AIT-082, a novel purine derivative with neuroregenerative properties.

Preclinical and clinical evidence support the effectiveness of neurotrophins in Alzheimer's disease (AD) and neurodegenerative diseases. Delivery of neurotrophins to target sites in the brain remains a major obstacle for their use in humans. Development of orally active agents that mimic the effects of nerve growth factor and other neurotrophins provides a promising alternative therapeutic strategy. AIT-082, a purine analogue, has been shown to reverse age-induced memory deficits in mice and is a growth factor-mimetic agent. It is orally active, rapidly penetrates the blood-brain barrier and induces the production of multiple growth factors at the appropriate target site in the central nervous system (CNS).

Neurosteroids block the memory-impairing effects of ethanol in mice.

Using a win-shift foraging paradigm to assess working memory in C57BL/6 mice, the memory-enhancing effect of low doses of the neurosteroids 5-pregnen-3 beta-ol-20-one [pregnenolone (PE)], 5-pregnen-3 beta-ol-20-one sulfate [pregnenolone sulfate (PS)], 5-androsten-3 beta-ol-17-one [dehydroepiandrosterone (DHEA)], and 5-androsten-3 beta-ol-17-one sulfate [dehydroepiandrosterone sulfate (DHEAS)] were demonstrated. The neurosteroids 5 beta-pregnan-3 alpha-ol-20-one [pregnanolone (PA)] and 5 beta-pregnan-3 beta-ol-20-one [epipregnanolone (EPI)] disrupted memory in this paradigm. PE, PS, DHEA, DHEAS, and PA were also capable of blocking the memory-impairing effect of 0.5 g/kg ethanol. EPI prevented PA from blocking the effect of ethanol. The influence of these compounds on memory and their interactions on this behavior are consistent with their actions on the GABAA system.

A behavioral animal model of posttraumatic stress disorder featuring repeated exposure to situational reminders.

The purpose of this study was to evaluate an animal model of posttraumatic stress disorder (PTSD) in mice. The model featured repeated exposures to situational reminders of a traumatic stress, which consisted of a brief electric shock, and included assessment of two behavioral parameters and the startle reflex. The findings indicated an initial, but unsustained, increase in locomotor activity in a neutral environment due to traumatic stress. Exposure to situational reminders was associated with a persistent bidirectional abnormal behavioral pattern in a fear-provoking environment and a progressive increase over time in the magnitude of the startle reflex. Exposure to situational reminders also produced an increase in aggressive behavior. This animal model appears to produce behavioral changes analogous to those seen in patients with PTSD.

Pregnenolone and pregnenolone sulfate, alone and with ethanol, in mice on the plus-maze.

The neurosteroids pregnenolone and pregnenolone sulfate were tested for anxiogenic/anxiolytic effects in mice on the elevated plus-maze. Pregnenolone in a dose of 0.01 micrograms/kg increased motor activity and caused an anxiogenic response, i.e., a decreased number of entries onto the open arms of the plus-maze. Pregnenolone sulfate had no effect on motor activity in the doses tested but showed a biphasic response on the plus-maze: at 10.0 and 1.0 micrograms/kg pregnenolone sulfate caused an anxiogenic response but at 0.1 microgram/kg it produced an anxiolytic response. When administered with 1.5 g/kg ethanol, neither neurosteroid altered the depression in motor activity caused by ethanol. However, all doses of pregnenolone tested blocked the anxiolytic effect of ethanol on the plus-maze while one dose of pregnenolone sulfate, 1.0 microgram/kg, attenuated the response to ethanol. These results support the suggestion that these neurosteroids could play a role in the initial response to stress and indicate that further work needs to be done to determine the mechanism for the interaction with ethanol.

Dehydroepiandrosterone is an anxiolytic in mice on the plus maze.

Dehydroepiandrosterone (DHEA) and its sulfate (DHEAS) are neurosteroids that have been shown to interact with the GABA system. The present study examined the effects of these compounds in mice on motor activity and behavior in the elevated plus maze. Doses of 0.5 mg/kg and above of DHEA reduced motor activity. This effect was blocked by diazepam, RO15-1788, pentylenetetrazole (PTZ), and ethanol. Both DHEA and DHEAS showed anxiolytic activity in the plus maze test, with DHEA being effective over a very wide range of doses (5 micrograms/kg to 1.0 mg/kg). Both RO15-1788 and PTZ blocked the anxiolytic effect of DHEA, there was no interaction with diazepam, and ethanol enhanced the anxiolytic effect of DHEA. At 1.0 mg/kg, DHEAS blocked the anxiolytic effect of ethanol. These results support the hypothesis that neurosteroids could be involved in the termination of a stress response.

The interaction of ethanol with the cognitive enhancers tacrine, physostigmine, and AIT-082.

Because tacrine, a cognitive enhancing agent, was being considered for approval for use in Alzheimer's disease, its possible interaction with ethanol, a commonly used substance to which elderly individuals are generally more sensitive than younger individuals, was explored. For purposes of comparison, two other drugs, which have also been shown to improve memory in mice, at doses which had activity in a working memory paradigm, were evaluated for an interaction with ethanol. Ethanol-induced sedation in mice was increased by tacrine and decreased by physostigmine, whereas AIT-082 did not alter sedation. However, tacrine had no effect on body temperature or on ethanol-induced hypothermia. Neither tacrine nor physostigmine had any effect on the rate of ethanol elimination from the blood. As tacrine comes into clinical use in the treatment of Alzheimer's disease, adverse interactions with ethanol should be explored further.

Effect of AIT-082, a purine analog, on working memory in normal and aged mice.

Because working memory is the primary type of memory which is disrupted by Alzheimer's disease and stroke and during aging, any therapeutic drug for these conditions should improve and/or restore working memory. The win-shift memory paradigm has been shown to be an excellent model of working memory. In the present study, we examined the effects of a novel purine derivative, 4-[[3-(1,6-dihydro-6-oxo-9-purin-9-yl)-1- oxopropyl]amino]benzoic acid (AIT-082) and physostigmine (PHY) on working memory. Both AIT-082 and PHY improved memory in young mice and restored memory in mice with mild age-induced memory deficits; however only AID-082 was also effective in subjects with moderate deficits. Neither drug improved memory in mice with severe memory deficits. AIT-082 exhibited effectiveness over a broad dose range (0.5-60 mg/kg), and the effects lasted for seven days after a single high-dose drug administration. AIT-082 was devoid of any effects on performance variables and has not shown any toxic side effects, thus making it an interesting potential treatment for working memory deficits associated with aging, strokes, and Alzheimer's disease.

Prevention and reversal of dopamine receptor supersensitivity by cyclo(leucyl-glycyl) (CLG): biphasic dose-response curves.

Chronic administration (21 days) of haloperidol (HAL) (IP, 1.0 mg/kg/day) induced a behavioral supersensitivity (stereotypic sniffing) to dopamine (DA) agonists (apomorphine) and upregulation (increased Bmax for sulpiride-inhibitable [3H]spiroperidol binding) of striatal and limbic D2 DA receptors (DAr). Coadministration of cyclo(leucyl-glycyl) (CLG; 8mg/kg, SC; every third day, every other day, but not every day) with HAL attenuated the behavioral supersensitivity. D2-DAr binding assays showed 1) that CLG-induced changes in Bmax parallel these behavioral changes and 2) that the biphasic CLG dose-response curve may involve CLG failure at high cumulative doses to lower Bmax. CLG also reversed an already established D2 DAr supersensitivity/upregulation (i.e., when CLG was injected daily for four days after the withdrawal of HAL). CLG alone did not alter behavior or binding. CLG's ability to both prevent and reverse D2 DAr upregulation/supersensitivity in animal models suggests that CLG may be useful, within a therapeutic window, in clinical disorders that are thought to involve upregulated DAr (e.g., tardive dyskinesia, L-DOPA-induced dyskinesias, and schizophrenia).

A low dose of ethanol impairs working memory in mice in a win-shift foraging paradigm.

Using a test of working memory which is not influenced by learning, the win-shift foraging paradigm, ethanol was found to produce amnesia in mice. This effect was produced by very low doses of ethanol, 0.5 and 1.5 g/kg, which did not alter motivation as measured by latency to leave the start box. These doses of ethanol also did not alter performance in the maze as measured by the time to traverse the maze. These data indicate that low doses of ethanol directly impair working memory--that is, the effects are not due to an alteration in learning. The win-shift foraging paradigm, which is free of some of the problems inherent in passive avoidance paradigms, may be useful in exploring the mechanism of this effect of ethanol.

Effect of age and strain on working memory in mice as measured by win-shift paradigm.

Working memory is disrupted in Alzheimer's disease and stroke; therefore, any therapeutic drug should restore deficits in working memory. The win-shift foraging paradigm has been demonstrated to be a model of working memory in rats. In the present study, this paradigm was adapted to mice because of the greater ease and economy of testing potential drugs in mice and the wider availability of strains of aged mice with naturally occurring working memory deficits. This study has demonstrated strain differences in the working memory trace and that age induces a deficit that can be detected at 11 months of age in mice. Tacrine and physostigmine enhance the memory trace in normal mice and physostigmine can reverse age-induced working memory deficits in subjects with mild and moderate deficits but not in subjects with severe deficits.

Dehydroepiandrosterone enhances the hypnotic and hypothermic effects of ethanol and pentobarbital.

Recent reports have indicated that the neurosteroid dehydroepiandrosterone (DHEA) and its sulfate (DHEAS) interact with the GABAA receptor complex. Because many of the behavioral effects of ethanol and pentobarbital are due to activity at this complex, DHEA and DHEAS were tested for their ability to interact with the hypnotic and hypothermic effects of ethanol and pentobarbital. DHEA, but not DHEAS, causes a dose-dependent increase in the sleep time induced by either ethanol or pentobarbital. At 20 mg/kg, DHEA and DHEAS themselves cause a fall in body temperature. DHEA enhances the hypothermic effect of both ethanol and pentobarbital. DHEAS enhances the hypothermic effect of ethanol, but with pentobarbital it only delays the return of body temperature to baseline levels. Neither DHEA nor DHEAS affects the metabolism of ethanol.

The effects of cyclo(leucyl-glycyl) on nigrostriatal dopaminergic supersensitivity--inhibition of apomorphine-induced climbing.

In a previous study we showed that cyclo(leu-gly) (CLG) prevents the behavioural supersensitivity induced in the mesolimbic dopamine (DA) tract (in mice) by chronic haloperidol (HAL). In the current study, we evaluated the effects of CLG on supersensitivity to DA agonists in the nigrostriatal DA tract induced by chronic HAL (1.0 mg/kg, i.p. x 21 days--Experiment 1) or by acute injection of a high dose of apomorphine (APO) (Experiment 2). In Experiment 1 CLG was given at doses of either (a) 0 mg/kg/day (b) 1 mg/kg every third day (30 minutes prior to HAL), (c) 1 mg/kg every day, or (d) 8 mg/kg every third day. In Experiment 2 the dose of CLG was 8 mg/kg, s.c., given 24h after APO. Co-administration of CLG with HAL attenuated the development of HAL-induced supersensitivity in both paradigms (b) and (c) above, although the attenuation was significantly greater in (c) compared to (b). This biphasic dose response (D-R) curve for CLG in Experiment 1 indicates that a therapeutic window exists for CLG (bell-shaped D-R curve) and is similar to previous curves for CLG effects on the mesolimbic DA tract. In Experiment 2, CLG attenuated the DA receptor supersensitivity caused by acute high dose APO. The capacity of CLG to down-regulate DA receptors and attenuate dopaminergic supersensitivity in these experiments suggests a potential therapeutic use in the prevention of tardive and/or L-dopa-induced dyskinesias.

A living systems approach to understanding the concept of stress.

In this article, the basic concepts used in conceptualizing stress and classifying its related phenomena are defined and a general conceptual framework for understanding the interrelationships among these concepts is proposed. The conceptual scheme described is intended to be applicable across disciplines including the biological and the behavioral and social sciences. This model is based largely on the living systems theory developed by Miller (1978) and the work of Selye (1950). It provides a coherent conceptual framework in which studies in the various disciplines engaged in stress research can be organized and integrated. Its use across disciplines will facilitate the sharing of information and prevent needless duplication of effort by researchers in different fields. It will also facilitate the generation and formulation of empirically testable hypotheses (including specific cross-level hypotheses), the development of more focused research designs and a clearer interpretation of findings. By understanding at which point in the stress pathway a system is when observed, a clearer picture of both the biological mechanisms involved and the psychosocial ramifications of stress may be obtained. As a consequence of this type of information becoming available, more optimal treatments for patients with stress-related disorders can be developed.

Differential down-regulation of delta opioid binding sites during physical dependence on methionine enkephalin in the rat.

Post-synaptic receptor modulation is thought to be one important mechanism involved in the adaptation of a neuronal system during chronic exposure to a drug. However, initial studies of opioid receptor regulation following chronic in vivo administration of narcotic agonists, such as morphine, reported no down-regulation in the number of opioid receptors in the brain. Subsequent studies, employing in vitro preparations, have reported evidence of opioid receptor down-regulation under specific conditions. It remains to be determined whether the in vitro phenomena of opioid receptor plasticity is relevant to the intact mammalian central nervous system. The data in this report shows that chronic in vivo administration the opioid peptide methionine enkephalin, results in a significant, regionally specific down-regulation of delta opioid receptors in rat brain: 30% decrease in receptor density in the striatum; no change in hypothalamus.

Cyclo (Leu-Gly) attenuates the striatal dopaminergic supersensitivity induced by chronic morphine.

Cyclo(Leu-Gly) (CLG), a diketopiperazine analog of Pro-Leu-Gly-NH2 (MIF), has direct effects on dopamine (DA) mediated behaviors as well as on D-2 DA receptors. Endogenous opioids, as well as morphine have also been implicated as neuromodulators of dopaminergic function. We studied these interactions in an animal model in which chronic morphine administration induces a dopaminergic supersensitivity that can be detected during the 48 hour (h) period following withdrawal of morphine. At 24 h following morphine withdrawal, there was a 3.5-fold increase in stereotypic behavior in rats following a challenge dose of apomorphine (APO) (0.5 mg/kg). By 48 h this effect had disappeared. Co-administration of CLG (8 mg/kg s.c.) with morphine attenuated the development of the behavioral supersensitivity to APO. D-2 DA receptor binding analysis indicated that parallel molecular changes occurred. There was a morphine-induced increase in the affinity (+167 percent) in antagonist (i.e. 3H-spiroperidol displaced by butaclamol) binding at 24 h after withdrawal. Co-administration of CLG with morphine attenuated these DA receptor changes at 24 hours which is consistent with the peptide's effect on stereotyped behavior. However, antagonist binding parameters did not parallel changes in behavior at 48 h. Agonist binding was then studied by examining DA displaceable 3H-spiroperidol (75 pM) binding to the D-2 DA receptor. Two receptor subpopulations D-2-HI and D-2-LO were revealed. Morphine caused an increase in the affinity for agonist binding to the D-2-HI site (83-fold increase). Affinity changes at the D-2-HI site correlated positively and strongly with the behavioral changes in all groups at both 24 and 48 h. We conclude that changes in agonist binding to D-2 DA receptors rather than antagonist binding is more consistent with the behaviors induced by morphine and CLG.

Radio-frequency analysis of the effect of haloperidol and cyclo (leucyl-glycyl) on apomorphine-induced stereotypy.

Our previous studies indicated that the peptide cyclo(leucyl-glycyl) (cLG) prevents the development of supersensitivity to dopamine in several animal models at both biochemical and behavioral levels. We therefore tested cLG in a paradigm more commonly used to model tardive dyskinesia, namely chronic haloperidol administration to rats. We found that cLG administered subcutaneously at a dose of 8 mg/kg, blocked about 50% of the supersensitizing effects of of haloperidol on apomorphine-induced stereotypic behaviors. Further, we used a novel method, radio-frequency analysis, that quantifies sniffing and other stereotypic movements. Unlike methods that rely on visual observation of stereotypy and utilize an ordinal scale, these measurements are rated by an automatic motility monitor and utilize a ratio scale. Unlike other automated motility monitors, this device can distinguish between various forms of stereotypic behaviors. Since parametric statistics can be used, there is a significant improvement in the efficiency of the task of rating and comparing stereotypy scores.

In vivo down-regulation of rat striatal opioid receptors by chronic enkephalin.

Administration of methionine enkephalin (ICV) to rats for 5 days resulted in the development of physical dependence as exemplified by a hypothermic response which peaked 2-8 hours after initiation of withdrawal. Twenty-four hours post-withdrawal, opioid receptor binding was determined in the striatum using a selective delta receptor ligand. These studies revealed a decreased in the number of receptors. Bmax decreased from 193 +/- 20.4 fmoles/mg protein in controls to 136 +/- 9.7 fmoles/mg protein in enkephalin treated rats. This difference is significant at p less than 0.001. Existing evidence suggests that this decrease in binding is predominantly due to a decrease in delta receptors. Hence, the present findings indicate that delta receptor down-regulation in vivo may be an important mechanism in the adaptive response to chronic exposure to an endogenous opioid peptide.

The effect of chronic met-enk on various dopamine systems in the rat.

Morphine and enkephalins act preferentially via different receptors, mu and delta, respectively. Chronic administration of morphine or methionine enkephalin results in development of physical dependence. Data from our laboratory suggests that there are some differences in the effect of these two opioids with regards to certain neurochemical systems.

Neuropeptides differentially effect various forms of morphine tolerance.

Two different forms of tolerance to morphine were shown to develop. One form, environment dependent (ED), was associated with cues paired with the arrival of the drug. The second form of tolerance, environment independent (EI), was not dependent on any cues. ED tolerance was induced using a multiple injection model in which a cue (orange scent) preceeded each morphine injection (ip). After 12 days the animals were tolerant to the i.p. injection. However if morphine was injected i.c.v. tolerance was no longer evident. EI tolerance was induced by a pellet implant method. These animals were tolerant to morphine regardless of the route of administration. Peptides arginine vasopressin (AVP) and cyclo (Leu-Gly) (cLG) were tested for their ability to alter tolerance. AVP was found to facilitate the development of ED tolerance but had no effect on EI tolerance. On the other hand cLG blocked the development of EI tolerance without affecting ED tolerance. The determination of brain morphine levels indicated that the ED tolerance produced by this method is dispositional while EI tolerance is functional.

The effects of acute and chronic morphine on regional distribution of cardiac output in brain.

Both acute and chronic administration of morphine resulted in an increase in the percent cardiac output received by brain. However, various brain regions were affected differently by the drug treatments. The greatest increases in percent cardiac output received after chronic administration of morphine occurred in pons and cerebellum, while the greatest increases after acute administration occurred in cortex and midbrain. The changes found are in contrast with earlier studies which suggest that morphine has no effect on cerebral blood flow.