Agonist and antagonist binding to rat brain muscarinic receptors: influence of aging

The objective of the present study was to determine the binding properties of muscarinic receptors in six brain regions in mature and old rats of both sexes by employing direct binding of [3H]-antagonist as well as of the labeled natural neurotransmitter, [3H]-acetylcholine [( 3H]-AcCh). In addition, age-related factors were evaluated in the modulation processes involved in agonist binding. The results indicate that as the rat ages the density of the muscarinic receptors is altered differently in the various brain regions: it is decreased in the cerebral cortex, hippocampus, striatum and olfactory bulb of both male and female rats, but is increased (58%) in the brain stem of senescent males while no significant change is observed for females. The use of the highly sensitive technique measuring direct binding of [3H]-AcCh facilitated the separate detection of age-related changes in the two classes (high- and low-affinity) of muscarinic agonist binding sites. In old female rats the density of high-affinity [3H]-AcCh binding sites was preserved in all tissues studied, indicating that the decreases in muscarinic receptor density observed with [3H]-antagonist represent a loss of low-affinity agonist binding sites. In contrast, [3H]-AcCh binding is decreased in the hypothalamus and increased in the brain stem of old male rats. These data imply sexual dimorphism of the aging process in central cholinergic mechanisms.     

Muscarinic cholinergic receptor subtypes in cerebral cortex of Fisher 344 rats: a light microscope autoradiography study of age-related changes

The density and localization of muscarinic cholinergic M1-M5 receptor subtypes was investigated in frontal and occipital cortex of male Fisher 344 rats aged 6 months (young-adult), 15 months (mature) and 22 months (senescent) by combined kinetic and equilibrium binding and light microscope autoradiography. In 6-month-old rats, the rank order density of muscarinic cholinergic receptor subtypes was M1>M2>M4>M3>M5 both in frontal and occipital cortex. A not homogeneous distribution of different receptor subtypes throughout cerebrocortical layers of frontal or occipital cortex was found. In frontal cortex silver grains corresponding to the M1 and M2 receptor subtypes were decreased in 15- and 22-month-old groups. The M3 receptor density was remarkably and moderately decreased in layers II/III and V, respectively, of rats aged 15 and 22 months. A reduced M4 receptor density was observed in layer I and to a lesser extent in layer V of mature and senescent rats, whereas no age-related changes of M5 receptor were found. In occipital cortex a diminution of M1 receptor was observed in layers II/III and V of mature and senescent rats. The M2 receptor expression decreased in layer I of 15- and 22-month-old senescent rats, whereas M3-M5 receptors were unchanged with exception of a slight decrease of the M4 receptor in layer IV and of M5 receptor in layers II/III. These findings indicate a different sensitivity to aging of muscarinic receptor subtypes located in various cerebrocortical layers. This may account for the difficulty in obtaining relevant results in manipulating cholinoceptors to counter age-related impairment of cholinergic system.     

Muscarinic cholinergic receptor binding in bovine retina

Using [³H] quinuclidinyl benzylate ([³H] QNB) muscarinic cholinergic receptors have been demonstrated in crude membrane fractions of bovine retina. Specific [³H] QNB binding is saturable with a K_D of 0.5 nM and a maximal number of muscarinic agonists and antagonists for displacing specific [³H] QNB binding closely parallel the affinities for muscarinic receptors in rat brain and guinea pig ileum. The findings may explain atropine sensitive effects of muscarinic agonists on the electroretinogram and on retinal cells in vitro.     

Autoradiographic distribution of [3H]acetylcholine binding sites in the cervical spinal cord of man and some other species

The distribution of [3H]acetylcholine ([3H]ACh) and [3H]ACh co-incubated with 1-mM nicotine (muscarinic receptor), and [3H]ACh co-incubated with 1.5 microM atropine (nicotinic receptor) binding sites were studied in man and compared to monkey, cat and rat using quantitative in vitro autoradiography. The highest density of total [3H]ACh binding sites was found in laminae II-III, IX (motor neuron areas) and X close to the central canal. The distribution pattern of the muscarinic cholinergic binding sites was similar to that of the total cholinergic binding. In general the number of nicotinic binding sites in the spinal cord was relatively small. The largest number of such binding sites was found in laminae II-III of the dorsal horn and in laminae X around the central canal. It is evident that the spinal cord has a 2-3 times higher number of muscarinic than of nicotinic cholinergic receptors.     

Selective, unilateral cortical infarction increases striatal muscarinic receptor binding: potential evidence for cortical modulation of intrastriatal cholinergic transmission

The effects of unilateral cortical infarction on subcortical (striatal) muscarinic receptors in rat brain were studied by means of in vitro receptor autoradiography using [3H]quinuclidinyl benzylate ([3H]QNB) and [3H]pirenzepine. The cortical lesions could be produced without compromising subcortical structures. A dramatic (20 to 59 per cent) increase in striatal [3H]QNB binding was observed ipsilateral to the damaged cortex. The increase in binding was greatest in the caudate-putamen, but was also noted in the nucleus accumbens. [3H]Pirenzepine binding (labeling M1 receptors) was also increased but to a lesser degree, as was [3H]QNB binding in the presence of 100 nM (unlabeled) pirenzepine (an indirect means of labeling M2 receptors). The results show that unilateral cortical infarction results in an upregulation of striatal muscarinic receptors, and suggest that both the M1 and M2 subtypes contribute to this effect. These findings provide evidence for cortical modulation of intrastriatal cholinergic transmission.     

Aging but not estrogen alters regional [3H]5-HT binding in the CNS of female Fischer 344 rats

Age-related changes in serotonergic regulation of neuroendocrine function were investigated in female Fischer 344 rats; serotonin ([3H]5-HT) binding sites were characterized in several brain regions. Neither the number (Bmax) nor the affinity (Kd) of [3H]5-HT sites were altered in the frontal cortex of reproductively young and senescent groups. However, a significant decline in receptor affinity was observed in the hypothalamus and midbrain dorsal raphe nucleus. An increase in the density of binding sites was also observed in the hypothalamus with advancing age. Acute 48 h exposure to estrogen failed to influence [3H]5-HT binding site characteristics in these brain regions. In summary, these results suggest that age-related changes in [3H]5-HT binding are regionally specific. Moreover, the observed changes in hypothalamic 5-HT function may underlie neuroendocrine aging events.     

Age-related changes in the cholinergic components within the central nervous system. II. Working memory impairment and its relation to hippocampal muscarinic receptors

Cognitive performance in aging Wistar rats was monitored using the radial arm maze and the latter was correlated with the density of muscarinic receptors in the CNS, using quantitative in vitro receptor autoradiography. Significant working memory deficits were observed in 12, 17 and 24-month-old rats as compared to 3-month-old animals. In addition, the number of the muscarinic receptors declined significantly with age (from 27 to 42% depending on the brain region sampled) utilising [3H]QNB and [3H]PZ receptor binding assays. The above trend became evident already at the age of 12 months. The present findings support the association of central cholinergic activity with memory processes.     

Muscarinic M2 receptor mRNA expression and receptor binding in cholinergic and non-cholinergic cells in the rat brain: a correlative study using in situ hybridization histochemistry and receptor autoradiography.

The goal of the present study was to identify the cells containing mRNA coding for the m2 subtype of muscarinic cholinergic receptors in the rat brain. In situ hybridization histochemistry was used, with oligonucleotides as hybridization probes. The distribution of cholinergic cells was examined in consecutive sections with probes complementary to choline acetyltransferase mRNA. Furthermore, the microscopic distribution of muscarinic cholinergic binding sites was examined with a non-selective ligand ([3H]N-methylscopolamine) and with ligands proposed to be M1-selective ([3H]pirenzepine) or M2-selective ([3H]oxotremorine-M). The majority of choline acetyltransferase mRNA-rich (i.e. cholinergic) cell groups (medial septum-diagonal band complex, nucleus basalis, pedunculopontine and laterodorsal tegmental nuclei, nucleus parabigeminalis, several motor nuclei of the brainstem, motoneurons of the spinal cord), also contained m2 mRNA, strongly suggesting that at least a fraction of these receptors may be presynaptic autoreceptors. A few groups of cholinergic cells were an exception to this fact: the medial habenula and some cranial nerve nuclei (principal oculomotor, trochlear, abducens, dorsal motor nucleus of the vagus). Furthermore, m2 mRNA was not restricted to cholinergic cells but was also present in many other cells throughout the rat brain. The distribution of m2 mRNA was in good, although not complete, agreement with that of binding sites for the M2 preferential agonist [3H]oxotremorine-M, but not with [3H]pirenzepine binding sites. Regions where the presence of [3H]oxotremorine-M binding sites was not correlated with that of m2 mRNA are the caudate-putamen, nucleus accumbens, olfactory tubercle and islands of Calleja. The present results strongly suggest that the M2 receptor is expressed by a majority of cholinergic cells, where it probably plays a role as autoreceptor. However, many non-cholinergic neurons also express this receptor, which would be, presumably, postsynaptically located. Finally, comparison between the distribution of m2 mRNA and that of the proposed M2-selective ligand [3H]oxotremorine-M indicates that this ligand, in addition to M2 receptors, may also recognize in certain brain areas other muscarinic receptor populations, particularly M4.     

Effects of nucleus basalis lesion on muscarinic receptor subtypes

The cholinergic system in the central nervous system is an important component of the neural circuitry of learning, memory and cognition. A decline of cholinergic innervation in the human brain is a characteristic feature of dementia of Alzheimer's type. In this study, changes in cholinergic markers were studied after a unilateral lesion of the nucleus basalis magnocellularis (nbM). Acetylcholinesterase (AChE) histochemistry showed a loss of cortical AChE-containing neurons, and choline acetyltransferase (ChAT) immunohistochemistry demonstrated a loss of cholinergic cells in nbM. The localizations of muscarinic Ml and M2 receptors using [³H]pirenzepine ([³H]PZ) and [³H]AF-DX 384, respectively, were studied by quantitative autoradiography 1, 2, 4 and 6 weeks following unilateral ibotenic acid lesion of nbM. A significant decrease in [³H]PZ binding sites was observed at postlesion week 1 in the parietal and temporal cortices. The decrease in [³H]AF-DX 384 binding sites on the lesioned side was observed throughout frontal, parietal and temporal cortices after postlesion week 1, with a significant increase after 6 weeks, possibly as result of loss of presynaptic receptors and upregulation of postsynaptic ones. Moreover, laminar distribution after nbM lesion shows that M1 and M2 receptor binding sites are more affected in superficial layers (I,II,III) than in the deep layers (IV,V,VI), depending on ligand, postlesion period and cortical region. Furthermore, nbM lesion causes a higher deficit of M2 receptors than of M1 receptors. These data suggest the existence of a presynaptic population as well as a postsynaptic population of M1 and M2 receptors which are differently affected after unilateral nbM lesion.     

Cholinergic innervation and topographical organization of muscarinic binding sites in rat brain: a comparative autoradiographic study

Employing [3H]hemicholinium-3 ([3H]HC), [3H]pirenzepine([3H]PZ) and [3H]quinuclidinyl benzilate ([3H]QNB), autoradiographic binding studies were performed to identify and quantitate the localization of high-affinity choline carriers, M1-subtype of muscarinic binding sites and a mixed population of M1- and M2-subtypes of muscarinic binding sites, respectively, in 38 anatomically defined areas of rat brain. Labelling of adjacent brain sections with [3H]HC, [3H]PZ and [3H]QNB revealed different topographical binding patterns. [3H]HC binding, which is supposed to reflect cholinergic innervation, was dense in the nucleus accumbens, olfactory tubercle, caudate putamen, basolateral amygdaloid nucleus and the interpeduncular nucleus. Moderate but heterogeneous binding was found in thalamic, hypothalamic, hippocampal and cortical areas. Maximal [3H]PZ binding was observed in the nucleus accumbens, olfactory tubercle and in discrete substructures of the hippocampus, e.g. CA1 and dentate gyrus. Binding to other hippocampal and cortical areas was intermediate, whilst minor binding was found in thalamic, hypothalamic and brain stem areas. The binding of [3H]QNB was more evenly distributed over the brain as compared to that of [3H]PZ. [3H]QNB clearly exceeded the binding of [3H]PZ in the thalamus, hypothalamus and brain stem. A relationship was found between the topography patterns of the [3H]PZ and [3H]QNB binding sites. However, some brain areas showed preference for one of the two ligands, pointing to a distinct localization of M1- and M2-subtypes of muscarinic binding sites. Although M1 sites appeared to predominate in the basal ganglia, hippocampus and cortex, some heterogeneity was observed indicative of the minor occurrence of M2 sites within these structures. There was no relationship between the density of the presumed cholinergic innervation and the binding capacity of either of the muscarinic sites in the various brain areas. However, a relationship was found between M2-selectivity and [3H]HC binding, pointing to a possible presynaptic localization of the M2-sites. In addition, it is suggested that distinct cholinergic cell groups might project their fibres to brain areas containing particular subsets of postsynaptic muscarinic binding sites.     

Effects of aging and cholinergic deafferentation on putative muscarinic cholinergic receptor subtypes in rat cerebral cortex

Despite a 34% decrease in the activity of choline acetyltransferase (ChAT) in the rat cerebral cortex following lesions of the nucleus basalis, there were no changes in the Bmax of the antagonist ligands [3H]quinuclidinyl benzilate ((-)-[3H]QNB) or (-)-[3H]N-methylscopolamine ((-)-[3H]NMS). Furthermore, this treatment produced no significant change in the proportions or affinities of muscarinic receptors having high and low affinity for pirenzepine or (-)-NMS. These data indicate that putative M2 muscarinic receptors are not restricted to ChAT-containing neurons in rat cerebral cortex. In senescent compared to mature rats there was no significant loss of ChAT activity although a significant reduction in the Bmax of both (-)-[3H]QNB and (-)-[3H]NMS binding was observed. However, no changes in the competition of pirenzepine or (-)-NMS for the remaining (-)-[3H]QNB binding sites were observed. Therefore, there is no evidence for any differential regulation of either putative muscarinic receptor subtype in response to cholinergic deafferentation or as a function of the natural aging process.     

Muscarinic receptors in basal ganglia in dementia with Lewy bodies,Parkinson's disease and Alzheimer's disease

Derivatives of the muscarinic antagonist 3-quinuclidinyl-4-iodobenzilate (QNB), particularly [123I]-(R,R)-I-QNB, are currently being assessed as in vivo ligands to monitor muscarinic receptors in Alzheimer's disease (AD) and dementia with Lewy bodies (DLB), relating changes to disease symptoms and to treatment response with cholinergic medication. To assist in the evaluation of in vivo binding, muscarinic receptor density in post-mortem human brain was measured by autoradiography with [125I]-(R,R)-I-QNB and [125I]-(R,S)-I-QNB and compared to M1 ([3H]pirenzepine) and M2 and M4 ([3H]AF-DX 384) receptor binding. Binding was calculated in tissue containing striatum, globus pallidus (GPe), claustrum, and cingulate and insula cortex, in cases of AD, DLB, Parkinson's disease (PD) and normal elderly controls. Pirenzepine, AF-DX 384 and (R,S)-I-QNB binding in the striatum correlated positively with increased Alzheimer-type pathology, and AF-DX 384 and (R,R)-I-QNB cortical binding correlated positively with increased Lewy body (LB) pathology; however, striatal pirenzepine binding correlated negatively with cortical LB pathology. M1 receptors were significantly reduced in striatum in DLB compared to AD, PD, and controls and there was a significant correlation between M1 and dopamine D2 receptor densities. [3H]AF-DX 384 binding was higher in the striatum and GPe in AD. Binding of [125I]-(R,R)-I-QNB, which may reflect increased muscarinic M4 receptors, was higher in cortex and claustrum in DLB and AD. [125I]-(R,S)-I-QNB binding was higher in the GPe in AD. Low M1 and D2 receptors in DLB imply altered regulation of the striatal projection neurons which express these receptors. Low density of striatal M1 receptors may relate to the extent of movement disorder in DLB, and to a reduced risk of parkinsonism with acetylcholinesterase inhibition.     

Cholinergic drug effects and brain muscarinic receptor binding in aged rats

Muscarinic systems are significantly altered in the brains of laboratory animals and man as a result of normal aging. Cholinergic neurotransmission in cerebral cortex and hippocampus is also severely impaired in a major age-related neurological disorder. Alzheimer's disease. The objective of these studies was to assess specific ³H-quinuclidinyl benzilate (³H-QNB) binding to brain muscarinic receptors in young, adult and senescent Fischer 344 rats, and to relate receptor changes to differences in the pharmacologic actions of cholinergic drugs. Muscarinic receptor density declined with advanced age in the frontal cortex, corpus striatum and hypothalamus, but no age-related changes in receptor affinity were observed. Specific binding of ³H-QNB in hippocampus was not significantly altered. In contrast, the in vivo effects of oxotremorine (hypothermia and antinociception) were markedly enhanced in aged rats, whereas scopolamine-induced locomotor activity was reduced. Hence, senescent rats were more sensitive to the pharmacologic actions of a cholinergic agonist, but less responsive than young rats to a muscarinic antagonist. These seemingly contradictory results of binding experiments and pharmacological studies could be due, in part, to changes in subtypes of brain muscarinic receptors with advanced age. Alternatively, the age-related differences in cholinergic drug effects may reflect a decreased ability of the senescent animal to adapt to changes in its environment.     

Binding of some organophosphorus compounds at adenosine receptors in guinea pig brain membranes

The organophosphorus compounds, sarin, tabun and soman are known to be potent inhibitors of cholinesterase. It was claimed that these agents also interact at neurotransmitter receptor sites. We were unable to detect any interaction at the acetylcholine muscarinic sites; we now focus our attention on the adenosine receptors. Binding studies showed that all three anticholinesterases inhibited the binding of [3H]L-phenylisopropyladenosine ([3H]L-PIA) to the brain adenosine receptors in a dose-dependent manner until receptor concentration became limiting. Soman was found to be 5 and 9 times more effective than tabun and sarin respectively in inhibiting [3H]L-PIA binding. These results suggested that these organophosphorus anticholinesterases could interact directly at the A1 adenosine receptors which could subsequently mediate changes in K+ permeability of synaptic membranes.     

Autoradiographic localization of muscarinic cholinergic receptors in visual areas of cat brain: variations in sensitivity of N-[3H]methylscopolamine binding sites to carbachol and pirenzepine

The distribution of muscarinic acetylcholine receptors (MChRs) was studied in visual areas of cat brain using in vitro quantitative autoradiography with 1 nM N-[3H]methylscopolamine ([3H]NMS) as a radioligand. The highest density of [3H]NMS binding was observed in lamina A of the lateral geniculate nucleus (LGN) and in layer II/III of the visual cortex. The lowest binding was seen in the stratum griseum intermediale of the superior colliculus (SC). The comparison of inhibition of [3H]NMS binding by 100 microM carbachol and 300 nM pirenzepine showed that the SC and LGN contain predominantly M2 sites. M1 sites constitute the main population of MChRs in the cortical areas studied.     

Distribution of muscarinic receptors in the caudal cerebellum and electrosensory lateral line lobe of gymnotiform fish

Muscarinic binding sites were found in the electrosensory lateral line lobe (ELLL) and vestibulo cerebellum (LC) of certain gymnotid fish; these binding sites were not present in significant numbers in the corpus cerebelli. Autoradiography of [3H]quinuclidinylbenzilate and [3H]propylbenzylcholine mustard binding confirmed these results and also demonstrated that, within the ELLL the region with muscarinic binding sites was coextensive with the region of cholinergic input. We did not find any evidence for nicotinic receptors (alpha-bungarotoxin binding) in ELLL, LC, or corpus cerebelli.     

Reduced number of [3H]nicotine and [3H]acetylcholine binding sites in the frontal cortex of Alzheimer brains

Nicotinic cholinergic receptors were measured in human frontal cortex using [3H]nicotine and [3H]acetylcholine (in the presence of atropine) as receptor ligands. A parallel marked reduction in number of [3H]nicotine (52%; P less than 0.01) and [3H]acetylcholine (-55%; P less than 0.05) binding was found in the frontal cortex of Alzheimer brains (AD/SDAT) when compared to age-matched control brains. As a comparison the number of muscarinic receptors was quantified using [3H]quinuclidinyl benzilate and found to be significantly increased (+23%; less than 0.01) in AD/SDAT compared to controls.     

Chronic treatment with simvastatin upregulates muscarinic M1/4 receptor binding in the rat brain

Statins are increasingly being used for the treatment of a variety of conditions beyond their original indication for cholesterol lowering. We previously reported that simvastatin affected the dopaminergic system in the rat brain. This study aims to investigate regional changes of muscarinic M1/4 receptors in the rat brain after 4-week administration of simvastatin (1 or 10 mg/kg/day). M1/4 receptor distribution and alterations in the post-mortem rat brain were detected by [(3)H]pirenzepine binding autoradiography. Simvastatin (1 mg/kg/day) increased [(3)H]pirenzepine binding, predominantly in the prefrontal cortex (171%, P<0.001), primary motor cortex (153%, P=0.001), cingulate cortex (109%, P<0.001), hippocampus (138%, P<0.001), caudate putamen (122%, P=0.002) and nucleus accumbens (170%, P<0.001) compared with controls; while lower but still significant increases of [(3)H]pirenzepine binding were observed in the examined regions following simvastatin (10 mg/kg/day) treatment. Our results also provide strong evidence that chronic simvastatin administration, especially at a low dosage, up-regulates M1/4 receptor binding, which is likely to be independent of its muscarinic agonist-like effect. Alterations in [(3)H]pirenzepine binding in the examined brain areas may represent the specific regions that mediate the clinical effects of simvastatin treatment on cognition and memory via the muscarinic cholinergic system. These findings contribute to a better understanding of the critical roles of simvastatin in treating neurodegenerative disorders, via muscarinic receptors.     

Nodose ganglionectomy selectively reduces muscarinic cholinergic and delta opioid binding sites in the dorsal vagal complex of the cat

The dorsal vagal complex of the medulla oblongata, comprising the nucleus tractus solitarii, the area postrema and the dorsal motor nucleus of the vagus nerve, is an important brainstem regulatory center for the autonomic nervous system. The major afferent input from abdominal and thoracic viscera to this region is via vagal sensory neurons which have their cell bodies in the nodose ganglion. Autoradiography has been used to study the effects of unilateral nodose ganglionectomy on receptor binding sites in this region of the brain for the neurotransmitters acetylcholine, norepinephrine, and opioids. Nodose ganglionectomy had no discernible effect on alpha 2 noradrenergic ([3H]p-aminoclonidine) or mu opioid [( 3H]Tyr-D-Ala-Gly-(NMePhe)-Gly-ol) binding sites. However, ganglionectomy did produce a 25% decrease in [3H]quinuclidinyl benzilate (muscarinic cholinergic) binding in the subnucleus gelatinosus of the solitary nucleus, and a marked decrease in [3H][D-Pen5]enkephalin (delta opioid) binding in the dorsomedial subnucleus of the nucleus tractus solitarii, ipsilateral to the lesion. These data suggest that muscarinic cholinergic and delta opioid receptors may be present on terminals of vagal afferent neurons that project to these specific brainstem regions. Since these vagal afferent neurons are known to arise, at least in part, from the gastrointestinal tract, it is possible that cholinergic and/or opioid receptors modulate specific autonomic functions associated with gastric sensory information such as satiety or nausea and emesis.     

Decreased carbachol-stimulated inositol 1,3,4,5-tetrakisphosphate formation in senescent rat cerebral cortical slices.

It is well established that muscarinic cholinergic receptors are linked to phosphoinositide hydrolysis in brain. Previous studies of muscarinic responses used Li+ to increase inositol phosphate accumulation and suggested little or no change during aging. Li+ disrupts certain aspects of the inositol phosphate metabolism and inhibits the formation of inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. Ins(1,3,4,5)P4 appears to have second messenger functions. To investigate the effects of aging on agonist stimulated Ins(1,3,4,5)P4 formation, young (6-8 months) and old (28-30 months) Fischer 344 rat cerebral cortical or hippocampal slices were challenged with various agonists known to stimulate phosphoinositide hydrolysis in brain using a recently developed assay that does not use Li+. Carbachol and quisqualate stimulated [3H]inositol trisphosphate ([3H]InsP3) and [3H]Ins(1,3,4,5)P4 formation in young and old rat cerebral cortical slices. Norepinephrine, 5-hydroxytryptamine, and vasopressin failed to stimulate [3H]Ins(1,3,4,5)P4 or [3H]InsP3 formation in either young or old rat cerebral cortical slices. In old rat cerebral cortical slices, the carbachol-stimulated [3H]Ins(1,3,4,5)P4 formation was reduced by 44%. Angiotensin II stimulated [3H]InsP3 was increased (219%) in old rats. There was no influence of aging either on the basal level or on the maximal response to carbachol or quisqualate in hippocampal slices. These studies suggest region-specific changes in phosphoinositide hydrolysis during aging.