Cocaine enhances one-way avoidance responding in mice.

We reported previously that posttraining cocaine injections enhance subsequent performance of an automated jump-up avoidance response and a trough avoidance response in rats. In the present study we examined the species generality of the cocaine enhancement by investigating the effects of posttraining cocaine injection on subsequent performance of a one-way active avoidance response in mice. Cocaine (30 mg/kg, IP) administered to mice immediately following completion of two escape-only trials on day 1 significantly enhanced avoidance response performance on day 2. Neither lidocaine nor cocaine methiodide, when administered in doses equimolar to the effective cocaine dose, altered performance on day 2. These data indicate that cocaine's enhancement of avoidance responding in mice probably is neither peripherally mediated nor attributable to its local anesthetic properties.

Differences in one-way active avoidance learning in mice of three inbred strains.

DBA/2J, C57BL/6J, and C3H/HeJ mice were given 10 one-way avoidance training trials per day, using an unconditioned stimulus intensity that provided equivalent motivation for learning to mice of all three strains, and were found to differ in their abilities to learn and retain the response. DBA/2J mice acquired the response in fewer days than did the mice of the other two strains, although C57BL/6J mice eventually reached a level of performance similar to that of DBA/2J mice. Both the rate of acquisition and the level at which avoidance performance stabilized were significantly lower in C3H/HeJ, than in DBA/2J or C57BL/6J, mice. In addition, DBA/2J mice showed a significantly greater task retention from one testing day to the next than did C57BL/6J or C3H/HeJ mice.

Enkephalin hydrolysis by mouse plasma in vitro.

Hydrolysis of [Leu]- and [Met]enkephalin was determined in samples of pooled whole mouse plasma in vitro by using HPLC-ECD to measure accumulation of Tyr-containing metabolites. More Tyr-Gly-Gly accumulated from [Met]enkephalin than from [Leu]enkephalin hydrolysis, and [Met]enkephalin's half-life in mouse plasma was approximately half that of [Leu]enkephalin. Comparisons of metabolite formation in the presence versus the absence of inhibitors with high selectivity for various peptidases demonstrated that a bestatin-sensitive aminopeptidase, presumably aminopeptidase M, as well as enkephalinase and angiotensin converting enzyme, participate in the hydrolysis of enkephalin in mouse plasma.

Opioid receptors are involved in an NMDA receptor-independent mechanism of LTP induction at hippocampal mossy fiber-CA3 synapses.

Long-term potentiation (LTP) of mossy fiber responses in area CA3 of the rat hippocampus in vivo is blocked by naloxone, an opioid receptor antagonist, in a stereospecific and dose-dependent manner. LTP of commissural afferents to the same population of CA3 pyramidal cells is not attenuated by naloxone. This suggests that opioid receptors are involved in a mechanism of LTP induction that is specific to mossy fiber synapses, and that endogenous opioid receptors are involved in a mechanism of LTP induction that is specific to mossy fiber synapses, and that endogenous opioid peptides, presumably released as a result of mossy fiber stimulation, may be necessary for the induction of mossy fiber LTP. The naloxone sensitivity is limited to the induction phase of LTP, since naloxone does not reverse previously established LTP. These data suggest that LTP at the mossy fiber-CA3 synapse constitutes an NMDA receptor-independent, opioid receptor-dependent, form of hippocampal synaptic plasticity.

Further characterization of the in vitro hydrolysis of [Leu]- and [Met]enkephalin in rat plasma: HPLC-ECD measurement of substrate and metabolite concentrations.

Hydrolysis of [Leu]- and [Met]enkephalin was determined in whole rat plasma in vitro by using HPLC-ECD to measure Tyr, Tyr-Gly and Tyr-Gly-Gly formation. Although [Leu]- and [Met]enkephalin did not differ in Tyr or Tyr-Gly accumulation, the amount of Tyr-Gly-Gly resulting from [Met]enkephalin hydrolysis was greater than that resulting from [Leu]enkephalin hydrolysis, and [Met]enkephalin's half-life in plasma was slightly shorter than that of [Leu]enkephalin. By comparing metabolite formation in the presence and absence of peptidase inhibitors with high selectivity for their respective enzymes, these studies demonstrated that aminopeptidase M and angiotensin converting enzyme are the major peptidases that hydrolyze enkephalins in rat plasma.

Hydrolysis of [Leu]enkephalin by chick plasma in vitro.

The in vitro hydrolysis of [Leu]enkephalin added to plasma collected from 2-day-old chicks was studied with two different techniques: thin-layer chromatography separation of intact [3H]-[Leu]enkephalin from its [3H]-Tyr-containing metabolites and high-performance liquid chromatography-electrochemical detection assay of [Leu]enkephalin disappearance and Tyr-containing metabolite accumulation. The radiometric assay evaluated enkephalin hydrolysis at close to presumed physiological concentrations of this peptide, whereas the liquid chromatography assay necessitated 100-fold higher peptide concentrations to achieve adequate sensitivity. Similar results were obtained with both techniques. We found that the in vitro hydrolysis of [Leu]enkephalin is more rapid in chick plasma (half-life, 0.7-1 min) than in rat (half-life, 2-2.5 min) or mouse (half-life, 9-14 min) plasma. Comparison of the rate of enkephalin hydrolysis and pattern of metabolite accumulation in the absence vs. the presence of various peptidase inhibitors suggested that a bestatin-sensitive aminopeptidase, probably aminopeptidase M, is the primary enzyme responsible for the hydrolysis of enkephalin by chick plasma, and that less than 1% of the total hydrolysis of [Leu]-enkephalin by chick plasma is attributable to dipeptidyl carboxy-peptidase activity. This pattern of enzyme activities differs from that which we identified previously in rat and mouse plasma.

Hydrolysis of [Leu]enkephalin by chick brain in vitro.

Using high-performance liquid chromatography with electrochemical detection to measure substrate disappearance and metabolite accumulation following addition of [Leu]enkephalin to samples prepared from chick brain in vitro, the following were found: 1. [Leu]enkephalin hydrolysis by whole forebrain homogenates is almost solely attributable to aminopeptidase MII activity. 2. [Leu]enkephalin hydrolysis by whole forebrain P2 membrane fractions is attributable to both aminopeptidase MII and dipeptidyl carboxypeptidase activity. 3. Differences are apparent in both [Leu]enkephalin disappearance and Tyr-Gly-Gly accumulation in P2 membrane fractions, but not in homogenate fractions, prepared from several regions of the chick brain.

Sensitive method for measuring hydrolysis of enkephalins in plasma, using high-performance liquid chromatography with electrochemical detection.

This paper describes a simple and sensitive method for detection of [Leu]- and [Met]enkephalin and their N-terminal tyrosine-containing metabolic fragments (Tyr, Tyr-Gly, Tyr-Gly-Gly, and Tyr-Gly-Gly-Phe), using high-performance liquid chromatography with electrochemical detection. The method employs a carbon graphite working electrode with increased working electrode surface area (40 mm2). The procedures were applied to assay of the activities of enkephalin-degrading enzymes in whole plasma collected from rats, mice, and chicks.

DPen2-[DPen5]enkephalin, a delta opioid receptor-selective analog of [Leu]enkephalin, impairs avoidance learning in an automated shelf-jump task in rats.

Both [Leu]enkephalin and DPen2-[DPen5]enkephalin, a delta opioid receptor selective analog of [Leu]enkephalin, impaired acquisition of an automated shelf-jump response in rats. A similar level of impairment was produced by equimolar doses of the two enkephalins. As is seen for [Leu]enkephalin when tested in a one-way active avoidance task, the dose-response function for the impairment produced by DPen2-[DPen5]enkephalin in the automated shelf-jump task is U-shaped. These results, together with our previous findings that DPen2-[DPen5]enkephalin and [Leu]enkephalin both impair acquisition of a one-way active avoidance response in mice, and that [Leu]enkephalin impairs acquisition of that same response in rats, support our suggestion that delta opioid receptors are implicated in the effects of [Leu]enkephalin on conditioning. In addition, these results indicate that the involvement of delta opioid receptors in acquisition impairment extends to two species of rodents and to two different avoidance conditioning tasks.

Plasma uptake and in vivo metabolism of [Leu]enkephalin following its intraperitoneal administration to rats.

To understand better how [Leu]enkephalin (LE) acts to modulate learning and memory in rats, the plasma uptake, disappearance, and metabolism of LE were investigated following its intraperitoneal administration. Concentrations of [3H]-LE and its radioactive metabolites were determined by thin layer chromatography in plasma samples withdrawn from rats at various times after injection of peptide. As measured in rats receiving an IP injection of a dose of LE (3 micrograms/kg) that impairs active avoidance conditioning, the LE was very rapidly metabolized, with greater than 95% of plasma [3H] in the form of metabolites by 1 min after injection. Despite this rapid metabolism, low but measurable quantities of intact LE were detectable in plasma at all sampling times. Consistent with a greater potency of D-Ala2-[D-Leu5]enkephalin (DADLE) than of LE in modulating avoidance conditioning, DADLE was less rapidly metabolized than was LE following its IP administration. The metabolism of DADLE and LE in vivo was more rapid than it was in plasma in vitro, suggesting a role for membrane bound enzymes in the metabolism of IP-administered enkephalins. The data demonstrate that, despite a rapid hydrolysis of LE in vivo, sufficient LE is present in plasma following IP administration of a behaviorally active dose to support a role of circulating intact LE in the modulation of avoidance conditioning.

Characterization of hydrolysis of [leu]enkephalin and D-ala2-[L-leu]enkephalin in rat plasma.

Based on differences in total metabolite accumulation in the presence or absence of selective peptidase inhibitors, rat plasma is found to have its own unique pattern of enkephalin hydrolysis. Approximately 85-90% of the hydrolysis of [leu]enkephalin is attributed to the combined action of aminopeptidase M and angiotensin converting enzyme, whereas "enkephalinase" and aminopeptidase MII activity against [leu]enkephalin are not detectable. Similarly, 80-90% of the hydrolysis of D-ala2-[L-leu] enkephalin (DALLE) is due to the combined action of aminopeptidase M and angiotensin converting enzyme, whereas aminopeptidase MII and enkephalinase activity against this substrate also could not be detected. This is in contrast to the high susceptibility to hydrolysis by enkephalinase, and the low susceptibility to aminopeptidase activity, for DALLE in brain tissue. Among other alternatives, it is suggested that enkephalin hydrolysis in plasma may appear to be unique because of differences in enzyme conformation and/or the availability of a substance(s) that competes with, or alters the binding of, [leu] enkephalin, DALLE or the inhibitors to the enzymes.

Behavioral assessment of forgetting in aged rodents and its relationship to peripheral sympathetic function.

Observation of age-related memory deficits in rodents is often dependent on the behavioral task used to assess these changes, rather than being universal to all memories. A review of studies using aversively-motivated multiple trial training paradigms suggests that the apparent acquisition deficits common to older animals may instead be due to a confounding tendency of these animals to behavioral rigidity or perseveration. Data obtained using single trial training paradigms, such as the one-trial passive avoidance task, indicate that young and old rodents can learn tasks with equal facility, that retention in young and old animals is similar at short training-testing intervals, but that retention is impaired in aged animals at longer training-testing intervals. We suggest that the adrenal medulla, a peripheral source of catecholamines, secretes catecholamines that act outside the blood-brain barrier to modulate memory processes. Further, we review evidence suggesting that the ability of the adrenal medulla to respond to the stresses of footshock during aversively-motivated training are impaired in aged rodents, and that this impairment may contribute to the rapid forgetting observed in senescent animals.

Enkephalin hydrolysis in plasma is highly correlated with escape performance in the rat.

A very high correlation was found in rats between latency to escape on the first trial of an active avoidance task and the rate at which [leu]enkephalin is hydrolyzed in plasma. In addition, the rate of [leu]enkephalin hydrolysis is significantly altered following the first training trial. The results suggest that a regulatory enzyme system exists for [leu]enkephalin in plasma and that this system may be important for modulating behavior.

Enkephalins and learning and memory: a review of evidence for a site of action outside the blood-brain barrier.

A series of studies indicate that enkephalins exert dramatic influences on learning and memory in rats and mice, when studied with conditioning tasks that are both negatively and positively motivated. Pharmacological analysis of these enkephalin actions on conditioning suggests that the [leu]enkephalin acts through a delta opioid receptor which is located outside the blood-brain barrier. Control studies indicate that enkephalins do not simply affect the performance of a conditioned response through actions on shock sensitivity or locomotor activity. Characterization of the peripheral enkephalin mechanism that affects behavior suggests an action through an enzymatic system that controls the concentrations of enkephalin present at its receptors in the periphery. This enzymatic mechanism is sensitive to experience, since its activity changes following conditioning, which suggests that it may be a regulatory mechanism for behavior.

Differential effects on active avoidance performance and locomotor activity of two major enkephalin metabolites, tyr-gly-gly and des-tyr-[leu]enkephalin.

We examined the effects of two enkephalin metabolites, des-tyr-[leu]enkephalin and tyr-gly-gly, on one-way active avoidance conditioning in mice. These metabolites are products of the two major enkephalin hydrolyzing enzymes in plasma, aminopeptidase and angiotensin converting enzyme. Like [leu]enkephalin from which it may be formed, tyr-gly-gly impaired avoidance acquisition, and its dose-response function for this effect was U-shaped. Also like [leu]enkephalin, tyr-gly-gly did not alter locomotor activity. On the other hand, des-tyr-[leu]enkephalin, at the doses tested, was without effect on avoidance conditioning but produced decreased locomotion. These data suggest that the tyrosine end of the enkephalin molecule may be important for its effects on conditioning. Because of their low opioid potencies, it is unlikely that the behavioral actions of tyr-gly-gly and des-tyr-[leu]enkephalin are mediated through opioid receptors.

Decreased locomotor activity produced by repeated, but not single, administration of murine-recombinant interferon-gamma in mice.

A single treatment with murine-recombinant interferon-gamma (murIFN-gamma; 30 micrograms/mouse), whether evaluated immediately after, or four hrs after, intraperitoneal injection, does not alter open field activity levels. On the other hand, repeated murIFN-gamma administration (30 micrograms/mouse/day for 5 days) results in decreased spontaneous locomotor activity and an increase in body weight.