Postnatal alterations in dopaminergic markers in the human prefrontal cortex

Neuroscience 144 (2007) 1109 –1119

POSTNATAL ALTERATIONS IN DOPAMINERGIC MARKERS IN THE HUMAN PREFRONTAL CORTEX C. S. WEICKERT,a M. J. WEBSTER,b P. GONDIPALLI,a D. ROTHMOND,a R. J. FATULA,a M. M. HERMAN,a J. E. KLEINMANa AND M. AKILa*

1991b; Lidow and Rakic, 1992; Perry et al., 1993; Tarazi et al., 1999). Of these neurotransmitter systems, dopamine (DA) is of particular interest in relation to the development of cognitive abilities subserved by the prefrontal cortex. The primate prefrontal cortex (PFC) receives a dense DA innervation (Levitt et al., 1984; Gaspar et al., 1989; Berger et al., 1991) and DA has been shown to be necessary for normal performance on working memory tasks in both human and non-human primates (Williams and GoldmanRakic, 1995; Dreher et al., 2002; Mattay et al., 2002; Gao and Goldman-Rakic, 2003). Alterations in DA markers in the PFC have been characterized during postnatal development in non-human primates. For example, refinements in the DA innervation of prefrontal pyramidal neurons, lamina-specific alterations in density of DA afferents as well as a dramatic increase in cortical DA content have been reported to occur during postnatal development in monkeys (Goldman-Rakic and Brown, 1982; Lewis and Harris, 1991; Rosenberg and Lewis, 1994). However, we know very little about changes in DA markers in the human PFC during postnatal development. In this study, we sought to address this question through the examination of presynaptic and postsynaptic markers of the DA system in the postmortem human PFC from infancy to old age. Tyrosine hydroxylase (TH) is the rate limiting enzyme in DA biosynthesis and TH immunoreactivity has been used as a marker of the density of DA afferents to the cerebral cortex in both human and non-human primates (Lewis et al., 1988; Gaspar et al., 1989; Williams and Goldman-Rakic, 1993; Zecevic and Verney, 1995; Akil et al., 1999). Alterations in the density of TH-immunoreactive axons during the postnatal development of monkey PFC have been reported (Rosenberg and Lewis, 1994; Lewis et al., 1998). However, changes in TH levels in the human dorso-lateral prefrontal cortex (DLPFC) as a function of postnatal development have not been described. The most obvious postsynaptic markers of the DA system are its receptors. There are five known G proteincoupled receptors (dopamine receptors (DAR) 1 through 5) expressed in the human PFC (for review see Missale et al., 1998). Although developmental changes in all DARs may occur, mRNA levels of three of these receptors DAR1, DAR2 and DAR4, have been shown to be the most abundant in the adult human PFC (Meador-Woodruff et al., 1996) and therefore lend themselves to quantitative comparisons across age groups. Moreover, these three receptors are of particular interest in psychiatric illness because of their known role in mediating executive function (Floresco et al., 2006) and their interaction with antipsychotic medications (Seeman, 1992; Hall et al., 1994; Beischlag et

a

Clinical Brain Disorders Branch, Intramural Research Program, National Institute of Mental Health, National Institutes of Health, 9000 Rockville Boulevard, Building 10, CRC6-5340, Bethesda, MD 20892, USA

b

Stanley Foundation Laboratory of Brain Research, Department of Psychiatry, Uniform Services University of Health Sciences, Bethesda, MD 20892, USA

Abstract—Dopamine in the prefrontal cortex plays a critical role in normal cognition throughout the lifespan and has been implicated in the pathophysiology of neuropsychiatric disorders such as schizophrenia and attention deficit disorder. Little is known, however, about the postnatal development of the dopaminergic system in the human prefrontal cortex. In this study, we examined pre- and post-synaptic markers of the dopaminergic system in postmortem tissue specimens from 37 individuals ranging in age from 2 months to 86 years. We measured the levels of tyrosine hydroxylase, the rate limiting enzyme in dopamine biosynthesis, using Western immunoblotting. We also examined the gene expression of the three most abundant dopamine receptors (DARs) in the human prefrontal cortex: DAR1, DAR2 and DAR4, by in situ hybridization. We found that tyrosine hydroxylase concentrations and DAR2 mRNA levels were highest in the cortex of neonates. In contrast, the gene expression of DAR1 was highest in adolescents and young adults. No significant changes across age groups were detected in mRNA levels of DAR4. Both DAR1 and DAR2 mRNA were significantly lower in the aged cortex. Taken together, our data suggest dynamic changes in markers of the dopamine system in the human frontal cortex during postnatal development at both pre-and post-synaptic sites. The peak in DAR1 mRNA levels around adolescence/early adulthood may be of particular relevance to neuropsychiatric disorders such as schizophrenia in which symptoms manifest during the same developmental period. © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: development, dopamine receptors, tyrosine hydroxylase, cerebral cortex, adolescence, aging.

During postnatal development, sweeping changes in neurotransmitter systems including glutamate, GABA, serotonin, dopamine and acetylcholine occur in the cerebral cortex of primates (Brooksbank et al., 1981, 1982; GoldmanRakic and Brown, 1982; Rakic et al., 1986; Lidow et al., *Corresponding author. Tel: ⫹1-301-451-1450; fax: ⫹1-301-402-2588. E-mail address: [email protected] (M. Akil). Abbreviations: DA, dopamine; DAR, dopamine receptor; DLPFC, dorso-lateral prefrontal cortex; PFC, prefrontal cortex; PMI, postmortem interval; TBS-T, Tris-buffered saline 1⫻, 0.05% Tween; TH, tyrosine hydroxylase.

0306-4522/07$30.00⫹0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2006.10.009

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al., 1995; Meador-Woodruff, 1995). Although patterns of gene expression of DARs have been described in the monkey (Lidow et al., 1998) and in the aged human PFC (Meador-Woodruff et al., 1996), they have not been examined in the human PFC throughout the lifespan. In this study, we used Western blotting and an antibody directed against TH and in situ hybridization with riboprobes specific for the DAR1, DAR2 and DAR4 transcripts in postmortem PFC specimens from six age groups: neonates, infants, adolescents, young adults, adults and aged. Characterizing changes in the levels of these pre- and post-synaptic markers of the DA system as a function of postnatal development in the human PFC is important for understanding the development of normal human cognition and how it might go awry in neuropsychiatric disorders with a neurodevelopmental component such as schizophrenia and attention deficit disorder.

EXPERIMENTAL PROCEDURES Tissue collection and processing Brain specimens were obtained through the Office of the Medical Examiners of the District of Columbia and were processed in the Section on Neuropathology of the Clinical Brain Disorders Branch as previously described (Kleinman et al., 1995). In each case, one cerebral hemisphere was cut coronally into 1–2 cm slabs and flash frozen in a mixture of dry ice and isopentane (1:1, V:V). The middle one-third (in the rostral– caudal dimension) of the middle frontal gyrus was selected from fresh frozen coronal blocks and sectioned on a cryostat at a thickness of 14 ␮m. Tissue sections were thaw-mounted onto gelatin-coated microscope slides and stored at ⫺80 °C. For protein extractions, 1 to 2 g of tissue immediately adjacent to the middle frontal gyrus was obtained. A pie-shaped wedge was carefully dissected to minimize the inclusion of white matter and the frozen tissue was thoroughly mixed by pulverization. In order to facilitate cytoarchitectural identification of Brodmann’s area 46 (BA 46 which we will also refer to as DLPFC), every 50th tissue section was stained for Nissl substance with Thionin. Tissue specimens in each set of experiments were selected according to tissue quality and availability. Consequently, not every case was included in every experiment. Brain pH was measured as previously described (Romanczyk et al., 2002).

Cohort description Nine neonates (4 months and younger), five infants (5–12 months), eight adolescents (14 –18 years), eight young adults (20 –24 years), eight adults (34 – 43 years) and seven aged individuals (63– 86 years) were included in this study (see Table 1). The cohorts for each experiment contained five cases or more per age group and did not show statistically significant group differences in either pH or postmortem interval (PMI) (ANOVA P⬎0.05). All available clinical information for each case was carefully and independently reviewed by two board-certified psychiatrists (M.A. and J.E.K.). Whenever possible, collateral information about the subjects was obtained from telephone interviews with surviving relatives of the deceased. Subjects with a history of neuropsychiatric disorders or substance abuse were excluded. Cases with significant neuropathological abnormalities or with neuropathology consistent with Alzheimer’s disease were also excluded.

Western blotting Pulverized frozen PFC tissue (100 –130 mg) was thawed and homogenized over ice using a handheld homogenizer filled with

extraction buffer (AEBSF 0.024%, aprotinin 0.005%, leupeptin 0.001%, pepstatin A 0.001%, glycerol 50%, Tris 0.6%) at the ratio of 1 g tissue to 10 ml buffer. Protein concentration was determined in each sample with the Bradford method (Bradford, 1976). Homogenized samples were aliquoted and stored at ⫺80 °C. Aliquots were defrosted on wet ice before use. In each case, equivalent amounts of protein (20 ␮g) were aliquoted from homogenates and prepared for blotting by adding 25% by volume Tris– glycine SDS sample buffer 4⫻ (Invitrogen Corporation, Carlsbad, CA, USA) and ddH2O to normalize the loading volume for experimental conditions. All samples were then loaded onto 10% Tris– glycine acrylamide gels and electrophoresed at 120 V with MES Running Buffer 1⫻. After transferring the protein from the gel to nitrocellulose membranes (80 V for 105 min), the membranes were placed on a rocking platform in blocking solution (Tris-buffered saline 1⫻, 0.05% Tween (TBS-T)), 6% normal goat serum (Vector Laboratories, Burlingame, CA, USA) and 5% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) for 40 min at room temperature. The membranes were then incubated with gentle agitation for 72 h in 4 °C refrigerator with a primary antibody directed against TH (Chemicon International, Temecula, CA, USA, MAB318) at 1:1000 concentration in TBS-T with 3% normal goat serum and 2% bovine albumin. Following primary incubation, the membranes were washed with TBS-T then placed in 1:10,000 goat anti-mouse IgG H&L secondary antibody (Chemicon International, AP124P) with 4% normal goat serum for two hours at room temperature. The membranes were again rinsed in TBS-T, developed with ECL-Plus (Amersham Biosciences, Piscataway, NJ, USA) according to manufacturer’s instructions and exposed to Kodak Bio-Max MR film (Eastman Kodak, Rochester, NY, USA) for several exposures times (ranging from 10 min to 1 h). Blots were rinsed and re-exposed with a primary antibody directed against ␤-actin (1:3000, Chemicon International, MAB1501) which served as a control for protein loading. Reported data are the average of two separate Western blotting experiments with the same subjects included in each experiment.

In situ hybridization Six slide-mounted tissue sections containing BA46 from each case were hybridized with 35S-labeled riboprobes for receptors DAR1, DAR2 or DAR4 (two tissue sections per riboprobe per case). We used cDNA templates for 1) DAR1395, a 395 base pair riboprobe corresponding to GenBank accession # BC074978.2 base pairs 489 – 883; 2) DAR2360, a 360 base pair corresponding to #NM000795.2 bp 826 –1185; 3) DAR4353, a 353 base pair riboprobe corresponding to GenBank accession #L12397 bp 3545–3898. All templates were sub-cloned from plasmids kindly provided by Olivier Civelli (UC Irvine). Inserts were sequenced and found to be 99 –100% homologous to the deposited sequences (BLAST search). In situ hybridization was performed as previously described (Meador-Woodruff et al., 1996). To control for between-experiment variations, tissue sections from all subjects were always processed in the same experiment. Also, sense strand control riboprobes for all three probes were hybridized under the same experimental conditions. After the in situ hybridization procedure, tissue slides, along with 14C standards (American Radiolabeled Chemicals, Inc., St Louis, MO, USA) were exposed to Kodak autoradiographic film (BioMax MR) for 1– 4 days.

Image analysis Autoradiographic films were scanned using a Hewlett Packard Scanjet Plus flatbed at 300 dpi resolution. Autoradiographic images were analyzed using NIH Image (Rasband, NIH, v1.61). Measures of optical density were conducted blind to diagnosis, within the boundaries of BA46, in a field that is cut perpendicular to the pial surface in order to minimize distortions in relative

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Table 1. Characteristics of cases Case #

Age

PMI

pH

Gender

Race

Cause of death

DAR

TH

20.0 100.0 40.5 56.0 18.5 37.0 64.0 56.0 61.0 50.3 25.1

6.35 5.98 6.25 6.76 6.69 5.93 6.30 6.47 6.45 6.35 0.3

F M F F F M F M M

AA AA AA AA AA AA AA AA AA

SIDS SIDS SIDS Undetermined Endocardial fibrolastosis SIDS Bronchopneumonia SIDS SIDS

X X X

X X X X X X X X

60.0 37.5 59.5 31.5 38.0 45.3 13.4

6.50 6.40 6.34 6.74 6.69 6.53 0.2

F M M F M

AA AA AA AA AA

SIDS Bronchiolitis Diphenhydramine intox SIDS Cardiomegaly

X X X X X

X X X X X

6.30 6.64 6.66 6.54 6.27 6.41 6.32 6.68 6.48 0.2

M M M M M M M M

AA AA AA AA AA AA AA AA

GSW to abdomen GSW to chest GSW to chest GSW to back GSW to chest GSW to back Stab wound to chest Multiple GSWs

X X X X X X X X

X X X X X X X X

43.5 38.0 12.5 54.5 32.5 28.5 16.5 21.5 30.9 14.2

6.69 5.85 6.59 6.48 6.39 6.18 6.77 6.50 6.43 0.3

M M M M M M M M

AA AA AA AA AA AA AA AA

GSW to torso GSW to torso Fibrinous pericarditis Pulmonary embolism Multiple GSWs Stab wound to chest Restrictive pericarditis GSW to chest

X X X X

X X

24.5 10.0 40.0 15.5 32.5 55.5 13.5 23.0 26.8 15.3

6.44 6.48 6.52 6.72 6.36 6.37 6.31 6.71 6.49 0.2

M M M M M M M M

AA AA AA AA AA AA AA AA

ASCVD Stab wound to chest Acute asthma attack GSW to chest Pulmonary embolism Multiple GSWs GSW to torso Hypertrophic cardiomegaly

X X X X X X X X

Neonates (n⫽9, age in months) 1N 2N 3N 4N 5N 6N 7N 8N 9N Mean ⫾SD

4.0 3.0 4.0 2.0 3.5 2.5 1.0 1.0 2.5 2.6 1.1

X X X X

Infants (n⫽5, age in months) 9I 10I 11I 12I 13I Mean ⫾SD

9.0 11.5 5.0 6.0 5.5 7.4 2.8

Adolescents (n⫽8, age in years) 15T 16T 17T 18T 19T 20T 21T 22T Mean ⫾SD

18 18 18 18 14 15 15 18 16.8 1.8

56.5 36.5 21.5 14.5 17.0 21.0 15.5 21.5 25.5 14.3

Young adults (n⫽8, age in years) 23Y 24Y 25Y 26Y 27Y 28Y 29Y 30Y Mean ⫾SD

24 22 24 22 20 21 23 24 22.5 1.5

X X X

X X X X

Adults (n⫽8, age in years) 31A 32A 33A 34A 35A 36A 37A 38A Mean ⫾SD

43 41 42 31 38 35 34 43 38.4 4.6

X X X X X X X

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Table 1. continued Case #

Age

PMI

pH

Gender

Race

Cause of death

DAR

TH

6.25 5.83 6.25 6.39 5.95 6.57 6.61 6.26 0.3

M M M M M F M

AA AA AA AA AA A AA

Undetermined Sepsis ASCVD Renal cell carcinoma Pulmonary embolism GSW to chest ASCVD

X X X

X

Aged adults (n⫽7, age in years) 39G 40G 41G 42G 43G 44G 45G Mean ⫾SD

86 73 71 74 83 78 68 76.1 6.5

45.0 36.5 40.5 42.0 66.5 23.0 58.0 44.5 14.2

X X X

X X X X X

Abbreviations: ASCVD, atherosclerotic cardiovascular disease; GSW, gunshot wound; SIDS, sudden infant death syndrome.

laminar thickness due to angle of cut. The boundaries of BA46 were delineated on Nissl-stained sections, applying the criteria described by Rajkowska and Goldman-Rakic (1995). The criteria used include: 1) the presence of a well-defined granular layer IV; 2) the columnar arrangement of pyramidal neurons in layer III; 3) the increase in size of pyramidal neurons in layer III with an increase in cortical depth; and 4) the presence of a clear transition from layer VI into the white matter. In each tissue section, three separate lines (170 ␮m wide each) were drawn perpendicular to the pial surface traversing the cortex from the pial surface to the edge of the white matter. Optical density, interpolated along the 14C standard curve, was sampled at 85 ␮m pixel intervals along these lines. These data were used to construct profile plots of ␮Ci/g of each DAR mRNA as it varied with gray matter depth. The profiles plots along each line were then linearly interpolated to a common anatomical scale in units of percent cortical depth (Romanczyk et al., 2002). The validity of using these cortical depths was confirmed by measuring laminar depths on a Nissl-stained section within 140 ␮m of the sections used in in situ hybridization experiments. In each case, the average value of six lines (three per section) was used in the statistical analysis, so that each individual had only one value at each cortical depth.

Statistical analysis One-way ANOVAs were performed to verify that the individuals in the age groups were sufficiently matched on demographic variables and to test whether TH or ␤-actin immunoreactivity varied with age group. Statistical comparisons of the regional expression of DAR1, DAR2 and DAR4 mRNA (dependent variable) between the age groups (independent variable) across all cortical layers were conducted by ANOVAs and ANCOVAs (with PMI as the covariate), followed by Fisher’s LSD post hoc tests. Pearson product moment correlations and Spearman correlations were performed to assess any significant relationships between DAR mRNA or TH protein levels with pH and PMI.

RESULTS Our measurements of TH immunoreactive bands via Western blotting were not significantly correlated with either pH or PMI. Similarly, there was no statistically significant correlation between brain tissue pH and DAR mRNA level for any of the three transcripts for all six layers (all between r⫽⫺0.06 and r⫽0.27, P⬎0.05) and age groups did not significantly differ with regard to brain pH (F⫽1.58, df⫽5, 35, P⫽0.19). We also did not find statistically significant relationships between DAR1, DAR2 and DAR4 mRNA

expression and PMI in any cortical layer (all between r⫽⫺0.15 and r⫽0.30, all P⬎0.05). Subsequent analyses using pH and PMI as a covariate did not affect the outcome of the statistical analyses. Age-related changes in TH immunoreactivity TH Western blots produced a discrete single immunoreactive band of approximately 59 kDa with minimal background signal for each age group examined (Fig. 1). Bands of TH immunoreactivity detected in the neonate and infant groups (less than 1 year of age) were visibly darker than in the other four age groups (over 14 years of age). Quantitative analysis demonstrated that neonates and infants had 400% and 250% higher TH protein levels, respectively, compared with the older age groups (Fig. 1, A and C). Statistical analysis of density measurements from the approximately 59 kDa immunoreactive band confirmed that TH immunoreactivity varied significantly according to age group (F⫽10.34, df⫽5, 34, P⬍0.001). Post hoc LSD analysis revealed that neonates had significantly elevated levels of TH immunoreactivity as compared with infants (P⫽0.04), teens, young adults, adults and aged (all P⬍0.001). Infants also had higher levels of TH-immunoreactivity as compared with the older age groups (all Pⱕ0.02). We noted a marked decline in TH-immunoreactivity levels in the first year of life; from fourfold that of adults in neonates to 2.5-fold in infants. In order to control for any potential systematic differences in protein quantification, loading, or transfer, we measured levels of ␤-actin in the same age groups by re-probing the Western blots used for TH. As predicted, a single dark band of immunoreactivity at approximately 45 kDa corresponding to ␤-actin was visualized in each age group with no difference in intensity between groups (F⫽1.068, df⫽5, 32, P⫽0.39) (Fig. 1, B and D). The difference in TH immunoreactivity across the age-groups remained highly statistically significant when expressed as a ratio of TH immunoreactivity/␤-actin (F⫽7.30, df⫽5, 32, P⬍0.001). Co-varying with either pH or PMI did not alter the statistical significance of the age group effect on TH protein levels (both P⬍0.001).

C. S. Weickert et al. / Neuroscience 144 (2007) 1109 –1119

A.

B. N

I

N

ADOL YA AD AG

I ADOL YA AD AG

49 –

62 –

C.

D. 200

700

β -Actin Protein (% Adult)

600 500

160 120

.

400 .

TH Protein (% Adult)

1113

300 200 100 0

N

I

ADOL

YA

AD

AG

80 40 0

N

I

ADOL

YA

.

.

Age Group

Age Group

AD

AG

Fig. 1. Autoradiographic film from Western blots showing one intense 58 – 60 kDa immunoreactive band in each age group detected with the anti-TH antibody (A) and 49 kDa immunoreactive bands detected with the anti-␤ actin antibody (B). The position of the 62 kDa and 49 kDa molecular weight markers is shown. Note the decrease in intensity of TH immunoreactivity with age in panel A and the unaltered intensity of ␤-actin immunoreactivity across age groups in the same blot in panel B. Optical density of TH immunoreactive bands and ␤-actin immunoreactive band in the PFC of individuals in each of the age groups is shown as scatterplots in panels C and D. Each triangle depicts one subject and each bar depicts a group mean. Note the marked decline of TH level in all other age groups compared with neonates and infants. No change was apparent when comparing levels of the ⬃45 kDa ␤-actin in the DLPFC of the same groups and under the same experimental conditions. Neonates (N), infants (I), adolescents (ADOL), young adults (YA), adults (AD) and aged (AG).

Age-related changes in DAR1 mRNA In the human PFC, the DAR1 gene was expressed in a layer-specific pattern (Fig. 2) consistent with previous reports in adults (Meador-Woodruff et al., 1996). Signal intensity was highest in layers II and deep V–superficial VI, absent in layer I and moderate in the remainder of layers III through IV. While the intensity of signal throughout the cortical depth varied with age, the relative laminar distribution did not. The peak of signal intensity was present in the adolescents and young adults groups, declined in the adult group and declined further in the aged group (Fig. 2) As expected, both pyramidal and non-pyramidal cellular profiles showed accumulations of silver grains in emulsioncoated slides (Fig. 5A and B). Statistical analysis by ANCOVA with PMI as a covariate, showed that DAR1 expression varied significantly according to age group (F⫽2.53, df⫽5, 34, P⬍0.05) and according to cortical layer (F⫽48.17, df⫽5, 175, P⬍0.001). DAR1 mRNA levels increased by 40% from infants to young adults and the highest expression of DAR1 mRNA occurred in the adolescent/young adult groups in all cortical layers. Compared with young adults, DAR1 mRNA was reduced by 38% in the adult group and 49% in the aged group (Fig. 2). Post hoc LSD analysis on the overall main

effect of age on DAR1 mRNA across cortical layers, demonstrated that the adult and aged group had lower DAR1 mRNA levels compared with the adolescent (P⫽0.07 and P⫽0.03, respectively) and young adult groups (P⫽0.04 and P⫽0.02, respectively). The young adults also had higher DAR1 mRNA levels compared with two youngest ages examined and this reached a trend level of significance when compared with the infants (P⫽0.06). The interaction between cortical layer and age was not statistically significant (F⫽0.96, df⫽25, 175, P⫽0.52). This also suggests that all layers changed similarly with age. The post hoc analysis for the main effect of cortical layer showed, as predicted, that the DAR1 mRNA levels were significantly less in layer I compared with all other layers (all P⬍0.001). DAR1 mRNA levels in mid-cortical layers III and IV were not statistically different from one another, but were significantly lower than mRNA levels in the deeper cortical layers, V and V1 (P⬍0.001, P⬍0.01 respectively) and significantly lower than superficial cortical layer II P⬍0.001). Age-related changes in DAR2 mRNA DAR2 mRNA was present in layers II through VI with the highest signal intensity in layer II, layer V and deep layer

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Fig. 2. Top panel: representative autoradiograms illustrating the gene expression of DAR1 in the human PFC of a neonate, a young adult, an adult, and an aged subject. Note the peak in signal intensity in young adults followed by a gradual decline with age. Scale bar⫽2 mm. The bar graphs in the bottom panel represent mean optical density of DAR1 mRNA plotted across layers II–VI of the PFC (error bars show S.E.M.). * Significant difference relative to aged, ‡ significant difference relative to both adults and aged (P⬍0.05).

VI. All age groups showed an overall moderate level of expression except for a visible decline in the aged group that reached significance in statistical analyses (Fig. 3). Similar to the cellular distribution of DAR1 mRNA, both pyramidal and non pyramidal cellular profiles appeared to express DAR2 mRNA as demonstrated by accumulations of silver grains in emulsion-coated slides (Fig. 5C and D). We detected significant difference in DAR2 gene expression as a function of age (F⫽2.43, df⫽5, 33, P⫽0.05, Fig. 3). However, unlike DAR1, DAR2 mRNA levels were highest in neonates and lower in the infant group compared with neonates (P⫽0.04). Levels of DAR2 mRNA appeared to increase in the adolescent group but this did difference did not reach statistical significance. The most robust change in DAR2 mRNA occurred in the aged group which displayed a 27%–35% reduction in DAR2 mRNA as compared with the neonate (P⫽0.005), the adolescent (P⫽0.02) and the adult groups (P⫽0.04). ANOVA also revealed that DAR2 mRNA levels varied significantly across cortical layers (F⫽171.12, df⫽5, 165, P⬍0.001). DAR2 mRNA levels in layer II were higher than layer III and layer IV (P⬍0.001), however, cortical layers V and VI were found to contain the highest levels of DAR2 mRNA compared with all other layers (P⬍0.001). In addition to these

two main effects of age group and cortical layer, a trend toward a significant interaction between cortical layer and age on DAR2 mRNA was detected (F⫽1.54, df⫽25, 165, P⫽0.06) suggesting that certain layers show a more robust change with age. Age-related changes in DAR4 mRNA Compared with DAR1 and DAR2, DAR4 mRNA was less intensely expressed in all age groups, but was clearly present in layers II through VI with the highest signal intensity visible in layer V. Minimal variations in DAR4 mRNA levels were visible across age groups (Fig. 4). DAR4 mRNA was most commonly expressed by presumptive non-pyramidal neurons and glia with some examples of pyramidal profiles showing accumulations of silver grains in emulsion-coated slides (Fig. 5E and F). Unlike the age-specific changes in DAR1 and DAR2 gene expression, we did not find significant age-related changes in DAR4 mRNA (F⫽0.72, df⫽5, 34, P⫽0.61, Fig. 4). DAR4 mRNA levels in the frontal cortex remained relatively stable and fluctuated only slightly from neonates through aged adults. The interaction between cortical layer and age on DAR4 mRNA was also not statistically signif-

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Fig. 3. Top panel: representative autoradiograms illustrating the gene expression of DAR2 in the PFC of a neonate, a young adult, an adult and an aged subject. Note that the highest level of expression is in the youngest age group. Scale bar⫽2 mm. The bar graphs in the bottom panel represent mean optical density of DAR2 mRNA plotted across layers II–VI of the PFC (error bars show S.E.M.). * Significant difference relative to infants; ‡ significant difference relative to neonates, adolescents, and adults (P⬍0.05).

icant (F⫽1.04, df⫽25, 170, P⫽0.42). We did detect a significant main effect of cortical layer on DAR4 mRNA (F⫽90.43, df⫽5, 170, P⬍0.001). The highest levels of DAR4 expression were found in the deep cortical layers V and VI as compared with all other layers (all P⬍0.001). Layers II also showed higher DAR4 mRNA levels than layers I and III (P⬍0.01).

DISCUSSION We report dramatic and significant changes in the levels of TH protein and DAR receptor mRNAs in the human PFC throughout postnatal life. To our knowledge, this is the first study examining these markers of the pre- and post-synaptic DA system in the human PFC during postnatal development. Most of the previously available information on age dependent changes in DA receptors in human and non-human primates has relied on ligand binding techniques (Seeman et al., 1987; Palacios et al., 1988; de Keyser et al., 1990; Rinne et al., 1990; Lidow and Rakic, 1992; Boyson and Adams, 1997; Montague et al., 1999). We noted two distinct peaks in levels of DA markers examined in this study in the human PFC; the first peak was in TH protein and DAR2 mRNA levels and occurred

very early in postnatal life, and the second peak was in the level of DAR1 mRNA and occurred during adolescence and early adulthood. In our study, the highest levels of TH immunoreactivity were present in the youngest group (neonates) and declined dramatically in ages 14 and above. This difference in TH levels was confirmed in two separate experiments and was not found in the control protein ␤-actin. Our finding is consistent with a report in non-human primates (Goldman-Rakic and Brown, 1982) of a decrease in DA content in the monkey PFC in the first six months of life. While a decrease in DA content may reflect a decrease in the level of the synthetic enzyme TH, as suggested by our study, changes in DA content may have also resulted from changes in DA degradation (Brust et al., 2004; Tunbridge et al., 2005). Unlike DA levels, the number of TH immunoreactive contacts on pyramidal neurons in the monkey PFC was reported to increase gradually from birth and peak in adolescence (Lambe et al., 2000). Similarly, Rosenberg and Lewis (1994) reported an increase in THpositive axon length in layer III of the monkey PFC during adolescence with modest decreases in TH-positive axon length in layers I/II and VI. Since our TH measures were

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Fig. 4. Top panel: representative autoradiograms illustrating the gene expression of DAR4 in the PFC of a neonate, a young adult, an adult and an aged subject. Fluctuations in the levels of DAR4 mRNA are not statistically significant. Scale bar⫽2 mm. The bar graphs in the bottom panel represent mean optical density of DAR4 mRNA plotted across layers II–VI of the PFC (error bars show S.E.M.).

conducted on homogenates of PFC tissue blocks, we may have missed lamina-specific changes or cell specific changes similar to those reported in monkey PFC. Our study established that DAR1, DAR2 and DAR4 mRNAs were expressed in the human PFC from birth on, and that changes in the gene expression during postnatal life are DAR subtype-specific. For example, the peak in DAR1 gene expression in the human PFC in adolescence and early adulthood is not found in DAR2, DAR4 mRNA or in the housekeeping control gene, cyclophilin (Webster et al., 2002) in the same cohort. This differential effect of age on different transcripts suggests that this finding is not a result of non-specific confounds like pH or PMI. Marked increases in DAR1 mRNA levels from birth to puberty similar to those we find in the human have been reported in the frontal cortex of rodents (Leslie et al., 1991; Tarazi et al., 1999). Interestingly, this peak in DAR1 mRNA levels in adolescence and early adulthood coincides with changes in several anatomical and functional indices of DA during the same developmental period in non-human and human primates. It is consistent with the peak in the density of TH-positive afferents in layer III of the monkey PFC and with the lamina-specific increase in the density of DA afferents to the PFC around puberty (Rosenberg and Lewis, 1994; Lambe et al., 2000). It also corresponds to the matu-

ration of PFC neurons active during the delay period of working memory tasks and the emergence of competence on these same tasks in monkeys (Goldman-Rakic, 1987). The ability of PFC pyramidal neurons to maintain persistent activity or form “upstates” is enhanced after puberty (O’Donnell, 2003; Tseng and O’Donnell, 2005), and likely dependent on synergism between DAR1 and NMDAR signaling (Wang and O’Donnell, 2001). Similarly, 50% reduction in DAR1 levels, blocks the DAR1-mediated increase in maintenance of long term potentiation, suggesting that DAR1 is key in modulating synaptic plasticity of prefrontal cortical neurons (Huang et al., 2004). Finally, in humans, the acquisition of adult-level competencies on working memory tasks occurs around late adolescence and young adulthood (Welsh et al., 1991; Luciana et al., 2005). Therefore, the peri-pubertal increase in DAR1 expression may be an important component of the development of mature cognitive abilities subserved by the PFC. This has clinical implications, particularly for schizophrenia, which usually becomes manifest in late adolescence and early adulthood and is accompanied by a decline in cognitive functions subserved by the PFC and DAR1. In this study, levels of DAR2 mRNA are significantly higher in the neonatal group. This is consistent with a previous report of high perinatal binding of DAR2 receptors in the monkey PFC (Lidow et al., 1991a). Interestingly,

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Fig. 5. High magnification brightfield (panels A, C and E) and darkfield (panels B, D and F) photomicrographs showing DAR1 (panels A and B), DAR2 (panels C and D) and DAR4 (panels E and F) mRNA in layer III of adult subjects. Silver grains are clustered over large pyramidal-shaped profiles (arrows), smaller round profiles presumed to be non-pyramidal neurons (arrowheads) in all panels. Note that silver grains corresponding to DAR4, but not DAR1 or DAR2, are also found clustered over very small and intensely Nissl-stained cells (presumed to be glial cells) in panels E and F (*). Scale bar⫽2 ␮m.

DAR2 mRNA has been found to be expressed in the fetal human temporal cortex in differentiated neurons of the cortical plate and cortical sub-plate (Gurevich et al., 2000). Taken together, these studies suggest that DAR2 may play a role in early developmental events in the human telencephalon. We find no significant differences in DAR4 mRNA levels across age groups and since DAR3 and DAR5 expression in the PFC was not examined, we cannot comment on their differential expression in postnatal development. We report significant reductions in both DAR1 mRNA and DAR2 mRNA levels in the DLPFC of the aged group.

Our findings are in agreement with other studies showing that binding to DAR decreases in the aged cerebral cortex of humans (de Keyser et al., 1990; Wang et al., 1998; Hemby et al., 2003; Moore et al., 2005). Molecular imaging studies in living humans have shown significant decreases in cortical DAR1 binding in the aged occipital cortex (Wang et al., 1998) and frontal cortex (Suhara et al., 1991). Additionally, studies in monkeys show an age-related alteration in working memory in response to drugs that bind to DAR1 (Arnsten et al., 1994) and show that memory span performance correlates with DAR1 binding in the frontal

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cortex (Moore et al., 2005). Since both DAR1 and DAR2 receptors are found on spines, it is worth noting that mRNA levels of neurotrophin receptors trkB and trkC, also found on spines, decrease with aging in the normal human PFC (Romanczyk et al., 2002; Beltaifa et al., 2005). Moreover, expression of the spine associated marker, spinophilin, also decreases with age (Weickert et al., 2004). Since there does not appear to be an appreciable loss of neurons in the normal aged human cortex (Morrison and Hof, 1997), the age-related changes in DAR1 and DAR2 may be related to loss of spines consistent with decreases in the neuropil of the frontal cortex of aged monkeys (Peters et al., 1998) and aged humans (Jacobs et al., 1997). Overall, alterations in synaptic spines and more specifically the DA receptors in the PFC have been implicated in age-related cognitive decline (de Keyser et al., 1990; Arnsten et al., 1994, 1995; Okubo et al., 1997).

CONCLUSION In summary, we have found changes in both presynaptic and postsynaptic indices of DA signaling in the human PFC throughout the lifespan. Delineating changes in DArelated mRNAs and proteins in humans may provide a better framework for the interpretation of DA-related cognitive changes, psychopathological tendencies and therapeutic interventions during certain epochs of postnatal life. Acknowledgments—The authors thank Rick Dreyfuss and Sherry Metzget for their technical assistance.

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(Accepted 5 October 2006) (Available online 22 November 2006)