proton mrs of early post-natal mouse brain modifications in vivo

NMR IN BIOMEDICINENMR Biomed. 2006; 19: 180–187Published online 8 February 2006 in Wiley InterScience (www.interscience.wiley.com). DOI:10.1002/nbm.997

Proton MRS of early post-natal mouse brainmodifications in vivo

Pierre Larvaron,1,2 Guy Bielicki,1 Odile Boespflug-Tanguy2 and Jean-Pierre Renou1*1QuaPA/STIM, INRA Theix, 63122 Saint Genes Champanelle, France2INSERM UMR 384, 28 place H. Dunant, BP 38, 63001 Clermont-Ferrand cedex, France

Received 22 August 2005; Accepted 22 September 2005

ABSTRACT: NMR provides a non-invasive tool for the phenotypic characterisation of mouse models. The aim of the

present study was to apply reliable in vivo MRS techniques for non-invasive investigations of brain development in normal

and transgenic mice, by monitoring metabolite concentrations in different brain regions. The conditions of anaesthesia,

immobilisation and respiratory monitoring were optimized to carry out in vivo MRS studies in young mice. All the

experiments were performed in normal mice, at 9.4 T, applying a point-resolved spectroscopy (PRESS) sequence

(TR¼ 2000ms; TE¼ 130ms). We obtained reproducible in vivo 1H NMR spectra of wild-type mouse brains as early as

post-natal day 5, which allowed us to follow brain maturation variations from post-natal days 5 to 21. The survival rate of

animals was between 66 and 90% at post-natal days 5 and 21, respectively. Developmental changes of metabolite

concentrations were measured in three brain regions: the thalamus, a region rich in cell bodies, the olfactory bulb, rich in

fibre tracts actively myelinated during brain maturation, and the cerebellum. The voxel size varied from 2 to 8 mL according

to the size of the brain structure analysed. The absolute concentrations of the total creatine, taurine, total choline, N-

acetylaspartate and of the glutamate/glutamine pool were determined from 1H NMR spectra obtained in the different brain

regions at post-natal day 5, 10, 15 and 21. Variations observed during brain development were in accordance with those

previously reported in mice using ex vivoMRS studies, and also in rats and humans in vivo. Possibilities of longitudinal MRS

analysis in maturing mice brains provide new perspectives to characterise better the tremendous number of transgenic

mutant mice generated with the aim of decrypting the complexity of brain development and neurodegenerative diseases but

also to follow the impact of environmental and therapeutic factors. Copyright # 2006 John Wiley & Sons, Ltd.

KEYWORDS: post-natal mouse brain development; localized in vivo 1H MRS; relaxation time; absolute metabolite

quantification; thalamus; cerebellum; olfactory bulb

INTRODUCTION

The mammalian central nervous system develops througha series of complex cellular processes which may begrouped into two broad phases (1,2). The first cytogenesisand histogenesis phase, in which neurons are formed andelaborate the primary neuritic processes, is largely com-pleted during the first half of gestation. The second,growth and differentiation, is a phase in which neuronsincrease in overall size, elaborate terminal dendritic andaxonal arborisations and form connections. This phaseis closely coordinated with the development of non-neuronal cell populations which elaborate the myelininvestments of axons and play critical structural, suppor-tive and metabolic roles. This phase extends over a longperiod: most rapidly during the second half of gestation

and during the first period of post-natal life, it thencontinues at a slower rate up to post-pubertal life.Impairment of these processes of growth and differentia-tion in humans may lead to disturbances of motor devel-opment and learning, language disorders, autism andother behavioural disorders and epilepsy. Identifyingthe genetic and environmental factors which influencethese complex developmental events is becoming animportant challenge for the development of preventiveand curative therapeutic strategies.Proton magnetic resonance spectroscopy (1H MRS)

provides regional in vivo measurements of brain metabo-lite levels. Modulations of major metabolite pools havebeen observed in human brain during childhood andadolescence (3) and in various pathological processes(4–6). At a higher field (9.4 T), by in vivoMRS, in 63 mLvoxel, quantification of 18 metabolites has been per-formed in adult rat (7) and mouse (8) brains. Morerecently, developmental changes of metabolite concen-trations have been determined in various areas (11 mL) ofhealthy rat brains (9).During the last decade, research on the identification of

molecular events leading to central nervous system

Copyright # 2006 John Wiley & Sons, Ltd. NMR Biomed. 2006; 19: 180–187

*Correspondence to: J.-P. Renou, QuaPA/STIM, INRA Theix, 63122Saint Genes Champanelle, France.E-mail: [email protected]

Abbreviations used: Ach, acetylcholine; CHESS, chemical shift-selec-tive water suppression; CNS, central nervous system; Glx, glutamate/glutamine pool; HPLC, high-performance liquid chromatography; NAA,N-acetylaspartate; PRESS, point-resolved spectroscopy; Tau, taurine;tCho, total choline; tCr, total creatine; VOI, volume of interest.

(CNS) development has been transformed by the produc-tion of transgenic animals. In mammals, the animal ofchoice for this technique is the mouse and a tremendousnumber of transgenic mutant mice have been generated tostudy brain maturation and neurodegenerative diseases(10). 1H NMR provides a non-invasive and non-destruc-tive tool for studying longitudinally the effect of geno-type on anatomy, physiology and behaviour andultimately, the occurrence of pathological processes andtherapeutic strategies. However, the challenge is to per-form in vivomeasurements with mice as early as possibleafter birth in very small brain areas.We adapted 1H NMR techniques in order to carry out

studies of wild-type mouse brains as early as post-natalday 5 and to follow the developmental changes ofmetabolite concentrations in three easily defined regionsof the brain: the thalamus, a nucleus-rich region, theolfactory bulb, with a high fibre content, and the cere-bellum.

MATERIALS AND METHODS

Animal monitoring

The study was performed according to European legisla-tive administrative and statutory measures concerning theprotection of animals used for experimental or otherscientific purposes (86/609/EEC). C57Bl6-SJL pregnantmice were monitored to determine precisely the day ofbirth of the newborns. Twenty-two animals were used forrelaxation time determination at various post-natal days:five (3.3� 0.3 g) at day 5 (P5), six (4.9� 1.3 g) at day 10(P10), six (6.5� 0.9 g) at day 15 (P15) and five(9.0� 1.2 g) at day 21(P21). Forty-two animals wereused for metabolite quantification: nine (3.0� 0.2 g) atday 5 (P5), 12 (4.7� 0.5 g) at day 10 (P10), 12(6.5� 0.9 g) at day 15 (P15) and 14 (9.4� 1.3 g) at day21(P21).Animals were anaesthetized through a mask by spon-

taneous inhalation of 1–2.5% isoflurane and air (300mL/min) using a Univentor 400 anaesthesia unit (Univentor,Malta). The probe was thermostated at 39�C by warm aircirculation. Mice were monitored for changes in theirrespiratory rate in order to adjust the anaesthetic con-centration. During the 1.5 h of MR acquisition, theanimals breathed freely. Recovery of mice after theinterruption of anaesthesia at the end of the procedurewas rapid (< 10min).

MRS acquisitions

All experiments were performed at 9.4 T on an Bruker(Ettlingen, Germany) Avance DRX 400 micro-imagingsystem with a wide-bore vertical magnet and an activelyshielded gradient coil. Mice were carefully secured in a

Bruker MicroMouse 2.5 animal handling system with thehead centred in a 20mm diameter birdcage radiofre-quency coil used for both excitation and signal reception.Maximum field homogeneity was set by adjusting the

manual shimming of the total proton signal to obtain ahalf-height linewidth of the water signal of < 120Hz.Localisation was achieved using a T2 weighted sequencewith a 10ms echo time; 32 slices of 0.5 mm thicknesswere acquired. The volumes of interest (VOIs) of differ-ent sizes depending on structures were centred in theolfactory bulb (1.25� 1.25� 1.25mm), cerebellum(1.5� 1.5� 1.5mm) and thalamus (2� 2� 2mm)(Fig. 1). The in vivo proton spectra were acquired atP10, P15 and P21 for the three brain structures. At day 5,acquisition was limited to the cerebellum and thalamusowing to the small size of the olfactory bulb by this post-natal day. Shimming was performed by optimising theproton signal of water in each VOI. Normally, a half-height linewidth of the water signal of 12Hz on thethalamus and around 20Hz for the other structures wasachieved. A point-resolved spectroscopy (PRESS) se-quence was used for signal acquisition. The water signalfrom the voxel was suppressed using a standard chemicalshift-selective water suppression (CHESS) pulse scheme.For metabolite quantification, each spectrum representedan average of 512 scans for thalamus and 1024 scans forolfactory bulb and cerebellum, with a 130ms echo timeand 2000ms recycle time.Relaxation times T1 and T2 of N-acetylaspartate

(NAA), total creatine (tCr), total choline (tCho), taurine(Tau) and water (T2 only) were calculated with spectrumacquired in voxel (2� 2� 2mm) placed on thalamus;each spectrum represented an average of four or 128scans for water or metabolite signal acquisitions with a130ms fixed echo time and five different recycle times(TR1–5¼ 1000/2000/4000/8000/10 000ms) for longitudi-nal recycle time measurement or 2000ms fixed recycletime and 10 different echo times (TE1–10¼ 17/20/30/40/60/80/100/130/180/250ms) for transversal recycle timemeasurement.

Water content

Ten animals at P5, four at P10, 4 at P15 and three at P21were killed to evaluate the variation in whole brain watercontent during development. Percentages of water werecalculated by using the difference of weight betweenfreshly removed whole brain and its residue after dehy-dration.

MRS analysis

NMR spectra were fitted by jMRUI software using thenonlinear algorithm AMARES. The signal of residualwater was filtered with HLSVD preprocessing and an

MOUSE BRAIN DEVELOPMENT BY IN VIVO 1H MRS 181


apodisation (10Hz) was applied to increase the signal-to-noise ratio of the spectrum acquired with watersuppression.For T1 and T2 measurements, the diminution of areas of

major peaks versus TE or TR values was fitted by amonoexponential function. A biexponential decay wasobserved on the graph with NAA resonance at 2.01 ppmversus TE; the shortest T2 was associated with macro-molecule compounds and the longest one was attributedto an NAA compound. To overcome the J-modulationeffect on T2 of the taurine signal, only areas at TE equal to16, 130 and 250ms were taken to estimate the T2 of Tau.For the glutamate/glutamine pool, no correction wasneeded due to J-modulation and lack of a low signal ofthese metabolites at early developmental stages.Resonances of the methylene of Tau and of the three

methyls of Cho close to 3.21–3.24 ppm were not easilydistinguishable and gave a unique peak. Therefore, thetCho signal was always calculated by the subtraction ofthe methylene signal of Tau at 3.42 to the composite peakat 3.21–3.24 ppm.

Absolute quantification

Metabolite levels were expressed as a function of internalwater content obtained from the unsuppressed watersignal. Corrections were performed to take into accountthe T2 and T1 effects. Results obtained by jMRUI soft-ware using the nonlinear algorithm AMARES wereexpressed as mean� SD. Measurements were compared

by analysis of variance (ANOVA), followed by a New-man–Keuls post hoc test to determine the significance(Statistica, StatSoft). The level of significance was set atp< 0.05.

RESULTS

Animal survival

Under our experimental conditions, the survival rateduring the week after anaesthesia was 66 and 90% atP5 and P21, respectively. The mouse weight in eachgroup was homogeneous and the development of thesurviving animals was normal.

Measurement of in vivo 1H NMR spectraat different post-natal days

As shown in Fig. 2(A) for the thalamus, in 5-, 10-, 15- and21-day-old mouse brains, the different metabolites wereeasily recognisable: the methyl resonance of NAA at2.01 ppm, methylene multiple signals of glutamate/glutamine pool (Glx) at 2.33–2.35 ppm, methyl andmethylene resonances of tCr at 3.02 and 3.90 ppm,respectively, the threefold methyl signal of tCho at3.21 ppm and methylene of Tau at 3.24–3.42 ppm. Inthe thalamus, brain maturation changes concern all thesemetabolites: the decrease in tCho and Tau was paralleledby an increase in NAA and Glx. Similar metabolite

Figure 1. Sagittal (A) and coronal (B) images of P15 mouse brain obtained with a T2 weightedsequence (TE¼10 ms; 32 slices thickness¼0.5mm). Typical VOI localisation and size selected for 1HMRS olfactory bulb (1.25�1.25� 1.25mm) (1); thalamus (2� 2�2mm) (2); cerebellum(1.5� 1.5�1.5mm) (3).

182 P. LARVARON ET AL.


profiles were observed in the olfactory bulb and cerebel-lum, as shown in Fig. 2(B) at P21.

Quantification of in vivo T1and T2 relaxation times

No significant maturation change was observed in thelongitudinal relaxation times (T1) of the different meta-bolites, as shown for the thalamus in table 1. The lowestT1 values (< 1 s) were obtained for NAA and tCr and thehighest for tCho (1.3 s) and Tau (1.5 s) in both groups atP5 and P21, with a transitory and non-significant increasebetween P10 and P15. However a decrease in tCho T1values was observed between P10 and P21.The transverse relaxation time (T2) of water (Table 1)

decreased significantly during brain maturation: T2 va-lues obtained in mice before 2 weeks (P5 and P10) werehigher (> 50ms) than in older animals (P15 and P21)(p< 0.001) and T2 values decreased significantly be-tween P15 and P21 from 41 to 36ms (p< 0.01). Incontrast, T2 values of brain metabolites tend to increasebetween P5 and P21. Although the increase in T2 valuesof tCr (from 104 to 133ms) and Tau (from 82 to 132ms)was not statistically significant, tCho T2 values increasedby 1.7 between P10 and P15 (from 79 to 138ms)(p< 0.001) and NAA T2 values by 2.5 between P5 andP21 (from 110 to 134ms) (p< 0.05). At day P21, tCr,tCho and Tau had similar T2 values (133ms), whichrepresented half the NAA T2 value (279ms).

Determination of absolute metaboliteconcentration in the different brain regionsby in vivo 1H NMR

The percentage of water in the mouse brain at 5 (87.5%),10 (86.5%), 15 (81.7%) and 21 post-natal days (80.4%)was used to calculate the absolute levels of 1H NMR-detectable metabolites in the different brain regions.At P21 (Fig. 3), thalamus showed the highest absolute

concentrations for all metabolites compared with theother structures. tCr, NAA and Glx were highly discri-minant (p< 0.001) with a difference which was alreadysignificant at P15 between thalamus and cerebellum orolfactory bulb. Cerebellum and olfactory bulb showedsimilar metabolite concentrations except for the tChovalues, higher in the cerebellum at the development stageP10 (p< 0.001).During thalamus maturation, the tCr, NAA and Glx

concentrations increased (Table 2) with a significantdifference for values obtained at P21 in comparisonwith those at younger ages (p< 0.001). The tCr increaseoccurred even earlier with a significant differencealready between P15 and P10 (p¼ 0.01). The Tauconcentration exhibited a transitory but significantvariation with a lower value at P10 than P5 (p< 0.01)

Figure 2. Typical 1H MRS spectra (PRESS, TR¼ 1.5 s,TE¼ 130ms, NS¼ 512, VOI¼ 8 mL; apodisation: LB¼ 10).(A) In thalamus between P5 and P21. The apparent decreasein the tCho and Tau resonances is paralleled by the apparentincrease in the tCr, NAA and Glu resonances; (B) comparisonat P21 between thalamus (T), olfactory bulb (OB) andcerebellum (Cb).



Table 1. Longitudinal (T1) and transverse (T2) relaxation times (in ms) as a function of post-natal developmentalstages (P5 to P21) in thalamus (Tha): values with a different superscript letter (a, b or c) on the same line indicate asignificant difference (p< 0.05) between ages

P5 P10 P15 P21

T1 tCr 886� 189 985� 197 1158� 170 932� 103NAA 990� 165 1328� 337 1012� 198 915� 175Tau 1520� 424 1803� 412 1864� 427 1580� 531tCho 1308� 362 1354� 182 1200� 209 1053� 150

T2 H2O 55� 3a 52� 5a 41� 3b 36� 5c

tCr 104� 49 112� 4 122� 33 133� 13NAA 110� 19a 157� 34a 255� 100b 279� 106b

Tau 82� 27 99� 27 109� 9 133� 54tCho 76� 13a 79� 7a 138� 22b 134� 12b

Figure 3. Absolute concentrations of selected metabolites at P21 according to the brain structureanalysed: thalamus (Tha), cerebellum (Cb) and olfactory bulb (OB). Tha shows the highest absoluteconcentrations for all metabolites compared with the other structures. tCr and NAA are highlydiscriminant related to the high-energy metabolism and neuronal cell body content of thisstructure. Error bars denote standard deviations; asterisks indicate a significant difference betweenTha and the other structures: *p< 0.05; **p< 0.01 and ***p< 0.001; §indicates a significantdifference between Tha and OB (p< 0.05).

Table 2. Absolute metabolite concentrations (in mm) as a function of post-natal developmental stages (P5 to P21)in three brain areas: thalamus (Tha), cerebellum (Cb) and olfactory bulb (OB): values with a different superscriptletter (a, b or c) on the same line indicates a significant difference (p< 0.05) between ages

P5 P10 P15 P21

Tha tCr 7.4� 2.0a 7.3� 3.7a 13.4� 1.6b 22.5� 2.8c

Glx 7.6� 2.3a 5.9� 2.9a 10.1� 4.6a 24.8� 4.4b

NAA 2.8� 1.3a 4.6� 2.1a 5.2� 1.7a 9.8� 2.6b

Tau 20.7� 4.3a 8.2� 5.9b 15.0� 1.3a 19.3� 4.7a

tCho 4.7� 1.4a,b 7.1� 2.8a 2.9� 0.5b 5.0� 0.9a,b

Cb tCr 7.5� 1.7 10.7� 2.0 7.6� 2.4 8.3� 0.7Glx 5.4� 2.6 4.4� 1.7 3.7� 1.1 7.7� 2.3NAA 2.2� 0.9 4.3� 0.7 2.7� 1.1 3.1� 1.6Tau 12.1� 5.0 15.2� 2.5 9.3� 3.3 8.7� 2.7tCho 5.4� 1.0a 12.8� 2.5b 2.0� 0.7c 2.2� 0.8a,c

OB tCr — 6.4� 2.6 8.9� 2.9 4.2� 0.9Glx — 3.1� 0.2a 7.6� 2.5a,b 11.2� 5.4b

NAA — 2.9� 0.7 3.8� 2.2 3.0� 1.0Tau — 11.1� 5.6 15.2� 1.9 9.7� 3.8tCho — 6.3� 2.0a 2.8� 0.7b 1.5� 0.4b



and P15 (p< 0.05). tCho was characterized by avariable modulation of its concentration with amaximum content measured at P10 (p< 0.01 vs P15).The tCho content at P10 was even significantly higherthan those at P5 and P15 (p< 0.001) in the cerebellum(Fig. 4). The olfactory bulb also showed a significantvariation in tCho content between P10 and the latterdevelopmental stages. Furthermore, the Glx concentrationincreased significantly between P10 and P21 in this laststructure.

DISCUSSION

The aim of the present study was to develop reliablein vivoMRS techniques for non-invasive investigations ofbrain development in normal and transgenic mice.The specific systems for young mouse anaesthesia,

immobilisation and respiratory monitoring developedfor in vivo MRS techniques allows the analysis of brain1H NMR spectra as early as 5 days post-natal. Thecomplete recovery of mice obtained after the procedure,with a good survival rate observed even at P5, allowslongitudinal MRS analysis. Mouse models of humandisease protocols using longitudinal follow-up are parti-cularly useful (a) to reduce the number of transgenicanimals needed, (b) to evaluate the individual variations

observed during the evolution and (c) to reproduce theclinical uses of MRS in human pathology.The technique that we used allowed us to obtain

reproducible in vivo 1H NMR spectra to evaluate meta-bolite variations in different brain regions and duringbrain maturation as early as the fifth post-natal day. Toour knowledge, the results presented here constitute thefirst report of brain metabolite analysis in vivo duringmouse brain maturation.Relaxation times measured in the thalamus of our older

mice group (P21) were in the same range as thosecalculated in the rat (7) and mouse (8) brains. Thelongitudinal relaxation time does not seem to vary duringearly post-natal brain development; nevertheless, a non-significant increase in T1 near P10 and P15 for tCho andTau clearly indicates that the brain maturation processescan influence this parameter. The significant increases inthe transverse relaxation time of tCho and NAA betweenP10 and P15 can reflect the active myelinogenesis whichoccurs parallel to axonogenesis and synaptogenesis atthis stage of development. To determine absolute meta-bolite concentrations, correction by T2 values has to beperformed. Variations of water content and water T2 inbrain must be taken in account.Concentrations of numerous metabolites vary during

brain maturation. Changes in Cho, Tau, NAA and gluta-mate content during brain maturation have frequentlybeen reported in humans and, more recently, in rats. Theabsolute concentrations of these brain metabolites re-ported in the present study are in the same range as thosefound in previous studies, in vivo in rats (9) and humans(3) and ex vivo in mice (11).The peak at 3.02 ppm, assigned to tCr, reflected the sum

of the native and phosphorylated forms of creatine. Phos-phocreatine is a donor of phosphate to adenosine dipho-sphate (ADP) for the production of adenosine triphosphate(ATP), which plays a major role in the energy metabolism.The tCr concentration increases substantially during thethird week of post-natal brain maturation to reach at P21its highest value in thalamus, a grey matter structure rich inneuronal cell bodies coupled with high-energy metabo-lism. The significant increase in tCr content found in thethalamus between P10 and P21 concurs with data reportedin rats (8).Identically, the NAA concentration increases signifi-

cantly from P5 to 21 only in thalamus. These results arein accordance with the fact that NAA is an amino acidessentially produced by neurons during post-natal life,which increases during normal brain maturation (3,9).The presence of a normal high level in mature brains is anindication of good neuronal vitality.Acetylcholine (Ach) is an essential neurotransmitter

in the CNS. At P21, the tCho concentration is moreimportant in the thalamus than in the cerebellumand olfactory bulb, in accordance with the higher densityof Ach neuronal contacts in basal ganglia. Moreover,choline plays an important role in the synthesis of

Figure 4. Absolute concentrations of total choline in cere-bellum as a function of age. The highest peak is observed atP10 when the phosphatidylcholine is highly produced to beincorporated, during the active phase of myelination, in themyelin phospholipids. Error bars denote standard deviations;***indicates a significant difference between P10 and allthe others ages (p< 0.01); §indicates a significant differencebetween P5 and P15 (p< 0.05).



phosphatidylcholine, one of the major components of cellmembrane phospholipids in white matter. Therefore, thismetabolite represents an indirect indicator of myelinsheath synthesis, which mainly occurs during the secondpost-natal week in mice. The total choline concentrationsat P21 were four- and sixfold lower than at P10 inthe olfactory bulb and the cerebellum, respectively.These results agree with previous results obtained inthe rat brain (8) and also during human white matterdevelopment (3) and are related to the progressive inte-gration of choline within the extensive cell membranesproduced during myelination.In our study, the developmental Tau concentration

relies on the brain region. Between P5 and P21, it tendsto decrease in the cerebellum and olfactory bulb andshowed a significant but transitory diminution at P10 inthe thalamus. Furthermore, the Tau content in thalamus istwofold higher than in other structures. When the Tauconcentration was measured ex vivo by high-performanceliquid chromatography (HPLC) during brain maturationin these three regions (12–14), its values decreasedbetween birth and the young adult stage. Similarly toour in vivo MRS study, HPLC analysis indicated that thetaurine content in the thalamus was twofold lower at D9and D11 compared with those at D7 and P15 and at D19,twofold higher compared with the cerebellum and olfac-tory bulb. Comparison of ex vivo HPLC levels with thoseobserved in vitro, in histotypic cerebellar cultures, de-monstrated a content which was fivefold lower in vitrothan ex vivo (13), suggesting that the high proportion ofendogenous taurine observed in cerebellum could comefrom an extrinsic origin. In the rat brain, specific anti-bodies of high-affinity Tau transporters showed a pre-dominant localisation in the cerebellum, in the neuronalPurkinje cell and in Bergmann glial cells (15). Therefore,during development, the Tau distribution results fromboth active transport and endogenous production. Irregu-lar variations of the concentration of this metabolitecould also be due to its inhibitory neurotransmitterfunction. Taurine regulates calcium fluxes in nerve brainterminals and it may play a role in dendritic and synapticprocesses (16).A long-TE PRESS sequence was used to suppress

macromolecule contributions in metabolite resonancesignals. Therefore, the separation of glutamate and glu-tamine resonances, which requires the application of ashort-TE MRS sequence, could not be achieved in ourstudy. In a normal rat brain, the variation of the Glxduring development is mainly due to the increase in theglutamate part (8). Glutamate is an amino acid involvedin protein synthesis, and it also represents the majorexcitatory neurotransmitter of the central nervous system.During mouse brain development, we found a relativeincrease in glutamate/glutamine concentration in thethree regions analysed, with significant variations in theolfactory bulb and especially in the thalamus. In this laststructure at P21, the Glx content was three- and twofold

higher than those at P5 and P15, respectively. In vivo 1HMRS analysis of rat brain showed a 2.5-fold increase inglutamine content between days 2 and 28 with a max-imum at day 21 in hippocampus, striatum and cortex (8),whereas no difference was observed in human brainanalysed between groups 0–1 to 18–39 years of age (3).The increase in Glx detected in mouse thalamus andolfactory bulb and rat hippocampus, striatum and cortexcould correspond to a progressive increase in neuronalactivity.

CONCLUSION

The reported protocol, based on controlled anaesthesia,immobilisation and respiratory monitoring of very youngmice, demonstrated that very small voxels (2–8 mL) couldbe successfully used to follow and measure, by in vivo 1HNMR spectroscopy, maturation changes of brain meta-bolites. Possibilities of longitudinal MRS analysis inmaturing mice brains provide a non-invasive and non-destructive tool to characterise better the physiologicaland pathological processes of brain development and tofollow the impact of environmental and therapeuticfactors.

Acknowledgements

This work was supported by grants from the EuropeanLeucodystrophy Association (ELA) and the Fondationpour la Recherche Medicale (Foundation for MedicalResearch) (ARS 2000). Pierre Larvaron is a fellowmember of ELA.

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proton mrs of early post-natal mouse brain modifications in vivo

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