D-Amphetamine reinstates behavior previously maintained by food: importance of context.

The drug self-administration reinstatement procedure provides an important animal model of relapse. While the procedure is widely used, there has been little investigation of the basic processes involved. This experiment determined the specificity of reinstatement by examining reinstatement of food seeking by D-amphetamine. During training, 24 rats pressed levers for food. Eight rats received 3.0 mg/kg D-amphetamine before and saline after sessions. Eight rats received saline before and after sessions. The final eight rats received saline before and 3.0 mg/kg D-amphetamine after sessions. All rats then experienced saline injections and extinction. During a reinstatement test, all rats received 3.0 mg/kg D-amphetamine. D-Amphetamine significantly increased lever pressing for rats with prior exposure to amphetamine as a predictive cue for food (pre-session) and for rats with no prior exposure to amphetamine. The effect was larger for rats with pre-session exposure to amphetamine than for rats with no previous exposure. Rats with exposure to amphetamine but not as a predictive cue for food (after sessions) did not show significant reinstatement of lever pressing. Therefore, the reinstating effects of amphetamine are not restricted to behavior previously maintained by amphetamine self-administration. In animal models of relapse, reinstatement of drug seeking could be due, in part, to discriminative and direct effects of self-administered drug.

Ethanol-maintained responding of rats is more resistant to change in a context with added non-drug reinforcement.

Alternative non-drug reinforcers reliably decrease drug-maintained responding in self-administration procedures. Studies of the resistance to change of food-maintained behavior, however, have found that responding in the presence of a stimulus associated with an alternative reinforcer is more resistant to disruption. This increase in persistence occurs despite lower response rates when the alternative reinforcer is present. The present experiment examined if, in addition to decreasing response rates, an alternative non-drug reinforcer also increases the persistence of drug-maintained responding. Rats self-administered oral ethanol in a multiple schedule of reinforcement in which responding was reinforced in two components signaled by different stimuli. In one component, response-independent food was delivered in addition to the earned ethanol. The effects of the alternative food reinforcer on response rates and resistance to extinction in the two components were examined. As in previous experiments on the resistance to change of food-maintained operant behavior, response rates were lower, but more resistant to extinction in the presence of the stimulus associated with the alternative reinforcer. These findings suggest that all the reinforcers obtained in a context in which drugs are consumed may contribute to the persistence of drug seeking in that context. This increase in persistence may occur even if the alternative reinforcers interfere with drug seeking.

Behavioral economics of human drug self-administration: progressive ratio versus random sequences of response requirements.

Progressive-ratio (PR) schedules have been used widely to examine the relationship between drug consumption and drug price (i.e. demand curves) in the study of the behavioral economics of drug abuse. Sequential effects produced by the increasing response requirements of progressive-ratio schedules might influence the shape of demand curves for drug reinforcers. This study compared progressive ratio schedule and random sequences of ratio requirements, each incremented across sessions in a within-subject design, to determine if they produced similar behavioral economic and traditional measures of reinforcer efficacy. Self-administration of standardized cigarette puffs (70 cc each) was studied with eight smokers. Puffs were available at nine ratio requirements (e.g. 3, 100, 300, 600, 1500, 3000, 6000, 12000, 24000 responses/three puffs), presented in ascending (progressive-ratio schedule) or random sequence across daily sessions. The parameter estimates obtained on measures of reinforcing efficacy (e.g. breakpoint, peak response rates, elasticity of demand) were similar for both methods of incrementing prices. We found no evidence that PR and random sequences of fixed-ratio (FR) schedules, incremented across daily sessions, resulted in different demand curves.

Sensitivity of nicotine-containing and de-nicotinized cigarette consumption to alternative non-drug reinforcement: a behavioral economic analysis.

A previous report from our laboratory showed similar measures of reinforcing efficacy for nicotine-containing and de-nicotinized cigarettes when each cigarette type was presented alone. The present experiment further compared the reinforcing efficacy of nicotine-containing and de-nicotinized cigarettes by assessing the effects of alternative non-drug reinforcement on self-administration of both cigarette types. Eight human subjects responded on a progressive-ratio schedule in which the number of plunger pulls required for standardized cigarette puffs increased across sessions. Responding for the two types of cigarette was examined when each was available alone and when the concurrent opportunity to earn money was available. Consumption of nicotine-containing and de-nicotinized cigarettes was decreased by both increases in price and by the concurrent availability of money. The two cigarettes types did not differ in their sensitivity to price or alternative non-drug reinforcement. These results replicate our previous report of similar measures of reinforcing efficacy for the two cigarette types when each was presented alone, and extend our previous findings to a choice situation involving an alternative non-drug reinforcer. These data suggest the importance of further examination of non-pharmacological variables in the maintenance of drug taking and the sensitivity of drug taking to alternative non-drug sources of reinforcement. Factors potentially contributing to the maintenance of smoking the de-nicotinized cigarettes (i.e. conditioned reinforcement, primary reinforcement by respiratory stimulation, instructional control, demand characteristics) are also discussed.

Superstitious responding and reinforcement rate under concurrent variable-interval extinction schedules.

To examine superstitious responding, four pigeons key pecked under multiple concurrent variable-interval 45 s variable-interval 90 s concurrent variable-interval 90 s variable-interval 180 s schedules in the absence of a changeover delay. The two variable-interval 90 s schedules then were replaced by extinction, and key-peck responding during extinction was examined as a function of the prevailing reinforcement rate. During the first several sessions, extinction-key responding was maintained closer to baseline levels in the presence of the higher reinforcement rate, and this effect dissipated or even reversed with continued exposure to extinction. Although extinction-key responding generally decreased to near-zero levels after several sessions, in a few instances, it continued for 30 and 45 sessions. These results demonstrate how concurrent variable-interval extinction schedules can be used to investigate what often has been labeled superstitious responding.

Nicotine gum as a substitute for cigarettes: a behavioral economic analysis.

The present experiment attempted to identify a substitute for cigarette smoking in a laboratory analog of the behavioral economics of drug dependence. The interaction between cigarette consumption and nicotine gum consumption was examined with eight human smokers by increasing the price of cigarettes (i.e. the number of responses required to obtain puffs) across sessions, while the price of concurrently available nicotine gum remained constant. In another phase, the price of nicotine gum was increased while the price of concurrently available cigarettes remained constant. To determine whether the presence of concurrently available nicotine gum influenced cigarette consumption, we also examined the effect of increasing the price of cigarettes on cigarette consumption without available nicotine gum. When cigarettes and nicotine gum were concurrently available, increases in the price of cigarettes increased consumption of nicotine gum. When the price of nicotine gum increased while the price of cigarettes remained constant, smokers nearly exclusively consumed cigarettes. The presence or absence of nicotine gum did not affect the relation between cigarette consumption and cigarette price. The results suggest that nicotine gum can maintain operant behavior of smokers in the laboratory and can function, in a behavioral economic sense, as a weak substitute for cigarette puffs. As a result, nicotine gum may be useful in human laboratory studies of the behavioral economics of reinforcer interactions and their role in drug dependence.

Pigeons may not remember the stimuli that reinforced their recent behavior.

In two experiments the conditioned reinforcing and delayed discriminative stimulus functions of stimuli that signal delays to reinforcement were studied. Pigeons' pecks to a center key produced delayed-matching-to-sample trials according to a variable-interval 60-s (or 30-s in 1 pigeon) schedule (Experiment 1) or a multiple variable-interval 20-s variable-interval 120-s schedule (Experiment 2). The trials consisted of a 2-s illumination of one of two sample key colors followed by delays ranging across phases from 0.1 to 27.0 s followed in turn by the presentation of matching and nonmatching comparison stimuli on the side keys. Pecks to the key color that matched the sample were reinforced with 4-s access to grain. Under some conditions of Experiment 1, pecks to nonmatching comparison stimuli produced a 4-s blackout and the start of the next interval. Under other conditions of Experiment 1 and each condition of Experiment 2, pecks to nonmatching stimuli had no effect and trials ended only when pigeons pecked the other, matching stimulus and received food. The functions relating pretrial response rates to delays differed markedly from those relating matching-to-sample accuracy to delays. Specifically, response rates remained relatively high until the longest delays (15.0 to 27.0 s) were arranged, at which point they fell to low levels. Matching accuracy was high at short delays, but fell to chance at delays between 3.0 and 9.0 s. In Experiment 2, both matching accuracy and response rates remained high over a wider range of delays in the variable-interval 120-s component relative to the variable-interval 20-s component. The difference in matching accuracy between the components was not due to an increased tendency in the variable-interval 20-s component toward proactive interference following short intervals. Thus, under these experimental conditions the conditioned reinforcing and the delayed discriminative functions of the sample stimulus depended on the same variables (delay and variable-interval value), but were nevertheless dissociated.

Tyrosine kinase activation in response to fungal spores is primarily dependent on endogenous reactive oxygen production in macrophages.

Studies from our laboratory (Shahan, T. A., Sorenson, W. G., and Lewis, D. M. (1994) Environ. Res. 67, 98-104) demonstrated that spores from different fungal species differentially activate rat alveolar macrophages as detected by the measurement of superoxide anion and cytokine production (Shahan, T. A., Siegel, P. D., Sorenson, W. G., Kuschner, W. G., and Lewis, D. M. (1998) Am. J. Respir. Cell Mol. Biol. 18, 435-441). Spores from Aspergillus candidus stimulated production of the highest levels of superoxide anion (5.2 nmol/1.0 x 10(6) alveolar macrophages (AMs)/30 min), followed by those from Aspergillus niger (2.4 nmol/1.0 x 10(6) AMs/30 min) and Eurotium amstelodami (0.4 nmol/1.0 x 10(6) AMs/30 min). The mechanism of this differential activation was studied. Our data demonstrate that the tyrosine kinases p56(Hck), p72(Syk), p77(Btk), p62(Yes), p56(Lck), and p59(Fyn) were specifically activated in response to spores from A. candidus, whereas spores from either A. niger or E. amstelodami activated p56(Hck), p72(Syk), and p77(Btk). Kinetic analysis of specific tyrosine kinases demonstrated that p56(Hck), p72(Syk), and p77(Btk) were activated faster and to a greater extent by spores from A. candidus as compared with spores from E. amstelodami. These data suggest a relationship between reactive oxygen species and tyrosine kinase activation. Treatment of AMs with H(2)O(2) (1 mM) caused the activation of p72(Syk) only, whereas treatment with superoxide dismutase and catalase before treatment with the spores had no effect on tyrosine kinase activation. Incubation with NADPH oxidase inhibitors inhibited both superoxide anion production and the activation of p56(Hck), p72(Syk), and p77(Btk) in response to fungal spores. These data indicate that endogenous reactive oxygen species are necessary for the activation of p56(Hck), p72(Syk), and p77(Btk) by spores; they also indicate that some species of spores are capable of activating tyrosine kinases independent of superoxide anion.

Regulation of tumor cell chemotaxis by type IV collagen is mediated by a Ca(2+)-dependent mechanism requiring CD47 and the integrin alpha(V)beta(3).

Studies from our laboratories demonstrated that synthetic peptides from the non-collagenous (NC-1) domain of the alpha3 (IV) chain of type IV collagen (COL IV) enhanced tumor cell adhesion (Han, J., Ohno, N., Monboisse, J. C., Pasco, S., Borel, J. P., and Kefalides, N. A. (1997) J. Biol. Chem. 272, 20395-20401). We have isolated the receptors for the alpha3(IV)185-203 peptide from melanoma and prostate tumor cells and identified them as CD47/integrin-associated protein and the integrin alpha(V)beta(3) (Shahan, T. A., Ziaie, Z., Pasco, S., Fawzi, A., Bellon, G., Monboisse, J. C., and Kefalides, N. A. (1999) Cancer Res. 59, 4584-4590). In the present study we have examined the effect of CD47 and the integrin alpha(V)beta(3) on in vitro tumor cell chemotaxis and Ca(2+)(i) modulation in response to COL IV, from the anterior lens capsule (ALC-COL IV) and peptides from its NC-1 domain. COL IV as well as the alpha3(IV) peptide promoted tumor cell chemotaxis with an immediate increase in intracellular [Ca(2+)]. Treating tumor cells with CD47 and integrin alpha(V)beta(3)-reactive antibodies reduced chemotaxis as well as the rise in [Ca(2+)](i) in response to ALC-COL IV or the alpha3(IV)185-203 peptide but not to Engelbreth-Holm-Swarm-COL IV or fibronectin. The alpha3(IV)185-203 synthetic peptide stimulated an increase in calcium from intracellular stores exclusively, whereas ALC-COL IV, Engelbreth-Holm-Swarm-COL IV, and fibronectin stimulated Ca(2+) flux from both internal and external stores. Furthermore, treatment of the cells with Ca(2+) chelator bis-(O-aminophenoxyl)ethane-N,N,N',N'-tetraaceticacid- acetomethoxy ester inhibited chemotaxis toward both ALC-COL IV and the alpha3(IV)185-203 peptide. These data indicate that CD47 and integrin alpha(V)beta(3) regulate tumor cell chemotaxis in response to COL IV and the alpha3(IV)185-203 peptide through a Ca(2+)-dependent mechanism.

Choice, changing over, and reinforcement delays.

In three experiments, pigeons were used to examine the independent effects of two normally confounded delays to reinforcement associated with changing between concurrently available variable-interval schedules of reinforcement. In Experiments 1 and 2, combinations of changeover-delay durations and fixed-interval travel requirements were arranged in a changeover-key procedure. The delay from a changeover-produced stimulus change to a reinforcer was varied while the delay between the last response on one alternative and a reinforcer on the other (the total obtained delay) was held constant. Changeover rates decreased as a negative power function of the total obtained delay. The delay between a changeover-produced stimulus change had a small and inconsistent effect on changeover rates. In Experiment 3, changeover delays and fixed-interval travel requirements were arranged independently. Changeover rates decreased as a negative power function of the total obtained delay despite variations in the delay from a change in stimulus conditions to a reinforcer. Periods of high-rate responding following a changeover, however, were higher near the end of the delay from a change in stimulus conditions to a reinforcer. The results of these experiments suggest that the effects of changeover delays and travel requirements primarily result from changes in the delay between a response at one alternative and a reinforcer at the other, but the pattern of responding immediately after a changeover depends on the delay from a changeover-produced change in stimulus conditions to a reinforcer.

Comparing the reinforcing efficacy of nicotine containing and de-nicotinized cigarettes: a behavioral economic analysis.

RATIONALE: Smoking-related respiratory stimuli produced by de-nicotinized cigarettes may function as conditioned reinforcers, but behavioral data on such reinforcing effects are limited. OBJECTIVES: The present experiment compared the reinforcing efficacy of cigarettes that provided only smoking-related stimuli (de-nicotinized cigarettes) and cigarettes that provided both smoking-related stimuli and nicotine. METHODS: Eight human subjects responded on a progressive-ratio schedule in which the number of plunger pulls required for standardized cigarette puffs increased across sessions. In one phase, the breakpoints, number of puffs earned per session, peak response rates, ratio producing peak response rates, and the elasticity of demand for cigarette puffs were compared for nicotine-containing and de-nicotinized cigarettes when each cigarette type was the only one available. In another phase, subjects chose between the two cigarette types at some of the prices examined in the previous phase. RESULTS: Nicotine-containing and de-nicotinized cigarettes produced similar measures of reinforcing efficacy when each was presented alone, but there was a strong preference for nicotine-containing cigarettes when subjects were given a choice. CONCLUSIONS: These data support suggestions that smoking-produced sensory stimuli may function as conditioned reinforcers and that the relative reinforcing efficacy of cigarettes is determined by the combined effects of the nicotine/conditioned reinforcing complex provided by smoking.

Identification of CD47/integrin-associated protein and alpha(v)beta3 as two receptors for the alpha3(IV) chain of type IV collagen on tumor cells.

Previous studies from our laboratories demonstrated that a peptide from the noncollagenous domain of the alpha3 chain of basement membrane collagen (COL IV), comprising residues 185-203, inhibits polymorphonuclear leukocyte activation and melanoma cell proliferation independently of its ability to promote cell adhesion; these properties require the presence of the triplet -SNS- at residues 189-191 (J. C. Monboisse et al., J. Biol. Chem., 269: 25475-25482, 1994; J. Han et al., J. Biol. Chem., 272: 20395-20401, 1997). More recently, we demonstrated that native COL IV and -SNS-containing synthetic peptides (10 microg/ml) added to culture medium inhibit the proliferation of not only melanoma cells but also breast, pancreas, and stomach tumor cells up to 82% and prostate tumor cells by 15%. This inhibition was shown to be dependent on a COL IV- or peptide-induced increase in intracellular cAMP (T. A. Shahan et al., Connect. Tissue Res., 40: 221-232, 1999). Attempts to identify the putative receptor(s) on tumor cells led to the isolation of five proteins (Mr 33,000, 52,000, 72,000, 95,000, and 250,000) from melanoma and prostate cells by affinity purification with the alpha3(IV)179-208 peptide. The Mr 52,000, 95,000, and 250,000 proteins were shown to be CD47/integrin-associated protein(IAP), the integrin beta3 subunit, and the alpha(v)beta3 integrin complex, respectively. The Mr 33,000 and 72,000 proteins have not yet been identified. To confirm the specificity of ligand binding to the receptors, cell membranes from either melanoma or prostate tumor cells were pretreated with the unlabeled ligand alpha3(IV)187-191 (-YYSNS-); alternatively, the peptide was pretreated with a peptide-reactive monoclonal antibody (A5D7) before receptor isolation. These treatments inhibited the purification of CD47/IAP, the integrin beta3 subunit, and the alpha(v)beta3 integrin complex from tumor cells. Furthermore, cells treated with CD47/IAP- or the alpha(v)beta3 integrin-reactive antibodies prevented the alpha3(IV)185-203 peptide from inhibiting cell proliferation and the subsequent rise in intracellular cAMP. Pretreating cells with the alpha3(IV)187-191 (-YYSNS-) peptide also inhibited their adhesion to the alpha3(IV)185-203 peptide substrate, whereas the inactive alpha1(IV)185-203 peptide, from the same region of the alpha1 chain as the alpha3(IV)185-203 peptide, had no effect. Incubation of cells with either CD47/IAP and/or alpha(v)beta3 integrin-reactive antibodies inhibited their adhesion to the alpha3(IV)185-203 peptide, whereas antibodies to the beta1 and beta2 integrin subunits were without effect. These data suggest that ALC-COL IV, through its alpha3(IV) chain, inhibits tumor cell proliferation using the receptors CD47/IAP and the alpha(v)beta3 integrin.

Inhibition of tumor cell proliferation by type IV collagen requires increased levels of cAMP.

Previous studies from our laboratories demonstrated that a peptide from the noncollagenous domain of the alpha3 chain of basement membrane collagen (COL IV), comprising residues 185-203, inhibits polymorphonuclear leukocyte activation and melanoma cell proliferation; this property requires the presence of the triplet -SNS- in residues 189-191 (Monboisse et al., J. Biol. Chem., 269, 25475, 1994; Han et al., J. Biol. Chem., 272, 20395, 1997). In the present study, we demonstrate that whole native COL IV and -SNS- containing synthetic peptides (10 microg/ml) added to culture medium inhibit the proliferation of not only melanoma cells, but also breast-, pancreas- and stomach-tumor cells up to 67%, and prostate tumor cells by 15%. ALC-COL IV at 5 microg/ml was shown to inhibit melanoma cell proliferation maximally at 69% and the alpha3(IV)185-203 peptide inhibited proliferation (62%) maximally at 10 microg/ml. Treatment of the alpha3(IV)185-203 peptide with either a specific mAb or a polyclonal antibody, prepared against the sequence alpha3(IV)179-208, decreased the ability of the peptide to inhibit cell proliferation by 97%, while treatment of ALC-COL IV with the same antibodies inhibited proliferation by 44%. Exposure of the above tumor cells to COL IV or the peptides resulted in an increase of intracellular cAMP that was inhibited by prior treatment of the protein with the above antibodies. To investigate the role of cAMP in the inhibition of cell proliferation, cAMP analogs and inhibitors were used. cAMP analogs mimicked the inhibitory effect of the peptide. Rp-cAMPS, a cAMP competitive inhibitor, suppressed the inhibitory effect of ALC-COL IV and of the cAMP analogs. The protein kinase-A inhibitor H-89 blocked the ability of ALC-COL IV and of the alpha3(IV)185-203 peptide to inhibit tumor cell proliferation. These data suggest that ALC-COL IV, through its alpha3(IV) chain, inhibits tumor cell proliferation utilizing a signal transduction pathway which includes cAMP and cAMP-dependent protein kinase(s).

Cell wall preparations from environmental yeasts: effect on alveolar macrophage function in vitro.

Organic dust toxic syndrome (ODTS) is associated with inhalation of high concentrations of organic materials and is a noninfectious illness characterized by fever, malaise, myalgia, and neutrophilic inflammation of the lower respiratory tract. Studies in our laboratory of fungi in fresh lumber have demonstrated that yeasts may predominate and have raised the issue of potential exposure of sawmill workers to yeasts. Zymosan, a cell wall preparation from Saccharomyces cerevisiae, is a potent stimulator of alveolar macrophages (AM). In the present study, preparations from the cell walls of Pichia fabianii, Candida sake, Trichosporon capitatum, Rhodotorula glutinis, and Cryptococcus laurentii were compared with zymosan and beta-1,3-glucan for their ability to stimulate AM and activate complement. All species activated complement. P. fabianii, C. sake, T. capitatum, R. glutinis, C. laurentii, as well as zymosan and glucan, stimulated superoxide anion and leukotriene B4 production in a dose-dependent fashion, but R. glutinis and C. laurentii were much less active. Zymosan, glucan, P. fabianii, and R. glutinis treatment of AM resulted in increased phagocytosis of labeled sheep RBCs, whereas there was no effect with C. sake or C. laurentii and T. capitatum significantly inhibiting phagocytosis. These results suggest that exposure to high concentrations of yeast could provoke pulmonary inflammation resulting in an episode of ODTS.

Concentration- and time-dependent upregulation and release of the cytokines MIP-2, KC, TNF, and MIP-1alpha in rat alveolar macrophages by fungal spores implicated in airway inflammation.

Inhalation of fungal spores has been shown to cause primary or secondary infection and respiratory inflammation and diseases such as allergic alveolitis, atopic asthma, and organic dust toxic syndrome, which are rarely reported in the absence of predisposing factors. Biochemical and molecular markers of inflammation were measured in rat bronchial alveolar lavage cells (> 95% macrophages) following stimulation with fungal spores isolated from pathogenic and nonpathogenic fungi that have been implicated in airway inflammation. The results of this study demonstrate that mRNA transcripts for the C-X-C branch of the PF4 superfamily are differentially upregulated over those of the C-C mediators in a time- and concentration-dependent manner. Macrophage inflammatory protein (MIP)-2 and KC were differentially upregulated over the acute phase inflammatory cytokines MIP-1alpha and tumor necrosis factor-alpha (TNF-alpha) in rat alveolar macrophages stimulated with fungal spores from Aspergillus candidus, Aspergillus niger, Eurotium amstelodami, and Cladosporium cladosporioides. Spores from Aspergillus terreus and Penicillium spinulosum failed to stimulate an increase of any cytokine mRNA, whereas those from Aspergillus fumigatus stimulated the upregulation of MIP-2, KC, TNF-alpha, and MIP-1alpha mRNAs. Over time, A. fumigatus stimulated increasing KC production until 24 h, when production levels increased slightly, then leveled off when measurements ceased at 36 h. Latex spheres stimulated modest amounts of MIP-2 and transforming growth factor-beta only. These observations suggest that the inflammatory cytokines MIP-2 and KC may be involved in the inflammation arising from the inhalation of fungal spores in a time- and concentration-dependent manner.

Mechanisms underlying the effects of unsignaled delayed reinforcement on key pecking of pigeons under variable-interval schedules.

Three experiments were conducted to test an interpretation of the response-rate-reducing effects of unsignaled nonresetting delays to reinforcement in pigeons. According to this interpretation, rates of key pecking decrease under these conditions because key pecks alternate with hopper-observing behavior. In Experiment 1, 4 pigeons pecked a food key that raised the hopper provided that pecks on a different variable-interval-schedule key met the requirements of a variable-interval 60-s schedule. The stimuli associated with the availability of the hopper (i.e., houselight and keylight off, food key illuminated, feedback following food-key pecks) were gradually removed across phases while the dependent relation between hopper availability and variable-interval-schedule key pecks was maintained. Rates of pecking the variable-interval-schedule key decreased to low levels and rates of food-key pecks increased when variable-interval-schedule key pecks did not produce hopper-correlated stimuli. In Experiment 2, pigeons initially pecked a single key under a variable-interval 60-s schedule. Then the dependent relation between hopper presentation and key pecks was eliminated by arranging a variable-time 60-s schedule. When rates of pecking had decreased to low levels, conditions were changed so that pecks during the final 5 s of each interval changed the keylight color from green to amber. When pecking produced these hopper-correlated stimuli, pecking occurred at high rates, despite the absence of a peck-food dependency. When peck-produced changes in keylight color were uncorrelated with food, rates of pecking fell to low levels. In Experiment 3, details (obtained delays, interresponse-time distributions, eating times) of the transition from high to low response rates produced by the introduction of a 3-s unsignaled delay were tracked from session to session in 3 pigeons that had been initially trained to peck under a conventional variable-interval 60-s schedule. Decreases in response rates soon after the transition to delayed reinforcement were accompanied by decreases in eating times and alterations in interresponse-time distributions. As response rates decreased and became stable, eating times increased and their variability decreased. These findings support an interpretation of the effects of delayed reinforcement that emphasizes the importance of hopper-observing behavior.