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Societies and journals in psychology encourage use of confidence intervals (CIs) on effect sizes. Gaining a maximum of precision of the CI at a minimum cost is desirable. Methods are available to calculate a sample size to provide some percent "assurance" that the final CI will be no wider than a desired value under the fixed-sample rule (FSR), in which a sample size must be decided a priori. Such assurance is expensive, and still subject to failure. The desired width can be specified either in standardized or unstandardized units, and this article focuses on unstandardized widths. A sequential stopping rule (SSR) can generate a CI for a mean difference that is always the desired width, and the average use of subjects is about the same as the FSR sample size without assurance. Improper use of sequential sampling can lead to a CI that has degraded coverage - the interval may contain the population value only 90% of the time for a nominal 95% CI. SSR methods are available to deliver CIs that are within a certain tolerance of the nominal coverage or that are at least the nominal coverage on average. These methods can be assessed by simulations, and they involve a manipulation of the minimum sample size, the desired width, or the practical confidence coefficient used to calculate CIs during the SSR experiment. As with FSR, more exact CIs are generated when the population standard deviation can be estimated accurately., (© 2022. The Psychonomic Society, Inc.)

Different methods have been suggested for calculating "exact" confidence intervals for a standardized mean difference using the noncentral t distributions. Two methods are provided in Hedges and Olkin (1985, "H") and Steiger and Fouladi (1997, "S"). Either method can be used with a biased estimator, d, or an unbiased estimator, g, of the population standardized mean difference (methods abbreviated Hd, Hg, Sd, and Sg). Coverages of each method were calculated from theory and estimated from simulations. Average coverages of 95% confidence intervals across a wide range of effect sizes and across sample sizes from 5 to 89 per group were always between 85 and 98% for all methods, and all were between 94 and 96% with sample sizes greater than 40 per group. The best interval estimation was the Sd method, which always produced confidence intervals close to 95% at all effect sizes and sample sizes. The next best was the Hg method, which produced consistent coverages across all effect sizes, although coverage was reduced to 93-94% at sample sizes in the range 5-15. The Hd method was worse with small sample sizes, yielding coverages as low as 86% at n = 5. The Sg method produced widely different coverages as a function of effect size when the sample size was small (93-97%). Researchers using small sample sizes are advised to use either the Steiger & Fouladi method with d or the Hedges & Olkin method with g as an interval estimation method., (© 2021. The Psychonomic Society, Inc.)

The variable criteria sequential stopping rule (vcSSR) is an efficient way to add sample size to planned ANOVA tests while holding the observed rate of Type I errors, α o , constant. The only difference from regular null hypothesis testing is that criteria for stopping the experiment are obtained from a table based on the desired power, rate of Type I errors, and beginning sample size. The vcSSR was developed using between-subjects ANOVAs, but it should work with p values from any type of F test. In the present study, the α o remained constant at the nominal level when using the previously published table of criteria with repeated measures designs with various numbers of treatments per subject, Type I error rates, values of ρ, and four different sample size models. New power curves allow researchers to select the optimal sample size model for a repeated measures experiment. The criteria held α o constant either when used with a multiple correlation that varied the sample size model and the number of predictor variables, or when used with MANOVA with multiple groups and two levels of a within-subject variable at various levels of ρ. Although not recommended for use with χ 2 tests such as the Friedman rank ANOVA test, the vcSSR produces predictable results based on the relation between F and χ 2 . Together, the data confirm the view that the vcSSR can be used to control Type I errors during sequential sampling with any t- or F-statistic rather than being restricted to certain ANOVA designs.

Background: Many humans suffering with chronic neuropathic pain have no objective evidence of an etiological lesion or disease. Frequently their persistent pain occurs after the healing of a soft tissue injury. Based on clinical observations over time, our hypothesis was that after an injury in mammals the process of tissue repair could cause chronic neural pain. Our objectives were to create the delayed onset of neuropathic pain in rats with minimal nerve trauma using a physiologic hydrogel, and characterize the rats' responses to known analgesics and a targeted biologic. Methods: In mature male Sprague Dawley rats (age 9.5 months) a percutaneous implant of tissue-derived hydrogel was placed in the musculofascial tunnel of the distal tibial nerve. Subcutaneous morphine (3 mg/kg), celecoxib (10 mg/kg), gabapentin (25 mg/kg) and duloxetine (10 mg/kg) were each screened in the model three times each over 5 months after pain behaviors developed. Sham and control groups were used in all screenings. A pilot study followed in which recombinant human erythropoietin (200 units) was injected by the GEL™ neural procedure site. Results: The GEL group gradually developed mechanical hypersensitivity lasting months. Morphine, initially effective, had less analgesia over time. Celecoxib produced no analgesia, while gabapentin and duloxetine at low doses demonstrated profound analgesia at all times tested. The injected erythropoietin markedly decreased bilateral pain behavior that had been present for over 4 months, p ≤ 0.001. Histology of the GEL group tibial nerve revealed a site of focal neural remodeling, with neural regeneration, as found in nerve biopsies of patients with neuropathic pain. Conclusion: The refined NeuroDigm GEL™ model induces a neural response resulting in robust neuropathic pain behavior. The analgesic responses in this model reflect known responses of humans with neuropathic pain. The targeted recombinant human erythropoietin at the ectopic neural lesion appears to alleviate the persistent pain behavior in the GEL™ model rodents., Competing Interests: Competing interests: MRH is an officer and stockholder in NeuroDigm Corporation.

Background: Many humans suffering with chronic pain have no clinical evidence of a lesion or disease. They are managed with a morass of drugs and invasive procedures. Opiates usually become less effective over time. In many, their persistent pain occurs after the healing of a soft tissue injury. Current animal models of neuropathic pain typically create direct neural damage with open surgeries using ligatures, neurectomies, chemicals or other forms of deliberate trauma. However, we have observed clinically that after an injury in humans, the naturally occurring process of tissue repair can cause chronic neural pain. Methods: We demonstrate how the refined biomimetic NeuroDigm GEL™ Model, in the mature male rat, gradually induces neuropathic pain behavior with a nonsurgical percutaneous implant of tissue-derived hydrogel in the musculo-fascial tunnel of the distal tibial nerve. Morphine, Celecoxib, Gabapentin and Duloxetine were each screened in the model three times each over 5 months after pain behaviors developed. A pilot study followed in which recombinant human erythropoietin was applied to the GEL neural procedure site. Results: The GEL Model gradually developed neuropathic pain behavior lasting months. Morphine, initially effective, had less analgesia over time. Celecoxib produced no analgesia, while gabapentin and duloxetine at low doses had profound analgesia at all times tested. The injected erythropoietin markedly decreased bilateral pain behavior that had been present for over 4 months. Histology revealed a site of focal neural remodeling, with neural regeneration, as in human biopsies. Conclusion: The refined NeuroDigm GEL™ Model induces localized neural remodeling resulting in robust neuropathic pain behavior. The analgesics responses in this model reflect known responses of humans with neuropathic pain. The targeted recombinant human erythropoietin appears to heal the ectopic focal neural site, as demonstrated by the extinguishing of neuropathic pain behavior present for over 4 months., Competing Interests: Competing interests: MRH is an officer and stockholder in NeuroDigm Corporation.

To obtain approval for the use vertebrate animals in research, an investigator must assure an ethics committee that the proposed number of animals is the minimum necessary to achieve a scientific goal. How does an investigator make that assurance? A power analysis is most accurate when the outcome is known before the study, which it rarely is. A 'pilot study' is appropriate only when the number of animals used is a tiny fraction of the numbers that will be invested in the main study because the data for the pilot animals cannot legitimately be used again in the main study without increasing the rate of type I errors (false discovery). Traditional significance testing requires the investigator to determine the final sample size before any data are collected and then to delay analysis of any of the data until all of the data are final. An investigator often learns at that point either that the sample size was larger than necessary or too small to achieve significance. Subjects cannot be added at this point in the study without increasing type I errors. In addition, journal reviewers may require more replications in quantitative studies than are truly necessary. Sequential stopping rules used with traditional significance tests allow incremental accumulation of data on a biomedical research problem so that significance, replicability, and use of a minimal number of animals can be assured without increasing type I errors., (Copyright 2011 by the American Association for Laboratory Animal Science)

The variable-criteria sequential stopping rule (SSR) allows an investigator to use a few subjects at a time to determine whether a planned experiment is worth pursuing without increasing the rate of false discoveries (type I errors). The SSR is appropriate whenever testing a null hypothesis if the experiment can be conducted in stages. The investigator adds a predetermined number of subjects at each stage and tests repeatedly for significance until the experiment is stopped because: (1) a significant effect is detected; (2) the effect is clearly not going to be significant; or (3) the predetermined maximal number of subjects has been reached. Two crucial features of the SSR are that it holds the probability of a type I error constant and maintains excellent power. The method is more efficient than is performing a typical significance test after a power analysis because SSR can require 30% fewer subjects to achieve the same power. The variable-criteria SSR provides a formal method for assuring the use of a minimal number of animals. This article provides practical examples of how to use the SSR in combination with a t test, one-way ANOVA, one-way ANOVA with a planned contrast as the focus of the stopping rule, or, in limited circumstances, multifactorial ANOVA., (Copyright 2011 by the American Association for Laboratory Animal Science)

The variable-criteria sequential stopping rule (SSR) is a method for conducting planned experiments in stages after the addition of new subjects until the experiment is stopped because the p value is less than or equal to a lower criterion and the null hypothesis has been rejected, the p value is above an upper criterion, or a maximum sample size has been reached. Alpha is controlled at the expected level. The table of stopping criteria has been validated for a t test or ANOVA with four groups. New simulations in this article demonstrate that the SSR can be used with unequal sample sizes or heterogeneous variances in a t test. As with the usual t test, the use of a separate-variance term instead of a pooled-variance term prevents an inflation of alpha with heterogeneous variances. Simulations validate the original table of criteria for up to 20 groups without a drift of alpha. When used with a multigroup ANOVA, a planned contrast can be substituted for the global F as the focus for the stopping rule. The SSR is recommended when significance tests are appropriate and when the null hypothesis can be tested in stages. Because of its efficiency, the SSR should be used instead of the usual approach to the t test or ANOVA when subjects are expensive, rare, or limited by ethical considerations such as pain or distress.

Sequential stopping rules (SSRs) should augment traditional hypothesis tests in many planned experiments, because they can provide the same statistical power with up to 30% fewer subjects without additional education or software. This article includes new Monte-Carlo-generated power curves and tables of stopping criteria based on the p values from simulated t tests and one-way ANOVAs. The tables improve existing SSR techniques by holding alpha very close to a target value when 1-10 subjects are added at each iteration. The emphasis is on small sample sizes (3-40 subjects per group) and large standardized effect sizes (0.8-2.0). The generality of the tables for dependent samples and one-tailed tests is discussed. SSR methods should be of interest to ethics bodies governing research when it is desirable to limit the number of subjects tested, such as in studies of pain, experimental disease, or surgery with animal or human subjects.

Hyperosmotic intravenous infusions of NaCl are more potent for inducing drinking and vasopressin (AVP) secretion than equally osmotic solutions of glucose or urea. The fact that all three solutes increased cerebrospinal fluid osmolality and sodium concentration led the investigators to conclude that critical sodium receptors or osmoreceptors for stimulating drinking and AVP secretion were outside the blood-brain barrier (BBB) in the circumventricular organs (CVOs). We tested an obvious prediction of this hypothesis: that all three solutes should increase c-Fos-like immunoreactivity (Fos-ir) inside the BBB, but that only NaCl should increase Fos-ir in the CVOs. We gave intravenous infusions of 3.0 Osm/l NaCl, glucose, or urea to rats for 11 or 22 min at 0.14 ml/min and perfused the rats for assay of Fos-ir at 90 min. Controls received isotonic NaCl at the same volume. Drinking latency was measured, but water was then removed. Drinking consistently occurred with short latency during hyperosmotic NaCl infusions only. Fos-ir in the forebrain CVOs, the subfornical organ, and organum vasculosum laminae terminalis was consistently elevated only by hyperosmotic NaCl. However, all three hyperosmotic solutes potently stimulated Fos-ir in the supraoptic and paraventricular nuclei of the hypothalamus inside the BBB. Hyperosmotic NaCl greatly elevated Fos-ir in the area postrema, but even glucose and urea caused moderate elevations that may be related to volume expansion rather than osmolality. The data provide strong support for the conclusion that the osmoreceptors controlling drinking are located in the CVOs.

Circulating angiotensin II is crucial for the activation of salt appetite after sodium depletion. We tested if angiotensin (ANG) II infused intravenously at 50 ng/kg/min overnight (chronic) can mimic the rapid salt appetite similar to furosemide and overnight sodium depletion. In experiment 1, rats received chronic ANG II or vehicle infusions all night with access to water and chow but no saline solution. In the morning, the infusions continued, but half of the vehicle-infused group was switched to ANG II (acute). Thirty minutes after the switch, all rats received 10 mg/kg furosemide SC. One hour later they were provided water and 0.3 M NaCl to drink. Rats infused with vehicle or acute ANG drank little, but the chronic ANG group drank 11+/-1 ml of saline in 90 min. In experiment 2, the furosemide was omitted, and a group receiving a chronic infusion of phenylephrine at 6.25 microg/kg/min was included. The chronic ANG group drank 10+/-1 ml saline in 90 min, but the phenylephrine group, which also incurred a significant negative sodium balance overnight, drank little. Thus, an overnight infusion of ANG II is sufficient to mimic the robust expression of salt appetite as observed after furosemide and overnight sodium depletion.

Cumulative responses are important for the study of ingestive behavior. The total amounts consumed at several time points after experimental or control treatments are often analyzed using a mixed-model analysis of variance. The method used by some investigators to calculate the significance of this difference is invalid and may lead to serious errors of inference. Direct analysis of cumulative data can lead to grossly erroneous p-values for certain effects because data from early time points contribute disproportionately to the outcome. Analysis of uncumulated data yields the appropriate levels of significance and all of the same hypotheses can be tested.

We previously reported that an intracerebroventricular (icv) injection of the oxytocin receptor antagonist ornithine vasotocin (OVT) caused water and saline intakes, a pressor response, and Fos-like immunoreactivity (Fos-IR) in the median preoptic nucleus of the rat brain. In the present report, rats receiving an icv injection of isotonic saline vehicle followed by an icv injection of 10 microg of OVT 20 min later drank 5.5+/-1.1 ml of total water and saline intake in 60 min after the OVT; rats receiving 10 microg of losartan before the OVT drank only 0.9+/-0.3 ml of total fluid. In a separate study, rats were treated as above except that they were not allowed to drink and were perfused for analysis of Fos-IR in the median preoptic nucleus at 90 min. Fos-IR in the dorsal part of the median preoptic nucleus was significantly suppressed from 2.69+/-0.57 cells per 10,000 square mum in vehicle-treated rats to 0.89+/-0.20 in losartan-treated rats. Losartan alone had no effect on Fos-IR. Losartan did not reduce intake of saccharin in a dessert test. This suggests that the OVT-induced drinking may result from an activation or disinhibition of angiotensin type AT1 receptors in the median preoptic nucleus.

The lamina terminalis was severed by a horizontal knife cut through the anterior commissure to determine the effects of a disconnection of the subfornical organ (SFO) on drinking and Fos-like immunoreactivity (Fos-ir) in the rat brain in response to an intragastric load of hypertonic saline (5 ml/kg of 1.5 M NaCl by gavage). After an initial load, knife-cut rats drank significantly less water than sham-cut rats, thus confirming a role for the SFO in osmotic drinking. After a second load at least 1 wk later, the rats were not allowed to drink after the gavage and were perfused for analysis of Fos-ir at 90 min. Compared with sham-cut rats, the knife-cut rats displayed significantly elevated Fos-ir in the main body of the SFO, in the dorsal cap of the organum vasculosum laminae terminalis, and in the ventral median preoptic nucleus after the hypertonic load. The knife cut significantly decreased Fos-ir in the supraoptic nucleus. Fos-ir was expressed mainly in the midcoronal and caudal parts of the area postrema of sham-cut rats, and this expression was greatly reduced in knife-cut rats. These findings strengthen the case for the presence of independently functioning osmoreceptors within the SFO and suggest that the structures of the lamina terminalis provide mutual inhibition during hypernatremia. They also demonstrate that the Fos-ir in the area postrema after intragastric osmotic loading is heavily dependent on the intact connectivity of the SFO.

In many previous studies, one or the other forebrain circumventricular organ, the subfornical organ (SFO) or organum vasculosum laminae terminalis (OVLT), was lesioned to test whether it was critical for the behavioral or physiological responses to sodium depletion and hypernatremia. These studies conflict in their conclusions. The present study was designed to create discrete lesions of both the SFO and OVLT in the same animals and to compare these with rats having a lesion of only the SFO or OVLT. Both the OVLT-lesioned group and the combined SFO + OVLT-lesioned group drank significantly more water and saline on a daily basis than Controls or SFO-lesioned rats. In both sodium depletion and hypertonic saline testing, rats with SFO lesions displayed transient deficits in salt appetite or thirst responses, whereas the rats with single OVLT lesions did not. In the sodium depletion test, but not in the hypernatremia test, rats with lesions of both the SFO and OVLT exhibited the largest deficit. The data support the hypothesis that a combined lesion eliminates redundancy and is more effective than a single lesion in sodium depletion tests. The interpretation of the OVLT lesion-only data may have been complicated by a tendency to drink more fluid on a daily basis, because some of those animals drank copious water in addition to saline even very early during the salt appetite test.

Central injection of ANG II has been proposed to have dual effects on salt appetite including a direct stimulatory effect and an indirect inhibitory effect through an activation of central oxytocinergic neurons. The inhibition was demonstrated by pretreating rats with central ornithine vasotocin (OVT; oxytocin antagonist) 30 min before a central ANG II injection. The OVT pretreatment produced a large increase in ANG II-induced saline intake. The present paper reports a failure to replicate that influential experiment. However, we also report for the first time that OVT by itself: 1) provokes drinking of both water and saline solution with a latency almost as short as that produced by ANG II; 2) produces a mild pressor response; and 3) increases c-Fos expression in the organum vasculosum laminae terminalis (OVLT) and the median preoptic nucleus (MnPO). Oxytocin activity may provide an inhibitory control of drinking responses as has been suggested, but the inhibition is tonic and includes both water and saline drinking. Inhibition of this tonic activity may stimulate drinking by increasing neural activity in the OVLT and MnPO.

There are several indications that neurons in the rat's subfornical organ (SFO) are sensitive to internal calcium status. We investigated the role of the SFO in regulating calcium intake by comparing the consumption of 30 mM CaCl2 by rats with (a) lesions of >90% of the SFO, (b) lesions that left the SFO mostly intact but disconnected its rostroventral stalk, (c) misplaced lesions that spared most of the SFO, or (d) a sham lesion procedure. In one experiment involving calcium-replete rats, these four groups had similar CaCl2 intakes. In another experiment involving calcium-deprived rats, those with lesions of the SFO or its rostroventral stalk consumed less CaCl2 than did those with missed or sham lesions. The SFO therefore appears to play a role in the calcium appetite that accompanies calcium deprivation in rats, most likely through its rostroventral efferents, but it is not important for need-free calcium intake.

Circumventricular organs such as the subfornical organ (SFO) may mediate the effects of circulating angiotensin (ANG) II on salt appetite under conditions of sodium depletion in the rat. We studied the effects of an electrolytic lesion of SFO on salt appetite after adrenalectomy (ADX) in Long-Evans rats. The SFO lesion had no effect on saline intake, but it did abolish water intake after acute peripheral treatments with 2 mg/kg of captopril or a 10 mg/kg of furosemide. These findings contrast with other recent data from this laboratory demonstrating large reductions in salt appetite in adrenal-intact rats with lesions of either SFO or the organum vasculosum laminae terminalis during acute iv infusions of ANG II. Thus, the SFO may contribute to the salt appetite response to circulating ANG II, but it is not essential for the response to adrenalectomy., (Copyright 2002 Elsevier Science B.V.)

The subfornical organ (SFO) may act as a sodium- or osmoreceptor that drives hypothalamic and other nuclei to secrete vasopressin and to elicit drinking. However, in response to mild doses of hypertonic saline, Fos-like immunoreactivity (Fos-ir) is absent in the SFO whereas it is well expressed in the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei. This suggests that the hypothalamus may be activated in advance of the SFO. In this study, the fibers connecting the SFO and hypothalamus were disconnected by a wire knife cut so that Fos-ir could be examined in both the SFO and hypothalamus after an intragastric (ig) load of 0.5% of body weight of 0.6 M NaCl. Compared with Fos-ir in isotonic-loaded rats, Fos-ir after the hypertonic load was not significantly elevated in the SFO or median preoptic nucleus in sham-cut or knife-cut rats and was only slightly elevated in the OVLT in sham-cut rats. However, the hypertonic load in sham-cut rats greatly elevated Fos-ir in the SON and in the entire PVN, but this expression was reduced significantly by 30-50% in knife-cut rats. Thus, the connectivity between SFO and the hypothalamus is critical for the full expression of Fos-ir in the hypothalamus during moderate ig hypertonic saline loading even when the SFO itself does not yet express Fos-ir.

Two circumventricular organs, the subfornical organ (SFO) and organum vasculosum laminae terminalis (OVLT), may mediate salt appetite in response to acute intravenous infusions of angiotensin (ANG) II. Fluid intakes and mean arterial pressures were measured in rats with sham lesions or electrolytic lesions of the SFO or OVLT during an intravenous infusion of 30 ng/min ANG II. Beginning 21 h before the 90-min infusion, the rats were depleted of sodium with furosemide and given a total of 300 mg/kg captopril in 75 ml/kg water in three spaced gavages to block the usual salt appetite and to hydrate the rats. No other food or fluids were available for ingestion. Sham-lesioned rats drank 9.3+/-1.2 ml if 0.3 M NaCl alone was available and drank 8.9+/-1.6 ml of saline and 3.7+/-1.6 ml of water if both were available. Either SFO or OVLT lesions reduced the intakes of saline to <5 ml in both conditions and of water to <1 ml. Mean arterial pressure did not differ among the groups and was maintained above 100 mmHg after the depletion and captopril treatments because of the large doses of water. Thus, a full expression of salt appetite in response to an acute intravenous infusion of ANG II requires the integrity of both the SFO and OVLT., (Copyright 2002 Elsevier Science B.V.)

If receptors in the gut relay information about increases in local osmolality to the brain via the vagus nerve, then vagotomy should diminish this signaling and reduce both thirst and brain Fos-like immunoreactivity (Fos-ir). Water intake in response to hypertonic saline (i.p. or i.g., 1 M NaCl, 1% BW; i.g., 0.6 M NaCl, 0.5% BW) was reduced during 120 min in rats with subdiaphragmatic vagotomy (VGX) compared to sham-VGX rats. Brain Fos-ir was examined in response to both i.g. loads. After the smaller load, VGX greatly reduced Fos-ir in the supraoptic nucleus (SON) and the magnocellular and parvocellular areas of the paraventricular nucleus (PVN). Fos-ir in the subfornical organ (SFO) and nucleus of the solitary tract (NTS) was not affected. After the larger load, VGX significantly reduced Fos-ir in the parvocellular PVN and in the NTS, but not in the other regions. Thus, decreased water intake by VGX rats was accompanied by decreased Fos-ir in the parvocellular PVN after the same treatments, indicating a role for the abdominal vagus in thirst in response to signaling from gut osmoreceptors. The decreased water intake in the VGX group was not reflected as a decrease in Fos-ir in the SFO. Absorption of the larger i.g. load may have activated Fos-ir through more rapidly increasing systemic osmolality, thereby obscuring a role for the vagus at this dose in the SON and magnocellular PVN.

Rats in which a ligation of the bile duct (BDL) was paired with a saccharin taste developed a persistent conditioned taste aversion in both preference and taste reactivity tests. All BDL animals regardless of pairing had increased c-Fos-like immunoreactivity (FLI) in the area postrema and the nucleus of the solitary tract. This FLI may reflect the illness associated with BDL, but there was no evidence of conditioned FLI.

A depletion of sodium may increase sodium intake by increasing the synthesis of angiotensin (ANG) II in the blood and by stimulating ANG II receptors in the subfornical organ (SFO) of the rat. Lesions of SFO reportedly reduce these intakes. The present experiments tested the effects of SFO lesions on salt appetite after three successive depletions. After a furosemide-induced natriuresis, Long-Evans rats had free access to water- and sodium-deficient diet for 22 h. Water and 0.3 M NaCl were given for 2 h, and then the rats received regular chow, water, and 0.3 M NaCl until the next injection 5 or 7 days later. SFO lesions reduced water intake 1-2 h after each furosemide injection but not during the overnight periods. The lesions did not affect salt appetite the next day, 24-26 h after furosemide, but they did prevent the expected increase in the chronic daily 0.3 M NaCl intake after repeated depletions. The second experiment was similar to the first except that three subcutaneous injections of 100 mg/kg captopril were given at 1, 18, and 20 h after furosemide for the second depletion only. After the first depletion, the results were similar to those of the same condition of the first experiment. After the second depletion, captopril greatly reduced water intake and salt appetite in all rats including those with SFO lesions. Thus, we found that the lesion reduced chronic intake, but we did not replicate results showing large effects of SFO lesions on acute salt appetite. This residual acute appetite after SFO lesion remains dependent on the synthesis of ANG II.

A lesion of the subfornical organ (SFO) may disrupt drinking after a meal of dry chow as it does drinking after intragastric administration of hypertonic saline. Food and water intakes of SFO-lesioned (SFOX) and sham-lesioned rats were measured during 90-min tests following various lengths of food deprivation. During the tests, all rats began eating before they began drinking. After 20-24 h of food deprivation, latency to begin drinking after eating had started was longer for SFOX than for sham-lesioned rats. Plasma osmolality was elevated by 2-3% in both lesion groups at 12 min, the latency for sham-lesioned rats to drink, but SFOX rats nevertheless continued eating and delayed drinking. Eating after shorter 4-h food deprivations and ad libitum feeding produced more variable drinking latencies and less consistent effects of SFO lesion. During 24 h of water deprivation, SFO lesion had no effect on the suppression of food intake and did not affect food or water intakes during the first 2 h of subsequent rehydration. These findings indicate that the SFO is involved in initiating water intake during eating and in determining drinking patterns and the amount of water ingested during a meal.

A lesion of the subfornical organ (SFO) may reduce sodium depletion-induced salt appetite, which is largely dependent on ANG II, and yet ANG II infusions directly into SFO do not provoke salt appetite. Two experiments were designed to address this apparent contradiction. In experiment 1 sustained infusions of ANG II into SFO did not produce a sustained elevation of blood pressure, and neither a reduction of blood pressure alone with minoxidil and captopril nor a reduction of both blood pressure and volume with furosemide and captopril enhanced salt appetite. Infusions of ANG II in the organum vasculosum laminae terminalis (OVLT) did evoke salt appetite without raising blood pressure. In experiment 2 knife cuts of the afferent and efferent fibers of the rostroventral pole of the SFO abolished water intake during an infusion of ANG II into the femoral vein but failed to reduce salt appetite during an infusion of ANG II into the OVLT. We conclude that 1) hypertension does not account for the failure of infusions of ANG II in the SFO to generate salt appetite and 2) the OVLT does not depend on its connectivity with the SFO to generate salt appetite during ANG II infusions.

The induction of c-Fos-like immunoreactivity (c-FLI) in the nucleus of the solitary tract (NTS) has been shown to be correlated with behavioral expression of a conditioned taste aversion (CTA). However, because this cellular response is also dependent on an intact amygdala, it may represent the activation of a stress-related autonomic response. The present experiments addressed this possibility by evaluating the correlation between c-FLI in the intermediate division of the NTS (iNTS) and 2 measures of conditioned fear: freezing and changes in mean arterial pressure (MAP) and heart rate (HR). Exposure to the taste conditioned stimulus (CS) resulted in a marked induction of c-FLI in the iNTS, whereas exposure to a fear CS did not. Further, exposure to a taste CS did not selectively lead to increases in MAP or HR. Results suggest that induction of c-FLI in the iNTS may reflect the activation of a cell population whose function is unique to the CTA paradigm.

A ligation of the common bile duct (BDL) produces cholestasis and hypotension and increases the daily ingestion of sodium chloride solutions in rats. Low-dose captopril (CAP) treatment also modifies the ingestion of water and sodium in naive rats, and may do so in cholestatic rats. This study examined whether the elevated ingestion of saline by Long-Evans rats after BDL is associated with increased plasma renin activity (PRA), and whether treatment with a low dose of the angiotensin converting-enzyme inhibitor CAP further exacerbates fluid intake and PRA after BDL. In these experiments water and 0.3 M saline intake and PRA and plasma aldosterone (PA) were measured in naive and CAP-treated BDL and sham-ligated rats. We found that BDL elevated rats' daily saline intake 2 weeks after the ligation procedure but had no effect on PRA. CAP (0.1 mg/mL) placed in the drinking water of some BDL rats further increased saline intake. Both PA and hematocrits tended to be reduced in BDL rats, whereas PRA was elevated in both BDL and sham-ligated rats receiving CAP in the drinking water or by gavage (0.1 mg/mL in 10 mL/kg). The data suggest that the ingestion of saline by rats can be modified by BDL and CAP administration, but that exaggerated saline intake in BDL rats is not associated with excessive renin secretion.

After a chronic ligation of the common bile duct (BDL), Long-Evans rats are hypotensive and have elevated saline intake during both sodium-depleted and nondepleted conditions. We tested whether BDL rats have exaggerated hypotension during sodium depletion or an elevated dipsogenic response to angiotensin II (ANG II) that might help to explain the saline intake. After 4 wk of BDL, rats were hypotensive at baseline and developed exaggerated hypotension during acute furosemide-induced diuresis. Without saline to drink, BDL rats increased water intake during depletion equal to sham-ligated rats. However, with saline solution available at 22 h after sodium depletion, the BDL rats drank more water and saline than did sham-ligated rats. This rapid intake temporarily increased their mean arterial pressure to equal that of sham-ligated rats. Intravenous infusion of ANG II induced equal drinking responses despite reduced pressor responses in the BDL rats relative to sham-ligated rats during both ad libitum and sodium-depleted conditions. Thus BDL rats have exaggerated hypotension during diuresis, and their hypotension is corrected by drinking an exaggerated volume of saline, but they do not have an increased drinking response to ANG II.

Water intake was elevated in sodium-depleted rats during a daytime salt appetite test, but other rats drank a similar amount of water when saline was not available for drinking during the test. This water intake stimulated by sodium depletion was blocked by an inhibition of angiotensin (ANG) II synthesis with a high dose of captopril (100 mg/kg, sc). Captopril did not reduce water intake by causing hypotensive shock or uremia, because water and saline intakes were increased rather than decreased after a low dose of captopril (5 mg/kg) that also reduced blood pressure and elevated blood urea nitrogen. The water intake, but not salt appetite, induced by sodium depletion was greatly reduced by a lesion of the subfornical organ (SFO) in one-bottle tests, and this was not clearly related to any effects of the lesion on blood pressure. A physiological role for ANG II in water intake induced by sodium depletion has recently been disputed, but the simplest explanation for the data remains that elevated levels of circulating ANG II bind to receptors in the SFO to generate daytime water drinking during sodium depletion.

This study examined the hypothesis that the subfornical organ (SFO), a circumventricular organ with both osmosensitive elements and dipsogenic receptors for circulating angiotensin (ANG) II, is important for the water drinking response that follows an intragastric (ig) load of hypertonic NaCl. A 2-ml saline load was administered ig at 300, 900, or 1200 mOsm/kg to rats with sham lesions or lesions of the SFO, and intake was measured periodically for 2 h. Hypertonic loads caused sham-lesioned rats, but not SFO-lesioned rats, to drink earlier in the test or to drink more water than did the isotonic load. Inhibition of ANG II synthesis in unoperated rats with 100 mg/kg of captopril reduced water intake only during the initial 15 min after a gavage of 1200 mOsm/kg saline. Loads of 900 and 1200 mOsm/kg both increased plasma osmolality and sodium concentration by 15 min after gavage without greatly affecting hematocrit or plasma protein concentration. Thus, the SFO is important for the osmotically-induced water drinking response after acute ig administration of hypertonic saline. With the possible exception of the first 15 min, this drinking response is independent of the peripheral synthesis of ANG II., (Copyright 1998 Elsevier Science B.V. All rights reserved.)

We examined whether a learned aversion to saline could account for the reduction in saline intake produced by bile duct ligation (BDL) in rats and whether increased saline intake by BDL rats was associated with hypotension. In three experiments, rats were given continuous access to water in choice with saline after surgery. In Experiment 1, rats were deprived of food and fluid for 24 h and then given 2-h access to either 0.15 or 0.3 M saline. Rats received a BDL or sham-ligation immediately (paired) or 48 h after (nonpaired) the 2-h bout of saline ingestion. The results show that nonpaired BDL rats increased their daily saline intake relative to nonpaired sham-ligated or paired BDL rats approximately 1-4 weeks after surgery. In Experiment 2, when water and either cherry or grape Kool-Aid (0.05% w/v) dissolved in 0.15 M saline to distinguish the flavor of the solution was offered prior to surgery, BDL rats reduced their ingestion of grape-flavored saline after surgery regardless of whether they were exposed to grape- or cherry-flavored saline prior to surgery. In Experiment 3, when rats were offered water and 0.3 M saline 48 h after surgery, BDL rats ligated for 4 weeks increased their saline intake relative to sham-ligated controls and this elevation in saline intake by BDL rats was associated with hypotension. The results suggest that the symptoms associated with the BDL surgery can serve as effective unconditioned stimuli in the acquisition of learned flavor aversions, and that hypotension may play a role in the elevated intake of saline by BDL rats.

The authors investigated whether a depletion of sodium with furosemide enhanced the water and 0.3 M NaCl intakes of rats with experimental cholestasis, portal hypertension or congestive heart failure. These were induced, respectively, by bile duct ligation (BDL), portal vein constriction or vena cava constriction. BDL alone increased daily saline intake. In BDL rats, but not in sham-ligated controls, experience with a prior depletion of sodium enhanced the 2-h saline intake and the retention of water and sodium after a subsequent depletion. Chronic cava constriction, but not portal constriction, enhanced sodium intake and retention after sodium depletion during a 2-h test and enhanced water intake overnight after the test. The results suggest that the ingestion of sodium by BDL and cava-constricted rats may share a common mechanism.

The effect of a ligation of the common bile duct (BDL) on the chronic free-selection intake of ethanol was investigated. Rats were given a choice between water and a solution of either 6% (v/v) ethanol, 0.06% (w/v) sodium saccharin, or a mixture of both ethanol and saccharin. In different experiments, solutions were first presented either 3 weeks before surgery, about the time of surgery, or 2 weeks after surgery. Reductions in ethanol or saccharin intake were observed in BDL rats whenever the solutions were first presented either 3 weeks before or shortly after the surgery. No differences attributable to BDL were seen when ethanol solutions were first presented 2 weeks after surgery. The contingent nature of the effect suggests that the reduction results from a conditioned taste aversion rather than from differences in ethanol metabolism, sensitivity, or neurohormones such as angiotensin. The findings urge caution in the monitoring of the dietary habits of patients with a rapidly developing biliary obstruction.

The authors tested whether the level of hydration after furosemide diuresis and 22 hr of sodium depletion affects the amount of water or 0.3 M NaCl solution consumed by rats with intact brains or with lesions of the subfornical organ (SFO). Rats received 2 (underhydrated) or 10 (euhydrated) ml/kg water by gavage as the only fluid input 2, 4, and 20 hr after 10 mg/kg furosemide. These hydration treatments had little or no effect on the amount of saline consumed in 2 hr by intact rats. SFO lesions reduced water intake regardless of hydration condition. Euhydrated, SFO-lesioned rats drank a normal amount of saline, but underhydrated, lesioned rats drank less saline than any other group. Thus, euhydration may facilitate salt appetite in SFO-lesioned rats, and the deficits in salt appetite noted in SFO-lesioned rats may result from deficits in water ingestion rather than from a destruction of angiotensin II receptor sites that directly provoke salt appetite.

A role for the renal renin-angiotensin system in the direct stimulation of salt appetite in the rat remains controversial because attempts to elicit the behavior by intravenous administration of angiotensin II (ANG II) have been unconvincing. We recently demonstrated that depletion-induced salt appetite was attenuated by selective blockade of peripheral ANG II synthesis with an intravenous dose of converting enzyme inhibitor [captopril (Cap)] that does not block the synthesis of ANG II inside the blood brain barrier. We now show that intravenous ANG II at 30 ng/min rapidly reestablishes salt appetite in Cap-blocked rats. The mean arterial blood pressure (MAP) of unblocked, sodium-depleted rats was normal, but Cap-blocked, depleted rats had low MAP. An intravenous infusion of ANG II in Cap-blocked rats brought MAP into the normal range and elicited water and salt drinking within 90 min. Phenylephrine also normalized MAP but failed to elicit fluid intake in Cap-blocked, sodium-deficient rats. Sodium and water balances tended to be more positive during ANG II than during phenylephrine infusions. Thus circulating ANG II may stimulate both thirst and salt appetite by a direct action on the brain and not by causing natriuresis or by raising the blood pressure.

Thirst elicited by the beta-adrenergic agonist isoproterenol in rats depends in part on the secretion of renin, the consequent synthesis of angiotensin II (ANG II), and the binding of circulating ANG II to dipsogenic receptors in the brain. These receptors probably reside in either of two forebrain circumventricular organs, the subfornical organ (SFO) or organum vasculosum laminae terminalis (OVLT). Experiments determined that lesions of the SFO, but not of the OVLT, reduced drinking induced by isoproterenol treatment. Competitive ANG II-receptor antagonism with sarthran reduced isoproterenol-induced drinking when the blocker was infused into the SFO but not when it was infused into the OVLT or into the lateral ventricles at a 25-fold greater dose. The findings confirm the widely held belief that renin-dependent thirst elicited by isoproterenol relies on ANG II binding to receptor sites at a circumventricular organ in the brain. The results demonstrate that this site is the SFO and not the OVLT.

The prevailing theory of sodium depletion-induced salt appetite states that angiotensin (ANG) of cerebral origin, not of renal origin, causes the behavior. This assertion depends partly on an experiment that suppressed salt appetite using an intravenous infusion of a dose of angiotensin-converting enzyme (ACE) inhibitor (captopril, 2.5 mg/h) that supposedly blocked both central and peripheral enzyme (17). The present experiments used the same dose of captopril to suppress salt appetite both in 24-h and in 3-h models of sodium depletion-induced behavior. The captopril-infused rats 1) increased water intake normally after central injections of ANG I given immediately after each salt appetite test, 2) had no arterial pressor response after intravenously injected ANG I, and 3) had normal arterial pressor responses after either intravenously injected ANG II or centrally injected ANG I. Thus, although the peripheral ACE was completely blocked in captopril-infused rats, the central ACE was not, because the central thirst and arterial pressor responses to ANG I were indistinguishable from controls. This demonstrates that ANG of renal origin is necessary for the expression of sodium depletion-induced salt appetite.

Chronic peripheral treatments with low doses of the angiotensin-converting enzyme inhibitor, captopril, enhance daily intakes of dilute ethanol solutions in rats as they do the intakes of water and saline solutions. Placing captopril into the drinking water or infusing it SC increases daily intake of 6% (v/v) ethanol from 30-100% over 4-12 days of treatment. The present study examined the effects of electrolytic lesions either of the subfornical organ (SFO) or of the organum vasculosum laminae terminalis (OVLT), on captopril-enhanced ethanol intake. Captopril was infused in minipumps at 5 mg/day for 14 days. The intake of 6% (v/v) ethanol was abolished by SFO lesions and was temporarily reduced by OVLT lesions. The SFO, in particular, is essential for the expression of enhanced ethanol intake during low-dose peripheral captopril administration. Local angiotensin II synthesis and receptor activation at the SFO appear to be the mechanism of the enhanced ethanol drinking during captopril.

Reportedly both angiotensin II (ANG II) and angiotensin-converting enzyme (ACE) inhibitors reduce ethanol intake when they are injected SC into certain chronic experimental conditions in the rat. The ACE inhibitors are suggested to reduce ethanol intake by increasing ANG II synthesis in the brain. The present results show that several different methods can produce opposite effects of ANG II and the ACE inhibitor captopril on ethanol intake. Continuous intraventricular infusions of ANG II for 7 days or low doses of oral or SC-infused captopril for up to 12 days increased the intake of ethanol. The only reduction of ethanol intake resulted from a universal blockade of all ACE in both the brain and periphery, a condition in which ANG II could not possibly mediate the decrease. The results contradict the hypothesis that ethanol intake is suppressed by centrally acting or centrally synthesized ANG II. ACE inhibitors may reduce ethanol intake only when they affect the brain as well as the periphery.

Sodium depletion in rats elicits a sodium appetite that results from a cerebral action of angiotensin II (ANG II) and aldosterone. Alcohol also activates the renin-angiotensin system, but the mechanism is poorly understood and not related to sodium excretion. In this study, 2.5 g/kg ip ethanol produced a 20% decline in plasma volume and plasma protein concentration in 1-2 hr and elicited salt appetite beginning in 3-4 hr. Blockade of ANG II synthesis in the brain and periphery with the angiotensin-converting enzyme inhibitor captopril eliminated the thirst and salt appetite. Peripheral captopril alone enhanced fluid intake, which indicated that alcohol elevated renin secretion. Ethanol-induced suppression of hepatic plasma protein secretion and the consequent fall in plasma colloid osmotic pressure apparently resulted in hypovolemia and renin secretion, which then produced thirst and salt appetite through an action of ANG II on the brain.

Lesions of the ventral ventral median preoptic nucleus (VVMnPO) enhanced daily salt appetite induced by subcutaneous (sc) injections of deoxycorticosterone acetate (DOCA) but did not affect acute salt appetite or water intake after sc injections of 5 mg/kg of the alpha-2-adrenoreceptor blocker yohimbine. Lesions of the subfornical organ (SFO) or its rostral fiber pathways had no effect on fluid intakes during DOCA treatments but significantly reduced water intake after yohimbine. These findings extend those of a previous report (Fitts, Tjepkes, & Bright, 1990) of enhanced DOCA-induced saline intake in VVMnPO-lesioned rats and demonstrate that the effect is specific to lesions of the VVMnPO. The mechanism of the thirst and salt intake elicited by yohimbine is still unclear, but the SFO and its fiber pathways appear to be important for the expression of the water drinking component. Neither lesion reliably affected yohimbine-induced salt appetite.

Angiotensin receptors in the most ventral part of the ventral median preoptic nucleus (VVMnPO) or organum vasculosum laminae terminalis appear to be important for salt appetite to angiotensin in rats. If so, then small lesions of this region should reduce salt appetite that is dependent on angiotensin. In separate experiments, the lesion greatly reduced salt appetite after treatments with chronic oral captopril or sodium depletion. On the other hand, the VVMnPO lesion actually enhanced salt appetite to deoxycorticosterone acetate. The lesion did not affect water intake to water deprivation, combined food-water deprivation, isoproterenol, or hypertonic saline, and basal plasma osmolality and sodium values were normal. These experiments suggest that VVMnPO lesions selectively affect angiotensin-induced salt appetite without producing the gross hydrational deficits that occur with larger lesions of the ventral forebrain.

Two experiments were designed to test whether angiotensin (ANG) synthesis or receptor activation in the ventral preoptic region is critical for ANG-induced salt appetite in rats. In Experiment 1, infusions of ANG into the subfornical organ (SFO) produced water drinking without saline intake, but infusions near the organum vasculosum laminae terminalis (OVLT) produced both water and saline drinking. Thus, forebrain areas that support water drinking to ANG do not all support salt appetite. In Experiment 2, rats were given oral captopril (CAP) to enhance daily intake of water and saline solution by increasing ANG II synthesis in the brain. CAP microinjected into the SFO reduced CAP-enhanced water drinking without affecting saline intake, but CAP in the OVLT reduced enhanced saline intake without affecting water drinking. Thus, ANG acting in the OVLT, the most ventral part of the median preoptic nucleus, or other nearby structures is important for ANG-induced salt appetite.

Three experiments tested whether the subfornical organ (SFO) could be a site of action for the antidipsogenic effects of atrial natriuretic peptide (ANP) in rats. Pretreatment with 100, 230, or 500 pmol ANP in the SFO reduced drinking induced by 10 pmol angiotensin II in the SFO. Drinking in response to water deprivation was reduced by ANP in rats having cannulas in or near the SFO, but not in rats having cannulas distant from the SFO or in the ventricles. Finally, ANP had no effect on eating or drinking after food deprivation, suggesting that the rats in the other experiments were not acutely incapacitated. The SFO may mediate the central effects of ANP on drinking induced by angiotensin or in response to water deprivation and could play a similar role in the central effects of ANP on salt appetite, diuresis, vasopressin secretion, and blood pressure.

Preference for NaCl solutions as a function of concentration was examined in three inbred strains of rats (Munich-Wistar, Buffalo, and Fischer-344) and outbred Wistar rats. Fischer-344 rats were found to differ markedly from the other strains in that they failed to prefer any concentration of NaCl solution to water. This difference did not appear to be due to an insensitivity to the taste of NaCl since they significantly avoided solutions at concentrations (greater than 0.7%) that were strongly preferred by other rat strains. Differences between Fischer-344 and other rat strains also did not appear to be due to a generally anomalous taste system since their intake and preference for prototypes of other basic tastes (sweet, sour, and bitter) were generally similar to those of other strains. Although Fischer-344 rats were somewhat slower than Wistar to excrete a sodium load, it is unlikely that this difference can provide a direct explanation of the aversion of Fischer-344 rats for NaCl.

Several experiments tested whether the subfornical organ (SFO) is necessary for water or NaCl intake arising from angiotensin-converting enzyme (CE) blockade with captopril (CAP) in the drinking fluids (0.1 mg/ml). Peripheral CAP was given to rats acutely following 24-h water deprivation or chronically over several days, either with water alone available for drinking or with 0.3 M NaCl in choice with water. Control rats drank more water, and NaCl when it was available, during CAP treatment, whereas rats with SFO damage increased NaCl intake only. CAP decreased urinary volume (UV) and increased urinary potassium excretion (UKV) during rehydration with only water available and increased urinary sodium excretion (UNaV) with NaCl present. Regardless of CAP treatment, rats with SFO damage had increased electrolyte concentrations and excretions during rehydration with only water available and increased UK with NaCl present, compared with controls. Oral CAP did not have an aversive taste at the dose used here. We conclude that the SFO is necessary for CAP-enhanced water, but not NaCl, intake and suggest that different neurological mechanisms control ingestion of each fluid during CAP.

Two methods of sodium loading were used to counteract the body fluid dilution resulting from natriuresis and water drinking during sustained lateral ventricular infusions of carbachol (CBC) or angiotensin II (ANG II) in rats. It was expected that preventing dilution would also prevent the precipitous decline of both drinking and natriuresis during the later hours of CBC infusion. In the first study, rats having isotonic saline as the sole drinking fluid during CBC infusions drank less fluid and had only slightly higher plasma osmolality and sodium concentration than rats drinking water, which showed extreme dilution. In the second study, rats with only water to drink were given intravenous infusions of 0.15, 0.45, or 1.00 M NaCl solutions at 1.8 ml/h concurrently with the intraventricular infusions. Significant dilution of plasma was found at the two lower rates but not at 1.00 M NaCl in CBC-infused rats. Only the latter group showed both persistent drinking and natriuresis throughout the 4-h infusion period, and this was not because of elevated plasma osmolality. Infusions of ANG II generated less severe body fluid dilution and more persistent drinking in both experiments. The study demonstrates that body fluid dilution may control the offset of both drinking and natriuresis during sustained infusions of CBC and that the more persistent drinking to ANG II vs. CBC probably occurs because of a lesser natriuresis and consequent fluid dilution.

These experiments were conducted to test whether drinking to ip captopril or to intraventricular carbachol requires an intact fiber system from the ventral subfornical organ (SFO). Wire-knife cuts were made through the wall of the third ventricle ventral to the SFO. Control rats had either sham lesions or histologically identified missed cuts. Rats with good cuts (a) drank less than either control group after ip injections of 4 mg/kg captopril, (b) drank normal amounts of 0.3 M NaCl solution when captopril was placed in the drinking water at 0.1 mg/ml, (c) drank less water but a normal amount of saline after 6 mg/kg captopril ip and 10 mg/kg furosemide diuretic ip, and (d) drank normal amounts of water after lateral ventricular injections of 1.2 or 4 nmol of carbachol. The results of the captopril experiments confirm predictions based on studies of SFO lesions and suggest that captopril causes water, but not saline, drinking via an angiotensin-related mechanism acting at the SFO. The carbachol experiment indicates either that the SFO is not a unique receptor site for ventricular carbachol or that the fibers mediating this response do not require the pathways through the ventral pole of the SFO.