ontogeny of non-nmda glutamate receptors in rat barrel field cortex: i. metabotropic receptors

Ontogeny of Non-NMDA GlutamateReceptors in Rat Barrel Field Cortex:

I. Metabotropic Receptors

MARY E. BLUE,1,2* LEE J. MARTIN,3,4 ELIZABETH M. BRENNAN,1

AND MICHAEL V. JOHNSTON1,2,5

1The Kennedy Krieger Research Institute, The Johns Hopkins University Schoolof Medicine, Baltimore, Maryland 21205

2Department of Neurology, The Johns Hopkins University School of Medicine,Baltimore, Maryland 21205

3Department of Neuroscience, The Johns Hopkins University School of Medicine,Baltimore, Maryland 21205

4Department of Pathology, The Johns Hopkins University School of Medicine,Baltimore, Maryland 21205

5Department of Pediatrics, The Johns Hopkins University School of Medicine,Baltimore, Maryland 21205

ABSTRACTThe ontogeny of metabotropic excitatory amino acid receptors (mGluRs) in rat barrel field

cortex was characterized by using receptor autoradiography and immunocytochemistry to testthe hypothesis that changes in mGluR expression coincide with the emergence of somatotopicpatterns in this region. On postnatal days 1 (P1) and 3, [3H]glutamate binding to mGluRs wasnot distributed in a somatotopic pattern. By P5, mGluRs exhibited a whisker-related pattern,with higher densities of mGluRs in barrel centers than in surrounding cortex. Between P5 andP14 and at P60, the overall binding density remained higher in barrels than in surroundingcortex. At P60, a somatotopic pattern of binding was not apparent. The majority of mGluRsites in the barrel field were blocked by the metabotropic agonist trans-1-aminocyclopentane-1,3-dicarboxylic acid but were not significantly displaced by quisqualate.

Immunocytochemical studies of phosphoinositide-linkedmGluRs,mGluR5 andmGluR1a,showed that the developmental expression of mGluR5 mirrored that of the pattern ofautoradiographically labeled mGluRs. The immature barrel field (ages P5–P14) was enrichedin mGluR5, with greater concentrations of mGluR5 immunoreactivity in barrels than insurrounding cortex. Within barrel centers, mGluR5 was localized within the neuropil, on thesurfaces of cell bodies and dendrites in layer IV. A somatotopic pattern of mGluR5immunoreactivity persisted into adulthood, although the pattern was less pronounced afterP14. In contrast, mGluR1a was never localized in a somatotopic pattern in barrel field cortex.We conclude from the developmental localization of mGluRs that the spatiotemporalregulated expression of these receptors may influence barrel maturation and plasticity. J.Comp. Neurol. 386:16–28, 1997. r 1997 Wiley-Liss, Inc.

Indexing terms: excitatory amino acid; somatosensory cortex; autoradiography: pattern formation;

development

The excitatory amino acid (EAA) neurotransmitters,glutamate and aspartate, exert their effects on neuronsthrough receptors that can be broadly classified into twogroups, ionotropic receptors, which are ion channels, andmetabotropic receptors (mGluRs), which are linked toG-proteins (see Young and Fagg, 1990; Bear and Dudek,1991; Schoepp, 1993; Pin and Duvoisin, 1995; for reviews).Ionotropic receptors include those activated by N-methyl-

Grant sponsor: N.I.H.; Grant number: NS29167 (M.E.B.), Grant number:NS34100 (L.J.M.), Grant number: NS2808 (M.V.J.), Grant number:HD24061 (Mental Retardation Research Center Grant).*Correspondence to: Mary E. Blue, Ph.D., Neuroscience Laboratory,

Kennedy Krieger Research Institute, 707 North Broadway, Baltimore, MD21205. E-mail: [email protected] 22 October 1996; Revised 18 February 1997; Accepted 18

March 1997

THE JOURNAL OF COMPARATIVE NEUROLOGY 386:16–28 (1997)

r 1997 WILEY-LISS, INC.

D-aspartate (NMDA), a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), and kainate (KA; seeMonoghan et al., 1989; Young and Fagg, 1990; Bettler andMulle, 1995; Cabelli et al., 1995; Jorgensen et al., 1995; forreviews). Metabotropic receptors, the focus of the presentstudy, have been classified into three groups based onamino acid identity, pharmacology, and transductionmechanisms. Group I mGluRs, which include mGluR1 andmGluR5, are coupled to phospholipase C and the phospho-inositol (PI) pathway, whereas both Group II (mGluR2 andmGluR3) and Group III (mGluRs 4, 6, 7, 8) are negativelycoupled to adenylate cyclase second messenger systemsbut differ in sequence homology and pharmacology (Pinand Duvoisin, 1995). Metabotropic receptor activation hasbeen shown to play a role in the induction of long-termpotentiation, different from the role played by NMDAreceptors (O’Connor et al., 1994; Bortolotto and Colling-ridge, 1995). In addition to this role, numerous studiessuggest that mGluRs have trophic or regulatory influenceson the developing brain. For metabotropic sites linked toPI signaling, it has been demonstrated that expression ofEAA-stimulated PI hydrolysis is heightened in the brainsof neonates (Dudek and Bear, 1989; Condorelli et al., 1992)and that alterations in the visual system, which modifynormal development, change mGluR expression (Dudekand Bear, 1989). A recent immunocytochemical study alsohas shown enhanced expression of the PI-linked mGluRproteins mGluR1a and mGluR5 during the critical periodin cat visual cortex (Reid et al., 1995).Recently, we showedwhisker-related patterns of [3H]glu-

tamate binding to mGluRs and [3H]AMPA binding toAMPAreceptors in the barrel field of immature rat somato-sensory (SI) cortex (Blue and Johnston, 1995). In contrast,[3H]glutamate binding to NMDA receptors did not exhibita somatotopic pattern. In the present study, we deter-minedwhether the dense, patterned expression of quisqual-ate-insensitive and quisqualate-sensitive metabotropicbinding sites in immature rat SI was either sustained ortransient. In addition, we identified by immunocytochem-istry the ontogeny of Group I mGluRs (i.e., mGluR1a andmGluR5 proteins) to determine whether one or both ofthese proteins linked to PI metabolism showed a pattern ofdevelopment similar to that of the autoradiographicallyvisualized mGluR sites.

MATERIALS AND METHODS

Autoradiographic experiments

Tissue preparation. All of the procedures for animaluse were reviewed and approved by the Animal Care andUse Committee at The Johns Hopkins University School ofMedicine. Sprague-Dawley albino rats at postnatal agesP1 (day of birth), P3, P5, P10, P14, P21, P30, and P60(adult) were used to examine the ontogeny of [3H]gluta-mate binding to mGluRs in barrel field cortex. Animalswere decapitated, and the brains were removed rapidlyand frozen on dry ice. To prepare ‘‘flattened’’ sections forexamination of barrels, cerebral hemispheres were sepa-rated, the brainstem was removed, and the remainingtissue was flattened between two slides and frozen. Brainswere kept at 280°C until the day of sectioning. Sectionswere cut on a Zeiss-Microm cryostat at 20 µm thickness,thaw mounted on gelatin-coated slides and stored at280°C prior to receptor labeling. For each brain, adjacentsections through layer IV were used to examine other

glutamate receptor subtypes (see accompanying paper);every sixth section was stained with cresyl violet.[3H]glutamate binding to mGluRs. [3H]glutamate

binding tomGluRs was assayed by standardmethods (Chaet al., 1990). Sections were prerinsed for 30 minutes in 50mM Tris HCl, 2.5 mM CaCl2 (pH, 7.4, 4°C) to wash offendogenous ligands and then were dried under cool air.Sections were then incubated in the same buffer to which82 nM [3H]glutamate, 10 µMAMPA, 100 µMNMDA, and 1µMKAwere added.After incubation, sections were rapidlyrinsed four times with cold buffer, followed by two fixationrinses of 2.5% glutaraldehyde in acetone and rapid dryingunder warm air. After drying, the slides were apposed toUltrofilm 3H (Leica) and stored in X-ray cassettes at 4°C.The films were developed photographically 1 or 2 weekslater, using D-19 and Rapid-fix (Kodak). Nonspecific bind-ing was determined in the presence of 2.5 µM quisqualate(QUIS) alone or 2.5 µM QUIS and 100 µM trans-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD), ahighly specific metabotropic agonist.Densitometric analysis. The autoradiographic films

were analyzed by densitometric analysis using a video-based image-analysis system (Imaging Research Inc., St.Catherines, Ontario, Canada). A set of 14C standards thathad been calibrated to brain paste samples with knownamounts of tritium was coexposed with the tissue sectionsfor quantitation of binding density. 14C standards wereused because they did not decay appreciably over time.The image analysis system reads a relative optical density(ROD) value for each radioactive standard and then fitsthe ROD values to an interpolation function. In thesestudies, the best fit was provided by a cubic spline functionwith variable smoothing. Through this function, opticaldensities of the autoradiographically labeled sections werecompared to densities produced by the radioactive stan-dards and calculated as picomoles per milligram protein.The Nissl-stained section was helpful for delineating bar-rels or the presumptive barrel area in those cases in whicha somatotopic pattern of expression of mGluRs was notdistinct (i.e., at P1 and P3). In the flattened sections, twozones were analyzed, barrel centers (centers plus walls,but not septa) and surrounding cortex, the region surround-ing barrels and zones containing other parts of the bodymap in somatosensory cortex (forelimb, hindlimb, mouth,etc.). For each section, 10–20 random readings were madeover barrel centers or in the surrounding cortex. Owing tothe facts that several receptor subtypes were examined foreach brain, that the extent of layer IV in barrel field cortexwas limited (200–300 µm) and that section to sectionvariability was small, only one section per animal wasanalyzed. Total specific binding was determined by sub-tracting the value for nonspecific binding from that foraverage total binding. The numbers of animals analyzedper age are listed in Tables 1–3. Values are presented asmeans 6 standard errors (s.e.m.). The statistical packageStatview was used to determine means and s.e.m and toperform analyses of variance (ANOVA) with a Fisher’sPLSD post hoc test.

Immunocytochemical studies

Tissue preparation. Rats at postnatal ages P5, P10,P14, P21, P30, and P60 (adults) were perfusion fixed andtheir brains prepared for immunocytochemical localiza-tion of mGluR1a andmGluR5 proteins. Rats were anesthe-tized either with ether (P5) or with sodium pentobarbital

ONTOGENY OF METABOTROPIC RECEPTORS IN BARRELS 17

Fig. 1. Pattern of [3H]glutamate binding to metabotropic excita-tory amino acid receptors (mGluRs) in flattened sections of rat barrelfield cortex at postnatal days P5, P10, P14, P21, and P60 (adult). Asomatotopic pattern of mGluR sites is present in the first 2 postnatalweeks, with higher densities of mGluR sites in barrel centers than insurrounding cortex. mGluR receptor density declines in the thirdpostnatal week. In the adult (P60), the whisker-related pattern is nolonger evident. Scale bar 5 2 mm.

18 M.E. BLUE ET AL.

(all other ages) and perfused transcardiallywith phosphate-buffered saline (PBS) followed by 4% paraformaldehyde in0.15 M phosphate buffer (pH 7.4); volume and speedsvaried depending on age. Brains were removed and post-fixed in the same paraformaldehyde solution for 4–8 hours,

depending on age (longer times for younger ages). Afterpostfixation, brains were cryoprotected in 30% sucrose inPBS and blocked to be cut in the coronal plane or forflattened sections (as described above). Blocks were storedat 280°C until the immunocytochemical labeling wasperformed.Immunocytochemistry. Sections (30–50 µm thick)

were cut frozen on a Zeiss-Microm sliding microtome andprocessed for mGluR1a and mGluR5 immunohistochemis-try as described previously (Martin et al., 1992), with theexception that the avidin biotin peroxidase (ABC) methodwas used for visualization of the antigen-antibody complexand that the resulting diaminobenzidine (DAB) reactionproduct was amplified by osmication (described in Blue etal., 1988). Primary antisera for mGluR1a and mGluR5were affinity-purified rabbit polyclonal antipeptide antibod-ies raised against the c-terminal of mGluR5 and mGluR1a(Martin et al., 1992, 1995). Controls included sections thatwere incubated in primary antiserum (0.5 µg/ml IgG) thathad been preabsorbed for 24 hours with an excess ofsynthetic peptide (10 µg/ml) or in similar concentrations ofrabbit IgG and cases in which the primary antiserum wasomitted.

Materials

L-[3H]glutamate was obtained from Amersham (specificactivity 45–56 Ci/mmole). Unlabeled QUIS, NMDA, KA,AMPA, and trans-ACPD were obtained from Tocris/Neuramin (Bristol, England) or Research BiochemicalsInternational (Natick, MA). ABC kits were obtained fromVector Laboratories, Inc. (Burlingame, CA), and DAB waspurchased from Sigma Chemical Co. (St. Louis, MO).

TABLE 1. [3H]glutamate Binding to Metabotropic Receptors(total binding)1

AgeBarrel center(pmol/mg)

Surround(pmol/mg) n P value

P1 1.047 6 0.096 0.955 6 0.133 3 0.718P3 1.025 6 0.013 0.744 6 0.031 3 0.270P5 1.581 6 0.104 1.056 6 0.042 10 ,0.001P7 1.751 6 0.087 0.994 6 0.054 8 ,0.001P10 2.349 6 0.112 1.621 6 0.109 12 ,0.001P14 2.157 6 0.041 1.701 6 0.032 14 ,0.001P21 1.670 6 0.113 1.498 6 0.088 14 0.145P30 1.571 6 0.112 1.332 6 0.096 9 0.106P60 1.966 6 0.143 1.510 6 0.113 11 0.001

1The density of [3H]glutamate binding to metabotropic receptors (mGluRs; pmol/mgprotein) in flattened sections of barrel field cortex based on the number (n) of animalsper observation. Total binding refers to the density of mGluRs observed when sectionswere incubated with 82 nM [3H]glutamate and 10 µM a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), 100 µM N-methyl-D-aspartate (NMDA), and 1 µMkainate (KA). The density of mGluR sites varied significantly between barrel centersand surrounding cortex between postnatal day 5 (P5) and P14 and at P60.

Fig. 2. Graph of the density of [3H]glutamate binding to mGluRs inrat barrel field cortex at postnatal days P5, P10, P14, P21, and P60(adult; means 6 s.e.m.; note: in some cases, the s.e.m. is not visiblebecause it is smaller than the data symbols). a: Barrel Center orSurround: Total binding refers to the total density of mGluRs. BarrelCenter or Surround-QUIS refers to the density of sites in barrelcenters or surrounding cortex after the quisqualate blank has beensubtracted. b: Barrel Center or Surround-trans-ACPD refers to thedensity of sites in barrel centers or surrounding cortex after thetrans-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) blankhas been subtracted. Asterisks indicate significant differences indensity between barrel centers and surrounding cortex.


RESULTS

Autoradiographic studies

The ontogeny of metabotropic receptors (mGluRs) inbarrel field cortex was examined quantitatively by autora-diographic binding of [3H]glutamate in the presence ofNMDA, AMPA, and KA displacers (total binding). QUIS-sensitive mGluR sites were identified by inclusion of 2.5µM QUIS; trans-ACPD-sensitive mGluR sites, which hada low affinity for QUIS, were labeled with [3H]glutamate inthe presence of NMDA, KA, QUIS, and trans-ACPD, aselective mGluR agonist. The quantitation of mGluR bind-ing was important to complement qualitative immunocyto-chemical results.

Total binding to mGluRs

A somatotopic pattern of expression of [3H]glutamatebinding to mGluRs was present at P5, when a higherdensity of mGluRs was observed in barrel centers than in

septa and surrounding tissue, thus forming a sensory mapof the rat whisker pad (Fig. 1). At earlier time points, onthe day of birth (P1) and at P3, the density of mGluRs inbarrel centers did not differ significantly from that inbarrel septa or surrounding cortex (Fig 2a, Table 1).Qualitatively, the highest densities of mGluR sites inbarrel centers were observed at P5 and P10. Quantita-tively, the density of mGluR sites was highest at P10 (Figs.1, 2a, Table 1). Significant differences in the density ofmGluRs between barrel centers and surrounding cortexwere observed at the time points examined between P5and P14 (Table 1). By the end of the third postnatal week,relative density differences between barrel centers andsurrounding cortex decreased so that the somatotopic mapwas less evident (Fig. 1). Between 1month and 2months ofage, the density of mGluRs increased significantly inbarrel centers, but not in surrounding cortex, so that, atP60, the overall binding density was higher in the zoneencompassing the barrels than in surrounding cortex(Figs. 1, 2a).

Fig. 3. Pattern of [3H]glutamate binding to mGluRs in rat barrel field cortex at P10 (flattenedsections). Left shows total binding, middle shows the pattern with 2.5 µM quisqualate added (1QUIS),and right shows the pattern with added trans-ACPD (1trans-ACPD). The whisker-related pattern ofmGluR sites is QUIS insensitive but blockable by trans-ACPD. Scale bar 5 2 mm.

TABLE 2. [3H]glutamate Binding to Metabotropic Receptors:QUIS-sensitive Sites (total binding 2 QUIS blank)1



P1 0.269 6 0.081 0.261 6 0.085 3 0.974P3 0.199 6 0.124 0.110 6 0.059 3 0.729P5 0.300 6 0.083 0.294 6 0.074 10 0.965P7 0.377 6 0.128 0.199 6 0.048 8 0.260P10 0.514 6 0.189 0.445 6 0.118 12 0.589P14 0.248 6 0.061 0.256 6 0.044 14 0.950P21 0.089 6 0.041 0.155 6 0.040 14 0.582P30 0.128 6 0.064 0.092 6 0.051 9 0.811P60 0.584 6 0.156 0.428 6 0.101 11 0.246

1The density of quisqualate (QUIS)-sensitive [3H]glutamate binding tomGluRs (pmol/mgprotein) in flattened sections of barrel field cortex based on the number (n) of animalsper observation. QUIS-sensitive binding refers to [3H]glutamate binding to mGluRsthat is displaceable by 2.5 µMQUIS.At all ages examined, the density of these sites doesnot vary significantly between barrel centers and surrounding cortex.

TABLE 3. [3H]glutamate Binding to Metabotropic Receptors:trans-ACPD-sensitive Sites (total binding 2 trans-ACPD blank)1



P5 1.615 6 0.134 1.003 6 0.070 5 0.003P10 1.922 6 0.160 1.293 6 0.185 6 ,0.001P14 1.643 6 0.071 1.296 6 0.059 6 0.056P21 1.388 6 0.098 1.184 6 0.086 6 0.258P30 1.402 6 0.154 1.021 6 0.165 3 0.136P60 1.351 6 0.125 0.921 6 0.087 11 ,0.002

1The density of trans-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD)-sensi-tive mGluRs (pmol/mg protein) in flattened sections of barrel field cortex based on thenumber (n) of animals per observation. trans-ACPD-sensitive binding refers to [3H]glu-tamate binding to mGluRs that is not displaceable by 2.5 µM QUIS but that is blockedby 100 µM trans-ACPD, a highly specific metabotropic agonist. The density oftrans-ACPD-sensitive mGluR sites varied significantly between barrel centers andsurrounding cortex between postnatal day 5 (P5) and P14 and at P60.

20 M.E. BLUE ET AL.

Age comparisons showed that total binding to mGluRsin barrel centers and in surrounding cortex varied signifi-cantly over time. For barrel centers, significant differencesin total binding to mGluRs was observed between P3 andP5 (P 5 0.013), P7 and P10 (P 5 0.001), P10 and P21 (P ,0.001), P14 and P21 (P , 0.001), and P30 and P60 (P ,0.02). For surrounding cortex, significant differences intotal mGluR binding site density only were observedbetween P7 and P10 (P , 0.001), P10 and P30 (P , 0.02),and P14 and P30 (P , 0.003).

QUIS-sensitive and trans-ACPD-sensitivebinding to mGluRs

The majority of mGluRs in barrel field cortex had a lowaffinity for QUIS but were blockable by the mGluR agonist

trans-ACPD (Figs. 2, 3). At all ages examined, the densityof QUIS-sensitive mGluRs in barrel centers did not differsignificantly from that in surrounding cortex (Figs. 2a, 3,Table 2). In terms of age-related changes, the QUIS-sensitive sites in barrel centers and surrounding cortexshowed a significant decline in density between P10 andP21 (P , 0.005) and a significant increase between P30and P60 (P , 0.01).In contrast to the case with QUIS-sensitive sites, the

density of trans-ACPD-sensitive sites (Figs. 2b, 3 andTable 3) was higher in barrel centers than in surroundingcortex at P5 (P , 0.002), at P10 (P , 0.001), and at P14(P 5 0.056). The density of trans-ACPD-sensitive sitesdecreased significantly in barrel centers between P10 andP21 (P , 0.01) but not between P21 and P60. In the

Fig. 4. Pattern of mGluR5 protein in flattened sections of rat barrelfield cortex at postnatal days P10, P14, P21, and P60 (adult). mGluR5immunoreactivity (IR) is more enriched in barrel centers than inbarrel septa and surrounding cortex. The somatotopic pattern ofmGluR5 IR persists throughout development, although, at later ages

(P21 and P60), the intensity of mGluR5 IR in barrel centers dependson whether the section is cut through the upper (lower density) ordeeper (higher density) part of layer IV (cf. Figs. 4 and 6). Scale bar 5200 µm.


surrounding cortex, the density of trans-ACPD-sensitivesites was not decreased significantly between P10 and P30but did differ significantly between P10 and P60 (P ,0.02). In the adult, the density of trans-ACPD-sensitivesites in barrel centers was significantly higher in barrelcenters than in surrounding cortex (P , 0.002).

Immunocytochemical studies

An immunocytochemical examination of mGluR1a andmGluR5 proteins in the barrel field revealed that thedevelopmental expression of mGluR5 was similar to thatof the autoradiographically localized mGluR sites. As withthe overall pattern of [3H]glutamate binding to mGluRs,mGluR5 showed a heightened expression in the immaturesomatosensory cortex, with greater enrichment of mGluR5immunoreactivity (IR) in layer IV and specifically in barrelcenters than in septa and surrounding cortex (Figs. 4, 5).Similarly, the somatotopic expression of mGluR5 emergedbetween P3 and P5. The somatotopic pattern of mGluR5staining was most distinct at P10; mGluR5 was enrichedin the neuropil of barrel centers, with IR present at thesurfaces of cell bodies and dendrites in layer IV of thebarrel field (Fig. 5). Unlike the case with autoradiographi-cally visualized receptors, the somatotopic pattern ofmGluR5 IR was maintained throughout development,although the density of staining in layer IV decreased afterthe second postnatal week (Figs. 4, 6). However, thedensity of mGluR5 staining in barrel centers varied,depending on whether the barrels were cut through theupper part of layer IV (lower density of mGluR5 sites; cf.Fig. 4, P21, and Fig. 6, P21 and P60) or lower part of layerIV (higher density of mGluR5 sites; cf. Fig. 4, P60, and Fig.6, P60).In coronal sections (Fig. 6), beginning at P5, the inten-

sity of mGluR5 IR was greater in layer IV than in otherlayers. At P10, barrel rows were stained densely withmGluR5 antibodies. At P14, the intensity of mGluR5 IRwithin layer IV barrel rows was diminished relative toearlier ages, whereas mGluR5 IR wasmore dense in layersI–III and layer V. This staining pattern persisted intoadulthood.At P60, mGluR5 IR in the upper part of layer IVwas low compared to that in layers I–III and the lowerparts of layers IV and V.In contrast to the enrichment of mGluR5 in layer IV and

the well-defined, whisker-related pattern of mGluR5 IR inthe barrel field at P10, mGluR1a showed a weak, somato-topic distribution at P5 and P10 (Figs. 7, 8). The diffuse,nonparticulate barrel pattern identified with mGluR1aantibodies in layer IV of somatosensory cortex at P5 andP10 could not be completely competed away with antigen(Fig. 9). In contrast, sections incubated with mGluR5antibody that were preabsorbed with antigen did not showa somatotopic pattern of expression (Fig. 9). However, byP14, discrete, particulate mGluR1a IR increased in den-sity, and patterns of dendritic staining emerged (Figs. 8,10). The dendritic mGluR1a IR was distributed through-out the cortex at P14. A few nonpyramidal cells were alsomGluR1a immunoreactive. In the adult (P60), althoughthe dendritic localization of mGluR1a IR was observedthroughout all layers of somatosensory cortex, the densityof mGluR1a sites was highest in layers V and VI.

DISCUSSION

Our results reveal a somatotopic pattern of expression ofmGluRs in barrel field cortex. These findings are consis-

tent with a possible role for these receptors in the forma-tion and maintenance of barrels. The somatotopic pat-terned expression of [3H]glutamate binding to mGluRsemerges at a time that closely parallels that of thecytoarchitectonic formation of barrels (Rice et al., 1985).Although it is uncertain whether the spatiotemporal pat-terns of mGluR expression reflect functional receptors, theemergence of the whisker-related patterns occurs at thetime when NMDAand non-NMDAreceptors become activeat thalamocortical synapses (Agmon and O’Dowd, 1992).The pattern of mGluR5 staining, which offered enhancedresolution over that of autoradiographic labeling, exhib-ited a time course similar to that observed for [3H]gluta-mate binding to mGluRs. The changing pattern of mGluRexpression in barrel field cortex throughout postnataldevelopment reflects the ongoing somatodendritic matura-tion of neurons within barrels. It is likely that the whisker-related patterns of mGluR5 and of [3H]glutamate bindingto mGluRs reflect postsynaptic sites on layer IV cells at the

Fig. 5. mGluR5 is localized in dense whisker-related patches inlayer IV of somatosensory cortex of P10-day-old rat (top). At highermagnification (bottom), mGluR5 is enriched within these patchesdiffusely labeling the neuropil, forming net-like structures around cellbodies and dendrites in layer IV of the barrel field (coronal section;arrows demarcate mGluR5 staining in the same barrel row). Scalebar 5 200 µm for top, 25 µm for bottom.

22 M.E. BLUE ET AL.

Fig. 6. Pattern of mGluR5 protein in coronal sections of rat barrelfield cortex at postnatal days P5, P10, P14, and P60 (adult). Theenrichment of mGluR5 IR is greater in layer IV than in other layers atP5. At P10 and P14, the somatotopic localization of mGluR5 is mostdistinct, with dense mGluR5 IR in layer IV forming whisker-related

patches overlying barrel rows. These patches are not evident in theadult (P60), in coronal sections, but are evident in flattened sections(see Fig. 4). At this age, the density of mGluR5 staining in the upperpart of layer IV is low compared to that in layers I–III and in the lowerparts of layers IV and V. Scale bar 5 200 µm.

time when the neurons become organized into barrels.This idea is consistent with the observations that mGluR5is predominantly postsynaptic (Shigemoto et al., 1993;Romano et al., 1995). Furthermore, the ontogenetic pat-tern of mGluR expression closely matches that of thebarrels themselves. mGluR5 antibodies distinctly labelindividual barrel centers at a time when individual barrelsare most distinct. Likewise, as the barrels graduallychange with time and the distinction between barrelcenters and sides disappears (Rice et al., 1985), the patternof mGluR5 receptors in the barrel field changes such thatrows rather than individual barrels are labeled.mGluR1a exhibited a pattern of ontogeny in rat barrel

field cortex contrasting with that of mGluR5. UnequivocalmGluR1a IR was not observed in barrel field cortex untilP14, followed by an increase in mGluR1a IR over time. Inthe adult, mGluR1a sites were localized to dendriticprocesses in lower cortical layers. Our results are consis-tent with those of other studies showing increasing levelsof mGluR1mRNAand protein during development (Shige-moto et al., 1992; Martin et al., 1993). The postnatalincrease in mGluR1a site density contrasts with a re-

ported decline in mGluR1a labeling in cat visual cortex(Reid et al., 1995). The discrepancy could result fromspecies differences, insofar as the same antibody for themGluR1a protein was used in both studies.Densitometric analysis indicated that the majority of

metabotropic sites in barrel centers had a low affinity forQUIS but were blockable by the metabotropic agonisttrans-ACPD. The trans-ACPD-sensitive sites exhibited asomatotopic pattern of expression, whereas the QUIS-sensitive mGluRs did not. Our immunocytochemical datashowed high densities of mGluR5 proteins in barrel cen-ters. mGluR5 is a member of the Group I mGluRs (Riedel,1996). Interestingly, mGluR1 which is also a Group Imetabotropic receptor, did not exhibit a somatotopic pat-tern of expression. In preliminary immunocytochemicalstudies, we also have observed that antibodies to theGroup II sites (i.e., mGluR2,3 receptors) are selectivelypresent in barrel centers at P10, in a pattern identical tothat we observed for mGluR5 site (data not shown).Previous combined receptor autoradiographic and in situhybridization studies of mGluR subtypes have shown thatboth mGluR3 and mGluR5 subtypes are prominently

Fig. 7. Comparison of the pattern of mGluR5 and mGluR1a IR in adjacent flattened sections of barrelfield cortex at P10 and P60. Although a somatotopic pattern of mGluR5 IR is present at P10, it dissipateswithmaturation. mGluR1a IR shows a weak somatotopic pattern at P10 that is absent at P60. Scale bar5200 µm.

24 M.E. BLUE ET AL.

Fig. 8. Pattern of mGluR1a protein in coronal sections of rat barrelfield cortex at postnatal days P5, P10, P14, and P60 (adult). A diffuse,nonparticulate pattern is observed in layer IV of the barrel field at P5and P10 that is not blockable by normal IgG or by preabsorbed serum(see Fig. 9). By P14, the pattern of cortical staining with mGluR1a

antibodies has changed; cell bodies and dendritic profiles are stained.In the adult (P60), dendritic labeling of mGluR1a sites is distributedthroughout the cortex, with the highest densities in lower corticallayers. A higher magnification view of the P60 section is shown inFigure 10 (asterisk). Scale bar 5 200 µm.


expressed in developing cerebral cortex, mirroring thepattern of the trans-ACPD-sensitive metabotropic bindingsites (Catania et al., 1993, 1994). These sites appear to beidentical to a portion of the developmentally expressedNNKQ sites (not displaceable by QUIS, KA, or NMDA;Greenamyre et al., 1987, 1990). The results of the presentstudy showing a prominence of mGluR5 in the immaturesomatosensory cortex and the ontogenic pattern of themGluR3 and mGluR5 subtypes observed by Catania et al.(1994) suggests a potential role for Group I and II metabo-tropic receptors in cortical pattern formation. In thefuture, this hypothesis can be directly tested with thegeneration of mGluR2, mGluR3, and mGluR5 knockout

mice, which could have anomalous barrel fields if thishypothesis is correct.The distribution of mGluR5 in the barrel field suggests

that these sites are closely associated with thalamocorticalafferents. The onset of a somatotopic pattern of expressionof mGluR5 occurs after thalamocortical segregation inbarrel centers (Erzurumlu and Jhaveri, 1990; Senft andWoolsey, 1991) and at the end of the critical period forstructural and functional plasticity of barrels (Jeanmonodet al., 1981; Fox, 1992; reviewed in Fox, 1995). Thus,

Fig. 9. Peptide and IgG controls for mGluR1a and mGluR5 stain-ing in barrel field cortex of a P10-day-old rat (cf. Fig. 7). The diffusesomatotopic staining with the mGluR1a antibody is not blocked by theaddition of an excess of synthetic peptide (10 µg/ml; top). In contrast,mGluR5 staining is blocked by synthetic peptide (middle). Sectionsincubated in rabbit IgG at concentrations similar to that of the mGluRantibodies do not exhibit a somatotopic pattern of expression (bot-tom). Scale bar 5 200 µm.

Fig. 10. Pattern of mGluR1a protein in coronal sections of ratbarrel field cortex at postnatal days P14 and P60 (adult). The P60section shows the cellular detail of the same section in Figure 8(asterisk). At P14, mGluR1a stains nonpyramidal cell bodies anddendritic profiles throughout the cortical depth. In the adult, thedensity of dendritic labeling of mGluR1a sites is highest in layers Vand VI. Scale bar 5 200 µm.

26 M.E. BLUE ET AL.

mGluR5 receptors could play a role in the signaling theend of the critical period for thalamocortical afferents.Alternatively, the enrichment of mGluR5 in barrels duringearly development could signify the time of maximalthalamocortical synaptogenesis. Unlike the casewithmanyintrinsic and axonal markers of barrels, the somatotopicexpression of mGluR5 receptors persisted into adulthood.Thus, mGluRs, which continue to show a whisker-relatedpattern in adulthood, could maintain their associationwith thalamocortical axons throughout life. Furthermore,the continued prominence of mGluRs in rat barrel fieldcortex suggests that these receptors also could play a rolein adult models of plasticity, where the physiologicalproperties of barrel neurons are altered after peripheralmodifications (Welker et al., 1989, 1992; Levin and Dunn,1991; Diamond et al., 1993; Land et al., 1995; Melzer andSmith, 1995; Dolan and Cahusac, 1996).The timing of the first expression of a somatotopic

pattern by mGluR5 appears to correspond to the middle ofthe critical period for long-term potentiation (LTP) ofthalamocortical afferents in barrels (Crair and Malenka,1995), providing further support for an influence bymGluR5 on the physiology of barrel neurons. Similarly, theresults of an immunocytochemical study of the ontogeny ofmGluR5 sites in cat visual cortex (Reid et al., 1995) showedthat mGluR5 immunoreactivity was densely expressed inlayer IV during the critical period. mGluRs have beendemonstrated to play critical roles in the induction of LTPin slices of the CA1 area of hippocampus (Bortolotto et al.,1994) and in the induction of LTP (Riedel and Reymann,1993) in the dentate gyrus, or its enhancement (O’Connoret al., 1994). Given the roles of mGluRs in LTP andlong-term depression (LTD) plasticity paradigms, mGluRscould critically influence the mechanisms of synaptic plas-ticity. Moreover, the period of peak expression of mGluRsin barrel centers in the second postnatal week matches thesensitive period for LTP induction in cortical slices ofsomatosensory cortex (Vilagi et al., 1992).Whereas the ontogenic pattern of mGluR1a expression

and the relative abundance in barrel field cortex suggestthat these mGluRsmay not influence the early maturationof barrel cortex, the increasing prominence of mGluR1a IRin barrel field cortex over time supports a possible role formGluR1a in the maintenance, stabilization, and plasticityof barrel field cortex in adult animals. Studies examiningthe role of mGluR1 in synaptic plasticity have shown thatmGluR1 antibodies block LTD in cultured Purkinje cells(Shigemoto et al., 1994) and that mice deficient in mGluR1exhibit motor in-coordination and spatial learning deficits(Conquet et al., 1994). Electrophysiological abnormalitiesin the mGluR1 knockout mice include impaired LTD in thecerebellum and defective LTP in hippocampal mossy fi-bers.The ontogenetic pattern of Group I mGluRs, mGluR1a,

and mGluR5 IR in the barrel field suggests an associationbetween these receptors and PImetabolism in the develop-ment- and experience-dependent plasticity of rat barrelfield cortex. Previous studies have demonstrated increasedlevels of PI metabolism during early postnatal develop-ment (Dudek et al., 1989; Palmer et al., 1990; Schoepp andHillman, 1990; Sortino et al., 1991; Condorelli et al., 1992).For kitten visual cortex, it has been shown that thedevelopmental increase in PI turnover can be prevented byrearing the kittens in complete darkness (Dudek and Bear,1989). A recent study found a transient expression of

mGluR-stimulated PI turnover in rat somatosensory cor-tex during the first 2 postnatal weeks (Bevilacqua et al.,1995). In this previous study, a peak in the PI turnoverresponse was observed at P10, which corresponded to thepeak in mGluR expression seen in our experiments. Inaddition to increased PI metabolism during development,PI turnover is enhanced in response to NMDA-mediatedinjury (Chen et al., 1992) or hypoxia-ischemia (Chen et al.,1988) in immature rats. Taken together, these studiessuggest that PI-derived second messenger signaling maybe involved in regulating not only development and synap-tic plasticity but also the response to neonatal injury.

ACKNOWLEDGMENTS

We thank Ms. Susan B. Plano for providing technicalassistance and Ms. Karen Smith-Connor, Ms. Mary S.Lange, and Ms. Sarah A. Wallace for assisting with thephotography.

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ontogeny of non-nmda glutamate receptors in rat barrel field cortex: i. metabotropic receptors

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