Elsevier

Experimental Gerontology

Volume 39, Issue 2, February 2004, Pages 217-223
Experimental Gerontology

Age-related changes of muscarinic cholinergic receptor subtypes in the striatum of Fisher 344 rats

https://doi.org/10.1016/j.exger.2003.10.016Get rights and content

Abstract

Striatum expresses a cholinergic system involved in the regulation of its activity and changes in striatal cholinergic receptors may be related to cognitive impairment. This study has investigated muscarinic cholinergic M1–M5 receptor subtype expression in striatum of Fischer 344 rats aged 6 (young), 15 (adult) and 22 months (senescent) to assess the contribution of different muscarinic cholinergic receptor subtypes in age-related changes of striatal cholinergic neurotransmission. Western blot analysis revealed the expression of the M1–M5 muscarinic receptor subtytpes in the striatum of rats of the three age groups investigated. Both radioligand binding assay and light microscope autoradiography showed in young rats a M4>M1>M2>M3>M5 rank order of receptor density. With the exception of M1 receptor, the density of which is similar in the dorsal (motor) and ventral (limbic) striatum, other receptor subtypes were more abundant in ventral than in dorsal striatum. M1 receptor expression was unchanged between young and adult rats and decreased in senescent animals both in dorsal and ventral striatum. In dorsal striatum M2 and M5 receptor expression did not show age-related changes, whereas in ventral striatum it was slightly decreased in adult rats compared to young or senescent cohorts. M3 receptor expression did not show age-related modifications, whereas a progressive age-related decrease of M4 receptor was found, both in dorsal and ventral striatum. These data indicate a heterogeneous response to age of different muscarinic receptor subtypes. Striatal cholinergic markers are thought to correlate with cognitive impairment in aged rats. In view of this, the identification of age-related changes of striatal muscarinic receptor subtypes may contribute to develop cholinergic strategies to counter cholinergic neurotransmission changes occurring with aging.

Introduction

Muscarinic receptors regulate several important basic physiologic functions such as heart rate, motor and sensory control, parasympathetic system as well as more complex behaviors including arousal, memory and learning (van der Zee and Luiten, 1999). Loss of muscarinic receptor number or function has been implicated in the pathophysiology of neurological disorders including Alzheimer's and Parkinson's disease and Down's syndrome (Felder, 1995). Muscarinic receptors transduce their signals by coupling with G-proteins, which then modulate the activity of a number of effectors enzymes and ion channels. Five subtypes of muscarinic receptors have been identified so far. The use of molecular cloning techniques has allowed to obtain important information on distribution, properties and structure of the five subtypes of muscarinic cholinergic receptors (Wei et al., 1994). Muscarinic receptors characterized with molecular biology techniques were defined as m1–m5 subtypes. Data on the pharmacological characterization of muscarinic receptor subtypes, indicated with capitalized M are less complete, being limited to M1–M4 receptor subtypes (Mei et al., 1989). The lack of M5 selective compounds has made difficult the characterization of this receptor subtype with pharmacological techniques (Eglen and Nahorski, 2000).

In aged rodents, cholinergic system is impaired in several brain structures including striatum (Aubert et al., 1995) and alterations of striatal cholinergic markers have been suggested to be related with cognitive impairment in aged rats (Bratt et al., 1994, Aubert et al., 1995, Lazaris et al., 2003, Packard and McGaugh, 1992). Several investigations have assessed age-related changes in striatal muscarinic receptor densities (Biegon et al., 1988, Biegon et al., 1989, Araujo et al., 1990, Yamagami et al., 1992, Ogawa et al., 1994, Bratt et al., 1994, Aubert et al., 1995, Tice et al., 1996, Gill and Gallagher, 1998), but none of these studies has investigated the five cholinergic receptors subtypes, or was extended to senescence (Tice et al., 1996).

The present study was designed to investigate M1–M5 cholinergic receptor subtypes in striatum of Fischer 344 rats of three different ages, from maturity to senescence, using a combined kinetic and equilibrium binding protocol recently developed for the labeling of the five muscarinic cholinergic receptor subtypes (Flynn et al., 1997, Reever et al., 1997). Immunochemical techniques (Western blot analysis) were also used to confirm the expression of the five muscarinic receptor subtypes in striata of rats of different age groups.

Section snippets

Animals and tissue treatment

Fisher 344 rats of 6 months (young, n=12, body weight 427±23 g), 15 months (adult, n=12, body weight 555±18 g, p<0.05 vs. young) and 22 months (senescent, n=12, body weight 529±20 g, p<0.05 vs. young) were used. Animals were handled according to internationally accepted principles for care of laboratory animals (European Community Council Directive 86/609, O.J. No. L358, Dec. 18,1986).

Six rats were anesthetized with pentobarbital sodium (50 mg/kg, i.p.) and perfused with a fixative solution

Results

Western blot analysis revealed the expression of the M1–M5 muscarinic receptor subtytpes in the striatum of rats of the three age groups investigated. In young rats, striatal membranes exposed to anti-M1 receptor IgGs were bound to a band of approximately 53 kDa (Fig. 1). Membranes exposed to anti-M2 receptor IgGs were bound to a band of approximately 55 kDa (Fig. 1), those exposed to anti-M3 receptor IgGs were bound to a band of approximately 85–90 kDa (Fig. 1), those exposed to anti-M4

Discussion

The present results provide direct evidence that in striatum of Fisher 344 rats muscarinic cholinergic receptors undergo age-dependent changes, affecting to a different extent M1, M2, M4 and M5 receptor subtypes, but not M3 receptor. For assessing quantitatively changes of receptors under investigation, our study has used a recently developed combined kinetic and equilibrium binding protocol that allows labeling and quantification of all five muscarinic cholinergic receptor subtypes (Flynn et

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