Mahanandeeshwar Gattu

Cognitive impairment in spontaneously hypertensive rats: Role of central nicotinic receptors. Part II

The adult spontaneously hypertensive rat (SHR) has been shown to exhibit a decrease in the expression and nicotine-stimulated function of brain nicotinic acetylcholine receptors, factors that could play a role in the impaired ability of this strain in the performance of learning and memory-related tasks. The purpose of this study was to determine whether either or both the impaired task performance and the loss of nicotinic receptors is directly related to the presence of the hypertensive state. To address this issue, two experimental approaches were taken. In the first series, 4-week-old pre-hypertensive SHR were tested in two phases of a water maze (spatial memory) task, and their performance was compared with that of two age-matched normotensive strains, Wistar Kyoto (WKY) and Wistar rats. During phase 1, SHR and WKY rats were not different in their ability to learn the task. In contrast, during phase 2 (subsequent series of trials after a 4 day inter-phase period), where rats were required to find a new platform location, SHR exhibited significantly impaired performance compared to both WKY and Wistar normotensive controls. In a single trial passive avoidance paradigm, SHR again displayed significantly reduced avoidance behavior as compared with both WKY and Wistar rats. In consecutive coronal sections the density of [3H]cytisine binding sites was decreased in pre-hypertensive SHR by up to 18% in about 40% of the brain regions examined, with the deficits particularly apparent in frontal cortex (layers 4-6), posterior subiculum, several thalamic regions, and the interpeduncular nucleus. In the second series, age-matched SHR and WKY were treated with the antihypertensive agent hydralazine administered in the drinking water beginning at 4 weeks of age. Hydralazine prevented the development of hypertension in adult SHR, but did not forestall the reduced expression of brain nicotinic receptors, nor the impairment in learning- and memory-related tasks normally observed in untreated adults with established hypertension. Moreover, the magnitude of nicotine-stimulated rubidium efflux from cortical and striatal synaptosomes in vitro was significantly reduced in samples derived from hydralazine-treated SHR as compared with those from hydralazine-treated, or untreated WKY. These results support the contention that the hypertensive state does not directly contribute to the reduced expression of nicotinic receptors in SHR. Therefore, the SHR may provide an important genetic model for the study of the role of central nicotinic receptors in cognitive and learning abnormalities.

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 Alzheimers 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.

Lobeline and structurally simplified analogs exhibit differential agonist activity and sensitivity to antagonist blockade when compared to nicotine

In the present study, lobeline and two structurally simplified analogs were evaluated for activity in muscarinic and nicotinic binding assays, a functional assay for nicotinic receptor activation (86Rb+ efflux from striatal synaptosomes) and an acetylcholinesterase (AChE) assay. Lobeline displaced [3H]cytisine binding to rat cortical membranes with a mean inhibition constant (KI) value of 16.0 nM, while the lobeline analogs CRM-I-13-1 and CRM-I-32-1 exhibited values of 15.0 and 5.4 microM, respectively. [3H]methylscopolamine was displaced by lobeline with a mean KI value of 37.0 microM while CRM-I-13-1 and CRM-I-32-1 exhibited values of 55.0 and 16.0 microM, respectively. While nicotine stimulated 86Rb+ efflux from striatal synaptosomes in a mecamylamine reversible manner at each concentration tested, lobeline slightly increased 86Rb+ efflux at lower concentrations and reduced efflux at higher concentrations. Further, none of the lobeline effects were reversed with mecamylamine. Although less potent, the two lobeline analogs exhibited a similar pattern of activity. These data may suggest that lobeline and structurally similar compounds bind with different subtype selectivity than nicotine, or exert their agonists effects through non-nicotinic mechanisms. All of the compounds tested were at least several hundred times less potent than physostigmine as AChE inhibitors. While some differences were apparent between the lobeline analog which contained the 2-keto-ethyl portion of lobeline and the analog which contained the phenyl 2-hydroxy-ethyl moiety, each compound was much less active than lobeline in most parameters assessed.

Reversal of Scopolamine-Induced Deficits in Navigational Memory Performance by the Seed Oil of Celastrus paniculatus

Celastrus paniculatus (CP), a medicinal plant from India has been reputed to be useful as a pharmaceutical aid for learning and memory. We investigated the effects of the seed oil of CP on the 6 day performance of young adult rats in a navigational memory task-the Morris water maze. Chronic oral (gavage) daily treatment with CP. (50, 200, or 400 mg/kg) for 14 days completely reversed the scopolamine (0.5 mg/kg)-induced task performance deficit. On the other hand, acute treatment (single injection prior to scopolamine treatment) with CP (200 mg/kg) did not significantly reverse the scopolamine-induced impairment in maze performance. Alone, CP produced a slight, but significant improvement in maze performance on the first day of testing. Acute treatment or chronic 14 day treatment with CP resulted in no significant alteration in normal locomotor activity in an open field. Moreover, CP did not alter the scopolamine-induced increases in locomotor activity. Chronic treatment with CP did not alter brain acetylcholinesterase levels and no signs of cholinergic overstimulation were ever noted during or after treatment. Thus, the seed oil of CP, when administered chronically, selectively reversed the impairment in spatial memory produced by acute central muscarinic receptor blockade, supporting the possibility that one or more constituents of the oil may offer cognitive enhancing properties. The neural mechanism underlying the reversal of scopolamines mnemonic effects by CP is not yet known, but it is not related to an anticholinesterase-like action.

Autoradiographic comparison of muscarinic M1 and M2 binding sites in the CNS of spontaneously hypertensive and normotensive rats

Spontaneously hypertensive rats (SHR) respond with exaggerated pressor responses of central origin in response to pharmacologic stimulation of brain muscarinic receptors when compared with those to normotensive Wistar Kyoto (WKY) rats. At least part of the enhanced response to central muscarinic stimulation may be due to alterations in the expression of one or more of the five subtypes of muscarinic receptors. SHR are also known to exhibit regional alterations in the levels of mRNA encoding the M1, M2 and M4 receptors. In this study, we estimated the number of specific muscarinic receptor binding sites in 12-week-old SHR and WKY by measuring the binding of M1- and M2-selective ligands. Using standard autoradiographic techniques, coronal sections obtained from 12-week-old SHR and WKY were incubated with [3H]pirenzepine or [3H]AFDX 384 to label M1 and M2 receptors, respectively. Although both strains exhibited similar distribution patterns for both binding sites, sections derived from SHR expressed a significant increase in the number of [3H]pirenzepine binding sites compared to normotensive WKY in caudate putamen, CA3 region of the hippocampus, cingulate cortex, substantia nigra, posterior hypothalamic area and tuberomammillary nucleus. An increased number of [3H]AFDX 384 binding sites in SHR were observed in the olfactory tubercle, nucleus accumbens, basolateral amygdaloid nucleus, rostroventrolateral medulla and nucleus paragigantocellularis. Decreases in the number of [3H]AFDX 384 binding sites in SHR were also observed in the parietal cortex, medial geniculate, and lateral hypothalamic area. Statistically significant site-selective differences in binding densities between strains ranged from 4.0% to 35.5% of WKY means. These alterations in the expression of M1 and M2 binding sites in cardiovascular regions may contribute to the strains hyper-responsiveness to cholinergic drugs and possibly to the appearance of other autonomic or behavioral phenotypes exhibited by SHR, including the hypertensive state itself.

Prevention of precipitated withdrawal symptoms by activating central cholinergic systems during a dependence-producing schedule of morphine in rats

Previous studies in this and other laboratories have suggested an important role for central cholinergic neurons in the expression of morphine withdrawal symptoms. This study was designed to determine whether the symptoms of withdrawal could be mitigated by normalization of the effect of morphine on cholinergic neurons. Since this effect is generally inhibitory, we used centrally acting cholinergic agonists to augment central cholinergic tone during chronic morphine infusion. Rats were made dependent following the intra-arterial (i.a.) infusion of increasing concentrations (35-100 mg kg(-1) day(-1)) of morphine over 5 days. I.a. injection of 0.5 mg/kg of naloxone precipitated a profound withdrawal response that included a dramatic increase in mean arterial pressure (MAP) which was maintained over the 60-min observation period, a short duration increase in heart rate (HR), and characteristic opiate withdrawal symptoms. In separate groups of rats, non-toxic doses (50 and 250 microg/kg) of the acetylcholinesterase (AChE) inhibitor, diisopropylflurophosphate (DFP) were administered as single daily injections concomitant with the morphine infusion. DFP treated rats, exhibited significantly reduced expression of the naloxone-evoked pressor response. The apparent anti-withdrawal effect of DFP was not reproduced by the selective peripherally acting AChE inhibitor, echothiophate, although both compounds effectively reduced the expression of certain other withdrawal symptoms. The centrally acting muscarinic cholinergic receptor agonist, arecoline, resulted in an even more impressive suppression of withdrawal symptoms. While not all symptoms associated with morphine withdrawal are mediated via central cholinergic pathways, these results suggest that physical dependence on morphine can be suppressed to a significant degree by the augmentation of central cholinergic activity during morphine administration.

Isoarecolone-induced enhancement of delayed matching to sample performance in monkeys: role of nicotinic receptors.

&NA; Nicotine enhances memory performance in young and aged monkeys performing a version of the delayed matching to sample (DMTS) task. However, the beneficial actions of nicotine are often limited by debilitating side effects. Isoarecolone is a piperidine derivative with nicotine‐like activity, but having a slightly different profile of behavioral actions in rodents. In young monkeys, isoarecolone produced a dose‐dependent enhancement of the DMTS task on the same day of testing, with a trend towards enhanced performance in animals tested 24 h after administration. Isoarecolone was about 1500‐fold less potent than nicotine in enhancing DMTS performance. The ability of isoarecolone to enhance DMTS performance was well correlated with its ability to interact with cortical nicotinic receptors in vitro.

Increased expression of M2 muscarinic receptor mRNA and binding sites in the rostral ventrolateral medulla of spontaneously hypertensive rats.

A significant body of evidence suggests that the development and maintenance of elevated blood pressure in the spontaneously hypertensive rat (SHR), a genetic model for essential hypertension, is due at least partly to a central hyper-cholinergic state. For example, this strain responds with an exaggerated pressor response to pharmacological stimulation of central muscarinic receptors in certain brain regions compared to normotensive Wistar Kyoto rats (WKY). At least part of the enhanced response to central muscarinic receptor stimulation in SHR is due to the altered expression of post-synaptic receptors. In the present study, the reverse transcriptase-polymerase chain reaction and autoradiographic techniques were used to estimate the relative levels of mRNA and density of receptor binding sites for the five subtypes of muscarinic receptors within the rostral ventrolateral medulla (RVL) of SHR and WKY. Adult (12-week-old) SHR exhibited an increase in the levels of both M2 muscarinic mRNA, and M2 receptor binding sites in RVL compared to age-matched normotensive WKY. Similarly, 4-week-old pre-hypertensive SHR exhibited increased levels of M2 mRNA in whole medulla oblongata, and an increase in the number of binding sites for M2 receptors in the RVL. Since the RVL is known to integrate tonic cholinergic sympathoexcitatory input, these results suggest that the increased expression of M2 muscarinic receptors in this region represents one neurochemical correlate for the maintenance of excessive central efferent sympathetic nervous activity in the SHR. Since the neurochemical change precedes the development of hypertension, the altered medullary M2 receptor expression may play a role as an initiating or predisposing factor for the development of hypertension in SHR.

A rapid microtechnique for the estimation of muscarinic and nicotinic receptor binding parameters using 96-well filtration plates

A microtechnique for the assay of muscarinic and nicotinic receptor binding parameters employing a 96-well filtration multiscreen system has been developed. Utilizing rat cerebral cortex as the receptor source, saturation analysis of [3H]NMS binding revealed a mean Bmax and Kd value of 1.95 pmol/mg protein and 0.71 nM, respectively. In competition studies with atropine a Ki value of 1.67 nM was determined. Nicotinic receptor binding studies were also performed with this technique with [3H]Cytisine. The binding constants for both ligands correlated well with established literature values; however, in the nicotinic assays concentrations of ligand at 20 times the Kd were required to develop adequate number of counts. Compared to presently available receptor binding techniques, this newly developed system for muscarinic and nicotinic binding is very simple and convenient to use and offers the advantages of speed, accuracy and reproducibility. In addition, a large number of samples can be assayed. This report also present the first use of the 96-well filtration multiscreen system for the estimation of muscarinic and nicotinic receptor binding constants. This methodology should prove to be useful for determining the binding characteristics of potential cholinergic ligands.