A 24-h access I.V. self-administration schedule of morphine reinforcement and the estimation of recidivism: Pharmacological modification by arecoline.

Central cholinergic neurons are known to play a role in the pharmacological actions of opiates. The purpose of the study was to determine whether the muscarinic receptor agonist arecoline, administered during morphine self-administration, would mitigate the subsequent return to self-administration behavior. Rats self-administered increasing concentrations of morphine in operant chambers according to a schedule that permitted unlimited access to lever-activated i.v. infusions on a continuous 24 h basis from 10 to 14 days. Abstinence was induced by discontinuation of the morphine solution and mild withdrawal symptoms were evident from 14 to 74 h. Thereafter the rats remained in their home cages for a 6-week period of protracted abstinence. They were then returned to the operant chambers where lever responding had no reward consequence. The cholinergic muscarinic agonist arecoline was administered twice daily (0.25 or 1 mg/kg, s.c.) throughout the self-administration schedule of morphine. Arecoline treatment partly decreased the self-administration of morphine, it prevented the abstinence-induced decrease in body weight, and it reduced lever responding after protracted withdrawal (by 56%). In animals already dependent on morphine, arecoline failed to alter ongoing self-administration behavior, but responding induced by lever reinstatement 6 weeks after withdrawal was significantly reduced (by 33%). There was a significant relationship between the degree of self-administration activity and the degree of lever responding during reinstatement after protracted abstinence. The results of this study support the role of cholinergic systems in self-administration behavior and context-induced post-withdrawal drug seeking.

Microtubule-associated targets in chlorpyrifos oxon hippocampal neurotoxicity.

Prolonged exposure to organophosphate (OP) pesticides may produce cognitive deficits reflective of hippocampal injury in both humans and rodents. Recent work has indicated that microtubule trafficking is also adversely affected by exposure to the OP pesticide chlorpyrifos, suggesting a novel mode of OP-induced neurotoxicity. The present studies examined effects of prolonged exposure to chlorpyrifos oxon (CPO) on acetylcholinesterase (AChE) activity, immunoreactivity (IR) of microtubule-associated proteins, neuronal injury, and tubulin polymerization using in vitro organotypic slice cultures of rat hippocampus and bovine tubulin. Cultures were exposed to CPO (0.1-10 microM) in cell culture medium for 1-7 days, a regimen producing progressive reductions in AChE activity of 15-60%. Cytotoxicity (somatic uptake of the non-vital marker propidium iodide), as well as IR of alpha-tubulin and microtubule-associated protein-2 (a/b) [MAP-2], was assessed 1, 3, and 7 days after the start of CPO exposure. As early as 24 h after the start of exposure, CPO-induced deficits in MAP-2 IR were evident and progressive in each region of slice cultures at concentrations as low as 0.1 microM. CPO exposure did not alter alpha-tubulin IR at any time point. Concentration-dependent injury in the cornu ammonis (CA)1 pyramidal cell layer and to a lesser extent, CA3 and dentate cells, was evident 3 days after the start of CPO exposure (>or=0.1 microM) and was greatest after 7 days. Tubulin polymerization assays indicated that CPO (>or=0.1 microM) markedly inhibited the polymerization of purified tubulin and MAP-rich tubulin, though effects on MAP-rich tubulin were more pronounced. These data suggest that exposure to CPO produces a progressive decrease in neuronal viability that may be associated with impaired microtubule synthesis and/or function.

The induction of surface beta-amyloid binding proteins and enhanced cytotoxicity in cultured PC-12 and IMR-32 cells by advanced glycation end products.

During aging the non-enzymatic glycation of proteins and other molecules increases significantly, leading to the accumulation of advanced glycation end-products (AGEs). These AGEs enhance inflammatory and autoimmune reactions with resultant cytotoxicity. We noted in an earlier study that individuals with Alzheimer's disease exhibit enhanced expression of the receptor for advanced glycation end-products (RAGE) on the surface of their leukocytes. In order to better understand the relationship between AGEs and the cell surface binding of amyloid-beta protein (Abeta) 42 we studied the effect of two AGEs: glycated bovine serum albumin (BSA), and epsilon-carboxymethyllysine-BSA (CML), a glycoxidation product, on the binding of Abeta42 to rat PC-12 and IMR-32 cells. We measured the expression of three potential cell surface receptors binding Abeta42: RAGE, beta-amyloid precursor protein (beta-APP), and the alpha7 subtype of the nicotinic acetylcholine receptor (alpha7nAChR) by using specific antibody probes. Incubation of PC-12 or IMR-32 cells with bovine serum albumin-advanced glycation end-product (BSA-AGE) or with CML induced small but significant concentration-dependent increases in the expression of beta-APP, RAGE, and alpha7nAChRs as measured by flow cytometry or by ELISA. Incubation of the cells with 48 microM of either of the AGEs combined with varying concentrations (138-1100 nM) of Abeta42 resulted in the enhanced binding of the Abeta42 to the cell surface as compared with cells not exposed to the AGE co-treatment. The combination of AGE and Abeta treatment also resulted in the heightened expression of all three potential Abeta binding sites as well as their gene precursors. Exposure of cells to the same regimen of AGE plus Abeta resulted in the production of reactive oxygen species and mitochondrial toxicity. These results are consistent with the ability of AGEs to enhance the cell surface expression of diverse Abeta42 binding sites, a factor that can lead to the enhanced binding of amyloid and subsequent cell death.

Effect of methyldopa on brain cholinergic neurons involved in cardiovascular regulation. A study in conscious spontaneously hypertensive rats.

Chemical stimulation of brain cholinergic neurons in many species can produce hypertension. Recent studies in this laboratory have demonstrated that clonidine inhibits this central cholinergic pressor response by inhibiting the biosynthesis of brain acetylcholine. This study was designed to determine whether methyldopa, like clonidine, could inhibit brain cholinergic neurons involved in cardiovascular regulation in freely-moving spontaneously hypertensive rats (SHR). Intravenous (i.v.) injection of methyldopa (50-200 mg/kg) produced a dose-related fall in blood pressure (29/15-54/33 mm Hg) in SHR. Intracerebroventricular (i.c.v.) injection of hemicholinium-3 (HC-3) in SHR evoked a fall in arterial pressure through inhibition of acetylcholine synthesis. Doses of HC-3 (10 micrograms, or 15 micrograms, i.c.v.) and methyldopa (50 mg/kg, i.v.) were administered to produce small reductions in arterial pressure in SHR (7-14 mm Hg diastolic, respectively). When the two agents were injected simultaneously, however, a greater than additive response was obtained (p less than 0.05). Central injection of echothiophate (a long-acting cholinesterase inhibitor) to potentiate brain cholinergic activity resulted in a sustained hypertensive response (greater than 40 mm Hg) in SHR for at least 150 minutes. Simultaneous injection of or pretreatment with methyldopa (100 mg/kg, i.v.) inhibited the pressor response to echothiophate over a time course similar to its antihypertensive response in untreated SHR. Methyldopa, however, was completely ineffective in altering the hypertensive response to central injection of carbachol (1 microgram, i.c.v.). This difference in methyldopa susceptibility between the indirect-acting (echothiophate) and direct-acting (carbachol) cholinergic agonists may be related to an inhibiting effect of methyldopa on brain acetylcholine release.(ABSTRACT TRUNCATED AT 250 WORDS)

Beneficial effects of nicotine administered prior to a delayed matching-to-sample task in young and aged monkeys.

Our earlier studies have demonstrated that administration of low micrograms/kg doses of nicotine to young adult monkeys prior to a delayed matching-to-sample (DMTS) task resulted in a centrally mediated improvement in performance of the task, particularly when delay intervals which most greatly challenged the animal's capabilities were involved. The present study confirmed these findings using a completely computer driven and automated procedure. In addition, performance on the DMTS was observed to be enhanced when animals were again tested 24 h after the dose of nicotine. Further analysis of the data indicated that the majority of enhancement to nicotine could be accounted for by a greatly increased performance at the least preferred stimulus color. Position preference (left vs. right stimulus) was not a factor in nicotine-induced enhancement. Two aged monkeys (34 years old Macaca mulatta) were significantly more difficult to train in the DMTS task and their longest delay capabilities were significantly shorter than the young animals (Macaca fascicularis). Nevertheless, the aged animals were essentially similar in most respects in their responses to nicotine administration. These data are consistent with a role for central nicotinic systems in memory performance and with the ability of nicotine to produce enhancement of selective features of mnemonic strategy in young and old monkeys. Furthermore, it is possible that either model, the aged animal, or the young animal stressed to his mnemonic capability may provide a good model for learning and memory disorders in humans.

Precursors of cholinergic false transmitters: central effects on blood pressure and direct interactions with cholinergic receptors.

The purpose of this study was to determine whether a series of analogs of choline depress central cholinergic function in a manner consistent with the activity of their acetylated derivatives. Intracerebroventricular infusion of monoethylcholine (MECh), diethylcholine (DECh) and triethylcholine (TECh) inhibited the pressor response of unanesthetized rats to the subsequent intravenous injection of physostigmine (a well-characterized muscarinic response). The order of blocking potency was TECh greater than DECh greater than MECh greater than choline, directly opposite to the order of potency for elicitation of a central pressor response by their associated acetylated derivative (i.e. ACh greater than AMECh greater than ADECh = ATECh; Aronstam, Marshall and Buccafusco, 1988). In contrast, there was little selectivity between the analogs of choline in terms of their affinity for muscarinic receptors in the brainstem or cortex; the Ki's for inhibition of the binding of [3H]quinuclidinyl benzilate ranged from 0.33 to 0.95 mM). In terms of their affinity for nicotinic receptors (from the electric organ of Torpedo californica) the following order of potency was obtained: choline greater than MECh = DECh greater than TECh. Choline and MEC stimulated the binding of [3H]phencyclidine to the nicotinic ion channel (EC50's = 79 and 115 microM, respectively). At greater concentrations, all of the analogs inhibited ligand binding to the channel (Ki's from 0.2 to 10 mM), with the following order of potency: TECh greater than DECh greater than MECh greater than choline. These findings suggest that the inhibitory actions of these analogs of choline are related to their synthesis and release as false cholinergic neurotransmitters.

Excitatory modulation by a spinal cholinergic system of a descending sympathoexcitatory pathway in rats.

Earlier studies have demonstrated that an excitatory cardiovascular response can be produced through activation of a spinal cholinergic system in rats. The present study was performed to determine whether this excitatory cardiovascular response was elicited through cholinergic potentiation of a descending spinal sympathoexcitatory pathway. Application of the cholinesterase inhibitor, neostigmine, directly to the surface of the spinal cord elicited increases in heart rate selectively when neostigmine was applied to upper thoracic segments (Th1-Th4), whereas all thoracic segments participated in the generation of the associated hypertensive response. The injection of a direct cholinergic agonist, carbachol, into an upper thoracic segment (Th2) produced tachycardic and pressor responses but only pressor responses when injected into a lower segment (Th11). Tachycardic responses occurred after injection of carbachol into the intermediolateral nucleus, a site between the intermediolateral nucleus and the central canal, the right lower dorsal horn and the ventral horn. At the Th11 level, carbachol increased blood pressure only when injected into sites between the intermediolateral nucleus and central canal. The pressor response, induced by electrical stimulation of the descending spinal sympathetic tract, was markedly potentiated at all stimulus strengths after intrathecal (i.t.) injection of neostigmine and this potentiation was antagonized by atropine. The accompanying tachycardic response to electrical stimulation was not affected when low stimulus strengths were employed but means increases in the heart rate, obtained in response to larger stimulus intensities, were eliminated after administration of neostigmine. These results are consistent with the presence of an intrinsic spinal cholinergic sympathoexcitatory pathway, possibly relegated to interneurons, which elicits cardiovascular responses, at least in part through potentation of descending sympathetic influences.

Adaptive changes in M1 muscarinic receptors localized to specific rostral brain regions during and after morphine withdrawal.

Morphine-dependent rats were allowed to undergo withdrawal by abrupt discontinuation of the drug. The regional expression of brain M1 muscarinic receptors was measured directly by autoradiographic determination with [(3)H] pirenzepine, and indirectly by quantifying the relative levels of M1 mRNA encoding the receptor protein. Patterns of receptor changes after morphine treatment were in general agreement using the two methods. Frontal cortical samples derived from morphine-dependent rats exhibited a 28% increase in M1 receptor mRNA measured at the end of the infusion. At the peak of the withdrawal, M1 mRNA levels for dependent rats were much lower (33.4%) than those for control rats. Hippocampal samples derived from morphine-dependent rats exhibited no changes in M1 mRNA levels after the morphine infusion. During the peak of withdrawal, however, hippocampal M1 mRNA levels were reduced (57%) compared with levels for controls. The M1 mRNA levels remained at this reduced degree of expression even after withdrawal symptoms had subsided. Addition of diisopropylflurophophate (DFP) to the morphine infusion schedule inhibited the adaptive changes in M1 mRNA levels induced by morphine. During the peak period of withdrawal, M1 mRNA levels in the hippocampus declined by only 18% as compared with 57% for the morphine control group. The adaptive decrease in hippocampal M1 receptors after withdrawal subsided may reflect prolonged heightened cholinergic activity in an area where such cholinergic innervation plays an important role in memory.

Role of spinal and supraspinal muscarinic receptors in the expression of morphine withdrawal symptoms in the rat.

Previous studies in this laboratory have demonstrated that prior intracerebroventricular (i.c.v.) administration of the muscarinic antagonist, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) in morphine dependent rats significantly attenuates the development of cardiovascular and certain behavioral responses precipitated by the opiate antagonist, naloxone. The purpose of this study was to determine whether both supraspinal and spinal cholinergic neurons are involved in the expression of withdrawal symptoms. Employing localized (i.c.v. or intrathecal, i.t.) infusions of muscarinic antagonists, it was determined that a significant antiwithdrawal action could be produced through both an inhibition of supraspinal and spinal cholinergic neurons. Pharmacological difference emerged regarding the antiwithdrawal potential of 4-DAMP and the partially M1 selective antagonist, pirenzepine. While our previous studies had revealed that pirenzepine had essentially no antiwithdrawal activity when administered by the i.c.v. route, in the present study, pirenzepine evoked a marked antiwithdrawal action by the i.t. route, significantly inhibiting both cardiovascular and behavioral signs of withdrawal. In contrast, 4-DAMP which was effective by the i.c.v. route (especially for the cardiovascular symptoms), elicited no antiwithdrawal action by the i.t. route. As a muscarinic antagonist (ability to block the pressor response to central injection of carbachol) 4-DAMP was equally active by i.c.v. or i.t. injection. However, pirenzepine was clearly more effective in this regard by the i.t. route. These results are consistent with ability of muscarinic antagonists to offer significant anti-morphine withdrawal activity at both supraspinal and spinal locations. They also suggest that different muscarinic systems, possibly different receptor subtypes, mediate the expression of morphine withdrawal symptoms within the two regions of the CNS.

Cholinergic false transmitters: physiological and biochemical actions in central and peripheral systems.

Three N-ethyl substituted analogs of acetylcholine (ACh) were evaluated for potential use as false neurotransmitters to decrease cholinergic transmission. This evaluation included (1) the elevation of arterial blood pressure upon central administration, (2) depression of blood pressure upon intravenous injection and (3) interactions with central muscarinic and peripheral nicotinic receptors. With respect to the central pressor response, ACh, acetylmonoethylcholine (AMECh) and acetyldiethylcholine (ADECh) were full agonists of decreasing potency; acetyltriethylcholine (ATECh) was a partial agonist of considerably lower potency. The duration of response paralleled potency. With respect to the peripheral depressor response, ACh and AMECh were full agonists of equal potency, and ADECh and ATECh were partial agonists of at least 100-fold lower potency. In terms of their affinity for central muscarinic receptors (brainstem and cerebral cortex), the following series was obtained: ACh greater than AMECh much greater than ADECh = ATECh. All of the agents had a greater affinity for muscarinic receptors in the brainstem compared to cortex. Acetylcholine and AMECh recognized multiple receptor binding conformations; the binding of ADECh and ATECh indicated interaction with a single set of equivalent sites. The affinity for nicotinic ACh receptors from the Torpedo electric organ was ACh = AMECh much greater than ADECh; ATECh had little affinity for these receptors. Acetylcholine, AMECh and ADECh stimulated the binding of [3H]phencyclidine to the ion channel of nicotinic receptor (potency series = ACh greater than AMECh = ADECh); ATECh was inactive. Acetylcholine, AMECh and ADECh also induced receptor conversion to a desensitized conformation; ATECh did not.(ABSTRACT TRUNCATED AT 250 WORDS)

Contribution of pre- and post-synaptic components to heightened central cholinergic activity in spontaneously hypertensive rats.

In several models of essential hypertension in the rat, the pressor response to central cholinergic stimulation is enhanced with respect to age-matched normotensive controls. Neurochemical evidence is available from previous studies to suggest that both pre- (transmitter synthesis and release) and post-synaptic (muscarinic receptors) components of cholinergic transmission may be enhanced in hypertensive rats and that such alterations might be responsible for the exaggerated pressor response to centrally-acting cholinergic agonists. The present study, employing pharmacological approaches, was designed to determine whether pre- or post-synaptic components of central cholinergic transmission were more important in this regard. The pressor response to intravenous injection of the indirect-acting agonist, physostigmine, but not to that of the direct-acting agonist, arecoline, was significantly reduced by pretreatment with hemicholinium-3 (to deplete acetylcholine in brain). The pressor response to physostigmine, but not to arecoline, was enhanced in adult, spontaneously-hypertensive rats, with respect to their normotensive controls. The pressor response to oxotremorine was partially inhibited by pretreatment with hemicholinium-3, but was only partially effective at inducing an exaggerated pressor response in spontaneously hypertensive rats. These results are consistent with the hypothesis that heightened cholinergic activity in spontaneously hypertensive rats is derived primarily through altered pre-synaptic mechanisms, and that the actions of oxotremorine may involve a multi-synaptic cholinergic pathway.

Sex dimorphisms in the cognitive-enhancing action of the Alzheimer's drug donepezil in aged Rhesus monkeys.

Brain acetylcholinesterase has been targeted for the development of novel treatments for memory deficits associated with Alzheimer's disease (AD) and other neurodegenerative disorders. The long-acting AChE inhibitor donepezil (Aricept) is used to improve memory and other aspects of cognition in AD patients. Because donepezil and other cholinesterase inhibitors are effective in a restricted population of AD patients, this study was to designed to determine whether aged females monkeys receive the same level of benefit to the mnemonic action of donepezil as do males. In this study, six male and six female rhesus monkeys (>20 years) who were proficient in the performance of a delayed matching-to-sample task each received an ascending series of four doses of donepezil (0.01-0.1 mg/kg) over 5 weeks. As a group, male subjects exhibited improvement in task accuracy across the three highest doses, with the maximum effect occurring after the 0.025 mg/kg dose. However, the females exhibited increased task accuracy only after the highest dose. When data were combined for sessions run 10 min after drug administration and for sessions run 24 h later (in the absence of drug), improvements in task accuracy were greater on average for males. Most of this difference was attributed to the fact that task accuracy by females actually declined during sessions run after the two lowest doses of donepezil. When task performance after donepezil was determined as the individualized Best Dose, as a group, males responded maximally to less than half the dose that was maximal for females. These findings support the concept that aged males and females respond differently to this class of agents, perhaps representing fundamental sex-related differences in memory processing, or in the manner that age affects these processes.

Structure-activity studies on 2-methyl-3-(2(S)-pyrrolidinylmethoxy) pyridine (ABT-089): an orally bioavailable 3-pyridyl ether nicotinic acetylcholine receptor ligand with cognition-enhancing properties.

2-Methyl-3-(2(S)-pyrrolidinylmethoxy)pyridine, ABT-089 (S-4), a member of the 3-pyridyl ether class of nicotinic acetylcholine receptor (nAChR) ligands, shows positive effects in rodent and primate models of cognitive enhancement and a rodent model of anxiolytic activity and possesses a reduced propensity to activate peripheral ganglionic type receptors. The profiles of S-4, its N-methyl analogue, and the corresponding enantiomers across several measures of cholinergic channel function in vitro and in vivo are presented, together with in vitro metabolism and in vivo bioavailability data. On the basis of its biological activities and favorable oral bioavailability, S-4 is an attractive candidate for further evaluation as a treatment for cognitive disorders.

Dahl salt-sensitive and salt-resistant rats: examination of learning and memory performance, blood pressure, and the expression of central nicotinic acetylcholine receptors.

Substantial human and animal data suggest a correlation between hypertension and memory impairment that may appear prior to overt manifestations of cerebrovascular pathology. It is unclear, however, whether hypertension plays a causal role in these memory deficits, whether hypertension and cognitive impairment are each based in family history and not interdependent, or whether a combination of these factors is important. The purpose of this study was to assess whether deficits in memory performance and nicotinic acetylcholine receptors were present in Dahl salt-sensitive rats (as observed previously in spontaneously hypertensive rats) and whether the presence of hypertension per se (induced with an 8% Na(+) diet) contributed to the deficits. Memory was assessed in a passive avoidance task, an eight-arm radial arm maze and in a water maze task, and nicotinic receptors were measured via quantitative receptor autoradiography utilizing [125I]alpha-bungarotoxin and [3H]epibatidine. Salt-sensitive rats exhibited impaired performance in both spatial learning tasks, but not the passive avoidance task, compared to controls (salt-resistant strain) and they exhibited reductions in nicotinic receptors labeled by [125I]alpha-bungarotoxin but not [3H]epibatidine in some brain regions, including some areas important for memory (e.g. the hippocampus and amygdala). In the radial arm maze, the degree of memory impairment and in binding studies the reduced expression of nicotinic receptors each failed to correlate with the highest blood pressures, and the salt-sensitive animals were impaired relative to controls whether or not the high Na(+) diet was administered. In contrast, higher blood pressures did correlate with inferior task performance in the water maze. These findings may suggest that the genetics of the subjects were critical for performance when appetitive drives were involved, but diet (and perhaps hypertension) were key to performance when memory did not involve appetitive drives or mechanisms. Overall, the data obtained from Dahl rats appear to support the role of family history (selective breeding in rats) as underlying the reductions in central nicotinic acetylcholine receptors, whereas both family history and hypertension may contribute to poor cognitive performance.

Potential cognitive actions of (n-propargly-(3r)-aminoindan-5-yl)-ethyl, methyl carbamate (tv3326), a novel neuroprotective agent, as assessed in old rhesus monkeys in their performance of versions of a delayed matching task.

(N-propargyl-(3R)-aminoindan-5-yl)-ethyl, methyl carbamate (TV3326), a known neuroprotective agent exhibiting the properties of both an inhibitor of monoamine oxidase (brain selective) and an inhibitor of acetylcholinesterase was administered to seven old rhesus monkeys well trained to perform versions of a delayed matching-to-sample (DMTS) task. An increasing dose regimen of TV3326 was administered orally according to a schedule that allowed the animals to perform the standard DMTS task and a self-titrating version of the DMTS task each week during the study. A distractor version of the task was administered during two of the doses of TV3326. Under the conditions of this experiment TV3326 failed to significantly affect accuracy on the standard DMTS task; however, the drug was very effective in improving the ability of subjects to titrate to longer-duration delay intervals in the titrating version of the task. The maximal drug-induced extension of the self-titrated delay interval amounted to a 36.7% increase above baseline. This increase in maximum delay duration occurred without a significant change in overall task accuracy. TV3326 also significantly improved task accuracy during distractor (interference) sessions. The compound was effective enough to return group performance efficiency to standard DMTS vehicle levels of accuracy. These results were independent of whether trials were associated with a distractor or non-distractor delay interval, and they were independent of delay interval. The lack of delay selectivity in task improvement by TV3326 may not be consistent with a selective effect on attention. TV3326 was not associated with any obvious side effect or untoward reaction of the animals to the drug. Thus, TV3326 may be expected to offer a significant positive cognitive outcome in addition to its reported neuroprotective action.

Deficits in spatial learning and nicotinic-acetylcholine receptors in older, spontaneously hypertensive rats.

Spontaneously hypertensive rats are often used as models of attention deficit hyperactivity disorder and to investigate the effects of hypertension on cognitive function. Along with the wide variety of cardiovascular anomalies, these animals as young adults also exhibit deficits in memory and attention and central nicotinic-acetylcholine receptor sites. These findings may have particular significance since nicotinic receptors appear to be involved in the regulation of cerebral circulation and mnemonic function. Furthermore, a lack of high affinity nicotinic receptors (in knockout mice) has also been shown to accelerate both the structural and cognitive degeneration associated with age, findings that may be especially relevant to age-related memory disorders such as Alzheimer's Disease where large deficits in nicotinic receptors are observed. Since spontaneously hypertensive rats appear to be both memory-impaired and deficient in nicotinic receptors at a young age (compared to the non-hypertensive phenotype, Wistar-Kyoto rats), we were interested to learn if these conditions were exacerbated in older animals with particular interest in specific nicotinic receptor subtypes in memory areas of the brain. Spatial learning was assessed in 15-month-old subjects of each phenotype (i.e. hypertensive and non-hypertensive) using a two-phase water maze paradigm, and nicotinic receptors were measured via autoradiography with [125I]-alpha-bungarotoxin and [3H]-epibatidine. In the water maze, both groups learned to locate a hidden platform as indicated by progressively shorter latencies across training days, however, Wistar-Kyoto rats were more efficient in both phases. While the number of both bungarotoxin and epibatidine binding sites was lower in the hypertensive rats across several brain regions, in the case of epibatidine binding, the magnitude of the difference and the number of areas affected was generally greater and included areas important for spatial learning (e.g. frontal and entorhinal cortex). In a direct comparison between 3-month-old and 15-month-old rats of each phenotype, epibatidine sites were markedly reduced by age (i.e. by greater than 50% in some cases) across multiple brain regions in both groups, although Wistar-Kyoto rats appeared to be more substantially affected by age. These data further support the use of the spontaneously hypertensive rat as model for studying learning-impairment and reduced central nicotinic receptors and also indicate that these characteristics persist and (in the case of high affinity nicotinic receptor cites) worsen with age.

Kinetics of [3H]choline and [3H]acetylcholine metabolism in several regions of rat brain following intracerebroventricular injection of [3H]choline. Effects of haloperidol.

Intracerebroventricular (icv.) injection of [3H]choline in conscious rats produced a rapid, efficient labeling of brain choline and acetylcholine (ACh) stores without altering steady-state levels of endogenous ACh. The kinetics of [3H]choline and [3H]ACh metabolism were measured in seven brain regions for up to 10 min following icv. administration of [3H]choline. The initial rate of formation of [3H]ACh varied in different brain areas, being greatest in the striatum and least in the hypothalamus. In contrast, the rate of [3H]choline metabolism was similar in all regions of the brain. Pretreatment of rats with haloperidol resulted in an increase in the apparent synthesis rate of ACh only in the striatum and rostral hypothalamus, pointing to possible dopaminergic-cholinergic interaction in these regions.

Opposing influences on behavior mediated by muscarinic and nicotinic receptors in the rat posterior hypothalamic nucleus.

Microinjection of carbachol into the posterior hypothalamic nucleus (PHN) of freely moving rats evoked marked behavioral changes characterized by an escape reaction. This response was quantitated by measuring locomotor activity. In contrast, cholinergic stimulation of the PHN with neostigmine produced sedation and inactivity. Local pretreatment with the nicotinic receptor blocking agent mecamylamine blocked the excitatory effects of carbachol while the muscarinic antagonist atropine abolished the inhibitory effect of neostigmine on motor activity. It is concluded that behavioral changes evoked through cholinergic stimulation of the PHN may be mediated by a muscarinic system which controls sedation and a nicotinic pathway which mediates arousal.

Chronic, low-level exposure to diisopropylfluorophosphate causes protracted impairment of spatial navigation learning.

Chronic, low-level exposure to cholinesterase inhibitor organophosphate (OP) insecticides or chemical warfare agents produces abnormalities in CNS acetylcholine (ACh) function, and in humans, may be associated with impaired cognitive function as well after withdrawal from such exposure. The purpose of the present study was to identify the severity of impairment in spatial learning of rats following protracted withdrawal from chronic, low-level exposure to the OP agent diisopropylfluorophosphate (DFP). Assessment of spatial learning began either 3 or 17 days after completion of a 14-day DFP treatment regimen (50, 250, or 500 micrograms/kg). During the 14-day treatment regimen, spontaneous activity and olfactory behaviors were suppressed, effects which subsided with repeated exposure to the 250 micrograms/kg dose regimen. In contrast, both behaviors were stimulated by exposure to the 50 micrograms/kg dose regimen, as was body weight gain. Performance of the spatial test of working memory was impaired for up to 21 days after withdrawal from treatment with a 250 micrograms/kg dose of DFP. AChE activity in the frontal cortex and hippocampus was suppressed to 42.58% and 50.35% of control levels, respectively, 3 days after completion of the DFP (250 micrograms/kg) treatment regimen. By 7 days after withdrawal from treatment, AChE activity in the cortex and hippocampus had recovered to 81.87% and 64.61% of control levels, respectively. These levels represent increases in activity of 39.29% and 14.26% in these regions, as compared to AChE activity in 3 days after DFP withdrawal. By 21 days after withdrawal from treatment, AChE in both brain regions had recovered to levels similar to those of controls. Chronic, low-level OP exposure, therefore, produces protracted impairment of working memory after drug withdrawal that is not associated with continued suppression of AChE activity. This impairment may, however, be associated with a decreased rate of AChE recovery in the hippocampus, relative to the cortex. This decreased rate of enzyme recovery may contribute to hippocampal toxicity underlying protracted impairment of working memory.

Velnacrine maleate improves delayed matching performance by aged monkeys.

Velnacrine maleate is a novel, orally active acetylcholinesterase inhibitor of the acridine class with a longer duration of action than physostigmine. Velnacrine has shown efficacy in the treatment of Alzheimer's disease and in improving both normal and experimentally impaired mnemonic function in animals and humans. To characterize this action further, the present study evaluated velnacrine for its ability to ameliorate the decline in short-term memory associated with aging in non-human primates at two time points after velnacrine administration: (1) 30 min and (2) 24 h. Initially, doses of 1, 2, 4, and 6 mg/kg, PO (free base corrected) were administered once to each of six aged (25-40 years), memory-impaired macaques that had been trained to perform a delayed matching-to-sample (DMTS) paradigm. The dose associated with the greatest improvement in session performance was administered three more times to the same individual. Four of the six monkeys showed improved DMTS performance during the repeated best dose phase (phase 2). Almost all of the improvement occurred during long-delay trials. Compared to placebo trials, velnacrine induced a significant improvement of long delay DMTS (58.0-66.7%, 13.4% of the placebo value). Long delay DMTS remained significantly improved during the test session conducted 24 h following velnacrine administration. Pharmacokinetic analysis following administration of 4 or 6 mg/kg velnacrine to three aged monkeys revealed peak plasma concentrations ranging from 27 to 166 ng/ml, 30-60 min after dosing. Six hours after dosing velnacrine plasma levels decreased to 5.1-11.8 ng/ml; and 24 h after dosing velnacrine plasma levels were less than the limit of quantitation (5 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)