Brain Research 931 (2002) 32–40www.elsevier.com/ locate /bres

Research report

Caloric restricted male rats demonstrate fewer synapses in layer 2 ofsensorimotor cortex

b , a b a,c*Lei Shi , Brandon Hollis Poe , Martha Constance Linville , William Edmund Sonntag ,a,bJudy Karen Brunso-Bechtold

aNeuroscience Program, Medical Center Boulevard, Wake Forest University School of Medicine, Winston-Salem, NC 27157-1010, USAbDepartment of Neurobiology and Anatomy, Medical Center Boulevard, Wake Forest University School of Medicine, Winston-Salem,

NC 27157-1010, USAcDepartment of Physiology and Pharmacology, Medical Center Boulevard, Wake Forest University School of Medicine, Winston-Salem,

NC 27157-1083, USA

Accepted 19 December 2001

Abstract

Previous studies have demonstrated an age-related decline in the density of presumptive inhibitory synapses in layer 2 of ratsensorimotor cortex [J. Comp. Neurol. 439(1) (2001) 65]. Caloric restriction has been shown to ameliorate age-related deterioration in avariety of systems and to extend life span. The present study tested the hypothesis that caloric restriction would prevent the previouslyreported age-related synaptic decline. Accordingly, synaptic density in layer 2 of sensorimotor cortex was compared between29-month-old male rats fed ad libitum and 29-month-old male rats that were caloric restricted (60% of ad libitum calories) from 4 monthsof age. In serial electron micrographs, the physical disector was used to determine the numerical density of presumptive excitatory andinhibitory synapses (those containing round or nonround vesicles, respectively) as well as that of neurons. Not only was the previouslyreported age-related decline in numerical density of presumptive inhibitory synapses not ameliorated by caloric restriction, the numericaldensity was significantly lower in caloric restricted than in ad libitum fed rats for total as well as for presumptive excitatory and inhibitorysynapses. Analysis further revealed no difference in the numerical density of neurons in this region. Relating synapse density to neurondensity as the ratio of synapses to neuron also demonstrated significantly fewer synapses per neuron in caloric restricted than in ad libitumfed old rats. Finally, synapse length was significantly less in caloric restricted rats. These results suggest that not only does caloricrestriction fail to prevent the age-related decline in presumptive inhibitory synapses, it results in fewer presumptive excitatory synapses aswell. 2002 Elsevier Science B.V. All rights reserved.

Theme: Development and regeneration

Topic: Aging process

Keywords: Dietary restriction; Aging; Quantitative ultrastructure

1. Introduction that improve quality of life for the aged. Caloric restrictionis the only nongenetic manipulation that has been shown to

Biological aging is associated with an increasing inci- produce a significant extension of life span in variousdence of functional deficits in multiple systems as well as a species [5,8,44,45,50,71,73–75]. While the exact mecha-growing risk of disease. One of the major focuses of nisms of the beneficial effects of moderate caloric restric-studies from aging individuals has been to understand the tion have not been determined, caloric restriction delaysunderlying factors that account for the physiological many age-related physiological changes in diverse organchanges associated with aging and to develop interventions systems [39,47,72,73]. For example, caloric restriction

retards age-associated fiber loss and fiber type changes inskeletal muscle and decreases the accumulation of dele-*Corresponding author. Tel.: 11-336-716-4388; fax: 11-336-716-tions in the mitochondrial genomes of rats [31], as well as4534.

E-mail address: lshi@wfubmc.edu (L. Shi). prevents the age-associated accumulation of oxidative

0006-8993/02/$ – see front matter 2002 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 02 )02249-7

L. Shi et al. / Brain Research 931 (2002) 32 –40 33

damage to mouse skeletal muscle mitochondria [30]. In the calories consumed by the ad libitum rats. The caloricaddition, caloric restriction improves thermotolerance, restriction paradigm was monitored at monthly intervalsthereby reducing hyperthermia-induced cellular damage in and was maintained exactly the same in our colony as atold rats [23], increases protein synthesis in heart and NCTR. Caloric restricted animals were fed every day justdiaphragm [16], and enhances the proteolytic capacity of before the end of the light cycle. The diet of caloricthe aging rat liver [69]. restricted rats was supplemented with vitamins and miner-

Caloric restriction appears not only to slow biological als to assure proper intake of essential nutrients. Dataaging, but also to retard the progression of diseases reported in this study were collected from animals thatassociated with aging. Specifically, the development of comprised the control groups of larger studies assessingchronic renal disease in rats is mitigated by caloric the effects of growth factors on behavior [37,38] andrestriction [21] as is the occurrence of granulocytic anatomy [57,58] and were infused intracerebroventricular-leukemia [67]. In addition, caloric restriction may slow ly with saline for 28 days through osmotic mini-pumps.neurodegenerative disorders such as Alzeheimer’s, Huntin- All ad libitum and caloric restricted animals receivedgton’s [40] and Parkinson’s diseases [10]. Previous studies saline infusion. Surgery for cannula placement was per-from this laboratory have shown an age-related decline in formed as described previously [63]. Briefly, rats werepresumptive inhibitory synapses [11,57] in layer 2 of anesthetized (ketamine 100 mg/ml:xylazine 20 mg/ml,sensorimotor cortex, an area associated with motor per- 1:1; 0.1 ml /kg body weight) and a small opening in theformance, cognition and memory in Brown Norway3 skull was made (21.0 mm Bregma, 2.0 mm mediolateral;Fischer 344 rats [35]. In light of the numerous biological [54]). At this location, a 27-gauge cannula was insertedbenefits of caloric restriction, the present study sought to through the cortex into the right ventricle. The cannula wasdetermine whether caloric restriction would ameliorate that attached to an Alzet 2002 osmotic mini-pump (Alza Corp.)age-related synaptic decline. Surprisingly, not only was the filled with saline. The mini-pumps were replaced after 14decline not ameliorated by caloric restriction, old caloric days.restricted rats demonstrated fewer synapses in this brainarea than age-matched ad libitum fed controls.

2.2. Tissue preparation

Twenty-eight days after implantation of the mini-pumps,2. Material and methods

animals were anesthetized deeply with an overdose ofpentobarbital and perfused transcardially with 1.3 M

2.1. Animalscacodylate buffer (pH 7.4; 20 ml) followed by fixative (2%paraformaldehyde/2% gluteraldehyde). The perfusate was

Eight male F1 Brown Norway3Fischer 344 rats (n54administered using a peristaltic pump with a flow rate of

ad libitum fed, n54 caloric restricted) were acquired from40 ml/min for a minimum of 30 min. Brains were

the National Center for Toxicology Research (NCTR)removed from the cranium and post-fixed overnight. The

(Jefferson, AK). While most studies concerning the effectfollowing day, brains were blocked to include anterior

of caloric restriction in the brain have focused only oncingulate cortex through the parietal association cortex and

males [22,33], in studies that have included both genders,sectioned coronally on a vibratome at 200 mm. Six to eight

caloric restriction tended to have similar effects on bothvibratome sections per animal contained the hindlimb area

males and females [2]. Since estrogen has been shown toof sensorimotor cortex when compared with stereotaxic

affect synapse number in the brain [1] and the currentmaps (21.8 to 23.3 mm Bregma, 1.5 to 4.0 mm mediola-

study quantified synaptic density only male rats wereterally) [77]. Trapezoidal blocks with faces of approxi-

included to avoid the confound of variations in estrogen 2mately 1–2 mm were cut from this area of cortex with thelevels. The animals were maintained in our colony for at

aid of a Nikon dissecting microscope. Blocks were pro-least 2 months prior to sacrifice at 29 months of age.

cessed for electron microscopy using a Reichert LynxAdequate measures were taken to minimize pain or

tissue processor (Leica) and embedded in Araldite 502discomfort. The rats were housed individually on a 12 h

plastic (Ted Pella).light /dark cycle in an animal facility fully accredited bythe American Association for Accreditation of LaboratoryAnimal Care. Experiments were carried out in accordance 2.3. Anatomical samplingwith the European Communities Council Directive of 24November 1986 (86/609/EEC). All experimental proto- Three blocks from each animal were selected randomlycols were approved by the institutional Animal Care and in order to provide independent samples of synapticUse Committee. The ad libitum fed rats were given free numerical density. Semithin (1 mm) full-face sections wereaccess to laboratory rat chow (Purina) throughout life. cut on a Nova ultramicrotome (LKB Broma). Blocks thenBeginning at 4 months of age, and over a 3-week period, were trimmed to include only cortical layers 1–3 and partthe intake of caloric restricted rats was reduced to 60% of of layer 4. Each block was sectioned through 100 mm, or

34 L. Shi et al. / Brain Research 931 (2002) 32 –40

half the thickness of the block, beginning randomly at the disector volume (see Statistics below). Since the˚ boundaries of specific cortical fields such as sensorimotorrostral or caudal face. Sections of 700 A (thin) or 1 mm

cortex are not absolutely identifiable, the reference volume(semithin) thickness were collected in alternating 10 mmof the anatomical space was not calculated and data aresectors, which allowed for systematic random sampling of

3presented as the number of synapses per mm for com-thin and semithin sections within coincident anatomicalparison between groups.space. Serial pairs of thin sections were collected at 20 mm

In the counts, synapses were classified by vesicle shapeintervals onto Formvar-coated slot grids. Thus, five serialand post-synaptic target. Synaptic terminals containingpairs of thin sections were collected through each block.round vesicles were classified as presumptive excitatoryTen serial 1 mm sections were collected through eachsynapses and those containing nonround vesicles weresemithin sector and mounted on glass slides.classified as presumptive inhibitory (Fig. 1; see also Refs.From each serial pair of thin sections, four photo-[11,56]). Ultrastructural studies also have classifiedmicrographs were taken (80003, Zeiss 10-C transmissionsynapses based on whether there is a symmetric or anelectron microscope), creating two physical disectors perasymmetric density (presumptive inhibitory or presumptiveserial pair and a total of 30 per animal. The photo-excitatory synapses, respectively) [20]. That classificationmicrographs included cortical layer 2, as determined fromscheme closely corresponds to the one used in the presentsemithin sections at the light microscopic level. Corre-study, specifically, symmetric synapses are associatedspondence of fields between serial pairs was established byprimarily with terminals containing nonround vesicles andcentering the images on a reliable landmark, such as aasymmetric synapses are associated primarily with termi-myelinated axon or a prominent mitochondrion. Negativesnals containing round vesicles. Immunocytochemistry(Kodak EM Film 4489) were scanned into an imagestudies at the ultrastructural level [24] have reported aanalysis computer station (PELCO Digital Darkroom, Tedclear correspondence between nonround (pleiomorphic)Pella) for stereological quantification.vesicle shape and GABA immunoreactivity as well asSerial semithin sections mounted on glass slides werebetween round vesicle shape and an absence of GABAstained with toluidine blue, coverslipped, and viewed on aimmunoreactivity. For each of the synapses included in theNikon Labophot microscope using a 203 objective. Acounts, the postsynaptic target was identified (dendriticdisector pair was composed of the first and fourth semithinspine, a dendritic shaft, or a soma) and length of thesections in a series. Thus, the disector height, h, was equaldensity was measured.to 4 mm.

Nv of neurons was determined manually on a lightmicroscope (Nikon Labophot) using the physical disector

2.4. Counting method technique. A counting frame of a known area was superim-posed on a field within the same area of cortical layer 2

Quantification was performed blindly in order to avoid where electron micrographs were taken in thin sections.investigator bias. Numerical density (Nv) of synapses in Counting units, the neuronal nuclei, were marked only iflayer 2 of sensorimotor cortex was determined using the they appeared in just one of the counting frames in thephysical disector method [64,70] and StereoInvestigator optical disector pair. Nv of neurons was calculated by

2software (MicroBrightField, Inc.) in a total of 240 photo- dividing Q by the volume of the disector using thegraphed pairs. Briefly, digitized photomicrographs were equation for Nv (see Statistics) and reported as the number

3overlaid with a counting frame consisting of two inclusion of neurons per mm . Since it is not possible to determineand two exclusion lines. Counting objects (i.e. a post- precisely the reference volume of sensorimotor cortex, thesynaptic density associated with a presynaptic element numerical density of synapses and the numerical density ofcontaining at least three vesicles) within the counting neurons were used to derive the ratio of synapses perframe or transected by either of the two inclusion lines neuron for each group.were marked while objects transected by either of the twoexclusion lines were not marked. Synapses marked on firstsection (the ‘reference’ section) but not on the corre- 2.5. Statisticssponding field from the adjacent section (the ‘look-up’

2section) were included in the count, denoted as Q . The The Nv of synapses and neurons for a block was2same procedure was repeated with the ‘look-up’ and calculated using the following equation: Nv 5 oQ /oa ? h

2‘reference’ sections reversed, effectively doubling the where Q is the number of unique counting objects in anumber of counting pairs from 240 to 480. These counting section, a is the area of the counting frame, and h is therules provided unbiased systematic random sampling of height of the physical disector [41]. Numerical densitiesobjects in the sampled space. The area of the counting reported are averages for all blocks in a group and areframe was recorded and the height, h, of the disector pair reported with the standard error of the mean. Data were

˚was verified by the Small fold method [69] to be 700 A. analyzed using InStat (Graph Pad). A Student’s t-test wasNv of synapses was calculated by dividing Q2 by the performed to compare ad libitum and caloric restriction

L. Shi et al. / Brain Research 931 (2002) 32 –40 35

Fig. 1. Photomicrographs of sensorimotor cortex in ad libitum fed (A) and caloric restricted (B) old Brown Norway3Fischer 344 rats. No qualitativedifferences were apparent in the overall health of neural tissue between the ad libitum and caloric restriction groups. Presumptive excitatory terminalscontained round (R) synaptic vesicles and presumptive inhibitory terminals contained non-round synaptic vesicles (NR). Bar50.5 mm.

groups on all measures with the null hypothesis rejected at evidence of gross damage that could be attributed either tothe P,0.05 level. surgery or the presence of the cannula.

3.1. Synapses3. Results

The Nv of all synapses in layer 2 of sensorimotor cortexThe present study quantified the numerical density (Nv) is illustrated as total synapses for ad libitum and caloric

of synapses and neurons in layer 2 of sensorimotor cortex restricted animals in Table 1 and represented graphically inof ad libitum fed old (29 months) rats in comparison with Fig. 2. A comparison of Nv in these two groups revealsage-matched rats caloric restricted beginning at 4 months that there are 32% fewer total synapses in caloric restrictedof age. Quantification was performed stereologically using rats compared to the ad libitum group (P,0.0007). Thethe physical disector method in electron micrographs for Nv of terminals containing round and non-round vesiclessynapses and in light microscopic fields for neurons. By also are shown in Table 1 and Fig. 2. As for total synapses,assessing synapses at the ultrastructural level, synapses there are significantly fewer synapses in caloric restrictedcould be classified by vesicle type, the postsynaptic than ad libitum rats for both terminals containing roundelement could be identified, and the length of each synaptic vesicles (P,0.0004) and those containing nonround vesi-density could be measured. cles (P,0.0306). Thus, caloric restricted rats showed

The animals used in this study were involved in studies significantly fewer synapses in all categories in thisof aging and growth factor effects on behavior and cortical area.anatomy [38] and therefore received intracerebroventricu- Because most of the electron micrographs analyzedlar infusion of saline as the control groups. The blocks that predominantly contained neuropil, a negligible number ofwere collected for study from the senrorimotor cortex were synapses contacted neuronal cell bodies. Within the neuro-never closer than 1 mm to the cannula tract and inspection pil, caloric restricted rats demonstrated strikingly fewerof the tissue at the light microscopic level revealed no synapses terminating on dendritic spines (37% decline) and

36 L. Shi et al. / Brain Research 931 (2002) 32 –40

Table 1Synapse density, neuron density, and ratio of synapses per neuron in ad libitum fed and caloric restricted rats

6 3 4Synapse density 310 synapses /mm (6S.D.) Neuron density 310 Synapses per neuron ratio (6S.D.)3neurons /mm (6S.D.)

Total Round Nonround Total Round Nonround

Ad libitum 485.0 368.3 116.7 9.280 5357.2 4065.5 1291.7(614.6) (611.1) (610.7) (60.448) (6305.1) (6255.8) (6136.3)

Caloric restriction 332.6 258.5 74.1 8.826 3860.3 3006.5 853.7(636.4) (623.6) (615.0) (60.492) (6379.7) (6255.1) (6159.2)

There is no significant difference between neuron density in the ad libitum and caloric restriction groups. However, for both comparisons of synapsedensity and synapses per neuron ratios, the values are significantly less in the caloric restriction group (P,0.05; P values for each comparison arepresented in the text).

on dendritic shafts (61% decline). The decline of spine and cut through the semithin sectors of the tissue blocks inshaft synapses in the round and nonround categories was which synapses were quantified. These data are shown insimilar to that for total synapses. Therefore, while caloric Table 1; there was no statistical difference between the Nvrestriction leads to a significantly lower Nv of synapses of neurons in old rats fed ad libitum throughout life andterminating on both dendritic spines and shafts, the impact that in rats calorically restricted beginning at 4 months ofof caloric restriction is greater for shaft than for spinous age (P.0.05, Student’s t-test).synapses.

In addition to determining the Nv of synapses, the 3.3. Synapse per neuron ratioslength of the synaptic density, regarded as the activecomponent of the synapse, was measured for all synapses The Nv of synapses provides a valuable index forcounted in both ad libitum and caloric restriction groups. comparison of treatment groups but does not take intoThe mean synaptic length for caloric restricted rats was consideration the possibility of differential tissue shrink-significantly less than that for ad libitum rats (3.1860.07 age. Accordingly, the ratio of synapses per neuron calcu-vs. 2.9660.07 mm, respectively; P,0.0384). Thus, caloric lated by dividing the Nv of synapses by Nv of neurons forrestriction leads to a significant decrease of synapse length each block was determined. This proportion not onlyin comparison to ad libitum feeding. relates the Nv of synapses to the Nv of neurons, but also is

independent of possible volumetric changes in the tissue.3.2. Neurons The ratios of synapses per neuron for each category of

synapse type in ad libitum and caloric restricted rats areThe Nv of neurons in layer 2 of somatosensory cortex shown in Table 1. There were significantly fewer total

was determined at the light microscopic level in sections synapses per neuron in caloric restricted than in ad libitumrats (P,0.0051). Furthermore, when classified accordingto synapse type, the ratios of both round and nonroundsynapses per neuron were significantly less in old caloricrestricted rats when compared with old ad libitum rats(P,0.0059 and ,0.0484, respectively). Thus, caloricrestricted rats demonstrated lower ratios of all categories ofsynapses per neuron in layer 2 of sensorimotor cortex inthis strain of rats.

4. Discussion

Results of the present study indicate that not only doescaloric restriction not prevent the age-related loss ofpresumptive inhibitory synapses in layer 2 of sensorimotorcortex of the Brown Norway3Fischer 344 rats, it insteadresults in 37% fewer presumptive inhibitory synapses and

Fig. 2. Numerical density of total, round, and nonround synapses in layer 30% fewer presumptive excitatory synapses. Moreover, the2 of sensorimotor cortex in ad libitum (filled bars) and caloric restricted synapses that remain are decreased significantly in length.(open bars) old Brown Norway3Fischer 344 rats. The Student’s t-test These synaptic changes take place in the absence of anyrevealed that caloric restriction led to a significant decrease in the

change in the numerical density of neurons in layer 2 ofnumerical density of total synapses (P,0.0007) as well as a decrease insensorimotor cortex. These observations raise the essentialthe numerical density of both round (P,0.0004) and nonround (P,

0.0306) synapses. question of why caloric restriction retards the progress of

L. Shi et al. / Brain Research 931 (2002) 32 –40 37

some age-related changes but fails to retard, or even dent for their maintenance on a factor or factors that areenhances, other changes. down-regulated by caloric restriction. On the other hand, if

the loss of presumptive inhibitory synapses that takes place4.1. Effects of caloric restriction on the nervous system between middle and old age in ad libitum animals is due to

an age-related decline in a factor or factors, caloricWhile the failure of caloric restriction to ameliorate restriction may exacerbate this decline, leading not only to

age-related synaptic changes may seem surprising given a greater loss of presumptive inhibitory synaptic terminals,the abundance of beneficial biological effects that have but also to a loss of presumptive excitatory terminals.been reported for caloric restriction, a close examination ofthe literature reveals two important points. First, far less is 4.2. Relationship between caloric restriction and trophicknown about the effects of caloric restriction on the factorsnervous system than on other organ systems [37,40]. Andsecond, not all existing studies support a protective effect While we do not yet have a full understanding of theof caloric restriction on the nervous system. It appears that effects of caloric restriction and its relationship to aging onthe most consistent effect on the brain may relate to its the brain, changes in the neuroendocrine system and/or inability to retard neurodegenerative diseases such as Al- trophic factor levels may be involved [32,40,61]. Onezheimer’s [40], Parkinson’s [40] and Huntington’s [10] trophic factor, insulin-like growth factor 1 (IGF-1), dem-diseases although not amyotrophic lateral sclerosis [55]. onstrates a clear age-related decline [62] and caloricPharmacological studies indicate that despite the ability of restriction has been shown to result in dramatic alterationscaloric restriction to retard the age-associated loss of in IGF-1 levels and related components of the neuroen-striatal dopaminergic receptors in rats [34], it fails to docrine axis [61]. Specifically, within 5 days of initiationproduce the same effect in mice [41]. Moreover, caloric at 4 months of age, moderate caloric restriction leads to anrestriction does not protect the retina in Fischer 344 rats increase in growth hormone and a striking decline infrom age-related thinning [52] despite its ability in senes- plasma levels of IGF-1 [61]. Since IGF-1 crosses thecent animals to reduce or reverse lipid changes in cerebral blood–brain barrier [3,4,53], the brain has access tocortex [66], to increase the production of protective stress circulating IGF-1 besides that synthesized in the brainproteins [2,76], to reverse the decline in vascularity of the [48]. And interestingly, it is not until 29 months of age thatcortical surface [61], and to attenuate increases in hip- plasma levels of IGF-1 in ad libitum animals decrease topocampal glial fibrillary acidic protein mRNA [36]. the levels present after 2 months of caloric restriction [62]

The present study is the first to quantify the effect of An effect of such decreases in IGF-1 levels on synapsescaloric restriction on synapse number. Surprisingly, old in caloric restricted animals is consistent with the role ofrats calorically restricted from 4 months of age demon- IGF-1 in synapse formation and maintenance that has beenstrated fewer synapses in layer 2 of sensorimotor cortex in demonstrated repeatedly in both the central and peripheralcomparison with old rats fed ad libitum throughout life. To nervous systems [12,13,17,18,26,51,59]. Moreover, IGF-1date, the only other study to have probed the effect of also is able to influence synaptic targets. In vitro slicecaloric restriction on synapses addressed the issue in preparations have demonstrated an effect of IGF-1 onisolated synaptosomes. In that study, an every-other-day dendritogenesis during early cortical development [49] andregimen of caloric restriction produced beneficial effects the present results suggest that the effect of caloricon glucose and glutamate transport and mitochondrial restriction is greater on dendritic shaft than spinousfunction in the synaptosomal fraction after exposure to synapses. Thus, the observed loss in synaptic contacts inbiochemical stressors which may reveal an improvement in caloric restricted rats compared with ad libitum rats couldthe function of the synapses present in caloric restricted be due to a primary reduction in the number of presynapticanimals [22]. elements or, on the other hand, represent a withdrawal or

The broad range of physiological benefits exhibited by atrophy of the postsynaptic element.caloric restricted animals raises the question of why there Besides IGF-1, other trophic factors in the CNS alsoare fewer synapses in these animals. In order to answer this have been shown to change in response to caloric restric-question, it is essential to keep in mind that caloric tion [33,40], including brain derived neurotrophic factorrestriction is not a simple manipulation, but rather a (BDNF), which has been shown to exert trophic effects oncomplex condition that may produce a wide variety of synapse formation and function [15,28,29,68]. In contrasteffects. In the present study, it is not known when the to the decrease of IGF-1 in caloric restricted rats compareddecrease in synapse level number begins. Although the with ad libitum fed animals, a recent study on miceinitial synaptogenesis is virtually complete when caloric suggested caloric restriction increases the mRNA levels forrestriction is initiated at 4 months [46,57], synaptic plas- several trophic factors, including BDNF [33]. Thus, whileticity in the central nervous system continues well after the caloric restriction decreases IGF-1 levels, it may increaseinitial period of synaptogenesis [60]. Thus, there may be BDNF levels. This raises the issue of how to resolve thean early critical period during which synapses are depen- seeming discrepancy between the levels and effects of

38 L. Shi et al. / Brain Research 931 (2002) 32 –40

these two trophic factors in caloric restricted animals. It animals, and the behavioral performance in these animalsmay be that as trophic factors have demonstrated compart- is the compromise of these two changes, which explainsmentalization in their effects on dendritic elaboration lack of sensorimotor and memory changes.[42,43,49], different synaptic populations may be depen- In summary, while the available data suggest that caloricdent on different trophic factors as well and that IGF-1 restriction produces numerous biological benefits for agingdemonstrates a stronger influence on synapses in sen- animals, the present results demonstrate that it leads tosorimotor cortex than does BDNF. fewer synapses (both excitatory and inhibitory) in layer 2

of sensorimotor cortex in old Brown Norway3Fisher 3444.3. Impact of synaptic loss in sensorimotor cortex on rats. Clearly, our understanding of this area is far frombehavior complete and further studies are needed to elucidate the

time course of this change and whether similar synapticThe observed loss of synapses in layer 2 of sensorimotor decreases also occur in other cortical areas.

cortex in caloric restricted old rats raises the issue ofwhether there is an associated behavioral deficit. As layer 2contains predominantly small pyramidal cells that form a Acknowledgementshorizontal system of cortico-cortical connection [27], thecurrent study focuses on an anatomical structure more The authors thank Dr David R. Riddle for carefulinvolved in cortical integration than in subcortical relay. reading of the manuscript and helpful suggestions. ThisSensorimotor cortex was chosen for the present study not work was supported by AG11370.only to facilitate comparison with our previous studies ofage-related synaptic decline in this region, but also becauseof its relationship to behavior. It has been shown to be Referencesrelated to motor /ambulatory functions such as perform-ance on the rotarod task, as well as to cognitive functions [1] M.M. Adams, R.A. Shah, W.G.M. Janssen, J.H. Morrison, Differentsuch as spatial learning and memory on the radial arm models of hippocampal plasticity in resxponse to estrogen in youngmaze and Morris water maze [35]. Despite the cognitive and age female rats, Proc. Natl. Acad. Sci. USA 98 (14) (2001)

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