Influence of age and splanchnic nerve on the action of melatonin in the adrenomedullary catecholamine content and blood glucose level in the avian group

Summary A single intraperitoneal (IP) melatonin injection (0.5 mg/100 g body wt.) caused an increase in norepinephrine (NE) fluorescence and elevation of NE content in newly-hatched pigeons (Columba livia), but a reduction of NE fluorescence and depletion of NE content in the adrenal medulla of newly-hatched crows (Corvus splendens) after 0.5 h of treatment. In contrast, in adults melatonin caused increase in NE fluorescence and elevation of NE content only in the parakeet (Psittacula krameri).Half an hour of IP melatonin treatment (0.5 mg/100 g body wt.) induced release of epinephrine (E) from the adrenal medulla of newly-hatched pigeon and parakeet. In contrast, in the adults melatonin caused more than a two-fold increase in E in the pigeon, and a significant increase in the crow.Single IP melatonin injection (0.5 mg/100 g body wt.) caused hypoglycemia in the newly-hatched parakeet and adult pigeon, and hyperglycemia in newly-hatched pigeon after 0.5 h of treatment. Melatonin failed to regulate glucose homoeostasis in newly-hatched and adult crow.Splanchnic denervation of the left adrenal gland was performed in the adult pigeon. The right adrenal served as the innervated gland. Melatonin-induced modulation of catecholamines following a single IP injection (0.5 mg/100 g body wt.) revealed significant increases in NE fluorescence and NE content at 4 and 12 h after treatment in the denervated gland only, which gradually approached normal levels 9 days after treatment. In contrast, E content showed more than a two-fold increase over the control value in both the innervated and denervated glands 0.5 and 24 h after treatment. At 9 days after treatment, E content showed significant depletion in the innervated gland.The results of this study indicate that melatonin modulates catechol hormone content in avian adrenal medulla, and also regulates glucose homoeostasis (except in the crow). The splanchnic nerve plays a vital role in the synthesis of NE but has no effect on E.     

Differential In Vivo Regulation of mRNA Encoding the Norepinephrine Transporter and Tyrosine Hydroxylase in Rat Adrenal Medulla and Locus Ceruleus

Abstract: To investigate the regulation of norepinephrine transporter mRNA in vivo, we analyzed the effects of reserpine on its expression in the rat adrenal medulla and locus ceruleus. First, PCR was used to clone a 0.5-kb rat cDNA fragment that exhibits 87% nucleotide identity to the corresponding human norepinephrine transporter cDNA sequence. In situ, the cDNA hybridizes specifically within norepinephrine-secreting cells, but in neither dopamine nor serotonin neurons, suggesting strongly it is a partial rat norepinephrine transporter cDNA. Reserpine, 10 mg/kg administered 24 h premortem, decreased steady-state levels of norepinephrine transporter mRNA in the adrenal medulla by ∼65% and in the locus ceruleus by ∼25%, as determined by quantitative in situ hybridization. Northern analysis confirmed the results of the in situ hybridization analysis in the adrenal medulla but did not detect the smaller changes observed in the locus ceruleus. Both analyses showed that reserpine increased tyrosine hydroxylase expression in the adrenal medulla and locus ceruleus. These results suggest that noradrenergic neurons and adrenal chromaffin cells can coordinate opposing changes in systems mediating catecholamine uptake and synthesis, to compensate for catecholamine depletion.     

Influence of age and splanchnic nerve on glucagon-induced changes of adrenomedullary catecholamine content and blood glucose level in the avian group

Summary Glucagon (0.1 mg · 100 g body wt^-1) increased norepinephrine (NE) content in adult pigeon (31%) and parakeet (58%), decreased NE content in the adrenal medulla of newly-hatched pigeon (36%), parakeet (52%), and crow (44%) 0.5 h after treatment. Epinephrine (E) content decreased to 26% and 59% of control values, respectively, in newly-hatched pigeon and parakeet 0.5 h after treatment. Glucagon produced hyperglycemia irrespective of age and species. The results indicate that aging modulates glucagon-induced changes of catecholamine (CA) content. In the innervated (I) adrenal gland of pigeon, glucagon caused a 31% increase of NE content 0.5 h after injection, a 46% decrease of NE content 12 h after injection, and a 192% increase of NE 24 h after injection. In the I gland of pigeons, glucagon also caused a 61% decrease of E content 4 h after injection, and brought about a 100% increase of E 24 h after injection. Glucagon-induced changes of CA content differ significantly between the I and denervated (D) glands. The results indicate that the splanchnic nerve regulates release and/or resynthesis of CA induced by glucagon.Abbreviations ANOVA analysis of variance - CA catecholamine - D denervated - E epinephrine - I innervated - MS mean sum of squares - NE norepinephrine - PNMT phenylethanolamine-N-methyl transferase - SS sum of squares - SV source of variation - TH tyrosine hydroxylase     

Role of dopamine in the modulation of adrenal gland responses in the osmotically stressed pigeons, Columba livia

Salt loading on pigeons (C. livia) had stimulatory effects on brain amines (dopamine and 5-hydroxytryptamine), corticosterone, norepinephrine and epinephrine contents of adrenal gland. Conjoint administration of dopamine with hypertonic saline restored the brain amines and corticosterone of adrenal gland, but had no effect on catecholamine (CAM) contents of adrenal medulla. The excessive release of CAM in the plasma indicates sympathetic stimulation after both the treatments.     

Central Regulation of Adrenal Tyrosine Hydroxylase: Effect of Induction on Catecholamine Levels in the Adrenal Medulla and Plasma

The effect of induction of adrenal tyrosine hydroxylase (TH) by various centrally acting drugs on catecholamine levels in adrenal and plasma was investigated in rats. All the drugs tested, namely oxotremorine, Piribedil, B-HT 920, and HA-966, produced significant increases in adrenal dopamine content and plasma epinephrine level. Denervation of the adrenal abolished the increase in adrenal dopamine as it did the induction of tyrosine hydroxylase. The results suggest that the induced increase of adrenal TH activity, as mediated by certain drugs, results in an elevation of the plasma epinephrine level and that the adrenal dopamine content is a better indicator of the catecholamine-synthesizing capacity of the adrenal medulla than are the other catecholamines.     

Effects of nitric oxide synthase inhibitor on tyrosine hydroxylase mRNA in the adrenal medulla of spontaneously hypertensive and Wistar Kyoto rats.

We studied the effects of N(G)-nitro-l-arginine methyl ester (L-NAME) on catecholamine levels, tyrosine hydroxylase (TH) activity, and TH mRNA levels in the adrenal medulla of spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY). L-NAME (100 mg/L in drinking water) and atropine (10 mg/L in drinking water) were administered for 2 weeks. Epinephrine and norepinephrine levels, TH activity, and TH mRNA levels in the adrenal medulla of L-NAME-treated WKY were significantly decreased. These parameters were not significantly altered in the adrenal medulla of L-NAME-treated SHR. Nitrite/nitrate levels in the adrenal medulla of L-NAME-treated WKY were significantly decreased; however, no significant change in L-NAME-treated SHR was observed. Ca(2+)-dependent nitric oxide synthase (NOS) activity in the adrenal medulla of SHR was significantly decreased compared to that of WKY. TH mRNA levels in L-NAME + atropine-treated and L-NAME-treated WKY were significantly lower than TH mRNA levels in control WKY. These results suggest that nitric oxide in the adrenal medulla may enhance the catecholamine biosynthetic pathway via increased TH mRNA expression. Our results also suggest that this effect is suppressed in SHR due to decreased NOS activity in the adrenal medulla.     

Role of TrkB expression in rat adrenal gland during acute immobilization stress

Expression of tyrosine receptor kinase B (TrkB), a receptor for brain‐derived neurotrophic factor (BDNF), is markedly elevated in the adrenal medulla during immobilization stress. Catecholamine release was confirmed in vitro by stimulating chromaffin cells with recombinant BDNF. We investigated the role of TrkB and the localization of BDNF in the adrenal gland during immobilization stress for 60 min. Blood catecholamine levels increased after stimulation with TrkB expressed in the adrenal medulla during 60‐min stress; however, blood catecholamine levels did not increase in adrenalectomized rats. Furthermore, expression of BDNF mRNA and protein was detected in the adrenal medulla during 60‐min stress. Similarly, in rats undergoing sympathetic nerve block with propranolol, BDNF mRNA and protein were detected in the adrenal medulla during 60‐min stress. These results suggest that signal transduction of TrkB in the adrenal medulla evokes catecholamine release. In addition, catecholamine release was evoked by both the hypothalamic–pituitary–adrenal axis and autocrine signaling by BDNF in the adrenal gland. BDNF–TrkB interaction may play a role in a positive feedback loop in the adrenal medulla during immobilization stress.     

Presence of the Novel Pituitary Protein „7B2” in Bovine Chromaffin Granules: Possible Co-Release of 7B2 and Catecholamine as Induced by Nicotine

We observed the presence of the novel pituitary protein "7B2" and its release in the bovine adrenal medulla. The 7B2 concentration (mean +/- SEM) in extracts of the bovine adrenal medulla was 952 +/- 155 pg/mg tissue (n = 6). 7B2 was distributed in the chromaffin granule fraction prepared from the bovine adrenal medulla and was released by high K+ and/or nicotine from cultured cells of the bovine adrenal medulla. Co-release of 7B2 with catecholamine induced by nicotine from the cultured bovine chromaffin cells was also observed. In an analysis of the bovine adrenal medulla chromaffin granule fraction on gel permeation chromatography, there was a major peak with an apparent molecular weight of 45,000, whereas a major peak with an apparent molecular weight of 20,000 was found in that on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On reverse-phase HPLC, a major peak with a retention time of 35 min was observed in the bovine chromaffin granule fraction and in the bovine anterior pituitary extract. These findings indicate that 7B2 is a secretory protein in the bovine adrenal medulla. The possibility that 7B2 might be released with catecholamine, possibly in response to stress, warrants investigation.     

Restoration of Catecholamine Content of Previously Depleted Adrenal Medulla In Vitro: Importance of Synthesis in Maintaining the Catecholamine Stores

The functional integrity of adrenal chromaffin storage vesicles was studied in the perfused rat adrenal gland subjected to intense exocytosis. Continuous perfusion with 55 mM K+-Krebs solution produced a large and uninterrupted secretion of catecholamines. Total amounts secreted within 45 min were 4.66 micrograms and represented almost 30% of the total tissue catecholamine content. If perfusion with excess K+ was extended to 90 min, the secretion increased further to 5.76 micrograms. Despite such a large secretory response, the catecholamine content of the K+-stimulated adrenal medulla was comparable to that of unstimulated control, suggesting an enhanced resynthesis to maintain the normal levels. Pretreatment of rats with alpha-methyl-p-tyrosine, and including this agent in the perfusion medium during stimulation with K+, caused a marked reduction in catecholamine content. The degree of depletion depended on the extent of stimulation with K+ (45% in 45 min and 60% in 90 min). Although depleted catecholamine stores did not show spontaneous recovery in 2 h, inclusion of tyrosine, L-3,4-dihydroxyphenylalanine or dopamine (but not epinephrine or norepinephrine) completely restored the catecholamine content of previously depleted adrenal medulla. Repletion achieved by tyrosine was time dependent (evident in 30 min and maximum in 2 h) and blocked by alpha-methyl-p-tyrosine but not by calcium deprivation. The ratio of epinephrine to norepinephrine remained constant during various stages of the experiment, suggesting both types of vesicles were equally affected by different treatments. The secretory response (10 Hz for 30 s) was unaffected even though tissue catecholamine stores were significantly depleted (50%). In summary, we have demonstrated that catecholamine content of the isolated perfused adrenal gland can be reduced by stimulation of exocytotic secretion in the presence of tyrosine hydroxylase inhibitor. Since the depleted stores can be fully refilled by synthesis of catecholamines from its precursors, it is suggested that chromaffin vesicles may be reutilized for the purpose of synthesis, storage, and secretion of adrenal medullary hormones.     

Modulation of catecholamine release by endogenous adenosine in the rat adrenal medulla

Adenosine was shown to inhibit norepinephrine (NE) release from sympathetic nerve endings. The purpose of this study was to examine whether endogenous adenosine restrains NE and epinephrine release from the adrenal medulla. The effects of an adenosine receptor antagonist, 1,3-dipropyl-8-(p-sulfophenyl) xanthine (DPSPX), on epinephrine and NE release induced by intravenous administration of insulin in conscious rats were examined. Plasma catecholamines were measured by HPLC with an electrochemical detector. DPSPX significantly increased plasma catecholamine in both control rats and rats treated with insulin. The effect of DPSPX on plasma catecholamine was significantly greater in rats treated with insulin. Additional experiments were performed in adrenalectomized rats to investigate the contribution of the adrenal medulla to the effect of DPSPX on plasma catecholamine. The effect of DPSPX and insulin on epinephrine in adrenalectomized rats was significantly reduced compared with that of the controls. Finally, we tested whether endogenous adenosine restrains catecholamine secretion partially through inhibiting the renin-angiotensin system. The effect of DPSPX on plasma catecholamine in rats pretreated with captopril (an angiotensin-converting enzyme inhibitor) was reduced. These results demonstrate that under basal physiological conditions, endogenous adenosine tonically inhibits catecholamine secretion from the adrenal medulla, and this effect is augmented when the sympathetic system is stimulated. The effect of endogenous adenosine on catecholamine secretion from the adrenal medulla is achieved partially through the inhibitory effect of adenosine on the renin-angiotensin system.     

Adrenal growth and ontogeny of adrenal substance P, enkephalins and catecholamines in the ovine fetus

Biologically active peptides have been identified in the adrenal glands of several adult mammalian species. Some of these peptides appear to modulate the nicotine-induced catecholamine release from the adrenal medulla. The present study was carried out to investigate the presence and ontogeny of the peptides substance P, met-enkephalin and leu-enkephalin in the ovine fetal adrenal gland from 70 to 140 days gestation (term = 145-150 days). Concurrently, the growth of the fetal adrenal as well as the gestational changes in catecholamine content were determined. The maternal adrenal glands were also studied for comparison. The ovine fetal adrenal gland increased in weight with advancing gestation at a single exponential rate. Total adrenal substance P content correlated with gestational age, while met-enkephalin, leu-enkephalin and total catecholamine contents correlated with adrenal weight. The adrenal content (normalized as per unit protein) of substance P was highest in the young fetuses at 70 days gestation, decreased progressively towards term and, in the adult levels were significantly lower than those measured in the fetuses. The contents of met-enkephalin and leu-enkephalin were low in the young fetuses at 70 days gestation, but reached high levels at 130 to 140 days gestation. Maternal adrenal contents of the two enkephalins were significantly lower than those measured in the near-term fetal adrenal. Total catecholamine content in the fetal adrenal medulla increased as the fetus matured. Norpinephrine was the primary catecholamine present in the medulla of fetuses at 70 and 80 days gestation, while epinephrine was the major one in the adult.(ABSTRACT TRUNCATED AT 250 WORDS)     

大鼠肾上腺髓质儿茶酚胺分泌的在体伏安法测定

应用碳纤微电极直接测定大鼠肾上腺髓质中儿茶酚胺浓度的在体伏安法。首次报道了大鼠肾上腺髓质中儿茶酚胺的基础浓度为０．１－０．５μｍｏｌ／Ｌ,缺血时髓质中儿茶酚胺的浓度显著增加,严重缺血时可达到５－３０μｍｏｌ／Ｌ。肾上腺髓质能自发分泌儿茶酚胺,缺血时自发分泌儿茶酚胺的幅度和频率都大大增加,提示缺血对大鼠是一种强烈的应激,测定结果还表明,乙酰胆碱能剂量依赖性地刺激肾上腺髓质嗜铬细胞分泌儿茶酚胺。     

Chromogranin A, the major catecholamine storage vesicle soluble protein. Multiple size forms, subcellular storage, and regional distribution in chromaffin and nervous tissue elucidated by radioimmunoassay

Chromogranin A (CgA), the major catecholamine storage vesicle (CSV) soluble protein, may index exocytotic sympathoadrenal secretion. To explore CgA in adrenergic tissues, we developed a radioimmunoassay for bovine CgA. Within adrenal medulla CSV, several minor chromogranins had similar amino acid compositions and peptide maps to that of CgA and also showed parallel, partial cross-reactivity in the CgA radioimmunoassay. CgA immunoreactivity represented 7 +/- 1% of total adrenal medulla cell protein and was localized to adrenal CSV, representing 46 +/- 2% of CSV soluble protein. In brain, there was 1000-fold less CgA than in adrenal medulla, with a widespread regional distribution (maximal in neocortex) and an unusual subcellular distribution (maximal in cytosol), both of which differ from reported catecholamine distribution. Brain chromogranin immunoreactivity also had a lower Stokes radius than adrenal CgA. Sympathetic nerve and serum had 6,000-fold and 30,000-fold less CgA than that in adrenal medulla. The results suggest a "family" of adrenal medulla chromogranins, similar structurally and immunoligically. Adrenal medulla and brain chromogranin differ in concentration, subcellular localization, and molecular size. Finally, CgA in serum may provide a useful tool for sympathoadrenal studies in intact organisms.     

Fetal adrenal VIP: distribution and effect on medullary catecholamine secretion

Vasoactive intestinal peptide (VIP) was found in the adrenal gland of ovine fetuses at 130-135 days gestation and was shown to stimulate catecholamine secretion. VIP was demonstrated by immunocytochemistry using the indirect antibody-enzyme method. VIP-immunoreactive nerve fibers were observed in the capsule, zona glomerulosa and inner layer of the cortex as well as in the medulla; furthermore small clusters of VIP-containing cell bodies were found at the corticomedullary border. To study the direct effect of VIP on catecholamine release, fetal adrenal medulla was dispersed into single cells and incubated in vitro with VIP for 6 hours. Catecholamine release into the medium was measured at 1, 3 and 6 hours. At 6 hours of incubation, VIP stimulated total catecholamine release from fetal adrenomedullary cells in a dose-dependent manner at concentrations ranging from 10(-8) to 10(-4) M. The release of norepinephrine and epinephrine, but not dopamine, was significantly enhanced. The presence of VIP in the fetal adrenal cortex and medulla, and the ability of VIP to stimulate catecholamine release from fetal adrenomedullary cells in vitro suggest that VIP may be an important modulator of medullary catecholamine secretion during fetal life.     

Effects of thyrotropin-releasing hormone (TRH) and MK-771 on schedule-controlled behavior of squirrel monkeys, rabbits and pigeons

The effects of TRH (0.001-10.0 mg/kg) and a more potent TRH analog, MK-771 (0.001-5.6 mg/kg), were studied on comparable schedule-controlled performances of squirrel monkeys, rabbits and pigeons. Responding was maintained in the presence of different stimuli by a multiple fixed-ratio (FR), fixed-interval (FI) schedule of food presentation (monkeys and pigeons) or 0.25% saccharin solution (rabbits). Generally, TRH and MK-771 produced decreases in responding under both schedules and in all three species. TRH and MK-771 were roughly equipotent in the squirrel monkey, whereas in the pigeon and rabbit MK-771 was approximately 20 times more potent than TRH in decreasing responding to 50 percent of control levels. The duration of action of doses of TRH and MK-771 that reduced responding to 50 percent of control was approximately 3 hr in the squirrel monkey; recovery of performance occurred twice as fast under the FR schedules. With the pigeon, TRH effects that produced 50 percent decreases in responding lasted over 6 hours, whereas behaviorally comparable doses of MK-771 lasted about 4 hours. With few exceptions, TRH and MK-771 appear to produce similar effects of schedule-controlled behavioral performances of the squirrel monkey, rabbit and pigeon. Compared to the effects of other behaviorally-active substances under these procedures, TRH and MK-771 exert a distinctive array of effects.     

Involvement of tyrosine hydroxylase up regulation in dexamethasone-induced hypertension of rats

We investigated the effect of dexamethasone (DEX) on tyrosine hydroxylase (TH) mRNA level, and TH activity and catecholamine levels in the adrenal medulla of the rat. DEX (1 mg/kg/day, s.c.) was administered for 2 days, and a control group was given corn oil. DEX significantly increased systolic blood pressure. TH mRNA level, TH activity, epinephrine level, and norepinephrine level in the adrenal medulla of DEX-treated rats were significantly higher than those of control rats. Also, epinephrine and norepinephrine levels in plasma were significantly higher in DEX-treated rats than in controls. alpha-Methyl-p-tyrosine prevented the DEX-induced blood pressure increase. These results suggest that the catecholamine synthetic pathway may be involved in DEX-induced hypertension.     

The effect of toluene inhalation exposure on catecholamine contents in rat sympathetic neurons

Adult male rats were exposed to toluene in short-term exposure by inhalation for 48 h (2000 ppm, continuously), and in long-term inhalation for 3 months (1000 ppm, 8 h daily). The formaldehyde-induced fluorescence (FIF) technique for histochemical demonstration of catecholamines (CA) was used to detect changes in the catecholamine stores. The concentration of CA in the sympathetic neurons of superior cervical ganglia and adrenal medulla was measured by the FIF technique combined with microfluorimetry. The short-term toluene exposure induced a statistically significant reduction of CA contents in sympathetic neurons. After long-term exposure, no change in the CA level could be demonstrated either in sympathetic ganglion or adrenal medulla. In electron microscopic studies no clear pathological changes were detected after toluene exposure.     

24 hour fasting and adrenoreceptor blocking agent influence on adrenal catecholamine synthesis rate changes induced by combined thermal and immobilization stress in rats.

The catecholamine (CA) depletion degree in rat adrenal medulla, the survival time (ST) and rectal temperature changes induced by combined thermal and immobilization stress were examined with the aim to prove tha alpha- and beta-adrenoreceptor sensitivity decrease implication in 24 hour fasting-induced changes of the above mentioned phenomena. The significant ST increase in strong stressful situations and the adrenal CA turnover augmentation in fed and fasted-propranolol or dihydroergotamine pretreated rats compared to untreated ones provided evidence that alpha- and beta adrenoceptor blockade causes adrenal CA turnover increase and ST prolongation similar to effects observed in our previous experiments. Thus the implication of fasting induced adrenoreceptor downregulation in adrenal CA turnover augmentation and ST prolongation of fasted rats in strong stressful conditions was suggested and the possible mechanisms of these phenomena have been discussed.     

Functional involvement of angiotensin AT2 receptor in adrenal catecholamine secretion in vivo.

The aim of the present study was to analyse modulations of adrenal catecholamine secretion from the adrenal gland of anesthetized dogs in response to locally administered angiotensin II (AngII) in the presence of either PD 123319 or CGP 42112, both of which are highly specific and selective ligands to angiotensin AT2 receptor. Plasma concentrations of epinephrine and norepinephrine in adrenal venous and aortic blood were quantified by a high performance liquid chromatography coupled with electrochemical detection (HPLC-EC) method. Adrenal venous blood flow was measured by gravimetry. Local administration of AngII (0.05 microg, 0.1 microM) to the left adrenal gland increased adrenal gland catecholamine output more than 30 times that found in nonstimulated states. Administration of either PD 123319 (0.085 microg (0.23 microM) to 8.5 microg (23 microM)) or CGP 42112 (0.005 microg (0.01 microM) to 5 microg (10 microM)) did not affect the basal catecholamine output significantly. The increase in adrenal catecholamine output in response to AngII was inhibited by approximately 80% following the largest dose of PD 123319. CGP 42112 significantly attenuated the catecholamine response to AngII by approximately 70%. PD 123319 and CGP 42112 were devoid of any agonist actions with respect to catecholamine output by the adrenal gland in vivo. Furthermore, both PD 123319 and CGP 42112 inhibited the increase in adrenal catecholamine secretion induced by local administration of AngII. The present study suggests that AT2 receptors play a role in mediating catecholamine secretion by the adrenal medulla in response to AngII receptor agonist administration in vivo.     

Behavioral effects and benzodiazepine antagonist activity of Ro 15-1788 (flumazepil) in pigeons

The selective benzodiazepine receptor antagonist, Ro 15-1788, produced behavioral effects in pigeons at doses at least 100 times lower than those previously reported to possess intrinsic pharmacological activity in mammals. In contrast to its effects in mammalian species, in pigeons, Ro 15-1788 does not exhibit partial agonist activity. Key-peck responses of pigeons were studied under a multiple fixed-interval 3-min, fixed-interval 3-min schedule in which the first response after 3-min produced food in the presence of red or white keylights. In addition, every 30th response during the red keylight produced a brief electric shock (punishment). Under control conditions, punished responding was suppressed to 30% of unpunished response levels. Ro 15-1788 (0.01 mg/kg, i.m.) increased unpunished response rates by 33% without affecting rates of punished responding. Doses of 0.1 to 1.0 mg/kg Ro 15-1788 produced dose-related decreases in both punished and unpunished responding. As is characteristic of other benzodiazepines, midazolam (0.1 and 0.3 mg/kg, i.m.) markedly increased punished responding but had little effect on rates of unpunished responding. Ro 15-1788 antagonized the increases in punished responding and also reversed the rate-decreasing effects of higher doses of midazolam. However, the effectiveness of Ro 15-1788 as a benzodiazepine antagonist was limited by its intrinsic activity: rate-decreasing doses of Ro 15-1788 were unable to completely reverse behavioral effects of midazolam. Midazolam was an effective antagonist of the behavioral effects of Ro 15-1788 (up to 0.1 mg/kg) but midazolam did not influence the rate-decreasing effects of 1.0 mg/kg Ro 15-1788 across a 100-fold dose range. In the pigeon, the behavioral effects of relatively low doses of Ro 15-1788 (0.01-0.1 mg/kg) appear to be related to benzodiazepine receptor mechanisms, whereas other systems appear to be involved in the effects of higher doses.