A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate drug-seeking behavior and prompt relapse in abstinent drug users. We have developed a procedure to study the behavioral and neural processes that mediate the impact of context on alcohol-seeking behavior in rats. Following acclimation to the taste and pharmacological effects of 15% ethanol in the home cage, male Long-Evans rats receive Pavlovian discrimination training (PDT) in conditioning chambers. In each daily (Mon-Fri) PDT session, 16 trials each of two different 10 sec auditory conditioned stimuli occur. During one stimulus, the CS+, 0.2 ml of 15% ethanol is delivered into a fluid port for oral consumption. The second stimulus, the CS-, is not paired with ethanol. Across sessions, entries into the fluid port during the CS+ increase, whereas entries during the CS- stabilize at a lower level, indicating that a predictive association between the CS+ and ethanol is acquired. During PDT each chamber is equipped with a specific configuration of visual, olfactory and tactile contextual stimuli. Following PDT, extinction training is conducted in the same chamber that is now equipped with a different configuration of contextual stimuli. The CS+ and CS- are presented as before, but ethanol is withheld, which causes a gradual decline in port entries during the CS+. At test, rats are placed back into the PDT context and presented with the CS+ and CS- as before, but without ethanol. This manipulation triggers a robust and selective increase in the number of port entries made during the alcohol predictive CS+, with no change in responding during the CS-. This effect, referred to as context-induced renewal, illustrates the powerful capacity of contexts associated with alcohol consumption to stimulate alcohol-seeking behavior in response to Pavlovian alcohol cues.     

Appetitive differential conditioning of the rabbit's jaw movement response. Effects of cue similarity and US magnitude

Two experiments examined appetitive differential conditioning of the rabbit's jaw movement response (JMR) in a two-phase procedure. The first phase entailed reinforced training with one conditioned stimulus (CS+), and the second phase involved intermixed presentations of CS+ and an unreinforced stimulus (CS-). In Experiment 1, CS+ was a 600-Hz tone, and CS- was either a 660-, 1,000-, or 2,100-Hz tone. In Experiment 2, the magnitude of the water unconditioned stimulus (US) paired with CS+ was either 1, 3, or 9 ml. The experiments revealed that 1) the level of responding to CS- rose for several days and then declined over the remainder of training; 2) the physical similarity between CS+ and CS- directly affected the level of responding to CS- but had no discernable effect on the level of responding to CS+; and 3) US magnitude positively affected the level of responding to CS+ and, to a lesser extent, CS-. The results are discussed in terms of their implications for Spence's gradient interaction theory and Pavlov's external inhibition hypothesis.     

Reaction time task as unconditional stimulus. On conditioning skin conductance responses and heart rate, using a nonaversive unconditional stimulus

The present study was conducted to demonstrate classic conditioning in electrodermal (ED) and heart rate (HR) responses by using a nonaversive reaction time (RT) task as unconditional stimulus (US). Three groups of 12 subjects each were studied to test the efficacy of this US procedure by varying the essential components of the RT task-US between groups. Eight seconds differential delay conditioning was applied in each group. Simple geometric features (square, cross) displayed on a TV screen were used as CS+ and CS-. RT task consisted of a nonaversive tone (72 dBA, 1000 or 1200 Hz) and a motor response (pressing a button with the left index finger). Subjects were asked to respond as soon as the tone stimulus was presented. The three groups received different stimulus sequences during the 16-trial acquisition phase only. In one group (Group C1), CS+ was followed by a tone to which subjects were to respond, whereas CS- was not followed by a tone. Similarly, in a second group (Group H), CS+ was followed by a tone, whereas CS- was not; however, subjects of Group H (habituation group) were not required to respond to the tone. In a third group, (Group C2) CS+ was followed by a tone to which subjects were to respond, while CS- was followed by a different tone requiring no response. According to analysis of Group C1 data, differential conditioning was obtained in each response measure. Group H displayed habituation in each response measure obtained. In Group C2, differential conditioning was obtained in the second latency window of ED responses only.(ABSTRACT TRUNCATED AT 250 WORDS)     

Discriminative behavior and Pavlovian conditioning in the mollusc Pleurobranchaea

The buccal motor system in the sea slug Pleurobranchaea californica is multifunctional; similar sets of neurons and muscles generate different behaviors through similar electrophysiological motor patterns. Such multifunctional systems compromise the traditional practice of identifying a motor pattern and then using that pattern to indicate the behavior in reduced preparations. We address this issue in a series of experiments leading to the comparison of differential Pavlovian conditioning in whole animals with the conditioned behavior of the same animals during electrophysiological recording. Because differential conditioning requires two conditioned stimuli (CSs), we show here that each of two CSs activated the conditioned response from animals after they received the stimulus (CS+) paired with an unconditioned stimulus (UCS). Conditioning sessions consisted of 5 training trials with a 2-h intertrial interval. In one study, experimental animals received a 60-s CS+, derived from beer (Sbr), paired with a 50-s electrical shock UCS whose onset occurred 10 s after the CS+ onset; control animals received the Sbr and UCS explicitly unpaired. In a second study, animals received similar procedures as in the first but with a CS+ consisting of squid homogenate (Ssq). Tests with both CSs showed that animals did not discriminate between Sbr and Ssq before beginning conditioning, but did so afterward. Experimental animals exhibited robust food aversion (withdrawal and suppressed feeding) to the CS+, but retained strong appetitive responses to the CS they did not receive in training; response thresholds to the CS+ changed as much as 1000-fold by comparison to the preconditioning values. Control animals exhibited similar though significantly smaller behavioral changes as the experimental animals. Both stimuli evoked associatively learned responses, but Sbr produced greater experimental-control differences than Ssq did. Two accompanying papers show the results of using both CSs in differential conditioning, and describe the behavioral/electrophysiological comparisons.     

Effects of nicotine and dietary salt on a learned blood pressure response in Dahl-S rats

We examined the effects of chronic nicotine exposure and dietary salt on the arterial blood pressure (BP) changes learned in response to an acute behavioral stress in the Dahl salt-sensitive rat. Four groups were tested: low salt + vehicle; low salt + nicotine; high salt + vehicle; and high salt + nicotine. Rats were fed a low-salt (0.08% NaCl) or a high-salt (8% NaCl) diet for 4 wk; 2.4 mg. kg(-1). day(-1) nicotine or vehicle was given via an implanted osmotic minipump for the last 2 wk. All rats were conditioned by following one tone (CS+) with a 0.5-s tail shock; another tone (CS-) was never followed by shock. CS+ in low salt + vehicle and high salt + vehicle-treated rats evoked an initial arterial BP increase (C(1)), a component of the startle response, and an ensuing, smaller, but more sustained, pressor response (C(2)), which is acquired with training. In these rats, both C(1) and C(2) evoked by CS- were significantly smaller than those to CS+, demonstrating that these groups discriminated between the two tests. Conversely, although the low salt + nicotine-treated rats had both the C(1) and C(2) components of the conditional arterial pressure response, they did not discriminate between CS+ and CS-. Finally, the high salt + nicotine group failed to both discriminate between tones and acquire (i.e., learn) the C(2) response. The unconditional response to shock did not differ between groups. We conclude that combined exposure to high salt and to nicotine inhibits the salt-sensitive animal's acquisition of a learned conditional BP response, perhaps because nicotine acts preferentially on those central processes required for associative learning versus those involved in orientating to external stimuli.     

Characterizing psychophysical measures of discrimination thresholds and the effects of concentration on discrimination learning in the moth Manduca sexta

What is the spatial and temporal nature of odor representations within primary olfactory networks at the threshold of an animal's ability to discriminate? Although this question is of central importance to olfactory neuroscience, it can only be answered in model systems where neural representations can be measured and discrimination thresholds between odors can be characterized. Here, we establish these thresholds for a panel of odors using a Pavlovian paradigm in the moth Manduca sexta. Moths were differentially conditioned to respond to one odor (CS+) but not another (CS-) using undiluted odorants to minimize salience-dependent learning effects. At 24 and 48 h postconditioning, moths were tested for the presence of a conditioned response (CR) with a blank, then the CS+ and CS- (pseudorandomly) across a 5-log step series of increasing concentration. Results identified discrimination thresholds and established that differential CRs to the CS+ and CS- increased with stimulus concentration. Next, 3 separate groups of moths were differentially conditioned at either one-log step below, at, or one log step above the identified discrimination threshold. At 24 and 48 h postconditioning, moths were tested sequentially with a blank, the concentration used for conditioning, and then undiluted odor. Conditioning at one log step below the discrimination threshold established a CR, indicating both stimulus detection and learning, but was insufficient to establish evidence of discrimination. Moths conditioned at the discrimination threshold were able to discriminate but only when stimulated with undiluted odors, indicating learning, but discrimination measures were hampered. When conditioned above the discrimination threshold, moths had no difficulty in discriminating. These results establish methods for psychophysical characterization of discrimination and indicate that differential conditioning at lowered concentrations biases threshold measures.     

Evidence for instantaneous e-vector detection in the honeybee using an associative learning paradigm

Many insects use the polarization pattern of the sky for obtaining compass information during orientation or navigation. E-vector information is collected by a specialized area in the dorsal-most part of the compound eye, the dorsal rim area (DRA). We tested honeybees' capability of learning certain e-vector orientations by using a classical conditioning paradigm with the proboscis extension reflex. When one e-vector orientation (CS+) was associated with sugar water, while another orientation (CS-) was not rewarded, the honeybees could discriminate CS+ from CS-. Bees whose DRA was inactivated by painting did not learn CS+. When ultraviolet (UV) polarized light (350 nm) was used for CS, the bees discriminated CS+ from CS-, but no discrimination was observed in blue (442 nm) or green light (546 nm). Our data indicate that honeybees can learn and discriminate between different e-vector orientations, sensed by the UV receptors of the DRA, suggesting that bees can determine their flight direction from polarized UV skylight during foraging. Fixing the bees' heads during the experiments did not prevent learning, indicating that they use an 'instantaneous' algorithm of e-vector detection; that is, the bees do not need to actively scan the sky with their DRAs ('sequential' method) to determine e-vector orientation.     

Eye Movements Index Implicit Memory Expression in Fear Conditioning

The role of contingency awareness in simple associative learning experiments with human participants is currently debated. Since prior work suggests that eye movements can index mnemonic processes that occur without awareness, we used eye tracking to better understand the role of awareness in learning aversive Pavlovian conditioning. A complex real-world scene containing four embedded household items was presented to participants while skin conductance, eye movements, and pupil size were recorded. One item embedded in the scene served as the conditional stimulus (CS). One exemplar of that item (e.g. a white pot) was paired with shock 100 percent of the time (CS+) while a second exemplar (e.g. a gray pot) was never paired with shock (CS-). The remaining items were paired with shock on half of the trials. Participants rated their expectation of receiving a shock during each trial, and these expectancy ratings were used to identify when (i.e. on what trial) each participant became aware of the programmed contingencies. Disproportionate viewing of the CS was found both before and after explicit contingency awareness, and patterns of viewing distinguished the CS+ from the CS-. These observations are consistent with “dual process” models of fear conditioning, as they indicate that learning can be expressed in patterns of viewing prior to explicit contingency awareness.     

Gut T1R3 sweet taste receptors do not mediate sucrose-conditioned flavor preferences in mice

Most mammals prefer the sweet taste of sugars, which is mediated by the heterodimeric T1R2+T1R3 taste receptor. Sugar appetite is also enhanced by the post-oral reinforcing actions of the nutrient in the gut. Here, we examined the contribution of gut T1R3 (either alone or as part of the T1R3+T1R3 receptor) to post-oral sugar reinforcement using a flavor-conditioning paradigm. We trained mice to associate consumption of a flavored solution (CS+) with intragastric (IG) infusions of a sweetener, and a different flavored solution (CS-) with IG infusions of water (23 h/day); then, we measured preference in a CS+ vs. CS- choice test. In experiment 1, we predicted that if activation of gut T1R3 mediates sugar reinforcement, then IG infusions of a nutritive (sucrose) or nonnutritive (sucralose) ligand for this receptor should condition a preference for the CS+ in B6 wild-type (WT) mice. While the mice that received IG sucrose infusions developed a strong preference for the CS+, those that received IG sucralose infusions developed a weak avoidance of the CS+. In experiment 2, we used T1R3 knockout (KO) mice to examine the necessity of gut T1R2+T1R3 receptors for conditioned flavor preferences. If intact gut T1R3 (or T1R2+T1R3) receptors are necessary for flavor-sugar conditioning, then T1R3 KO mice should not develop a sugar-conditioned flavor preference. We found that T1R3 KO mice, like WT mice, acquired a strong preference for the CS+ paired with IG sucrose infusions. The KO mice were also like WT mice in avoiding a CS+ flavor paired with IG sucralose infusions These findings provide clear evidence that gut T1R3 receptors are not necessary for sugar-conditioned flavor preferences or sucralose-induced flavor avoidance in mice.     

Reaction time task as unconditional stimulus. Comparing aversive and nonaversive unconditional stimuli

Nonaversive unconditional stimuli (USs) are seldom used in human classic conditioning of autonomic responses. One major objection to their use is that they produce deficits in electrodermal (ED) second- and third-interval response conditioning. However, a nonaversive reaction time (RT) task that includes feedback of success has been shown to be an effective US while avoiding this disadvantage (Lipp and Vaitl 1988). The present study compared this new RT task (RT-new) with a traditional RT task (RT-old) and with a standard aversive US (shock) in differential classic conditioning of ED, heart rate (HR), and digital pulse volume (DPV) responses. Eight-second-delay differential conditioning was applied in three groups of 12 subjects each. Simple geometric features (square, cross) displayed on a television screen served as conditional stimuli (CS+ and CS-). In acquisition, there were no statistically significant differences among the groups; differential conditioning did occur in HR, first- and second-interval ED responses, and first-interval DPV responses. Separate analyses within each group, however, revealed that there was no second-interval ED conditioning in the RT-old group. During extinction, neither DPV nor second-interval ED conditioning could be obtained, whereas HR and first-interval ED conditioning occurred in each group. In third-interval omission ED responses, RT-old and shock groups exhibited extinction, while response differentiation was maintained in the RT-new group throughout extinction. The RT task including feedback proved to be as reliable a US as a standard aversive US, whereas application of a traditional RT task again yielded some weaknesses in second-interval ED conditioning.     

A simple and reliable test of olfactory learning and memory in mice

The present paper describes a quick and efficient method for assessing olfactory discrimination learning in mice. In training mice received trials in which one odor (CS+) was paired with sugar and another odor (CS-) was paired with no sugar. When the mice were subsequently placed in a chamber with CS+ odor at one end and CS- odor at the other, they spent more time digging in CS+ than in CS- odor. In Experiment 2 mice trained with this procedure and tested after 60 days also spent more time digging in CS+ than CS- in the test phase, indicating that this olfactory discrimination task is effective for assessing long-term memory. In addition to the outbred strain of CD1 mice used in Experiments 1 and 2, C57Bl/6NCr/BR and DBA/2NCr/BR mice used in Experiment 3 also acquired this learned odor discrimination. Moreover, Experiment 4 showed that DBA animals were capable of acquiring this odor discrimination after receiving only two training trials (one exposure each to CS+ and CS-) per day for 4 days.     

Differential Pavlovian conditioning in the mollusc Pleurobranchaea

The present differential Pavlovian conditioning experiments on the sea slug Pleurobranchaea californica extend conditioning described in a preceding paper and provide the conditioning foundation for studies reported in another accompanying paper comparing learned behavior in whole animals with the behavior and motor patterns of electrophysiological preparations. All animals received two appetitive-conditioned stimuli (CSs), one derived from beer (Sbr) and the other derived from squid muscle (Ssq), in different temporal relationships to an electric shock unconditioned stimulus (UCS). Two groups of animals were run concurrently. One group (n = 19) received Sbr as the CS+ in close temporal pairing with the UCS, and Ssq as the CS- explicitly unpaired with the UCS (Sbr +/Ssq-). The second group (n = 20) received the opposite contingencies (Sbr-/Ssq+). All animals received only one day of conditioning involving 5 trials with an intertrial interval of 2 h. There were two replicate experiments, each involving about half of the total n, and each yielding similar results as the sum we report here. Before conditioning, animals exhibited feeding behavior (extension of the proboscis and bite-strike responses) to both stimuli at similar low thresholds. Conditioning produced long-term behavioral changes in all animals throughout the 4.5-day postconditioning observation period. However, only the Sbr+/Ssq- animals consistently exhibited the appropriate differentially conditioned food-aversion behavior which consisted of strong withdrawal and high-threshold feeding responses to Sbr, and low-threshold feeding responses to Ssq. We discuss the possibility that such differences between Sbr+/Ssq- and Sbr-/Ssq+ conditioning may arise either from inherent differences in the responses of the animals to Sbr and Ssq, or, as seems more likely to us, from training and testing effects produced by differences in the compositions of the two stimuli.     

Comparison of differential Pavlovian conditioning in whole animals and physiological preparations of Pleurobranchaea: implications of motor pattern variability

The present study compares differential Pavlovian conditioning in whole animals with the behavior of the same animals during electrophysiological recording. Untrained specimens of the sea slug Pleurobranchaea did not discriminate between two appetitive stimuli, one derived from an extract of beer (Sbr) and the other from a homogenate of squid muscle (Ssq). When animals received Sbr as the CS+ and Ssq as the CS- in a single day of five-trial, differential Pavlovian conditioning they learned to avoid selectively the Sbr but continued to exhibit appetitive responses to Ssq. Quantitative measures show that there was over a 1000-fold increase in the thresholds of the proboscis extension and bite-strike responses, many animals ceased all feeding behavior, and exhibited withdrawal responses to Sbr. We examined the behavior of the same trained animals immediately before preparing them for physiological recording and during the recording session. There was a close one-to-one relationship between these behavioral observations, showing that the qualitative and quantitative features of whole-animal Pavlovian conditioning persist into the physiological preparations. Unexpectedly, motor patterns from untrained preparations showed considerable variability both within the same preparation at different times and between preparations; conditioning appeared to increase such variability. Thus, it was not possible to state unequivocally the behavior of the animal by examining the electromyogram recording alone. Many of the trained preparations not only exhibited suppressed feeding behavior and withdrawal responses to Sbr, but, as a consequence of the multifunctional nature of the Pleurobranchaea buccal-oral system, also regurgitated previously ingested Ssq or squid meat when they were stimulated with Sbr. We discuss the findings with respect to self-organizing mechanisms that may establish motor patterns in multifunctional systems, and suggest that such mechanisms may lead to the generation of behaviors that are not specifically encoded by the conditioned cellular changes.     

Sugar and fat conditioned flavor preferences in C57BL/6J and 129 mice: oral and postoral interactions

C57BL/6J (B6) mice consume more sugar and fat solutions than do 129 mice in 24-h preference tests. Previous studies have attributed this observation to strain differences in taste responsiveness to these nutrients. We tested the hypothesis that differences in postingestive responsiveness contribute to the strain differences. In experiment 1, B6 and 129 mice were trained to associate consumption of a flavored solution (CS+) with intragastric (IG) infusions of 16% sucrose and a different flavored solution (CS-) with IG water infusions (22 h/day). They were then retrained with new flavors paired with IG infusions of 5.6% soybean oil and water. Although both strains developed preferences for the nutrient-paired CS+ solutions, the B6 mice displayed significantly stronger preferences. The B6 mice consumed more CS+ during training, which may have contributed to their enhanced preference. In a second experiment, training intakes were equated by giving B6 and 129 mice "isosweet" CS solutions prepared with different amounts of sucrose and saccharin. The B6 and 129 mice consumed more of the sugar- or fat-paired CS+ than the water-paired CS- during training. The two strains also displayed equally strong preferences for the CS+ over CS- in choice tests, indicating that they had similar postingestive responsiveness to the sucrose and soybean oil. We propose that B6 mice consume more sugar and fat than 129 mice because their stronger orosensory response stimulates greater intake, which leads to greater stimulation of postingestive nutrient detectors and further enhancement of consumption.     

Does Trapping Influence Decision-Making under Ambiguity in White-Lipped Peccary (Tayassu pecari)?

The white-lipped peccary (Tayassu pecari) is an endangered species whose bold anti-predator behaviour in comparison to related species may increase its vulnerability to hunting and predation. We used a judgement bias test to investigate whether captive peccaries that had recently experienced a trapping event made more ‘pessimistic’ decisions under ambiguity. If so, this would indicate (i) that the procedure may induce a negative affective state and hence have welfare implications, and (ii) that the species is able to adopt a cautious response style despite its bold phenotype. Eight individuals were trained to ‘go’ to a baited food bowl when a positive auditory cue (whistle; CS+) was given and to ‘no-go’ when a negative cue (horn A; CS-) was sounded to avoid a loud sound and empty food bowl. An ‘ambiguous’ auditory cue (bell; CSA) was presented to probe decision-making under ambiguity. Individuals were subjected to three tests in the order: T1 (control-no trap), T2 (24h after-trap procedure), and T3 (control-no trap). In each test, each animal was exposed to 10 judgement bias trials of each of the three cue types: CS+,CS-,CS_A. We recorded whether animals reached the food bowl within 60s (‘go’ response) and their response speed (m/s). The animals varied in their responses to the CS_A cue depending on test type. In all tests, animals made more ‘go’ responses to CS+ than CS_A. During control tests (T1 and T3), the peccaries showed higher proportions of ‘go’ responses to CS_A than to CS-. In T2, however, the animals showed similar proportions of ‘go’ responses to CS_A and CS-, treating the ambiguous cue similarly to the negative cue. There were differences in their response speed according to cue type: peccaries were faster to respond to CS+ than to CS- and CS_A. Trapping thus appeared to cause a ‘pessimistic’ judgement bias in peccaries, which may reflect a negative affective state with implications for the welfare and management of captive individuals, and also function to increase caution and survival chances following such an event in the wild environment.     

Memorization of contextual and CS conditioned fear response (freezing) in a one-trial acquisition paradigm.

In fear-conditioned Wistar rats freezing was induced by the delivery of a series of footshocks paired to tones (CS) in a specific conditioning chamber (context). CS and contextual fear were acquired in the same single conditioning session without preexposition to the conditioning chamber (day 1). Different groups of animals were conditioned employing three increasing US (footshock) intensities (0.25, 0.5, 0.75 mA). During the retention sessions context and CS conditioned freezing (fear response) were measured using a paradigm that fulfilled the following conditions: i) CS freezing retention was measured in a context different from the conditioning one; ii) CS and context freezing were measured at increased delays after the training session (days 3 and 4, 14 and 15, 28 and 29). The results show that there are significant differences between CS and context freezing retention, which are clearly related to delay after the initial session and to US intensity. In particular: 1) conditioned freezing to a discrete tone is better retained than conditioned freezing to context (irrespective of US intensity); 2) context freezing is directly related to US intensity much more than to tone freezing; 3) context freezing is easier to extinguish than tone freezing. The results are discussed in relation to previous ones and to their relevance to freezing genesis neural correlates.     

Structural characterization of the epitopes of the monoclonal antibodies 473HD, CS-56, and MO-225 specific for chondroitin sulfate D-type using the oligosaccharide library

The variation in the sulfation profile of chondroitin sulfate (CS)/dermatan sulfate (DS) chains regulates central nervous system development in vertebrates. Notably, the disulfated disaccharide D-unit, GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate), correlates with the promotion of neurite outgrowth through the DSD-1 epitope that is embedded in the CS moiety of the proteoglycan DSD-1-PG/phosphacan. Monoclonal antibody (mAb) 473HD inhibits the DSD-1-dependent neuritogenesis and also recognizes shark cartilage CS-D, which is characterized by the prominent D-unit and is also recognized by two other mAbs, CS-56 and MO-225. We investigate the oligosaccharide epitope structures of these CS-D-reactive mAbs by ELISA and oligosaccharide microarrays using lipid-derivatized CS oligosaccharides. CS-56 and MO-225 recognized the octa- and larger oligosaccharides, though the latter also bound one unique hexasaccharide D-A-D, where A denotes the disaccharide A-unit GlcUA-GalNAc(4-O-sulfate). The octasaccharides reactive with CS-56 and MO-225 shared a core A-D tetrasaccharide, whereas the neighboring structural elements located on the reducing and/or nonreducing sides of the A-D gave a differential preference additionally to the recognition sequence for each antibody. In contrast, 473HD reacted with multiple hexa- and larger oligosaccharides, which also contained A-D or D-A tetrasaccharide sequences. Consistent with the distinct specificity of 473HD as compared with CS-56 and MO-225, the 473HD epitope displayed a different expression pattern in peripheral mouse organs as revealed by immunohistology, extending the previously reported CNS-restricted expression. The epitope of 473HD, but not of CS-56 or MO-225, was eliminated from DSD-1-PG by digestion with chondroitinase B, suggesting the close association of L-iduronic acid with the 473HD epitope. Despite such supplemental information, the integral epitope remains to be isolated for identification and comprehensive analytical characterisation. Thus novel information on the sugar sequences containing the A-D tetrasaccharide core was obtained for the epitopes of these three useful mAbs.     

Purification and characterization of beta-cyanoalanine synthase and cysteine synthases from potato tubers: are beta-cyanoalanine synthase and mitochondrial cysteine synthase same enzyme?

beta-Cyanoalanine synthase (CAS; EC 4.4.1.9) and two kinds of cysteine synthases (CS; EC 4.2.99.8) have been purified from the particulate fraction of potato tubers. By DEAE Sephacel and Resource PHE chromatography, CAS activity was separated from two CS activities, designated as CS-1 and CS-2. The molecular masses of CAS, CS-1 and CS-2 were estimated to be 37, 39 and 34 kDa, respectively, by SDS-PAGE analysis. The purified CAS had CS activity, and both CS-1 and CS-2 had CAS activity. However, CAS and CSs had significant differences in kinetic characters. The antibody raised against purified CAS discriminated CAS from CSs, whereas the antibody raised against purified CS-2 recognized CS-1 and CS-2 but not CAS. The molecular mass and the partial amino acid sequence of CS-2 were similar to those of the cytosolic CS of potato, whereas the molecular mass of CS-1 was similar to that of the plastidic CS. The partial amino acid sequence of CAS was similar to those of CS isozymes, especially the mitochondrial CS isolated from spinach.     

Nicotine-evoked conditioned responding is dependent on concentration of sucrose unconditioned stimulus

Previous studies have shown that the interoceptive nicotine conditional stimulus (CS) functions similarly to exteroceptive CSs such as lights or environments. For instance, the appetitive conditioned response (CR) evoked when nicotine is repeatedly paired with sucrose presentations (the unconditioned stimulus; US) is sensitive to changes in training dose (CS salience) and the contiguity between the CS effects and sucrose. The current study was conducted to extend this research by examining the possible role of US intensity in CR acquisition and maintenance. Rats were trained using one of four sucrose concentrations: 0, 4, 16, or 32% (w/v). On nicotine sessions (0.4 mg base/kg), rats received 36 deliveries (4 s each) of their assigned concentration intermittently throughout the session; sucrose was withheld on saline sessions. In all groups, an appetitive goal-tracking CR was acquired at a similar rate. However, the asymptotic CR level varied with sucrose concentration. The magnitude of the CR was increased in rats trained with higher sucrose US concentrations. These findings are consistent with previous Pavlovian conditioning research, and extend the conditions under which the nicotine state functions as an interoceptive conditional stimulus.     

Comparing test–retest reliability and magnitude of conditioned pain modulation using different combinations of test and conditioning stimuli

This study aimed to compare the reliability and magnitude of conditioned pain modulation (CPM) by applying different test stimuli (TS) and conditioning stimuli (CS). Twenty-six healthy male participants were recruited in the study of two identical sessions. In each session, four TS (electrical, heat, handheld, and cuff pressure algometry) were applied before and during CS (cold pressor test (CPT) or cuff algometry). The same procedure was repeated with 45-min intervals, but with the other CS. Five thresholds were measured including four pain detection thresholds from four TS and pain tolerance threshold from cuff TS (cuff PTT). Intraclass correlation coefficient (ICC (3,1)) and coefficient of variation (CV) were calculated as measures of reliability. The reliability of TS before and during CS was good for all combinations (ICC: 0.60–0.96, CV: 2.2–22.9%), but the reliability of the CPM effect varied (ICC: 0.04–0.53, CV: 63.6–503.9%). The most reliable combinations were considered to be the handheld pressure pain threshold with CPT (ICC: 0.49, CV: 63.6%) and the cuff pressure pain threshold with CPT (ICC: 0.44, CV: 107.6%). Significant CPM effects were found for all combinations, except the combinations of electrical and heat pain thresholds with cuff CS, which indicates the novel classification of the CPM mechanism. The combinations of handheld pressure and heat pain threshold with CPT would provide the minimum sample size to detect the significant CPM changes in further studies. It is beneficial to provide and compare both ICC and CV to design further clinical trials.