Non-thermal microwave effects on protein dynamics? An X-ray diffraction study on tetragonal lysozyme crystals.

X-ray diffraction (XRD) was used to investigate the structural and dynamical effects of microwave fields on tetragonal single crystals of hen egg-white lysozyme at a resolution of 2.0 A. Using a modified slab-line waveguide allows on-line XRD to be carried out while the protein crystal is exposed to well defined microwave fields. High microwave power levels mainly lead to increased, but largely recoverable, lattice defects owing to the evaporation of crystal water. At lower microwave power levels, the presence of the microwave field results in localized reproducible changes in the mean-square displacements (B factors). At particular sites, it is found that the B factors even decrease with increasing microwave power. Most of these effects can be explained by a comparison of the data obtained under microwave irradiation with data obtained at elevated temperature which simulate heating owing to microwave absorption by unbound crystal water. The data show no indication of large microwave-driven displacements of structural subunits in the protein that would be expected if microwaves were to be absorbed resonantly by protein vibrations. Rather, the observed changes in the atomic mean-square displacements suggest that if microwaves couple non-thermally to globular proteins at hydration levels at which they still function, their effect on protein dynamics and structure is very small.

State-dependent effects of alcohol on recollective experience, familiarity and awareness of memories.

RATIONALE: Explicit memory (EM) is the memory for events which occurs with full awareness of where and how the recalled events took place, whereas implicit memory (IM) is the memory which is unfolded without any awareness of these events and usually becomes apparent when performance is facilitated by its presence. These two types of memory can be understood as different systems. Findings attempting to differentiate between the two systems in normal subjects have been controversial, with some researchers arguing that there is a single memory system and only the match in processes used during learning and later at retrieval can be important. OBJECTIVES: The present study compared the effects of alcohol (0.8 g/kg) or placebo administered prior to encoding and/or retrieval on measures of explicit and implicit memory in terms of recollective experience and familiarity. METHODS: At encoding subjects studied a list of 80 words presented in pairs. At retrieval, participants first carried out an implicit stem completion task, followed by an explicitly cued recall task (stem completion) which measured IM and EM respectively. After stem completion participants were required to indicate whether the items from the studied list were consciously recollected ("remember" response) or was known for a fact that were presented in the studied list ("know" response). In the IM task completed items from the studied list but not recognised by the subjects as such indicated memory without awareness. Studied items were of high and low associations. Forty-eight participants were tested in one of four drug conditions: alcohol-alcohol, placebo-placebo, placebo-alcohol, alcohol-placebo. RESULTS: In the implicit stem completion task, alcohol did not affect overall correct completion rates. However, participants who received alcohol prior to encoding reported lower awareness of correctly completed study items. In the cued recall task, alcohol also did not affect overall performance. However, participants in the same drug-state conditions (SS) reported greater recollection than familiarity with study material, whereas participants who encoded and retrieved material in different drug-state conditions (DS) reported recollection and familiarity to the same extent. In addition, DS participants showed more familiarity with study material compared to SS participants. Direct comparisons between IM and EM tasks demonstrated that alcohol at retrieval decreased the cued recall of items from high associations compared to placebo, but did not have any effect on implicit stem completion. CONCLUSIONS: In summary, these results demonstrate a dissociation of alcohol effects on measures of EM and IM. Alcohol administered prior to encoding reduced awareness of implicitly retrieved material, but did not impair IM per se, confirming previous findings with alcohol. In addition, the data provided new evidence for state-dependent retrieval effects on EM but not IM. It was also shown that for explicitly retrieved items, recollective experience benefits from same drug state, whereas familiarity benefits from different drug state between encoding and retrieval.

State-dependent effects of alcohol on explicit memory: the role of semantic associations.

RATIONALE: Memory performance can be facilitated when the context in which retrieval occurs matches the context in which learning initially took place in two separate ways, in form of interactive or independent context. In the present study, the differential effects of alcohol, as independent context, on the free recall of items of high or low semantic associations were investigated. The high and low associations offer different strengths of interactive context at stimulus input, i.e. context that influences what will be stored. METHODS: Using a state-dependent retrieval paradigm, alcohol (0.8 g/kg) or placebo was administered prior to encoding and/or retrieval and their effects were tested using measures of free recall. Forty-eight participants were tested according to a traditional state-dependent retrieval design where half of the subjects studied the items under alcohol (A), half under placebo (P) followed by retrieval of the items under A or P giving four groups (AA, AP, PA, PP). RESULTS: Delayed free recall was significantly impaired when alcohol was administered prior to both encoding and retrieval of study material (P<0.05). Alcohol administered prior to encoding and prior to retrieval decreased especially the recall of high association items (P<0.05). Participants in the same-state groups (AA, PP) recalled fewer low association items than participants in disparate state groups (AP, PA; P<0.05). This effect of drug state on low associations may reflect an inability of weaker cues to facilitate retrieval in the presence of stronger cues (i.e. high associations and drug). Indeed, participants in the same-state groups recalled a greater percentage of material in form of high association items than participants in disparate-state groups (P<0.05). CONCLUSIONS: These results demonstrate that alcohol given at encoding and at retrieval specifically impairs retrieval of high association items. However, if the drug state is the same at encoding and at retrieval, the items with the high associations represent a higher proportion of the total recalled items. These data suggest that alcohol may provide an internal context that can facilitate retrieval of information, acquired in semantic context, which otherwise would have been lost.

Excitotoxic lesions of the mediodorsal thalamic nucleus attenuate intravenous cocaine self-administration.

The present experiments investigated the effects of excitotoxic, axon-sparing lesions of the mediodorsal nucleus of the thalamus (MD) on locomotor activity and i.v. cocaine self-administration. Infusion of quinolinic acid into the MD using a glass micropipette produced well-defined neuronal loss restricted to medial and lateral portions of the MD, sparing adjacent areas such as the lateral habenula and paraventricular thalamic nucleus. MD lesions resulted in delayed habituation to activity cages. In addition, lesioned rats self-administered significantly smaller amounts of cocaine than controls during a 14-day acquisition period, and showed attenuated responding for cocaine doses on the descending limb of the dose-effect function. Since typical titrating patterns of responding were maintained in lesioned rats, and responding on the inactive lever did not differ from sham-operated animals, these present results indicate an enhanced sensitivity to the reinforcing effects of response-contingent cocaine in rats with excitotoxic lesions of the MD.

Effects of lesions of the nucleus basalis magnocellularis on the acquisition of cocaine self-administration in rats.

The nucleus basalis magnocellularis (NBM) is one element in the limbic cortical-ventral striatal circuitry that has been implicated in reinforcement processes. The present study examined the involvement of the cholinergic neurons of the NBM in mediating aspects of cocaine reinforcement. Lesions of the NBM were made by injecting 0.01 M AMPA into the subpallidal basal forebrain. Following 4 days' recovery, rats were implanted chronically with catheters in the jugular vein. In three separate experiments, rats were trained to acquire cocaine self-administration under a FR1 schedule of reinforcement at doses of 0.25, 0.083 and 0.028 mg/injection. A dose-effect function was also determined at the end of the acquisition experiments using five different doses of cocaine (0.009, 0.028, 0.083, 0.25, 0.50 mg/injection) and saline which were presented once daily in a Latin square design. There were no significant differences between groups in the acquisition of cocaine self-administration at any of the three doses studied (0.028, 0.083 and 0.25 mg/injection), although at the lowest dose, lesioned animals responded at greater levels on both active and inactive levers. However, a shift to the left in the cocaine dose-response function was observed revealing that the lesioned group self-administered significantly higher amounts of low doses of cocaine than control rats. These data suggest that the integrity of the NBM is not a critical determinant of the reinforcing effects of cocaine during the acquisition of self-administration of the drug, but that NBM-dependent cholinergic mechanisms may nevertheless interact with the neural substrates mediating the reinforcing properties of cocaine. The data are relevant to recent hypotheses of functional interactions between the dopaminergic system and the cholinergic NBM.

Substance dependence as a compulsive behavior.

A compulsion to take a drug combined with a loss of control in limiting intake is the defining feature of substance dependence or addiction, and is the conceptual framework for the criteria of substance dependence or addiction outlined by the World Health Organization and the American Psychiatric Association. However, defining exactly what constitutes loss of control and compulsive drug taking at the level of animal models is a daunting task, and it is clear that no validated animal model exists for the whole syndrome of addiction. The present discussion redefines loss of control as a narrowing of the behavioral repertoire toward drug-seeking behavior and suggests that there are many sources of reinforcement that contribute to this behavioral focus on drug seeking. Evidence is presented demonstrating separate animal models for many of these sources of reinforcement as well as for most of the criteria for substance dependence. Evidence is also presented showing that the brain neurochemical systems involved in processing drug reward are altered by chronic drug exposure to contribute additional sources of reinforcement. Challenges for the future involve not only elucidation of the neurobiological substrates of the different behavioral components of addiction, but better animal models of these components with which to effect such studies.

Effects of medial prefrontal or anterior cingulate cortex lesions on responding for cocaine under fixed-ratio and second-order schedules of reinforcement in rats.

Four experiments examined the effects of excitotoxic, axon-sparing lesions of the medial prefrontal cortex or anterior cingulate cortex in rats on responding under different schedules of intravenous cocaine self-administration and on the locomotor stimulant effects of cocaine. Experiment 1 tested the acquisition and maintenance of cocaine self-administration under a fixed ratio schedule. Rats with medial prefrontal cortex lesions showed facilitated acquisition and enhanced responding for low doses of the drug when lesions were induced before self-administration behaviour was established. Lesions of the anterior cingulate cortex did not affect cocaine self-administration. In experiment 2, rats were trained to respond under a second-order schedule of cocaine reinforcement, where responding during the fixed interval was reinforced by presentation of a cocaine-associated visual stimulus under fixed-ratio contingencies. In control rats, these schedule conditions were found to maintain high rates of responding and a scalloped pattern of responding over time. Omission of conditioned stimulus presentation during the fixed interval significantly disrupted response patterns, confirming that the stimulus served to maintain responding during the fixed interval. By contrast, rats with medial prefrontal cortex lesions showed higher rates and disrupted patterns of responding that were unchanged by stimulus omission. Rats with lesions of the anterior cingulate cortex responded at high rates throughout the fixed interval under all test conditions, indicating that the cocaine-associated stimulus did not serve to maintain temporal patterns of responding in these rats. Experiment 3 demonstrated the lack of effect of either lesion on the acquisition of responding for a non-drug reinforcer, sucrose. In experiment 4, measures of spontaneous and cocaine-induced locomotor activity revealed that rats in both lesion groups were significantly more active than controls regardless of test conditions. These data indicate that facilitated acquisition of cocaine self-administration and disrupted response patterns under second-order schedule contingencies may result from deficits in behavioural inhibition induced by medial prefrontal cortical lesions that contrast with deficits following damage to other limbic cortical regions, such as the basolateral amygdala or anterior cingulate cortex.

Effects of dopamine agonists and antagonists on cocaine-induced operant responding for a cocaine-associated stimulus.

The present study examined the effects of receptor subtype-selective dopamine agonists and antagonists on (i) cocaine-induced responding for a cocaine-associated stimulus and (ii) on responding for food and cocaine reinforcement. Rats implanted with intravenous catheters were trained to lever-press for food or cocaine reinforcers on an FR5-FR5 multiple schedule, which was preceded by a 5-min component during which only stimuli previously associated with the primary reinforcers were available response-contingently. (i) Non-contingent delivery of cocaine at the beginning of the stimulus component significantly increased responding for the cocaine-associated stimulus, compared to responding for the food-associated cue. Changes in the dose of cocaine administered non-contingently before the stimulus component resulted in an inverted U-shaped dose-effect curve in responding for the cocaine-associated cue. In subsequent experiments, pretreatment with the dopamine D2 receptor agonist bromocriptine (4.0-16.0 mg/kg IP) attenuated the cocaine-induced increase in responding for the cocaine-associated cue. In contrast, pretreatment with low doses of SDZ 208-911, a dopamine D2 partial agonist (0.025-0.1 mg/kg SC), further potentiated the cocaine-induced response. Pretreatment with low and medium doses of the dopamine D1 and D2 receptor subtype-selective antagonists SCH 23390 (D1; 5-10 micrograms/kg SC) and raclopride (D2; 100-200 micrograms/kg SC) blocked responding for cocaine-associated cues, with SCH 23390 acting more selectively than raclopride. At higher doses (SCH 23390: 20 micrograms/kg SC; raclopride: 400 micrograms/kg SC), both drugs produced non-selective effects by inhibiting responses for the food-associated cue. (ii) Varying the dose of cocaine self-administered during the multiple schedule resulted in an inverted U-shaped dose-effect curve during the cocaine components, while the number of food pellets earned remained unchanged. Pretreatment with bromocriptine selectively reduced the number of cocaine infusions obtained. The compensatory increases in responding for cocaine typically associated with SCH 23390, raclopride or SDZ 208-911 pretreatment were also observed under the present schedule conditions, although the effect did not reach statistical significance in the case of SCH 23390 and raclopride, possibly due to methodological constraints. The results indicate that the present rat model of cocaine-seeking behavior is sensitive to pharmacological manipulations and may yield important information regarding the neurobiological mechanisms underlying conditioned and unconditioned reinforcing aspects of cocaine addiction.

Schedule-induced polydipsia and the nucleus accumbens: electrochemical measurements of dopamine efflux and effects of excitotoxic lesions in the core.

The efflux of dopamine (DA) in the nucleus accumbens (NAcc) core during the acquisition of schedule-induced polydipsia (drinking in response to intermittent food presentation) was measured using rapid scan voltammetry. DA efflux increased throughout the SIP sessions, always reaching a peak after the session had terminated. There was, however, no relationship between the acquisition of the drinking response to intermittent food presentation and DA efflux. When water was absent from the test chamber, DA efflux still increased and reached a peak after food delivery was terminated, dissociating drinking and increased DA efflux. Taken in conjunction with previously presented data, these results suggest that the presence of DA in the NAcc core might be necessary for the development of SIP but that its efflux does not bear a systematic relationship to the acquisition of adjunctive behaviour. In a second experiment the effects of NMDA-induced lesions of the NAcc core on the acquisition and performance of SIP were examined. Lesioned rats did not differ to controls in terms of water intake, mean drinking bout length, latency to panel press for food or to begin drinking. The number of drinking bouts/min was reduced in lesioned rats, but did not reach statistical significance; the number of panel presses/min was significantly reduced in lesioned rats. These data demonstrate that the NAcc core is not necessary for the development of SIP but that elements of performance are affected. This suggests that the development of SIP can be fractionated and that different neural elements control different aspects of its expression. These data are used to support the hypothesis that the NAcc core is involved in focusing behaviour and regulating switching between response options.

Measures of cocaine-seeking behavior using a multiple schedule of food and drug self-administration in rats.

Animal models of human drug abuse measuring (i) cocaine-seeking behavior maintained by primary or secondary reinforcers, (ii) the time to extinction of cocaine-seeking behavior, and (iii) following extinction, reinstatement of operant responding resulting in presentation of a cocaine-associated stimulus were developed in rats. Animals were trained to respond on a multiple schedule of food and intravenous cocaine reinforcement during which either food or cocaine paired with auditory or visual stimuli were available during four alternating 30-min schedule components. At the beginning of each experimental session, a 10-min component (stimulus component) was introduced during which the stimuli associated with the primary reinforcers were made available response-contingently. Subsequent non-contingent presentation of food or cocaine at the beginning of the stimulus component produced a significant increase in lever-pressing resulting in presentation of the respective reinforcer-associated stimulus. Removal of cocaine and the associated stimulus during all schedule components led to extinction of drug-seeking behavior within six days. Lever-pressing resulting in presentation of the drug-associated stimulus was subsequently by non-contingent delivery of cocaine, but not by non-contingent presentation of the stimulus alone. These results suggest that different aspects of cocaine-seeking behavior, such as operant responding resulting in presentation of a cocaine-associated external stimulus, time to extinction in the absence of primary reinforcement, and reinstatement of responding following extinction can be measured in the rat. These tests may provide useful tools for the assessment of potential treatment drugs for human cocaine abuse.

Regulatory behaviour, exploration and locomotion following NMDA or 6-OHDA lesions in the rat nucleus accumbens.

The effects of bilateral, N-methyl-D-aspartate (NMDA)-induced lesions of the nucleus accumbens (N.Acc.) on regulatory and behavioural responding were studied in rats and compared with the effects of bilateral 6-hydroxydopamine (6-OHDA) lesions. After postoperative body weight, food and water intake had been monitored for a period of 4 weeks, rats were tested in an exploration-choice box. Spontaneous locomotion and the locomotor and stereotypy responses to different doses of dopaminergic agonists were measured subsequently. Detailed assessment of NMDA-induced lesion volumes showed that on average 81.53% of total N.Acc. area was damaged, depending on excitotoxin dose. Tyrosine hydroxylase immunohistochemistry was used to confirm loss of mesolimbic dopamine neurones following 6-OHDA. Analysis of the behavioural data showed that NMDA N.Acc. lesions significantly enhanced exploratory behaviour, spontaneous locomotor activity and the locomotor response to a low dose of D-amphetamine. By comparison, 6-OHDA lesions did not affect exploration and spontaneous locomotion but significantly attenuated the locomotor response to a low dose of D-amphetamine. Regulatory responses were unaffected 28 days after surgery, although NMDA-lesioned rats took longer to recover from postoperative hypodipsia. The results suggest that NMDA N.Acc. lesions induce a deficit in the control of general locomotor output and are consistent with the hypothesis that the N.Acc. functions as an interface between sensory input and locomotor output and that it is needed to channel activity levels appropriately.