Pharmacological adaptations and muscarinic receptor plasticity in hypothalamus of senescent rats treated chronically with cholinergic drugs

Receptor plasticity is an important compensatory process by which the central nervous system adapts to pathological insult or long-term exposure to drugs. Senescent animals may show an age-related impairment of muscarinic receptor up- or down-regulation after chronic exposure to cholinergic drugs. The purpose of this study was to assess biochemical and pharmacological endpoints of muscarinic receptor plasticity in young, adult and senescent animals. Male, Fischer 344 rats (ages 3, 9, and 27 months) were administered methylatropine or oxotremorine intracerebroventricularly (IVT) for 3 weeks and tested for their functional response to a muscarinic agonist. The density of hypothalamic, muscarinic receptors was also estimated from analysis of ³H-QNB binding isotherms. In young rats, parallel changes in muscarinic receptors and response were noted, but chronic administration of cholinergic drugs to senescent animals had no effect. Thus, ³H-QNB binding in hypothalamus of young and adult rats was increased (31% and 17%) after chronic IVT methylatropine and decreased (20% and 15%) after IVT oxotremorine. Also, young rats treated with IVT methylatropine were supersensitive to the hypothermic effects of a muscarinic agonist (oxotremorine), while young and adult animals administered chronic IVT oxotremorine exhibited marked tolerance. In contrast, identically treated senescent rats showed no changes in ³H-QNB binding or oxotremorine-induced hypothermia. These results demonstrate the impaired ability of senescent rats to up- or down-regulate brain muscarinic receptors and to exhibit functional adaptations seen in young animals treated chronically with cholinergic drugs.Presented, in part, at the Third Tokyo Symposium, Liver and Aging, 1986-Liver and Brain, Tokyo, 1986     

Dose-related effects of pentobarbital on the genetic differences seen between paired, Roman high- or low-avoidance rats in a shuttle box

The effects of low doses of pentobarbital (PB) were measured on the activity levels, shock-induced fighting and avoidance or escape behavior of paired rats of two psychogenetically-selected lines, in multiple shuttle box sessions, following shock-induced fighting or two-way avoidance training. Each pair served as its own control, by receiving drug injections only every second week. Independent of training conditions, the RLA/Verh pairs showed about 90% freezing behavior and no fighting, whereas all RHA/Verh pairs preferred avoidance or escape behavior to fighting. Although their intertrial (shuttling) responses (ITRs) were reduced at the higher doses of PB used, RHA/Verh rats were still capable of most behavioral responses even at 24 mg/kg, whereas all RLA/Verh rats slept at that dose. On the other hand, the ITRs and avoidance responses of the (less active) RLA/Verh rats were increased by injections of 8 and 16 mg/kg PB. The results, especially those pertaining to freezing behavior and changes in activity levels, were discussed in comparison to other selected rat strains which have shown certain similarities to the Roman lines in regard to "emotionality" and associated neurochemical status.     

A differential activation of dopamine output in the shell and core of the nucleus accumbens is associated with the motor responses to addictive drugs: a brain dialysis study in Roman high- and low-avoidance rats

Addictive substances like morphine and psychostimulants induce a preferential increase in dopamine (DA) output in the nucleus accumbens (NAC), a major terminal field of the mesolimbic dopaminergic projection. Two subregions of the NAC, the dorsolateral core and the ventromedial shell, are thought to subserve different functions related to the reinforcing properties of natural and drug rewards. The selective breeding of Roman high- (RHA) and low-avoidance (RLA) rats, respectively, for rapid vs. extremely poor active avoidance acquisition in a shuttle-box has resulted in two phenotypes that differ in their behavioural and neurochemical responses to addictive drugs. We used brain dialysis to assess whether such differences in the responsiveness to drugs of abuse are related to differences in mesolimbic DA neuron function. In RHA rats, morphine, cocaine, and amphetamine caused a larger increase in DA efflux in the NAC shell vs. the NAC core, whereas the profile for the drug-induced increases in DA output was almost completely superimposable in the NAC shell and NAC core of RLA rats. Moreover, morphine, cocaine, and amphetamine caused a larger increment in basal DA output in the NAC shell of RHA rats vs. the NAC shell of RLA rats. These drugs also elicited a more robust increase in locomotion, rearing, sniffing, and grooming in RHA than in RLA rats. These results demonstrate that genetically determined differences in the functional properties of DA neurons projecting to the NAC shell may critically influence the behavioural response patterns to addictive drugs that distinguish the Roman lines.     

Effects of prenatal diazepam on two-way avoidance behavior,swimming navigation and brain levels of benzodiazepine-like molecules in male roman high- and low-avoidance rats

Utilizing psychogenetically selected Roman high- and low-avoidance rats (RHA/Verh and RLA/Verh), the present experiments investigated the effects of prenatally administered vehicle and diazepam (1 and 3 mg/kg per day, SC) on the behavior and neurochemistry of adult, male offspring. Active, two-way avoidance behavior was analyzed in 96 rats, at 6 months of age, and swimming navigation in 68 others, at 11 months. Three weeks after testing, selected brain areas from the latter animals were immunoassayed for benzodiazepine (BZD)-like molecules. The 3 mg/kg dose of diazepam both decreased freezing behavior in the shuttle box and reduced the hippocampal content of BZD-like molecules in the RLA/Verh male rats. Swimming navigation (spatial learning), at which the RLA/Verh rats were more adept, was not specifically affected by prenatal diazepam in either rat line. The possibility exists that an increased hippocampal release of BZD-like substances may be necessary to alter shuttle box behavior in RLA/Verh rats.     

Chronic sympathetic denervation increases muscarinic cholinoceptor binding in the rat submaxillary gland

Summary Superior cervical ganglionectomy was found to produce a large decrease in the cocaine-sensitive accumulation of ³H-noradrenaline in the rat sub-maxillary gland, indicating an effective sympathetic denervation. Six weeks after unilateral denervation the muscarinic cholinoceptor binding of ³H-QNB was increased by over 50% compared to the contralateral, innervated gland. There were no differences in the Kd values between the innervated and denervated glands. These results suggest that changes in muscarinic cholinoceptor density might be in part responsible for the postsynaptic supersensitivity to cholinoceptor agonists observed after chronic sympathetic denervation.     

Regional 5-HT analysis in Roman high- and low-avoidance rats following MAO inhibition

5-HT and 5-HIAA levels were investigated in three brain regions of Roman high- and low-avoidance rats, following 10, 30 and 60 min of MAO inhibition (pargyline injection). Higher levels of 5-HT in the cortex at 30 and 60 min, as well as a higher disappearance rate of 5-HIAA in the midbrain/medulla region were exhibited by Roman high-avoidance rats, thus further characterizing the differences previously observed in whole brain 5-HT synthesis between these selected rat lines.     

Differential effects of early stimulation and/or perinatal flumazenil treatment in young Roman low- and high-avoidance rats

The effect of infantile handling-stimulation and/or perinatal flumazenil (3.7 mg/kg/day) administration on exploratory and emotional-related behavior was investigated using Roman high- and low-avoidance (RHA/Verh and RLA/Verh) rats. Postnatal handling increased exploration in 30-day-old rats of both psychogenetically selected lines when they were exposed to a hexagonal tunnel maze including an illuminated central arena. Likewise, postnatal stimulation decreased emotional reactivity in both lines of rats, as expressed by increased entry into the central arena, decreased defection and vocalization frequency, but these effects were more pronounced in the RLA/Verh line. There were interactions between perinatal flumazenil treatment and rat line, indicating that flumazenil enhanced entry into the maze central arena in handled-RLA/Verh rats, whereas a tendency toward the opposite effect was observed in drug-treated and handled-RHA/Verh animals. Thus, the present study emphasizes that the effects of environmental manipulations are partly dependent upon genetic factors, and that pharmacological effects also depend on both genetic and environmentally-induced predisposition.     

Limbic muscarinic cholinergic and benzodiazepine receptor changes with chronic intravenous morphine and self-administration

Muscarinic cholinergic and benzodiazepine receptor affinities and densities were evaluated in membranes from seven brain regions of rats intravenously self-administering morphine and in littermates receiving yoked-morphine or yoked vehicle infusions to identify neuronal systems potentially involved in mediating opiate reinforcement processes. Passive morphine infusion resulted in increases in muscarinic cholinergic receptor densities in the pyriform cortex and in decreases in the cingulate cortex while benzodiazepine receptor densities were decreased in both the hippocampal formation and entorhinal-subicular cortex compared to littermates receiving passive infusions of vehicle. Morphine self-administration resulted in decreased muscarinic cholinergic receptor densities in the frontal and entorhinal-subicular cortices and increases in the amygdaloid complex compared to littermates receiving yoked passive drug. These data are in agreement with acetylcholine turnover rate measurements in these animals and support the proposed role of cholinergic innervations of the frontal and entorhinal-subicular cortices and amygdaloid complex in opiate reinforcement processes.     

Sex and strain differences in benzodiazepine receptor binding in Roman rat strains

Specific [3H]flunitrazepam binding was determined for five brain regions in both sexes of the three Roman rat strains. Small strain differences and a large Sex-Region interaction were detected, benzodiazepine receptor binding being significantly higher in females in the two cortical areas and the cerebellum, but significantly lower in the striatum and hippocampus. These findings are discussed in the context of sex and strain differences in behavior and benzodiazepine sensitivity.     

Sex differences in muscarinic receptor binding after chronic ethanol administration in the rat

Male and female rats were administered ethanol (5% v/v) in a liquid diet for 18 weeks. Pair-fed control animals were fed the same diet except that dextrose was substituted isocalorically for ethanol. Normal controls received a commercial laboratory chow for the same duration. Results showed that, in females, chronic ingestion of an ethanol liquid diet significantly increased the number of muscarinic receptor binding sites compared to both control groups. In contrast, for males, there was no significant difference in the mean number of binding sites among the treatment groups. Furthermore, the mean maximum number of binding sites for males and females varied across brain areas. Males had a significantly greater number of receptor binding sites than females in the striatum, while females had a greater number in the cortex. It was suggested that the geuder differences observed in the present study could be mediated by hormonal effects on central muscarinic functioning.     

Differences between proximal and distal portions of the male rabbit posterior urethra in the physiological role of muscarinic cholinergic receptors

1. The aim of the present study was to elucidate functional differences between embryologically different portions of the posterior urethra of male rabbits in response to muscarinic acetylcholine receptor (mAChR) stimulation using in vitro isometric tension experiments and radioligand binding studies.
2. In the in vitro isometric tension experiments, carbachol, produced a dose-dependent contraction of the proximal portion under the resting state, but did not change the basal tone of the distal portion. Contraction of the proximal portion by 10⁻⁵ M noradrenaline (NA) was dose-dependently enhanced by carbachol either in the presence or absence of N^G-nitro-L-arginine (NOARG). In contrast, carbachol induced relaxation of the distal portion contracted by 10⁻⁵ M NA, which was reversed to dose-dependent contraction in the presence of NOARG.
3. Both portions of the urethra had a similar number of [³H]-quinuclidinyl benzilate ([³H]-QNB) binding sites (195.3±74.1 fmols mg⁻¹ protein for the proximal portion and 146.5±8.5 fmols mg⁻¹ protein for the distal portion) with similar affinities (115.0±45.4 pM for the proximal portion and 79.9± 2.9 pM for the distal portion).
4. The concentration-response curves to carbachol in both portions were shifted to the right in a parallel manner in the presence of pirenzepine (an M₁ antagonist), 11-[[2-[(diethylamino)methyl]-1-piperidinyl] acetyl]-5, 11-dihydro-6H-pyrido-2,3-b)-(1,4)-benzodiazepin-6-one (AFDX-116, an M₂ antgonist) and 4-diphenyl-acetoxy-N-methyl-piperidine (4-DAMP, an M₁/M₃ antagonist). The pA₂ values for pirenzepine, AFDX-116 and 4-DAMP were 7.5±0.1, 7.2±0.02 and 9.3±0.1 respectively for the contraction of the proximal portion, and 7.2±0.1, 7.1±0.2 and 9.1±0.2, respectively for the relaxation of the distal portion.
5. In conclusion mAChR subtypes distribute in a similar fashion throughout the length of the male rabbit posterior urethra with the discrepant responses to carbachol attributable to the differential involvement of the NO pathway in mAChR-generated reactions.

     

Impulsivity Characterization in the Roman High- and Low-Avoidance Rat Strains: Behavioral and Neurochemical Differences

The selective breeding of Roman high- (RHA) and low-avoidance (RLA) rats for rapid vs extremely poor acquisition of active avoidance behavior in a shuttle-box has generated two phenotypes with different emotional and motivational profiles. The phenotypic traits of the Roman rat lines/strains (outbred or inbred, respectively) include differences in sensation/novelty seeking, anxiety/fearfulness, stress responsivity, and susceptibility to addictive substances. We designed this study to characterize differences between the inbred RHA-I and RLA-I strains in the impulsivity trait by evaluating different aspects of the multifaceted nature of impulsive behaviors using two different models of impulsivity, the delay-discounting task and five-choice serial reaction time (5-CSRT) task. Previously, rats were evaluated on a schedule-induced polydipsia (SIP) task that has been suggested as a model of obsessive-compulsive disorder. RHA-I rats showed an increased acquisition of the SIP task, higher choice impulsivity in the delay-discounting task, and poor inhibitory control as shown by increased premature responses in the 5-CSRT task. Therefore, RHA-I rats manifested an increased impulsivity phenotype compared with RLA-I rats. Moreover, these differences in impulsivity were associated with basal neurochemical differences in striatum and nucleus accumbens monoamines found between the two strains. These findings characterize the Roman rat strains as a valid model for studying the different aspects of impulsive behavior and for analyzing the mechanisms involved in individual predisposition to impulsivity and its related psychopathologies.     

Differential response to cholinergic stimulation in psychogenetically selected rat lines

Male and female rats of two lines psychogenetically selected for bipolar extremes in shuttle box avoidance were evaluated for tremor, salivation, chromodacryorrhea, and hypothermia following treatment with the muscarinic cholinergic agonist oxotremorine. Roman Low-Avoidance (RLA/Verh) rats exhibited more pronounced oxotremorine-induced tremor, chromodacryorrhea, and hypothermia than Roman High-Avoidance (RHA/Verh) rats. There was a sex difference only for the chromodacryorrhea response, with femles exhibiting a greater response following oxotremorine than males. In a subsequent experiment using female rats of both rat lines, it was demonstrated that pretreatment with the cholinergic antagonist scopolamine blocked oxotremorine-induced tremor, salivation and chromodacryorrhea responses in both rat lines and reduced the hypothermic effect observed in RLA/Verh rats (but not the much weaker hypothermia found in RHA/Verh rats) after oxotremorine injection. Pretreatment with the peripherally active cholinergic antagonist methscopolamine significantly reduced oxotremorine-induced salivation and chromodacryorrhea and somewhat decreased tremor and hypothermic responses in both rat lines. These results stand in contrast to the results of earlier research in which RHA/Verh rats exhibited greater behavioral depression in a tunnel maze than RLA/Verh rats following cholinergic manipulations. In view of evidence that these rat lines do not differ in number of muscarinic brain receptors, the present results may be due to genetic differences in other aspects of cholinergic neurotransmitter function, differences in the function of other neurochemical systems, or differences in the absorption, distribution, or metabolism of oxotremorine.     

Muscarinic cholinergic receptors in the human right coronary artery: a receptor binding and autoradiographic study

Summary We used a combination of radioreceptor binding and autoradiographic techniques to study the pharmacological characteristics and anatomical localization of [³H]-quinuclidinyl benzilate (QNB) binding sites in the human right coronary artery. The ligand was bound to sections of the human right coronary artery in a manner consistent with the labelling of muscarinic receptors. The addition of pirenzepine or of carbachol to the incubation medium to generate displacement curves was indicative of the presence of M₁ and M₂ receptors in the right coronary artery. Autoradiography showed the localization of M₁ sites primarily in the medial layer of the right coronary artery. M₂ sites were located primarily in the adventitia. No [³H]-QNB binding sites were observed in the endothelium. A possible role of muscarinic receptors in the pathogenesis of coronary vasospasm is discussed.     

Behavioural effects of acute and repeated cocaine treatments: a comparative study in sensitisation-prone RHA rats and their sensitisation-resistant RLA counterparts

Rationale Dopamine (DA) transmission is critically involved in the motor effects of psychostimulants and opiates, as well as in the augmentation of these effects resulting from repeated drug administration—a process termed behavioural sensitisation. The latter is known to play a central role in the development and maintenance of drug addiction as well as in the high frequency of relapse observed in drug addicts following detoxification. The selective breeding of Roman high- (RHA) and low-avoidance (RLA) rats for extreme performances in the acquisition of avoidant behaviour has generated two phenotypes that differ in the functional properties of the mesocortical and mesolimbic DA systems and in their behavioural and neurochemical responses to the acute administration of psychostimulants and opiates. More recently, we showed that repeated morphine or amphetamine injections induce behavioural sensitisation in RHA, but not RLA, rats.Objective To further characterize the differences in the susceptibility to develop psychostimulant sensitisation between the Roman lines, we evaluated the behavioural effects of acute cocaine (5 and 10 mg kg^–1, i.p.) 1 day before and 8 days after repeated administration of saline (2 ml kg^–1, i.p.) or cocaine (10 mg kg^–1, i.p. for 14 consecutive days).Results We show that repeated cocaine administration elicits augmented behavioural responses to both challenge doses of the same drug only in RHA rats.Conclusions The Roman lines represent a useful model to investigate how, and to what extent, the genetic make-up influences the neural substrates of individual vulnerability to addiction.     

Pharmacokinetics of anticholinergic drugs and brain muscarinic receptor alterations in streptozotocin diabetic rats

We studied the effects of experimental diabetes on the pharmacokinetics of biperiden (BP) and scopolamine (SP) and brain muscarinic receptor alterations in rats after the injection of streptozotocin (STZ) (60mg kg^?1 i.v.). The serum levels of BP and SP differed significantly between the rats 14 weeks after the STZ treatment and age-matched control rats. The values of total body clearance (CL_tot) of BP and SP were significantly increased by STZ treatment. The values of volume of distribution (V_dss) of SP were slightly increased in the STZ-treated rats, although V_dss of BP was decreased. Because of the high lipophilicity of BP, V_dss of BP may be decreased due to the reduced fat tissue volume caused by STZ treatment. The density of the muscarinic receptors in whole brain was measured by a radioligand receptor binding assay using [³H]-quinuclidinyl-benzylate ([³H]-QNB). The density in the diabetic rats two weeks after the STZ treatment was significantly decreased compared to age-matched control rats. However in the diabetic rats 14 weeks after the STZ treatment, there was no difference in the density of muscarinic receptors. The IC₅₀ of muscarinic antagonist for the binding of [³H]-QNB to the receptor did not change on STZ treatment. Modulation of the receptor following repeated anticholinergic drug exposure was studied. In control rats, the number of muscarinic receptors in the brain increased by 6.9% on chronic treatment with BP for two weeks. When diabetic rats were treated with BP and SP, the number of muscarinic receptors in the brain increased by 9.6% and 33.8%, respectively.     

Muscarinic Receptor Density in the Rat Urinary Bladder after Denervation,Hypertrophy and Urinary Diversion *

Abstract: Parasympathetic denervation of the urinary bladder results in supersensitivity to muscarinic agonists and in bladder hypertrophy. In the present study, the effects of denervation on the muscarinic receptors in the rat bladder were investigated, using a receptor binding technique with (-)³H-QNB as radioligand. The density of muscarinic receptors was increased in denervated, hypertrophied bladders but it was decreased, below that in control bladders, when the development of hypertrophy was prevented by urinary diversion. A decreased receptor density was also found in innervated bladders after urinary diversion, whereas the receptor density was unaffected by hypertrophy alone. Competition experiments with methacholine revealed no changes in the agonist binding properties of the receptors. When the present data are combined with those in previous functional studies, it seems unlikely that the muscarinic receptors in the bladder are involved in the development of supersensitivity. It is suggested that the density of muscarinic receptors in the bladder may be related to the bladder function.     

Effects of scopolamine,pilocarpine, and oxotremorine on the exploratory behavior of two psychogenetically selected lines of rats in a complex maze

Rats of two psychogenetically selected lines received pretest IP injections of scopolamine hydrobromide (0.25, 1.0, or 4.0 mg/kg), pilocarpine hydrochloride (3.0, 6.0, or 12.0 mg/kg) or oxotremorine sesquifumarate (0.2, 0.4, or 0.8 mg/kg) and were subseqently placed in a complex enclosed maze of the Dashiell type that included a small, central, illuminated arena. Animals receiving pilocarpine or oxotremorine injections were pretreated with methscopolamine to counter the peripheral actions of these muscarinic cholinergic agonists. Following vehicle injections, Roman High-Avoidance rats (RHA/Verh) were significantly more active, explored more maze sectors, and required less time to activate the initial 24 different photocell units uniformly distributed throughout the maze than Roman Low-Avoidance rats (RLA/Verh). Scopolamine, pilocarpine, and oxotremorine depressed locomotor activity, reduced the explored area, and increased the time required to activate the initial 24 different photocell units within this complex maze for both RHA/Verh and RLA/Verh rats. Although the doses of scopolamine injected were approximately equally effective in both rat lines (except for total maze activity), the RHA/Verh rats exhibited significant alterations in several measures of maze patrolling after treatment with the lowest dose of pilocarpine, whereas the RLA/Verh rats did not. In contrast, most of the RLA/Verh rats exhibited very pronounced tremors following treatment with the highest dose of oxotremorine, but none of the RHA/Verh rats did. These results demonstrate that manipulation of the central cholinergic system with scopolamine, pilocarpine, or oxotremorine, despite their different pharmacological mechanisms, impair maze patrolling. Furthermore, the results suggest that the two psychogenetically bred lines of rats investigated are differentially sensitive to central cholinergic manipulation with the muscarinic receptor agonists pilocarpine and oxotremorine.     

Conditioned taste aversions produced by nicotine in Roman High and Low Avoidance strains of rats

Rats of the RHA/iop and RLA/iop strains have been compared in a conditioned taste aversion procedure using nicotine (0.4 mg/kg SC) as the UCS. The procedure utilised a balanced, within-subject design for assessing discriminative aversions to drug- and saline-paired flavoured solutions. Nicotine produced clear aversions in both strains and there were no detectable differences in acquisition. During extinction, rats of the RHA/iop strain consumed more of the drug-paired flavoured solution than rats of the RLA/iop strain, and this difference became greater as the number of extinction trials proceeded. Differences in total fluid intake were too small to account for these effects that were also shown by changes in proportional intake when both flavoured solutions were presented simultaneously. Aversion was, therefore, rather weaker in RHA/iop rats than in RLA/iop rats. These results suggest that rats of the two strains do not differ in learning ability in a general way, and support interpretations based on differences in emotionality.     

Divergent effect of the selective D<Subscript>3</Subscript> receptor agonist pd-128,907 on locomotor activity in Roman high- and low-avoidance rats: relationship to NGFI-A gene expression in the Calleja islands

Rationale

The inbred Roman high- (RHA-I) and low-avoidance (RLA-I) rats, differing in dopaminergic activity and novelty/substance-seeking profiles, may be a suitable model to study the implication of the dopaminergic system in vulnerability to drug abuse. Differences in D₃ receptor binding recently described between the two strains (Guitart-Masip M, Johansson B, Fernández-Teruel A, Cañete T, Tobeña A, Terenius L, Giménez-Llort L, Neuroscience 142:1231–1243, 2006b) may be important in shaping the aforementioned differences in novelty seeking.

Objective

The aim of the present work was to study the effect of D₃ receptor activation on novelty-induced locomotor activity in these two strains of rats.

Materials and methods

We administered saline and PD-128,907 (0.01 and 0.1 mg/kg), a putative D₃ receptor agonist, to the Roman rats and studied the locomotor activity when animals were placed in a novel environment. Thereafter, by means of in situ hybridization, nerve growth factor inducible clone A (NGFI-A) mRNA was measured in the striatum and the Calleja islands of these animals.

Results

We found that RLA-I rats showed stronger locomotor inhibition than RHA-I rats after PD-128,907 administration. Moreover, RLA-I rats showed stronger reduction of NGFI-A mRNA in the Calleja islands than RHA-I rats.

Conclusions

These results, together with previous findings, suggest that differences in D₃ receptor expression in the Calleja islands may contribute to the divergent behavioral effect of PD-128,907 administration in the two strains of Roman rats.