Ž .Developmental Brain Research 107 1998 299–307

Research report

NADPH-d positive neurons in the developing somatosensory cortex ofthe rat: effects of early and late environmental enrichment

Victor Fernandez a,), Hermes Bravo b, Miguel Sanhueza a, Oscar Inzunza b´a Physiology and Biophysics Program, Biomedical Sciences Institute, School of Medicine, UniÕersity of Chile, Santiago, Chile

b Department of Anatomy, School of Medicine, Catholic UniÕersity of Chile, Santiago, Chile

Accepted 20 January 1998

Abstract

Ž qThe effects of environmental enrichment upon the topographic arrangement of NADPH diaphorase-positive neurons NADPH-d. Žneurons was studied in the somatosensory cortex of 56 Sprague–Dawley albino rats during early stages of development 18th, 24th, 30th

.and 60th postnatal days . This diaphorase is easily demonstrable, providing a convenient marker for quantitative studies. Environmentalenrichment diminished the number of NADPH-dq neurons and exerted its maximal influence during lactation, a time of exceptionalcortical susceptibility. This implies that the magnitude of such effects on the density of NADPH-dq neurons is age-dependent.Furthermore, it was found that the experience-dependent cortical changes persisted after a subsequent period without environmentalstimulation. The effects of early environmental enrichment did not occur uniformly throughout the cerebral hemispheres but, instead, sucheffects were maximal in the latero-ventral sector of the cerebral cortex where a dramatic reduction in the number of NADPH-dq neuronswas observed. Particularly striking was the existence of a latero-medial sequence of NADPH-dq neurons in the infragranular layer and areversed distribution of labeled cells, in the supragranular layer. Both ontogenetic sequences of NADPH-dq neurons remained unchanged

Ž .during postnatal development in controls and enriched subjects 18th–60th postnatal days . q 1998 Elsevier Science B.V.

Keywords: Nicotinamide adenine dinucleotide phosphate-diaphorase neuron; Environmental enrichment; Persistence; Histogenetic gradient; Somatosensorycortex; Rat

1. Introduction

It is well-known that the complex cytoarchitectonicorganization of the cortical plate provides the substrate formotor skills, cognitive development and exploration of theenvironment. Emerging evidences indicate that the devel-oping cerebral cortex is sculptured by a highly sophisti-cated program of neuronal reorganization, influenced bysocial, nutritional and environmental factors. These obser-vations raise a number of question to both basic scientistsand neurologists, as: What types of mechanisms specifycortical ontogenesis? What is the time course of theseevents during pre- and postnatal development? Perhapsmost important, what are the functional implications of the

) Corresponding author. Physiology and Biophysics Program, Biomed-ical Sciences Institute, School of Medicine, University of Chile, P.O. Box70005, Correo 7, Santiago, Chile. Fax: q56-2-7776916; E-mail: vifer-nan@machi.med.uchile.cl

program that governs the normal development of the pal-lium?

During the last few years, it has become evident that thesequential gradients of histogenesis and cytodifferentiationpresent in the developing cortical plate depend on criticalinteractions that play an important role in the specificationof the cerebral cortex and provide clues about the underly-

w xing evolutionary strategies 7,8 . Consistent with this inter-pretation, recent advances suggest that gradients of recog-nition molecules are at the basis of some topographicallyorganized patterns of connectivity in the central nervous

w x 3system 35 . When H-thymidine is made available to theembryonic brain, latero-medial differences in the time of

w xorigin of the cerebral cortex are found 10 . Further, theŽpattern of histogenesis at a given embryonic stage E15–

.E25 is strikingly consistent with the sequence of neuronaldifferentiation observed later during the early postnatal

w xperiod in the rat visual cortex 13 and with findings of ourgroup that studied the packing density of neurons in the rat

w xoccipital cortex 34 . Moreover, recent results of our labo-

0165-3806r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.Ž .PII S0165-3806 98 00037-6

( )V. Fernandez et al.rDeÕelopmental Brain Research 107 1998 299–307´300

ratory indicate that the pattern of distribution of NADPH-dq neurons in the rat somatosensory cortex correlate wellwith the gradients of histogenetic development and with

w xthe pattern of maturation of cortical neurons 3 . Conse-quently, the present findings—that investigate the effectsof early and late environmental enrichment—may haveimportant implications to elucidate the role of NADPH-dq

neurons in the developing cerebral hemispheres. Althoughthe presence of synthesized oxides of nitrogen in mammals

w xwas suggested in 1916 25 , only recently has it beendemonstrated that NADPH-d, the enzyme which synthe-

Ž .sizes nitric oxide NO , is widely distributed in the nervousw xsystem 5 . In addition, a growing number of studies have

Ž .provided evidence that nitrogen monoxide NO play animportant role in both neuroprotective and neurodegenera-

w xtive mechanisms 22 , mediates neuronal communicationw xand synaptic plasticity 29 , is implicated in developmental

w xprocesses in the nervous system 4,14,32,41,42 and nu-merous other functions. On the other hand, there areconsiderable evidences that environmental enrichment sig-nificantly improves the functional outcome of laboratory

w xanimals after brain ischemia 17 , brain infarctionw x w x16,18,30 , cortical lesion 20 or traumatic brain injuryw x15 . Similar beneficial effects have been found with re-

w xgard to deficits associated with malnutrition 6,13 andw xenvironmental deprivation 11 .

In the light of the above data, the aim of the presentstudy was to elucidate whether the arrangement ofNADPH-dq neurons, present during the early postnatalperiod, may be modified under the influence of environ-mental enrichment. Further, our findings could give exper-imental support to the idea that the brains of rats reared incomplex habitats retain the structural changes after a pe-

Ž .riod without environmental stimulation persistence .

2. Materials and methods

2.1. Animals

Fifty-six Sprague–Dawley male albino rats from ourown breeding stock were used in the present study. Atbirth, complete litters of eight animals were assigned either

Ž .to standard conditions SC or to relatively enriched condi-Ž . Ž .tions EC Table 1 .

2.2. BehaÕioral manipulations

From day 3, pups in the enriched environment wereŽ .exposed to three 30-min sessions per day of free explo-

ration in a 70=70=40-cm wire mesh playbox furnishedwith a large variety of objects which were changed orre-arranged daily. These included: running wheels, tunnels,platforms, ladders, sand boxes, balls, rattle, leafy plants,long grass, flowers, tree branches, sawdust, furry and

Žrough surfaces, ‘toys’ of different sizes and textures wood,.metals and plastic . Recorded music was also provided.

Each pup also underwent gentle handling for 2 min beforeeach testingrtraining session, while 24 small lights flashedintermittently. Afterwards, the pups were gently placed ina water bath at 378C for at least 25 s during which timethey would swim. Rats in the standard condition werehandled only for routine maintenance. All animals wereraised on a 12-h dark–12-h light cycle, with food and

Žwater ad libitum relative humidity 50"5%, ts21".1.28C .

2.3. Morphological assessment

In order to study the distribution of NADPH-dq neu-rons and the effects of early enrichment, the somatosen-sory cortex of EC and SC pups was studied at postnataldays 18, 24, 30 and 60. In addition, to elucidate whetherthe effects of enrichment persist after a period withoutenvironmental stimulation, two other litters were analyzed:

Ž .EC-P 18–30 and EC-P 30–60 see Table 1 .At each postnatal developmental period, seven animals

per litter were deeply anesthetized with sodium pento-Ž .barbital 6 mgr100 g body weight and perfused through

the left ventricle with saline phosphate buffer 0.1 M, pH7.4, followed by 4% paraformaldehyde in 0.1 M phosphatebuffer, pH 7.4. The brains were removed from the skull,postfixed for 8 to 12 h at 48C and then transferred to 0.1 Mphosphate buffer, pH 7.4, in sucrose 30% at 48C, for 12 to24 h. The brains were sectioned serially in a freezingmicrotome at 50-mm intervals on a coronal plane. Every

Ž .other section was transferred to Tris HCl THCL buffer0.1 M, pH 8.0, following the histochemical protocol for

w xNADPH-diaphorase of Mizukawa et al. 26 . Briefly, thesections were incubated in 10 ml of 0.1 M THCL bufferpH 8.0, containing 10 mg of reduced nicotinamide adenine

Ž .dinucleotide phosphate NADPH , 2.5-mg nitroblue tetra-Ž .zolium Sigma and 1.5-mg of saccharose at 378C for 1 h.

After incubation, sections were rinsed in phosphate buffer,mounted in gelatin-coated slides, air-dried, counterstainedwith neutral red and dehydrated. Cover slips were appliedwith Permount.

In order to get data as comparable as possible, sectionsobtained at different postnatal developmental periods wereprocessed under identical conditions. Fourteen sections foreach experiment were selected. These sections correspondto a region situated between the anterior commissure and

Žthe anterior aspect of the hippocampus according to theStereotaxis Atlas of the developing rat brain of Sherwood

w xand Timiras 36 , this region is located from As6.2 mmto As4.1 mm for 21-day-old rats and from As7.0 mm

.to As5.0 mm for 39-day-old rats . The evaluations wereassessed by counting under camera lucida all the labelledneurons included in two vertical strips of cortex 800-mm

Ž .wide including layers I to VI . They were situated in thedorso-medial and ventro-lateral sectors of the somatosen-

Ž .sory cortex Sm , which included the disgranular sector ofw x w xSmI 38 and the lateral somatosensory cortex SmII 40 .

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Table 1Distribution of experimental groups submitted to environmental influences during the early postnatal period

Environmental Environmental Period without Corporal development Number of Number ofŽ .statusrpostnatal enrichment environmental mean"S.E.M. brains neurons

day postnatal days enrichment analyzed quantifiedŽ . Ž .Weight g Size mm

SC 18 y y 35.28"1.51 106.31"1.55 7 7044EC 18 3–18 y 27.35"0.32 104.20"0.89 7 5249SC 24 y y 49.33"1.73 129.00"1.60 7 7399EC 24 3–24 y 38.10"0.56 129.79"1.32 7 6655SC 30 y y 68.95"2.88 148.80"2.80 7 8716EC 30 3–30 y 55.72"1.12 142.80"0.74 7 6914EC-P 18–30 3–18 19–30 63.96"2.88 142.35"2.68 7 5295EC-P 30–60 3–30 31–60 208.38"4.76 173.38"3.87 7 6415

SCsStandard condition, ECsenriched condition, EC-Psenriched condition-persistence after a period without environmental enrichment.

( )V. Fernandez et al.rDeÕelopmental Brain Research 107 1998 299–307´302

3. Results

In order to analyze the distribution of NADPH-dq

neurons, the cortical layers were outlined and then themicroscopically identified labeled neurons were plottedinside the different laminae: I marginal layer, II–III supra-granular layers, IV granular layer, V–VI infragranularlayers. The mean NADPH-dq neuron numbers, obtainedfrom measurements performed within two radially ar-

Ž .ranged cortical sample columns 800-mm wide located atmedial and lateral somatosensory sectors in standard andenriched conditions, are shown in Fig. 1. It is important topoint out that the density of NADPH-dq neurons in layer Iis not depicted in the histograms since their presence was

Žextremely scarce e.g., six out of 7050 neurons in P18,.nine out of 7408 neurons in P24 and so forth . On postna-

Ž .tal day 18 standard condition A , a large number oflabeled neurons accumulates in the infragranular layers

q Ž .Fig. 1. Mean number of NADPH-d neurons per 800-mm wide bands X"S.E.M. found in the somatosensory cortex at the 18th, 24th and 30th postnataldays in pups grown under standard conditions or under enriched conditions. Cells of seven animals per experimental condition have been summed for each

Žlaminae represented. Means and standard deviations are given for individual laminae standard error lines too short to be visible are not depicted;. q

))) p-0.0001; )) p-0.005; ) p-0.05, ANOVA ; nsnumber of NADPH-d neurons; n snumber of animals per experimental condition.1Ž .Histograms showing Standard condition in our article Neuroscience Letters, Vol. 234 1997 103–106, are re-used with permission of Elsevier, Ireland.

( )V. Fernandez et al.rDeÕelopmental Brain Research 107 1998 299–307´ 303

Fig. 2. Mean number of NADPH-dq neurons per 800-mm wide bandsŽ .X "S.E.M. found in the somatosensory cortex at 30th and 60th postna-tal days in enriched rats after 12 and 30 days without environmentalenrichment, respectively. Cells of seven animals per experimental condi-tion have been summed for each laminae represented. Means and stan-

Ždard deviations are given for individual laminae standard error lines too.short to be visible are not depicted; ))) p-0.0001, ANOVA ; ns

number of NADPH-dq neurons; n snumber of animals per experimen-1

tal condition.

Ž .V–VI at the ventro-lateral aspect of the somatosensoryŽ .cortex VL . By contrast, this distribution appears reversed

in the supragranular layers II and III in which the amountof labelled neurons was larger in the dorso-medial sectorof the somatosensory cortex. The pattern of NADPH-dq

neuron distribution found at the 18th postnatal day wasstrikingly consistent with the patterns observed at later

Žstages 24th and 30th postnatal days, see standard condi-.tion B and C . This means that the patterns are sequential,

which confirms the method’s reliability and proves theorderly fashion in which histogenesis of NADPH-dq neu-rons takes place in the cerebral cortex, as was observedpreviously during the genesis and maturation of the corti-

w xcal plate 10,13,34 .The latero-medial topographic arrangement found in the

infragranular layers, as well as the opposite medio-lateralorganization, observed in the supragranular layers of pups

grown under standard conditions were also present in pupsŽ .reared under enriched conditions Fig. 1 . This indicates

the immutability of such topographic organization ofNADPH-dq neurons. The average amount of NADPH-dq

neurons found in enriched rats is presented for the totalcortical thickness and for individual laminae in A, B andC. For the whole cortical thickness, there was a decrease in

Žthe number of positively stained neurons from 7044, 7399

Fig. 3. A reconstruction of the number of NADPH-dq neurons atŽ . Ž .successive dorso-medial DM and ventro-lateral VL sectors of the rat

somatosensory cortex at the 18th, 30th and 60th postnatal days. For thesegraphs, the number of cells of seven animals per experimental conditionhave been summed for each point represented. SCsstandard condition;ECsenriched condition; EC-P enriched condition after a period without

Ž . Ž . Ž .environmental enrichment. A Supragranular layers II–III , B infra-Ž .granular layers V–VI . With the exception found at the 18th postnatal

Ž .day in the supragranular layers filled and open circles , EC pups clearlyshow a dramatic decline in the number of NADPH-dq neurons whencompared with SC pups. Note, in the infragranular layers, that after a

Ž .period without environmental enrichment EC-P , the number ofNADPH-dq neurons tends to recover but still remains under the levelsfound in SC subjects. However, in the ventro-lateral sector of the

Ž .somatosensory cortex area of higher level of susceptibility , the numberof NADPH-dq neurons steadily decreased until the 60th postnatal dayŽ .open squares in the supra and infragranular layers, 30-VL .

( )V. Fernandez et al.rDeÕelopmental Brain Research 107 1998 299–307´304

.and 8716 to 5249, 6655 and 6914 labelled neurons . Thenumber of NADPH-dq neurons is thus 18.74% lower inthe enriched pups.

Fig. 2 shows the persistent effects of early stimulationafter variable periods without environmental enrichment.

ŽAfter 12 days without environmental enrichment EC-P.18–30 and after 30 days without environmental enrich-Ž .ment EC-P 30–60 , a notable decrease in the number of

NADPH-dq neurons remains in the somatosensory cortexŽ .from 7044 and 8716 to 5295 and 6415 , indicating thatthe effects of early enrichment involve a persistent mor-phological expression. Further, it can be observed thatthere were no major re-arrangements in the topographicaldistribution of NADPH-dq neurons, since they developedidentical latero-medial and medio-lateral sequences.

The numerical distribution of NADPH-dq neuronsfound in supra and infragranular layers at the dorso-medialand ventro-lateral sectors of the somatosensory cortex

Ž .during normal development SC , after early enrichmentŽ . ŽEC and after a period without enrichment EC-P, persis-

.tence is summarized in Fig. 3. In the infragranular layers,a dramatic drop in the number of labeled neurons may be

Ž .noted after environmental enrichment EC . The drop ishighly pronounced during pre-weaning in the ventro-lateral

Žsector of the somatosensory cortex open circles, postnatal.day 18, 18-VL . In addition, Fig. 3 shows that environmen-

Žtal enrichment maintained during a long period of time 28.days determines, after a period without enrichment in

EC-P, a steady decline in the number of NADPH-dq

neurons situated in the ventro-lateral sector of both supraŽ .and infragranular layers open squares, 30-VL . These

findings are consistent with the higher level of susceptibil-ity to environmental influences previously observed in the

w xventro-lateral sector of the cortical plate 13 .

4. Discussion

This study analyzes the effects of early enrichmentupon the distribution of NADPH-dq neurons in the so-matosensory cortex of the rat. Our results show that thenumber of NADPH-dq neurons is lower in enriched rats.This indicates that there is a greater separation between theNADPH-dq cell bodies, a fact that may be due to ageneralized increase in the size and complexity of den-dritic trees along the cortical plate rather than to a loss ofneurons. A recent Golgi study from our laboratory sup-

w xports this interpretation 13 since it demonstrates a profusearborization of dendrites after environmental richness. Re-duction in neuronal density is a good general marker ofdendritic growth when it is reasonable to assume that thereis no experimentally-induced loss of neurons. To our

Žknowledge, no evidence of programmed cell death apop-.totic mechanisms induced by environmental enrichment

has been reported so far. However, we cannot rule out thepresence of other factors such as gliosis and angiogenesis.

The latter factor seems to be particularly relevant at theŽventro-lateral sectors of the somatosensory cortex see Fig.

.4D .In the present study, a combination of neonatal handling

and environmental enrichment was used. This point is ofparticular importance to explain the long lasting effects

q Žobserved in the packing of NADPH-d neurons per-.sistence, Fig. 2 since neonatal handling has been shown to

w xpermanently affect behavior and endocrine responses 27 .An interesting additional finding is that the topographicarrangement of NADPH-dq neurons, found along thecoronal plane of the cerebral hemispheres, is retained inenriched pups and persists after a relatively long period oftime without environmental enrichment.

It is obviously very difficult to link our histological dataand its physiological significance, especially during thecritical period of development. We have found, however,that the significance of NADPH-dq neurons developmen-tal arrangement in the infragranular layers may be ex-plained in several ways. The pattern of distribution ofNADPH-dq neurons in the somatosensory cortex of devel-oping pups coincides with regional and temporal specifici-ties found in the neurogenetic program of incoming thala-

w x w xmocortical axons 28 , with the histogenesis 10,24 andlater, with the levels of cytodifferentiation present in the

w xneocortex of the rat 2 . A recent study of our groupprovides, in addition, valuable data that appear to correlatethe sequence of ontogenetic development in layers V andVI and the levels of cortical susceptibility to environmen-

w xtal enrichment 13 . In agreement with the later report,NADPH-dq neurons counts in EC-P 30–60 showed thatcell number progressively decreases until the postnatal day

Ž60 in the ventro-lateral sector of the pallium area of. Žhigher level of susceptibility to sensorial inputs Figs. 2

.and 3 . The aforementioned result is entirely consistentwith ontogenetic and phylogenetic considerations previ-

w x w xously held by Cajal 33 , Karten 19 and Marin-Padillaw x23 in relation with the arrival of afferent fibers at thecortical plate. It is perhaps worth emphasizing that ourhistological observations also indicate a medio-lateral dis-tribution of NADPH-dq neurons in the supragranular lay-ers II and III. This topographic arrangement is closelyrelated with the pattern of neuronal migration through thelateral cortical stream. The neurons that constitute thisstream are formed during the late prenatal period and latermigrate inside-out and medio-ventrally to accumulate in

w xlayers II and III 1 .In our experimental paradigm, enrichment of the habitat

during the early postnatal period resulted in long-termdecrease of NADPH-dq neuronal density in the rat so-matosensory cortex. The evidence points to the notion thata common mechanism of neural plasticity might be in-volved in the response of the nervous system to differentenvironmental manipulations. In fact, morphometric stud-ies using different methods such as Nissl and Golgi stain-ing techniques have revealed that environmental enrich-

( )V. Fernandez et al.rDeÕelopmental Brain Research 107 1998 299–307´ 305

Fig. 4. Photomicrographs of NADPH-dq neurons found in the somatosensory cortex of enriched pups. It is important to point out that the form and size ofthe perikarya and the distribution of the dendritic arborizations vary considerably. Note that none of the neurons observed has features consistent with

Ž .pyramidal neurons. A Slender fusiform neuron having dendrites which arise from opposite ends of the cell body found in the dorso-medial sector of theŽ .supragranular layer at the 24th postnatal day. B Neurons with an ovoid tripolar soma located in the ventro-lateral sector of layer V at the 24th postnatal

Ž .day. C Polygonally shaped neuron having radiating dendrites with curly branches found in the ventro-lateral sector of the supragranular layer at the 30thŽ .postnatal day. D Multipolar neuron with radiating dendritic processes found in the ventro-lateral sector of layer V at the 30th postnatal day. Three of the

dendrites are in close association with branches of neighboring blood microvessels. Scale barss15 mm.

ment and training in motor tasks induce decreased neu-ronal density associated to increments in dendritic ar-

w xborization 9,12,39 , while environmental injuries such as

malnutrition and sensorial deprivation result in increasedneuronal density, reflecting a reduction in dendritic span

w xand branching 11,31 .

( )V. Fernandez et al.rDeÕelopmental Brain Research 107 1998 299–307´306

The finding that the effects of enrichment are maximalduring pre-weaning implies that the magnitude of suchmorphological effects are age-dependent. The role thatNADPH-dq neurons might play with regard to the ontoge-netic development of the brain is not certain. According to

w xLawrence and Jarrott 21 ‘‘NO appears to have importantneuromodulatory properties in cardiovascular control path-ways of the brain stem’’. The evidences presented heresuggest that NO may be associated with the regulation ofblood supply to the developing cortical plate. In the pre-sent study, many NADPH-dq cells appear closely relatedto blood vessels in the ventro-lateral sectors of the so-matosensory cortex. It seems reasonable to suppose thatthey may be involved in the mechanisms that regulateblood supply to the pallium. This is consistent with arecent study showing that ‘‘the resting tone of the cerebralvascular bed of the lamb fetus is under NO control, andNO mediates the cerebral vasodilatatory response to hy-

w xpoxia in the lamb fetus’’ 37 .

Acknowledgements

This research was supported by Grant FONDECYT1950649. The authors are grateful to Dr. Rodrigo O.Kuljis, Center for Cognitive Neuroscience, Department ofNeurology, University of Miami, for critical reading andhelpful comments on the manuscript, to Dr. Ruben Soto-´Moyano and to Dr. Alejandro Hernandez for their valuable´suggestions. We thank Dr. Emilio Decinti for assistance inthe statistical analyses.

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