Neonatal Handling Increases Sensitivity to AcuteNeurodegeneration in Adult Rats

Katalin M. Horvath,1 Tibor Harkany,1 Jan Mulder,1 Jaap M. Koolhaas,2

Paul G.M. Luiten,1 Peter Meerlo1,2

1 Department of Molecular Neurobiology, Graduate School of Behavioral and CognitiveNeurosciences, University of Groningen, 9750 AA Haren, The Netherlands

2 Department of Behavioral Physiology, Graduate School of Behavioral and CognitiveNeurosciences, University of Groningen, 9750 AA Haren, The Netherlands

Received 18 November 2003; accepted 16 January 2004

ABSTRACT: Environmental stimuli during theperinatal period can result in persistent individual dif-ferences in neural viability and cognitive functions. Ear-lier studies have shown that brief daily maternal sepa-ration and/or handling of rat pups during the first weeksof life reduces stress reactivity during adulthood andattenuates neuronal loss and cognitive decline duringaging. In the present study we examined whether neo-natal handling also affects the sensitivity of the adultbrain to an acute neurotoxic insult. Postnatally handledand nonhandled control rats were left undisturbed fromweaning onwards until the age of 11 months. At this age,the animals were subjected to a neurotoxic challenge byunilateral infusion of 60 mM of the glutamate analogueN-methyl-D-aspartate (NMDA) into the nucleus basalismagnocellularis (NBM). The brains were collected to

measure cholinergic cell and fiber loss. In the nonle-sioned side of the brain, cholinergic cell number in theNBM and fiber density in the cortex were not differentbetween postnatally handled and control rats. However,in the lesioned hemisphere handled animals exhibited asignificantly higher loss of choline-acetyltransferase-im-munoreactive and acetylcholinesterase-positive fibers inthe somatosensory cortex. The present results provideevidence for an enhanced vulnerability of postnatallyhandled rats to acute neurodegeneration in contrast tothe previously reported attenuation of spontaneous ag-ing-related neurodegenerative processes. © 2004 Wiley

Periodicals, Inc. J Neurobiol 60: 463–472, 2004

Keywords: postnatal development; maternal separation;cholinergic system; nucleus basalis magnocellularis; ex-citotoxicity; NMDA

INTRODUCTION

Adult physiology and behavior are not only shaped bygenetic factors but also by environmental factors and

life experiences, particularly during the early neonatalperiod (Cirulli et al., 2003; Levine, 2001; Meaney,2001; Nyakas et al., 1996). A well validated model tostudy early life influences on adult physiology ishandling of rat pups and separation from their motherfor a brief period of time, once a day during the firstfew weeks of life. It has been shown that this briefdaily removal of the young evokes an increase inmaternal care that strongly affects the development ofthe pups (Liu et al., 1997; Pryce et al., 2001). Long-lasting consequences of postnatal handling includereduced neuroendocrine stress reactivity and attenu-ated anxiety during adulthood (Hess et al., 1969;Levine et al., 1967; Meerlo et al., 1999; Plotsky andMeaney, 1993; Vallee et al., 1997). Neurobiological

Correspondence to: P. Meerlo (p.meerlo@biol.rug.nl).Contract grant sponsor: Graduate School of Cognitive and

Behavioral Neurosciences of the University of Groningen (K.M.H.and P.G.M.L.).

Contract grant sponsor: Netherlands Organization for ScientificResearch (NWO-ALW); contract grant number: 805-30-181 (P.M.and J.M.K.).

Contract grant sponsor: Hungarian National Science Founda-tion (OTKA); contract grant number: F035254 (T.H).© 2004 Wiley Periodicals, Inc.Published online 1 July 2004 in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/neu.20037

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mechanisms underlying the blunted stress responsive-ness have been the focus of extensive research inrecent years, describing the reduced expression ofcorticotropin releasing factor and up-regulation ofglucocorticoid receptors in specific brain regions asimportant hallmarks of postnatally handled animals(Avishai-Eliner et al., 2001; Meaney and Aitken,1985; Plotsky and Meaney, 1993). In addition, post-natal handling was found to result in a reduced aging-related hippocampal neuronal loss and cognitive de-cline (Escorihuela et al., 1995; Meaney et al., 1988),which may be partly due to a reduced exposure anddetrimental influence of stress hormones in the courseof life (Sapolsky et al., 1986).

Cortical cholinergic mechanisms are involved incomplex mental functions, such as arousal, attention,and memory. The decline of cognitive function inneurodegenerative diseases and aging is often par-tially attributed to defects in cholinergic transmission(Bartus et al., 1982; Coyle et al., 1983; Kasa et al.,1997). Numerous studies have investigated aging-related alterations in pre- and postsynaptic elementsof the cholinergic system and reported loss of basalforebrain cholinergic cells and a decrease in severalother cholinergic markers (Baxter et al., 1999;Springer et al., 1987; Strong et al., 1980). Interest-ingly, Liu and colleagues (2000) reported increasedhippocampal cholinergic activity in animals raisedwith active maternal care, the natural analogue ofpostnatal handling-induced changes in maternal care.

Overstimulation of N-methyl-D-aspartate (NMDA)receptors by excessive amount of glutamate is thoughtto be an important cause of cell death in severalneurological conditions associated with excitotoxicneuronal damage such as stroke, epilepsy, Alzhei-mer’s, and Parkinson’s disease (Choi, 1995; Coyleand Puttfarcken, 1993; Doble, 1999; Fountain, 2000;Harkany et al., 1999). The neurons of the nucleusbasalis magnocellularis (NBM), part of the cholin-ergic basal forebrain system, are particularly suscep-tible to excitotoxic insults in rat (Abraham et al.,2000; Harkany et al., 2000; Luiten et al., 1995;Stuiver et al., 1996). In addition, it has been demon-strated that the vulnerability of these cholinergicnerve cells is strongly affected by plasma levels ofcorticosterone (Abraham et al., 2000).

Because postnatal handling appears to attenuatespontaneous aging-related neurodegeneration in rats,we addressed the question of whether it also affectsthe sensitivity to acute degenerative processes. Hence,we studied the influence of early postnatal handlingon acute damage to the cholinergic system in adult-hood by an excitotoxic NMDA infusion into theNBM. The consequences of the manipulation were

evaluated by determining the loss of cholinergic cellsin the NBM and the loss of cholinergic fiber projec-tions to the somatosensory cortex by means of quan-titative histochemistry for two cholinergic markers,choline-acetyltransferase (ChAT) and acetylcholines-terase (AChE).

METHODS

Animals, Postnatal Handling, andExperimental Protocol

This study was performed with Wistar rats bred in our ownfacilities. The day after birth, nests were mixed to random-ize genetic variation and all nests were reduced to eightpups (six males and two females). Subsequently, half of thenewly arranged nests were left undisturbed except for cagecleaning once a week (control nests), while the other halfunderwent daily handling treatment from postnatal day 1 to21 (handled nests). The handling procedure was performedas described previously (Meerlo et al., 1999). In brief, allpups were removed from their nest and individually placedin glass jars lined with paper towel for 15 min. The jars werein a warm water bath (32–34°C) to prevent hypothermia ofthe pups. Each pup was handled for 3 min and after 15 minall pups were returned to their home cage. Daily handlingtook place at the end of the light phase. Importantly, al-though there is some variation in the handling procedurebetween studies and laboratories (e.g., the duration of han-dling or separation from the mother, frequency of handling,whether pups were isolated or with litter mates), our pro-cedure resulted in the same reduction of neuroendocrinestress reactivity and anxiety that has been reported by othergroups (Meerlo et al., 1999).

On postnatal day 24 the animals were weaned. Malepups remained group housed (six per cage) and were leftundisturbed except for the weekly cage cleaning. The ani-mals had free access to water and food and were kept undera 12:12 light/dark cycle (lights on at 08:00) in a tempera-ture-controlled environment (20 � 1°C). At an adult age of11 months, the animals were individually housed and sub-jected to unilateral lesion of the NBM (control n � 14;handled n � 11). Sham-lesioned animals were not includedin the present experiment, because our earlier results dem-onstrated that infusion of the vehicle solution into the NBMresults in a negligible reduction of cholinergic fiber projec-tions (Abraham et al., 2000; Luiten et al., 1995). Twelvedays after surgery, animals were deeply anesthetized andtheir brains were collected for histochemical analysis. Allanimal experiments were in accordance with the regulationsof the Ethical Committee for the Use of ExperimentalAnimals of the University of Groningen (DEC 2111/1998).

Surgical Procedure

For infusion of NMDA into the NBM we followed theprocedure as described previously (Luiten et al., 1995;

464 Horvath et al.

Stuiver et al., 1996; Horvath et al., 2000). The rats wereanesthetized with halothane and their heads were mountedin a stereotaxic frame (Narishige International, London,UK). Then, 60 nmol of NMDA (Sigma, St. Louis, MO)dissolved in a total volume of 1 �L phosphate-bufferedsaline (PBS; 0.01 M, pH 7.4) was infused into the rightNBM. The standard antero-posterior coordinate of the in-termediate NBM of �1.5 mm according to Paxinos andWatson (1986) was adjusted for each animal on the basis ofthe individual distance between the bregma and the inter-aural line. The lateral coordinate was set at 3.2 mm. TheNMDA solution was slowly injected at two dorso-ventralpositions (7.0 and 6.2 mm from the dura mater) at a rate of0.1 �L/min with a 5 min interval between the two depths.Body temperature during surgery was monitored with arectal probe and maintained at 36.5–37.5°C with a heatingpad. After the injection, the wound on the head was closedand 0.5 mL penicillin was injected subcutaneously(1.000.000 I.E./5 mL; Yamanouchi, Leiderdorp, The Neth-erlands). The animals were then returned to their homecages and observed for an additional hour.

Tissue Processing

Twelve days after NMDA infusion, the animals were deeplyanesthetized with ether and transcardially perfused withheparinized saline as a prerinse, followed by 300 mL ice-cold fixative composed of 4% paraformaldehyde in 0.1 Mphosphate buffer, pH 7.4 (flow-rate: 17 mL/min). The brainswere removed and postfixed for an additional 3 h in 4%paraformaldehyde and subsequently cryoprotected by stor-age in 30% sucrose in phosphate-buffer for 48 h at 4°C.Coronal sections were cut on a cryostat microtome at 20 �mthickness and series of sections spanning the damaged NBMand the somatosensory cortex were collected in 0.01 M PBS(pH 7.4). Directly after perfusion, the adrenal gland, thy-mus, and spleen were removed and wet tissue weights weremeasured.

Histochemistry andImmunocytochemistry

To visualize cholinergic cells and fibers, we performedimmunocytochemistry for the acetylcholine-synthesizingenzyme ChAT and histochemistry for the acetylcholine-metabolizing enzyme AChE. The stainings were performedon free-floating sections from all animals simultaneously.

For visualization of ChAT-immunoreactive (ir) cells andfibers two different protocols were applied. To achieve acortical fiber staining that could be reliably quantified, weused a higher concentration of the anti-ChAT antibody anda longer incubation time than we did for the NBM cellstaining. Furthermore, the fiber staining was visualized withnickel-enhanced 3,3�-diamonibenzidine (DAB), whereasthe cell staining was done with normal DAB. For bothstainings, sections were pretreated with 0.3% H2O2 in PBSfor 30 min. After several rinses in PBS, nonspecific bindingof immunoreagents was blocked with 5% normal rabbit

serum in PBS for 1 h. Then, sections were exposed toaffinity purified polyclonal goat antibody directed againstChAT (Chemicon, Harrow, UK). For staining of cholinergiccells in the NBM, sections were incubated with the ChATantibody (1:1000) overnight at 37°C, then thoroughly rinsedin PBS and exposed to the secondary antibody, rabbit anti-goat IgG (1:50; Sigma), for 4 h at room temperature. Fi-nally, the sections were incubated with goat peroxidase-antiperoxidase complex (1:300; Dakopatts, Glosstrup,Denmark) and developed with DAB as chromogen (30 mgin 100 mL Tris-HCl buffer) with 0.01% H2O2 (Horvath etal., 2000). For ChAT-ir fiber staining, the sections wereincubated with the ChAT antibody (1:500) for 3 days at4°C. Next, sections were thoroughly rinsed in PBS andincubated in biotinylated rabbit antigoat antibody (1:300;Vector, Temecula, CA) for 4 h at 4°C. After overnightwashing in PBS, the sections were incubated with ABC-kit(1:400; Vectastain Elite Kit; Vector Laboratories, Burlin-game, CA) for 2 h at room temperature. Finally, the stainingwas visualized with nickel-enhanced DAB (Ni-ammonium-sulfate 60 mg/100 mL; DAB 15 mg/100 mL Tris-HClbuffer).

For staining of AChE-positive fibers, brain sections werepostfixed by immersion in a 2.5% glutardialdehyde solutionin PB overnight at 4°C. AChE histochemistry was carriedout according to Hedreen et al. (1985) using a silver nitrateintensification procedure.

After the cytochemical procedures the sections wererinsed with Tris-HCl, mounted onto slides, air-dried, andcoverslipped with DPX (BDH Immuno, UK). Control ex-periments showed that there was no specific labeling in theabsence of the primary antibodies.

Quantification

All measurements were performed without prior knowledgeof the case condition. Effects of postnatal handling andNMDA infusion on ChAT-ir cell numbers in both the con-tra- and ipsilateral side of the brain were examined bycounting cells at four levels of the intermediate subdivisionof the NBM (Horvath et al., 2002). Briefly, the intermediatesubdivision of the NBM was analyzed over a length of 500�m measuring four sections (starting from Bregma �1.2,with 120 �m intersectional distance) in each animal. Cellcounting was carried out at 200X final magnification. Toavoid the inclusion of fragmented neuronal profiles, onlycells with clearly discernible nuclei of at least 7 �m indiameter (a) were included (Harkany et al., 1999; Horvath etal., 2000). The number of counted cells was multiplied withthe Floderus correction factor (f) (Floderus, 1944; Palkovitset al., 1971) to correct for the biased relationship betweenthe number of sectional profiles and the true object number.The correction factor was f � 0.7368, following calculationaccording to the formula: f � T1/[T1 � 2sqrt(r2 � (a/2)2)],where r corresponds with the radius of nuclei of intactneuronal somata and T1 the section thickness (20 �m).Subsequently, the counted area of the NBM was delineatedand its surface area measured by computer-assisted image

Neonatal Handling and Adult Neurodegeneration 465

analysis (Leica, Quantimet Q-600HR, Rijswijk, The Neth-erlands). A prerequisite for further data processing was thatthe surface area among the experimental groups was notdifferent. Subsequently, the cell counts from the four sec-tions were averaged for each side of the brain and the totalnumber of ChAT-ir cells per mm2 section area was calcu-lated. The effect of NMDA infusion was expressed bycalculating percentage ratios of cell densities in the lesionedand nonlesioned hemispheres.

Quantification of the ChAT-ir fiber density was per-formed in layers I–II and V of the posterior somatosensorycortex according to a standard protocol using a QuantimetQ-600HR image analysis system (Harkany et al., 1999;Horvath et al., 2000). Briefly, surface area density of corti-cal ChAT-ir fibers was measured in four sections (Bregma�0.5, �0.92, �1.4, �2.3; Paxinos and Watson, 1986). Foreach section, measurements were performed twice in layerI–II and layer V, both in the damaged and control hemi-sphere. After background subtraction and gray-scale thresh-old determination, the surface area of skeletonized ChAT-irfibers (the area covered by ChAT-ir cholinergic fibers di-vided by the total sampling area, given as percentages) wascomputed in each parietal cortical section using a 469.4 nmemission filter. Due to the unilateral cortical projections ofcholinergic NBM neurons, contralateral fiber density valuesserved as controls within each animal (Luiten et al., 1995).Fiber reduction was thus calculated as the percentage dif-ference between fiber densities (FD) at the injected andcontralateral sides of the brain: percentage decrease in FD� 100 � (FD injected side/FD control side � 100).

Quantification of the AChE-positive fiber density in thesomatosensory cortex was performed similarly as describedabove with the exception of a 599.5 nm emission filter.

Corticosterone Measurements

Two blood samples were taken for measurement of corti-costerone levels. The first sample was collected duringNMDA infusion by tail bleeding while the animals wereunder anesthesia. The second sample was taken from the leftventricle of the heart right before perfusion. In both cases,the blood samples were taken 30 min after removing theanimals from their housing environment to get an indicationof peak stress levels. The blood samples were collected inprechilled tubes containing EDTA as anticoagulant. Aftercentrifuging, the plasma was stored at �80°C until furtheranalysis. The plasma concentration of corticosterone wasmeasured by HPLC with UV light detection (Dawson et al.,1984).

Statistics

Data were analyzed with a one-way ANOVA test withpostnatal treatment as factor (postnatally handled rats vs.nonhandled controls). Within each group, NMDA lesion-induced changes on the lesioned side of the brain relative tononlesioned control side were determined by paired t-test.Furthermore, relationships between different parameters

were assessed by Pearson correlation. A p-value of �0.05was considered statistically significant for the tests. All dataare expressed as means �SEM.

RESULTS

There was no difference in body weight betweenpostnatally handled and nonhandled animals at anadult age of 11 months. Furthermore, wet tissueweights of the adrenal gland, thymus, and spleen werenot different between the two groups either (Table 1).Also, stress-induced “peak-levels” of plasma cortico-sterone as measured during surgery and perfusionwere similar in the postnatally handled and non-handled rats (Table 1).

For analysis of the NMDA-induced damage to theNBM, only those animals were included for which thelocation of the lesion was correct (control n � 13,handled n � 10). Determination of ChAT-ir cell den-sities on the control side of the NBM showed thatthere was no significant effect of the postnatal han-dling treatment compared to nonhandled controls(Fig. 1). NMDA infusion in the NBM resulted insevere loss of cholinergic cells in both groups [con-trol: T(12) � 8.07, p � 0.001; handled: T(9) � 13.50,p � 0.001]. Although ChAT-ir cell densities on thelesion side were not significantly different betweenhandled and control animals, expression of cell lossrelative to the control side showed a trend towardsincreased ChAT-ir cell loss in postnatally handledanimals [F(1, 21) � 3.23; p � 0.087].

Density of ChAT-ir fibers was measured in layerI–II and layer V of the somatosensory cortex, whichare the main target regions of cholinergic fibers orig-inating from the NBM (Eckenstein et al., 1988). In thenonlesion hemisphere, ChAT-ir fiber density was not

Table 1 Effect of Postnatal Handling on AdultPhysiology

Parameters Control Handled

Body weight (g) 570.2 575.6(12.6) (22.5)

Adrenal gland (mg) 53.3 55.7(1.8) (4.7)

Thymus (mg) 225.7 221.3(14.4) (14.3)

Spleen (mg) 870.4 912.5(42.5) (64.2)

Plasma CORT (�g/dL) lesion 29.1 31.3(2.7) (1.7)

Plasma CORT (�g/dL) perfusion 42.5 45.0(3.4) (3.1)

466 Horvath et al.

different between postnatally handled and control an-imals (Fig. 2). In the lesioned hemisphere there was asignificant loss of ChAT-ir fibers in both groups [con-trol animals: layer I–II: T(12) � 12.75, p � 0.001;layer V: T(12) � 9.50, p � 0.001; handled animals:layer I–II: T(9) � 13.02, p � 0.001; layer V: T(9)� 11.42, p � 0.001; Fig. 2]. In postnatally handledanimals, ChAT-ir fiber density in layer I–II of theipsilateral somatosensory cortex tended to be lowerthan in nonhandled controls [F(1, 21) � 3.38; p� 0.081]. Calculated as a percentage of cholinergicfiber loss in layer I–II, there was a significantly in-creased loss in handled animals [F(1, 21) � 9.10; p �0.007; Fig. 2]. A similar tendency for increased fiberloss was observed in layer V, which, however, did notreach statistical significance [control: 38.64 � 4.60%;handled: 46.91 � 3.88%; F(1, 21) � 1.81; p� 0.193]; notwithstanding a significant correlation

between fiber densities in layer I–II and layer V(control animals: contralateral side: r � 0.83, p �0.01; ipsilateral side: r � 0.91, p � 0.001; handledanimals: contralateral side: r � 0.63, p � 0.053;ipsilateral side: r � 0.92, p � 0.001).

Besides the measurement of ChAT-ir fibers, wealso measured the density of AChE-positive fibers inlayer I–II and layer V of the somatosensory cortex(Figs. 3 and 4). AChE-positive fiber densities in thesomatosensory cortex of the nondamaged hemispherecontralateral to the lesion did not differ between post-natally handled rats and nonhandled controls (Fig. 3).NMDA infusion in the NBM of the other hemisphereresulted in a severe loss of AChE-positive fibers inboth groups [control animals: layer I–II: T(12)� 11.48, p � 0.001; layer V: T(12) � 11.97, p� 0.001; and handled animals: layer I–II: T(9)� 10.26, p � 0.001; layer V: T(9) � 15.90, p �

Figure 1 Effect of postnatal handling on the loss of cholinergic cells in the intermediatesubdivision of the NBM induced by unilateral infusion of 60 mM NMDA during adulthood. Astrong reduction in numerical cell densities (number of cells/mm2 in �m thick sections) in bothgroups was determined following excitotoxic lesion. Data are expressed as means (�SEM). # trendwith p � 0.087.

Figure 2 Effect of postnatal handling on the loss of cortical ChAT-ir fibers induced by unilateral60 mM NMDA infusion into the intermediate subdivision of the NBM during adulthood. Postnatallyhandled animals (n � 10) had increased fiber loss in layer I–II of the somatosensory cortexcompared to controls (n � 13). Data are expressed as means (�SEM). *p � 0.05; # trend with p� 0.081.

Neonatal Handling and Adult Neurodegeneration 467

0.001; Fig. 3]. Comparison of fiber densities on thelesion side of the brain revealed lower densities inpostnatally handled animals compared to controls[layer I–II: F(1, 21) � 6.45, p � 0.018; layer V: F(1,21) � 3.88, p � 0.061]. Consequently, expression ofthe lesion-induced percentage decrease of AChE-pos-itive fibers showed significantly more extensive cho-linergic fiber loss in postnatally handled animals, ascompared to control rats both in layer I–II [F(1, 21)� 6.52; p � 0.018] and in layer V [F(1, 21) � 6.81;p � 0.016] of the somatosensory cortex (Fig. 3).

Correlation of AChE-positive and ChAT-ir fiberdensities revealed a strong relationship in both layerI–II and layer V of the lesioned hemisphere (in controlanimals: layer I–II: r � 0.65, p � 0.015; layer V: r� 0.80, p � 0.001; in handled animals: layer I–II: r� 0.87, p � 0.002; in layer V: r � 0.78, p � 0.013)as well as a strong relationship in percentage fiber loss

(in control animals: layer I–II: r � 0.58, p � 0.036;layer V: r � 0.63, p � 0.021; in handled rats: layerI–II: r � 0.86, p � 0.003; layer V: r � 0.93, p� 0.001).

DISCUSSION

Our results demonstrate that postnatal handling af-fects the vulnerability of cholinergic nucleus basalisneurons to acute excitotoxic damage during adult-hood. In particular, we found an increased loss ofChAT-ir and AChE-positive projection fibers in thesomatosensory cortex after NMDA infusion in theNBM.

The long-term effects of postnatal handling aregenerally interpreted as beneficial based on studies

Figure 3 Effect of postnatal handling on the loss of cortical AChE-positive fibers induced byunilateral 60 mM NMDA infusion into the intermediary subdivision of the NBM during adulthood.Quantitative analysis of area densities of AChE-positive fibers was performed in layer I–II (A) andlayer V (B) of the somatosensory cortex. In both cortical layers postnatally handled animals (n � 10)had significantly higher fiber loss compared to controls (n � 13). Data are expressed as means(�SEM). *p � 0.05; # trend with p � 0.061.

468 Horvath et al.

showing a reduced aging-related neurodegenerationand cognitive decline (Escorihuela et al., 1995;Meaney et al., 1988; Pham et al., 1997). In contrast,our results point to a neurodegeneration promotingeffect and thus suggest that long-term consequencesof postnatal handling in terms of neuronal vulnerabil-ity may depend on the nature of the degenerativeprocess (e.g., an acute toxic insult vs. aging-relateddegeneration) and the brain area investigated (e.g.,hippocampus vs. basal forebrain). The exact mecha-nisms that are responsible for an altered neurodegen-erative susceptibility during adulthood following neo-natal handling are unclear. In the first weeks afterbirth, the period in which our postnatal manipulationtook place, several neurotransmitter systems are stillactively developing and there is an on-going matura-tion of synapses (Clancy et al., 2001; Ullian et al.,

2001). Daily handling of pups or short-term separa-tion from their mothers evokes active maternal care,of which tactile stimulation of the pups is one of themost important components (Levine, 2001). The gen-eral increase in sensory input associated with maternalcare enhances neuronal activity, which subsequentlystimulates NGF synthesis and growth processes in thehippocampus, sensory-motor cortex, and other areas(Cirulli et al., 2003). Along this line, maternal careand postnatal handling may affect cholinergic neurondevelopment as well, especially because NGF recep-tors are expressed in cholinergic neurons in particular(Mufson et al., 1999). In agreement with these find-ings, Liu et al. (2000) showed an increased cholin-ergic activity in adult offspring of actively care-takingmothers, expressed by increased acetylcholine releaseand ChAT and AChE activities in the hippocampus.

Figure 4 Photomicrographs of AChE-positive fibers in layer V of the somatosensory cortex,contralateral to the lesion [(A) nonhandled; (B) handled] and ipsilateral to the lesion [(C) non-handled; (D) handled]. Excitotoxic lesion of the cholinergic NBM induced strong fiber loss in bothgroups. However, this was more pronounced in postnatally handled animals (B,D) than in non-handled controls (A,C). Scale bar in (D) � 50 �m.

Neonatal Handling and Adult Neurodegeneration 469

However, these differences were restricted to the hip-pocampus and were not evident in cortical regions. Inline with the latter, our study did not show basaldifferences in NBM cell numbers or cortical fiberprojections on the nondamaged side of the brain.Nevertheless, following a neurotoxic challenge to thecholinergic NBM neurons, we found increased corti-cal fiber loss in postnatally handled animals.

Interestingly, in both groups of rats, there appearedto be a more extensive loss of AChE-positive fibersthan ChAT-ir fibers in the somatosensory cortex. Thiscan be explained by the presence of intracortical cho-linergic interneurons immunopositive for ChAT butdevoid of AChE (Eckenstein et al., 1988). Therefore,the cortical AChE-positive fiber density in the ipsilat-eral hemisphere reflects the remaining projectionalfibers, whereas the ChAT-ir fiber density reflects boththe remaining fibers arising from the NBM and intactfibers of the intracortical interneurons.

One explanation for the increased NMDA-inducedneurotoxicity could be an alteration in the sensitivityto NMDA, which is supported by findings of long-term changes in NMDA receptor expression in pupsraised with active maternal care (Liu et al., 2000).Particularly, the expression of NMDA receptor sub-units 1, 2A, and 2B in the hippocampus is signifi-cantly increased by a high level of postnatal care. TheNR1 subunit is essential for the receptors’ channelfunction, and an increased incorporation of NR2Binto the receptor complex may result in longer exci-tatory postsynaptic effects (Monyer et al., 1992). Itmay thus be that similar alterations in the NBM ofpostnatally handled rats lead to an enhanced excitabil-ity of cholinergic neurons, as well as an enhancedvulnerability to overexcitation.

Besides direct effects of postnatal handling on thedevelopment of specific neurotransmitter systems,persistent changes in neuroendocrine stress systemsmay be another mechanism underlying increased sen-sitivity of basal forebrain neurons to acute neurode-generative processes. Several authors have describedattenuated corticosterone stress responses in postna-tally handled rats (Hess et al., 1969; Levine et al.,1967; Meerlo et al., 1999; Plotsky and Meaney, 1993;Vallee et al., 1997). Also in our own laboratory wefound an attenuated neuroendocrine stress reactivityin the adult offspring that were handled as neonates(Meerlo et al., 1999). Lower corticosterone responsesoccur especially under conditions of mild stress but incases of severe stress there may be a ceiling effectresulting in similar peak levels in postnatally handledand control animals (e.g., Vallee et al., 1997). In linewith this, although we found lower corticosteronelevels in response to mild novelty stress in a previous

study, we did not find a difference in “peak” cortico-sterone levels during surgery and perfusion in thepresent experiment. The complex alterations in theglucocorticoid system may play a significant role inthe increased sensitivity to excitotoxic injury in post-natally handled animals. Via GR, glucocorticoidshave an important neuromodulatory role in the regu-lation of neuronal and glial cell function in the brain(Abraham et al., 2001; Sapolsky et al., 1986). Underconditions of stress, this modulatory effect is a com-plex interaction of peak levels of corticosterone, thetotal amount of corticosterone released, and the sen-sitivity to corticosterone in terms of GR density invarious brain regions. In postnatally handled animals,particularly those brain regions with an increased GRexpression may be subject to increased corticosteroneinfluences, especially under conditions of severestress when peak levels of corticosterone are the sameas in control animals. Excess exposure to glucocorti-coids was shown to damage septo-hippocampal neu-rons (Tizabi et al., 1989) and enhance the vulnerabil-ity of both the septo-hippocampal and the corticopetalcholinergic systems to toxic insults (Abraham et al.,2001; Hortnagl et al., 1993). Regarding our lesionmodel it is important to note that, although cholin-ergic cells of the NBM express GRs, these receptorsare more abundantly present in the somatosensorycortex. The cortical target cells of cholinergic fibersproduce neurotrophins such as NGF, which via retro-grade transport play an important role in the mainte-nance of cholinergic cells (Mufson et al., 1999). Thus,potential modulatory effects of glucocorticoids maynot only involve direct effects in the NBM itself, butmay involve trans-synaptic pathways in the projectionareas as well. It remains to be established whetherpostnatally handled rats indeed have an increased GRexpression in the NBM and/or somatosensory cortex,as was reported for other areas.

In addition, acute brain injuries, like the one in ourstudy, trigger neuroinflammatory mechanisms, whichare protected from overshooting by an adequate cor-ticosterone feedback response (Kapcala et al., 1995).Although the attenuated corticosterone response inpostnatally handled rats is counterbalanced by in-creased GR expression in certain specific brain re-gions, this may not be true for all brain areas. Ablunted stress response in postnatally handled animalsmay be less effective in controlling neuroimmuneprocesses and may result in a potentiated immunereactivity (Solomon et al., 1968; Von Hoersten et al.,1995). Accordingly, Laban and colleagues (1995)found an increased susceptibility to experimental al-lergic encephalomyelitis in adult postnatally handled

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animals, as revealed by a higher incidence of thedisease and more severe clinical symptoms.

In conclusion, it is well known that the sensitivityto acute brain injuries and aging-related neurodegen-erative diseases varies between individuals. Ourpresent results, together with previously reported data,suggest that adult brain vulnerability may be affectedby early life events, for example, postnatal handling.However, the final outcome of postnatal handling, interms of a protective or endangering influence, willdepend on the adult environmental context and thenature of the neurodegenerative process. The presentresults provide evidence for an enhanced vulnerabilityof postnatally handled rats to acute neurodegenerationin contrast to the previously reported attenuation ofspontaneous aging-related neurodegenerative pro-cesses.

The authors acknowledge the expert help of Ibolya Bod-nar and Jan N. Keyser during the histochemical experi-ments.

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