss of slow wave activity. Most muscles became electrically quiescent after 3-4 weeks of exposure to ACK2. 5. In some muscles small clusters of Kit-immunoreactive IC-MY remained after culturing with ACK2. These muscles displayed slow wave activity but only in the immediate regions in which Kit-positive IC-MY remained. These data suggest that regions without Kit-immunoreactive cells cannot generate or regenerate slow waves. 6. After loss of Kit-immunoreactive cells, the muscles could not be paced by direct electrical stimulation. Stimulation with acetylcholine also failed to elicit slow waves. The data suggest that the generation of slow waves is an exclusive property of IC-MY; smooth muscle cells may not express the ionic apparatus necessary for generation of these events. 7. We conclude that IC-MY are an essential element in the spontaneous rhythmic electrical and contractile activity of gastric muscles. This class of ICC appears to generate slow wave activity and may provide a means for regeneration of slow waves.

On the mechanism of the positive feedback action of estradiol on luteinizing hormone secretion in the rhesus monkey.

In women and rhesus monkeys, both the negative and positive feedback actions of estradiol (E2) on gonadotropin secretion (inhibition followed by a surge) can be exerted directly at the level of the pituitary gland. We have tested the hypothesis that the positive feedback action of E2 represents but an "escape" from its negative feedback inhibition of gonadotropin secretion consequent to a desensitization of the gonadotropes occasioned by sustained exposure to elevated concentrations of the steroid. We have attempted to replicate such a desensitization by blocking the negative feedback action of E2 by the administration of a potent estrogen receptor antagonist devoid of any agonistic properties (ZM 182,780) to rhesus monkeys in the midfollicular phase of the menstrual cycle (n = 14). The estrogen antagonist, administered at a dose that in separate experiments completely blocked both the negative and the positive feedback effect of exogenous E2 on pituitary LH secretion, failed to produce a surge-like increase in serum LH concentrations. The present results do not support the hypothesis that the LH surge is the consequence of the removal of the negative feedback action of E2. Evidence is presented that ZM 182,780, in contrast to its inhibition of E2-induced LH surges, cannot block the inhibition of hypothalamic GnRH pulse generator activity by E2.

Mitigation of the somnolence of insulin-induced hypoglycemia by a vasopressin V1 receptor antagonist in the rhesus monkey.

Insulin-induced hypoglycemia causes somnolence in rhesus monkeys, a phenomenon usually considered an aspecific consequence of neuroglycopenia. Previous observations from our laboratory have raised the possibility that arginine vasopressin (AVP) may also play a role in this decrease in wakefulness. In the present study we tested this hypothesis by inducing hypoglycemia (approximately 40 mg/dl) in ovariectomized rhesus monkeys by intravenous administration of insulin in the presence of continuous intracerebroventricular infusions of the V1 receptor antagonist [deamino-Pen1, O-Me-Tyr2,Arg8]-vasopressin (180 micrograms/60 microliters per h) or of its vehicle alone (artificial cerebrospinal fluid, 60 microliters/h). Wakefulness was assessed by a scoring system by observers blinded to the experimental protocol. The AVP antagonist significantly attenuated the decrease in wakefulness observed in response to insulin-induced hypoglycemia (p < .03) without increasing blood glucose levels. These and previous findings suggest that the somnolence induced by a moderate degree of hypoglycemia may not entirely be the direct consequence of neuroglycopenia and that AVP may, directly or indirectly, be involved.

On the mechanism of lactational anovulation in the rhesus monkey.

The relative roles of infant suckling and of maternal prolactin (PRL) secretion in lactational anovulation were studied in ovary-intact and ovariectomized rhesus monkeys nursing young that had been removed from their natural mothers. Hypothalamic gonadotropin-releasing hormone (GnRH) pulse generator activity was monitored electrophysiologically in freely behaving animals by radiotelemetry. Serum luteinizing hormone, PRL, estradiol, and progesterone were also measured. Suckling inhibited GnRH pulse generator activity and ovarian cyclicity in all ovary-intact females but had no such effect on the pulse generator in long-term ovariectomized animals. When PRL secretion was suppressed by daily bromocriptine administration, GnRH pulse generator activity remained significantly inhibited and ovulation was prevented in four monkeys (6 trials), whereas in two females (6 trials) a rapid increase in pulse generator frequency and the resumption of ovarian cyclicity were observed although suckling activity was maintained. One monkey displayed both response types. Although these results indicate that suckling per se is able to restrain GnRH pulse generator activity in the absence of PRL, they also suggest that the relative importance of these determinants is variable depending on factors that remain to be determined. The present study also confirms the permissive role of the ovary in the lactational suppression of GnRH pulse generator activity.