Dopamine afferents from the mesencephalon appear to play acritical role in the normal development and cognitive functions ofmultiple areas of the primate cerebral cortex. In some regions, suchas the prefrontal and motor cortices, dopamine innervation changessubstantially during postnatal development. However, little is knownabout the postnatal maturation of dopamine afferents to the primaterostral entorhinal cortex, a periallocortical region that receives adense dopamine innervation in adults. In this study, we usedimmunocytochemical techniques and antibodies against tyrosinehydroxylase and the dopamine transporter to examine the postnataldevelopment of dopamine axons in the rostral subdivision ofmacaque monkey entorhinal cortex. Within animals, the axonslabeled with each antibody did not differ in overall density or laminardistribution. Across development, the density of dopamine axons inlayers I and VI did not change appreciably. In contrast, the density oflabeled axons in layer III significantly increased by a factor of threebetween birth and 5–7 months of age. The timing of this changediffers substantially from that observed in prefrontal cortex, wherepeak dopamine innervation occurs between 2 and 3 years of age.These findings, in concert with other data, suggest that develop-mental changes in the dopamine innervation of cortical regions mayparallel the functional maturation of those areas.

IntroductionThe entorhinal cortex (ERC), a periallocortical region located in

the rostral portion of the medial temporal lobe (Fig. 1A), is an

integral component of the hippocampal formation (Van Hoesen,

1982). The ERC receives inputs from multiple polymodal

sensory association areas of neocortex and relays this informa-

tion to the hippocampus (Van Hoesen and Pandya, 1975; Insausti

et al., 1987a; Witter et al., 1989; Witter and Amaral, 1991).

Hippocampal output is, in part, relayed back to neocortical

regions via the ERC (Van Hoesen, 1982). Thus, the ERC may be

regarded as a critical point of interface between the neocortex

and hippocampus, and consequently as an important mediator

of higher cognitive functions.

The ERC has also been implicated as a site of dysfunction in

schizophrenia. Multiple postmortem and in vivo imaging studies

have reported reduced volume of the medial temporal lobe in

subjects with schizophrenia compared to normal controls

(Bogerts et al., 1985; DeLisi et al., 1988; Falkai et al., 1988;

Altshuler et al., 1990; Young et al., 1991). In addition, prelim-

inary studies have revealed a decreased density of dopamine

(DA) axons in the rostral subdivision (ER) of the ERC in

schizophrenic subjects, an abnormality which was not present

in subjects with other psychiatric disorders (Lewis and Akil,

1997). This finding is of particular interest given the suggestion

that cognitive dysfunction in schizophrenia may be related to

diminished cortical DA neurotransmission (Weinberger et al.,

1988; Davis et al., 1991; Goldstein and Deutch, 1992; Dolan et

al., 1995; Okuba et al., 1997).

Since schizophrenia has been hypothesized to have a

neurodevelopmental origin (Weinberger, 1987; Walker, 1994;

Bunney et al., 1995), knowledge of the normal development of

the DA innervation of the primate ERC may provide insight into

the pathogenesis of this disorder. Previous studies have indicated

that the DA projections to many regions of primate neocortex

undergo extensive refinements postnatally. For example, in the

prefrontal cortex, another region implicated in the patho-

physiology of schizophrenia, biochemical analyses have revealed

that maximal tissue concentrations and synthesis rates of DA

are not achieved until 2–3 years of age in macaque monkeys

(Goldman-Rakic and Brown, 1982), the usual age of puberty

onset in this species (Plant, 1988). Similarly, the density of axons

immunoreactive for tyrosine hydroxylase (TH), the rate-limiting

enzyme in catecholamine synthesis, peaks in prefrontal cortex

around 2–3 years of age, before declining to reach stable adult

levels (Rosenberg and Lewis, 1995). Furthermore, these changes

in axon density are lamina-specific, with the greatest shifts

occurring in the middle cortical layers.

Given that the developmental changes in the DA innervation

of prefrontal cortex continue through late adolescence,

the typical age of onset of the symptoms of schizophrenia

(Waddington, 1993), these findings raise the question of

whether a similar timecourse of developmental changes in DA

axons occurs in the ERC. However, both the laminar pattern of

DA axons (Akil and Lewis, 1993, 1994) and the complement of

DA receptor subtypes (Meador-Woodruff et al., 1994) in the

adult primate ERC are different from those in neocortex. These

differences suggest that DA may play a distinct functional role in

the adult ERC, and consequently, that DA axons may exhibit a

different pattern of developmental changes in the ERC than in

neocortex. In order to test the latter hypothesis, we used

immunocytochemical techniques and antibodies against two

markers of DA axons, TH and the DA transporter (DAT), the

transmembrane protein involved in reuptake of DA, to examine

postnatal developmental changes in the DA innervation of the

rhesus monkey ERC.

Materials and Methods

Animals and Tissue Preparation

Twenty-three rhesus monkeys (Macaca mulatta) of both sexes, ranging

in age from 2 days to >18 years, were utilized in this study (Table 1). Four

of these animals had been subject to previous manipulations for a

different study. Two of these four animals (RH127 and RH119) had been

castrated <2 weeks after birth and two animals (RH144 and RH145) had

received two to four injections of gonadotropin-releasing hormone

(GnRH) >8 months prior to perfusion. Weekly measures of plasma

testosterone levels in the two GnRH-treated animals just prior to

perfusion revealed hormone levels indicative of prepubertal (RH144) or

late pubertal (RH145) patterns of secretion. These findings are consistent

with previous data showing that cessation of GnRH stimulation of the

hypothalamus results in a return to the animal’s previous stage of sexual

development (Gay and Plant, 1987, 1988). Tissue from 20 of these animals

Cerebral CortexJul/Aug 1998;8:415–427; 1047–3211/98/$4.00

Postnatal Development of TyrosineHydroxylase- and DopamineTransporter-immunoreactive Axons inMonkey Rostral Entorhinal Cortex

Susan L. Erickson1, Mayada Akil2, Allan I. Levey3 and

David A. Lewis1,2

1Department of Neuroscience and

2Department of Psychiatry,

University of Pittsburgh, Pittsburgh, PA 15213 and3Department of Neurology, Emory University, Atlanta,

GA 30322, USA

was utilized in a previous study of TH-labeled axons in frontal cortex

(Rosenberg and Lewis, 1995).

Animals were anesthetized with ketamine hydrochloride (25 mg/kg)

and pentobarbital sodium (30 mg/kg), and transcardially perfused with

cold 4% paraformaldehyde in phosphate buffer. Brains were removed,

blocked coronally and postfixed for 6 h in the same fixative. The blocks

were cryoprotected and maintained at –30°C in a storing solution of

glycerine (30%) and ethylene glycol (30%) in dilute phosphate buffer.

Storage under these conditions has previously been shown not to affect

immunoreactivity (Rosenberg and Lewis, 1994). Blocks were then

sectioned (40 µm) on a cryostat and a one in ten series of sections was

stained for Nissl substance.

Cytoarchitectonic Subdivisions of ERC

Seven distinct cytoarchitectonic regions have been identified in the

cynomolgus (Macaca fasicularis) monkey ERC (Amaral et al., 1987). In

the adult, the olfactory (EO) and rostral (ER) subdivisions are densely

innervated by DA axons (Akil and Lewis, 1993); the two lateral

subdivisions have a moderate innervation; and the intermediate, caudal

and caudal limiting subdivisions have a relatively sparse DA innervation.

Of the two subdivisions of the ERC that have a robust DA innervation,

subdivision ER has a more uniform distribution of DA axons, facilitating

comparisons across animals. Thus, subdivision ER was selected for study.

Due to the presence of some minor differences between cyno-

molgus and rhesus monkeys in the cytoarchitecture of the ERC, the

cytoarchitectonic criteria of Amaral et al. (1987) were modified for

rhesus monkeys and used to identify ER in each animal in this study. ER

was identified by the following features (Fig. 2): (i) layer II had islands of

cells separated by acellular gaps; (ii) layer III contained large, irregular

patches of cells, with cell-sparse zones often in registration with the gaps

in layer II; (iii) neurons in superficial layer III were larger than those in

deep III; (iv) layer IV (lamina dissecans) was not apparent, or if present,

was not prominent; (v) layers V and VI had large, darkly stained cells, and

sublaminae in these layers were not easily distinguished. Nissl sections

meeting these criteria were identified in animals of all ages (Fig. 2B,C).

Subdivision ER occupies a relatively small portion of the rostrocaudal

extent of the ERC (Fig. 1A), and an ideal match to the cytoarchitectonic

criteria could be identified on one or two sections from the one in ten

Nissl series for each animal. Note also that although the thickness of ER

increases from medial to lateral (Fig. 2A), the same cytoarchitectonic

features can be used to identify this region (Fig. 2B,C).

Immunocytochemistry

Random sections located within 360 µm of the selected Nissl sections

were processed for immunocytochemistry, and sections from animals of

different ages were processed together in the same experiment. After

removal from storage solution, sections were washed in 0.012 M

phosphate-buffered saline (PBS), and incubated for 30 min at room

temperature in PBS containing 0.3% Triton X-100 and 4.5% normal

donkey serum (NDS). Sections were then transferred to PBS containing

either a 1:20 000 dilution of mouse anti-TH primary antibody (kindly

Table 1Rhesus monkeys used in this study

Agegroup

Animal Age Weight(kg)

Sex Time in storingsolution(months)

1 RH142 2 days 0.3 M 58RH109 4 days 0.5 F 69RH106 22 days 0.6 M 79

2 RH131 37 days 0.7 F 45RH130 67 days 0.8 F 45RH117 72 days 0.6 F 53

3 RH134 5 months 1.2 F 43RH155 6 months 1.2 F 0RH128 7 months 1.3 F 49

4 RH133 1 year 1.9 F 43RH147 1.2 years 2.5 M 5RH149 1.4 years 2.6 M 4RH135 1.5 years 2.8 F 42

5 RH127a 2.3 years 3.3 M 50RH111 2.5 years 2.9 M 67RH118 2.8 years 3.2 M 53RH144b 2.9 years 3.6 M 20RH145b 3.0 years 6.0 M 20

6 RH119a 4.5 years 6.4 M 53RH114 5.7 years 4.4 F 55RH143 15.9 years 8.4 M 22RH112 16.7 years 7.0 F 65RH141 >18 years 9.0 M 23

aCastrate; bGnRH treatment.

Figure 1. Schematic drawing of the medial surface of a macaque monkey brain (A).Dashed line indicates location of ERC and solid area indicates approximate location ofsubdivision ER. Vertical line shows the approximate location of the coronal sectionillustrated in (B). The shaded area in (B) indicates the mediolateral extent of ER. (C)shows an enlargement of the shaded area in (B), and rectangular boxes indicate wherequantitative assessments of TH-IR axons were made. Open boxes indicate samplinglocations at mediolateral midpoint of ER (arrow), and solid boxes show representativelocations of sampling at regions of highest density of TH-labeled axons within 500 µmmedial to the midpoint of ER. Am, amygdala; cc, corpus callosum; RS, rhinal sulcus.Roman numerals indicate the cortical layers.

Figure 2. Brightfield photomicrographs of coronal sections stained for Nissl substance illustrating the cytoarchitectonic features used to identify subdivision ER. In (A), a low-powermicrograph from the 7 month old animal shows the ventrolateral and dorsomedial borders of subdivision ER (arrows). Note the substantial decrease in cortical thickness fromventrolateral (left side of micrograph) to dorsomedial (right side) within ER. Below, higher-magnification micrographs from the 7 month (B) and 5.7 year (C) old animals illustrate thatcytoarchitectonic features were matched across ages. The micrograph from the 7 month old animal was taken from a slightly more dorsomedial portion of ER than in the 5.7 yearold, and therefore the cortex appears thinner. RS, rhinal sulcus. Roman numerals indicate cortical layers, WM indicates white mattter. Scale bars = 600 µm (A), 200 µm (B,C).

416 DA Development in Monkey ERC • Erickson et al.

Cerebral Cortex Jul/Aug 1998, V 8 N 5 417

supplied by Dr G. Kapatos, Wayne State University, Detroit, MI) or a

1:2000 dilution of rat anti-DAT primary antibody, in addition to 0.3%

Triton X-100, 3% NDS and 0.05% bovine serum albumin. The tissue was

incubated in this solution for 48 h at 4oC, washed in PBS and immersed in

a solution of 0.5% biotinylated donkey anti-mouse or donkey anti-rat

secondary (Jackson Immuno Research, West Grove, PA). Sections were

then processed with either Standard (for TH) or Elite (for DAT)

avidin–biotin reagents (Vector Laboratories, Burlingame, CA) and

visualized with diaminobenzadine (DAB). Sections were mounted on

slides and the DAB reaction product was stabilized by immersion in

solutions of 0.005% osmium tetroxide and 0.5% thiocarbohydrazide.

The specificity of the TH antibody has been demonstrated in both

immunoblot and immunocytochemical experiments in monkey and

human tissue (Wolf et al., 1991; Akil and Lewis, 1993; Lewis et al., 1993,

1994). The DAT antibody was raised against a fusion protein containing

the N-terminus of the human DAT protein (Ciliax et al., 1995), and its

specificity has been demonstrated in both immunoblot and immuno-

cytochemistry studies using transfected cells and human tissue (Miller et

al., 1997). In addition, preadsorption of the antibody with the fusion

protein eliminated specific immunoreactivity, and replacement of the

primary antibody with normal rat serum resulted in an absence of

staining. Furthermore, the DAT antibody failed to label the neurons

of the locus coeruleus or raphe nuclei, confirming the absence of

cross-reactivity with the norepinephrine or serotonin transporters (Lewis

et al., 1998).

All tissue sections were coded to conceal the specimen number and

age. Initial qualitative evaluation and all quantitative measures were

conducted without knowledge of the age or age group of each specimen.

Quantification of TH-labeled Axons

For each brain, regional and laminar boundaries were determined on

Nissl-stained sections, and sampling was conducted on two sections

labeled for TH at the three laminar locations that had the highest density

of DA axons in adult monkey. At the border of layers I–II, in deep layer III

and in layer VI, two 50 × 100 µm sampling boxes were placed 100 µm

apart at the mediolateral midpoint of ER (Fig. 1C, open boxes). Because

labeled axons were distributed in a discontinuous fashion in deep layer

III, two additional boxes per section were sampled in this layer in the area

of highest density of immunoreactive structures located within 500 µm

medial to the midpoint of ER (Fig. 1C, solid boxes). Total axon length and

number of varicosities were recorded per 5000 µm2 of tissue by tracing

immunoreactive fibers with the Eutectic Neuron Tracing System at a

final magnification of 600× as previously described (Rosenberg and

Lewis, 1995). An axon was defined as a continuous labeled structure. A

varicosity was defined as a round or oval labeled structure greater than

the cursor size of 0.5 µm. Although using the cursor to define varicosities

limited our sampling to a subpopulation of the largest TH-immuno-

reactive terminals (Smiley and Goldman-Rakic, 1993; Sesack et al., 1995),

it ensured consistency of sampling across specimens.

Statistical Analyses

As in the previous study of the prefrontal cortex (Rosenberg and Lewis,

1995), the animals were divided into six age groups: <1 month (n = 3), 1–3

months (n = 3), 5–7 months (n = 3), 1–1.5 years (n = 4), 2–3 years (n = 5)

and >4 years (n = 5) (Table 1). For each animal, mean total axon length and

varicosity number per 5000 µm2 were calculated for each layer. One-way

(age group) analyses of variance (ANOVAs) were used to assess

differences among means in layers I and VI. In layer III, mean total axon

length and mean number of varicosities were calculated from the four

samples taken from the midpoint of ER as for the other layers, separate

mean values were calculated from the four additional measures obtained

by sampling in the area of highest density, and a two-way ANOVA (age

group, sampling method) was used for this layer. Because the two-way

ANOVA revealed no significant interaction between age group and

sampling method [F(5,34) = 0.19, P = 0.96], this analysis was used to

assess a main effect for age group. Tukey’s Studentized range test was

used for between-group comparisons. In order to assess the adequacy of

the sampling procedure, ANOVAs were used to compare the within- and

between-animal measures of total axon length and varicosity number for

each age group.

Results

Qualitative Observations

Fiber Morphology

Two different morphological types of TH-immunoreactive (IR)

axons were observed in all animals examined (Fig. 3A), as

previously described in adult monkey ERC (Akil and Lewis,

1993). The vast majority of labeled axons were very fine and

varicose, and were present in all cortical layers. In layers I and

superficial II, these fibers were predominantly oriented parallel

to the pial surface, and in the deeper layers they were oriented in

all directions. The less common type of TH-labeled axons were

thick, smooth and very intensely immunoreactive. These axons

were observed primarily in the deep half of layer I and in

superficial layer II, where they were oriented parallel to the pial

surface. This type of axon was rarely observed in the deeper

layers. Both axon types were included in quantitative measures

of fiber length.

The axons visualized with the DAT antibody were pre-

dominantly of a single morphological type (Fig. 3B). These axons

were thin and varicose, and similar to the majority of TH-labeled

Figure 3. Brightfield photomicrographs of TH-IR (A) and DAT-IR (B) axons in thesuperficial layers of monkey entorhinal cortex. Note that a population of thin, varicoseaxons is labeled with both antibodies, but a population of thick, smooth axons isvisualized with only the TH antibody. Scale bar = 70 µm and applies to (A) and (B).

418 DA Development in Monkey ERC • Erickson et al.

axons. However, in contrast to the TH antibody, no thick,

smooth DAT-positive fibers were observed.

Regional and Laminar Distribution

Comparisons across animals revealed that the relative density

and laminar distribution of TH-IR axons in ER changed sub-

stantially during development. The overall density of TH-IR

axons in the youngest animals was quite low (Fig. 4A) and then

increased steadily (Fig. 4B) to reach a peak at 7 months of age

(Fig. 4C). Over the next year of life, the density of TH-IR axons

appeared to decline to reach a level intermediate between that

of the newborn and 7 month old animals (Fig. 4D). The axon

density then remained stable through adolescence (Fig. 4E) and

adulthood (Fig. 4F).

The laminar pattern of TH-IR axons followed a similar

developmental time course, with the most marked changes

occurring between birth and 7 months of age, and with little

change evident between 1.5 years and adulthood. In animals <1

month of age, bands of TH-IR axons were present at the layer I–II

border and in layer VI, producing a bilaminar distribution of

labeled axons (Fig. 4A). By 3 months of age, an additional band

of TH-IR axons had become evident in deep layer III, giving a

suggestion of the trilaminar pattern characteristic of the adult

(Fig. 4B). By 5–7 months, the band of axons in layer III was

prominent, and the trilaminar pattern was quite striking (Fig.

4C). The density of axons in layer III declined over the next year

of life (Fig. 4D), but the trilaminar pattern was maintained

through adolescence (Fig. 4E) and into adulthood (Fig. 4F).

Animals that had been castrated or received GnRH treatment did

not appear to deviate from this developmental pattern. The

density of labeled axons was also qualitatively similar in male and

female animals that were close to each other in age.

The density and laminar pattern of DAT-IR axons were

identical to those of TH-IR axons at all ages. In the youngest

animals, the overall density of DAT-IR axons was low (Fig. 5A). In

these animals, immunoreactive fibers formed a band at the layer

I–II border that was oriented parallel to the pial surface. A lower

density band of labeled fibers was present in the deep layers,

giving a bilaminar appearance. Peak overall density of DAT-IR

axons and a trilaminar pattern of innervation were evident in the

5–7 month old animals (Fig. 5B). After this peak, overall density

of DAT-IR axons declined somewhat over the next year of life,

and remained stable through adulthood, but the trilaminar

pattern of axons persisted (Fig. 5C).

Quantitative Analyses

Length of TH-IR Axons

At the border of layers I–II, the total length of TH-IR axons

exhibited modest developmental changes that approached

statistical significance [F(5,17) = 2.66, P = 0.06]. The total length

of TH-IR axons increased by 60% from birth through the 5–7

month old group (Fig. 6A,B), and then remained stable through

adulthood. In contrast, total length of labeled axons in layer III

increased significantly [F(5,34) = 3.26, P = 0.016] by a factor of

three from birth to reach a peak in the animals 5–7 months of

age (Fig. 6D). To determine whether the within-group variability

in this layer was a consequence of biological differences

between animals rather than an artifact of the sampling

procedure, we compared the variability across sampling boxes

within animals to the variability across animals within an age

group using one-way ANOVAs. With the exception of age group

4 (1–1.5 years), the results indicated that the within-animal

variability was small compared to the across-animal differences

(P < 0.05), confirming that results were not confounded by

the sampling procedure. In layer VI, no significant changes

[F(5,17) = 0.52, P = 0.758] in axon length occurred during

postnatal development (Fig. 6E,F).

Number of TH-IR Varicosities

The developmental changes in the number of TH-IR varicosities

generally paralleled the refinements in total axon length. At the

border of layers I–II, the mean number of varicosities almost

doubled from birth through 5–7 months of age, declined by 25%

over the next year, and then remained stable through adulthood

(Fig. 7A,B). However, these changes were not statistically

significant [F(5,17) = 0.52, P = 0.756]. In deep layer III, the

number of varicosities significantly [F(5, 34) = 2.69, P = 0.038]

increased >3-fold from birth to 5–7 months of age. The number

of varicosities appeared to decline over the next year to

approximately half the peak value, but this difference did not

achieve statistical significance (Fig. 7C,D). As with the measures

of axon length, the differences in varicosity number across

sampling boxes within animals were smaller than the differences

between animals (P < 0.05) in all age groups, except the 1–1.5

year old group. In layer VI, no significant [F(5,17) = 0.69, P =

0.639] developmental changes in the number of varicosities

were observed (Fig. 7E,F).

DiscussionThe results of this study demonstrate that the total length of

TH-IR axons and number of labeled varicosities in subdivision ER

of rhesus monkey ERC changed in a laminar-specific manner

during postnatal development. The most prominent changes

occurred in layer III, where the density of TH-IR axons and

varicosities increased from birth to reach peak values in the

middle of the first year of life. An identical developmental

pattern was evident for DAT-IR axons.

Methodological Issues

The parallel developmental changes observed in the TH- and

DAT-IR axons strongly indicate that the findings of this study

reveal the postnatal maturation of the DA innervation of the

rostral subdivision of monkey ERC. Indeed, in a double-labeling

study, >95% of cortical axons labeled with an anti-TH antibody

were also labeled with the anti-DAT antibody used in this study

(Lewis et al., 1998), and DAT is known to be selectively

expressed in DA neurons (Nirenberg et al.,1996; Pif l et al.,

1996). These observations are consistent with previous reports

indicating that although TH protein is present in both norepin-

ephrine and DA neurons, antibodies against TH predominantly

label DA axons in primate cortex (Lewis et al., 1987; Noack and

Lewis, 1989). Within the monkey ERC, three lines of evidence

support this conclusion (Akil and Lewis, 1993). First, the laminar

pattern of axons immunoreactive for dopamine-β-hydroxylase

(DBH), a protein expressed in norepinephrine but not DA

neurons, is markedly different from that of TH-IR axons in adult

monkey ERC. Second, the TH antibody used in the present study

labels <2% of axons immunoreactive for DBH in monkey ERC.

Finally, the pattern of labeling seen with this TH antibody is in-

distinguishable from that observed with an antibody against DA.

Interpreting the results of the present study also depends

upon an understanding of whether differences in tissue storage

time may have contributed to differences in apparent fiber

density across animals. Although tissue storage time varied over

a considerable range (Table 1), it was not significantly correlated

Cerebral Cortex Jul/Aug 1998, V 8 N 5 419

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with measures of total axon length in layer I (r = 0.32, P = 0.14),

in deep layer III (r = 0.17, P = 0.42) or in layer VI (r = 0.10, P =

0.61). Similarly, there was no correlation between storage time

and number of varicosities in layer I (r = 0.14, P = 0.56), in deep

layer III (r = 0.03, P = 0.91) or in layer VI (r = 0.16, P = 0.47).

These findings are consistent with our previous observation that

the density and laminar distribution of labeled axons in a given

animal did not change with storage time (Rosenberg and Lewis,

1994).

Some of the developmental changes observed in the density of

DA axons may be confounded by gender differences, since two

of the groups contained only females, and one of the groups

contained only males. However, in the three age groups that

contained both sexes, the quantitative data for the male and

female animals were similar. For example, in the adult group, the

two females represented the two extremes in the range of total

axon length in layer III, and the three males were clustered in the

middle. The mean values for males and females (685 ± 100 and

739 ± 477 µm/5000 µm2 respectively) were not significantly

different from each other in this group (P = 0.89). Similarly, in

the one to 1.5 year old group, the mean values for total axon

length did not significantly differ (P = 0.92) between the males

and females (635 ± 170 and 655 ± 167 µm/5000 µm2

respectively). Although these data indicate that sex is not a

Figure 6. Total length (µm) of TH-IR axons (per 5000 µm2) in layers I–II (A,B), deep III (C,D) and VI (E,F) of area ER. Data are presented as mean values for individual animals (A,C,E)and as mean (±SE) values for the six age groups (B,D,F). Note the difference between layer III and layers I and VI in the magnitude of the developmental change. In (D), age groupsnot sharing the same letter (located above each bar) are significantly different at P < 0.05.

Cerebral Cortex Jul/Aug 1998, V 8 N 5 423

confounding factor in these age groups, we cannot rule out the

possibility that the peak values of DA axon density observed in

the 5–7 month old group ref lect developmental changes specific

to females.

Other factors that could have inf luenced our findings are the

previous experimental manipulations of some of the animals.

Two of the males in the adolescent group had received GnRH

treatment, and one animal each in the adolescent and adult

groups were castrates. However, for both of these conditions,

the measures of axon density and varicosities in the animals

in question were comparable to others in their age groups.

Although not of direct relevance to the present study, removal of

the ovaries has been reported to affect the number of TH-IR

axons in the frontal cortex of adult female monkeys (Kritzer,

1995), indicating that manipulation of sex steroids can alter the

mesocortical DA system in some cases.

The developmental changes observed in the density of DA

innervation of ER could potentially be consequences of other,

more general developmental phenomena that occur during early

postnatal development, such as increases in cortical thickness or

myelination. However, both of these factors would be expected

to result in a decrease in the density of DA axons during early

postnatal development, the former by diluting DA axon density

relative to other cortical elements, and the latter by interfering

Figure 7. Total number of TH-IR varicosities (per 5000 µm2) in layers I–II (A,B), deep III (C,D) and VI (E,F) of area ER. Data are presented as mean values for individual animals (A,C,E)and as mean (±SE) values for the six age groups (B,D,F). The greatest magnitude of developmental change occurred in layer III, in a pattern similar to that observed in the total lengthof TH-IR axons (Fig. 6). In (D), age groups not sharing the same letter (located above each bar) are significantly different at P < 0.05.

424 DA Development in Monkey ERC • Erickson et al.

with antibody penetration. Consequently, the increase in density

of DA axons observed during this time period seems likely to

represent an actual change in these axons rather than an

epiphenomenon.

The developmental variation in the density of TH-IR and

DAT-IR axons could be attributed to one of several factors

specific to these axons. The fact that two different markers of

DA axons changed in parallel throughout development could

indicate that DA axons grow into layer III during early postnatal

development and then regress later. However, concomitant

changes in the concentrations of TH and DAT proteins could

have affected their detectability by immunocytochemical

techniques, without a difference in axon number. In fact, several

lines of evidence suggest that levels of TH and DAT protein

may be up- or down-regulated in synchrony. For example,

TH and DAT mRNA first appear in rat mesencephalon at about

the same time, and reach adult patterns over a similar period

of development (Fujita et al., 1993). Similarly, levels of both

TH and DAT mRNAs decline with increasing age in adult

human substantia nigra (Bannon et al., 1992). Finally, in mice

lacking the gene which encodes for DAT, the absence of

DAT protein is accompanied by a 90% decrease in TH protein

levels (Jaber et al., 1997). Although the available data do not

permit a distinction between changes in axon number or

protein content, both interpretations are consistent with

substantial shifts during postnatal development in DA

neurotransmission in the ERC.

Regional Comparison

A previous study in these same animals also revealed extensive

postnatal refinements in the DA innervation of prefrontal areas 9

and 46 (Rosenberg and Lewis, 1995). Consistent with our

findings in the ERC, the greatest changes in the density of DA

innervation of the prefrontal cortex were observed in layer III,

and little or no change occurred in layers I or VI. However, the

timecourse of the postnatal changes in DA innervation of frontal

regions was substantially different from those in ERC. As shown

in Figure 8, the density of TH-IR axons and varicosities in layer III

of area 9 progressively increased to reach peak values at 2–3

years of age, much later than in the ERC. Thus, although the

laminar location of postnatal changes in DA innervation may be

similar across the cortical mantle, the timing of these changes

exhibits regional specificity. These anatomical and temporal

patterns of postnatal refinements in cortical DA innervation may

be associated with regionally distinct functional consequences.

Functional Implications

The laminar specificity of changes in DA axon density provides a

clue as to which elements of cortical circuitry may be most

affected by developing DA afferents. DA inputs are thought to

modulate the response of cortical cells to excitatory inputs

(Berger et al., 1987; Goldman-Rakic et al., 1989; Law-Tho et al.,

1995). Therefore, the marked developmental changes in DA

innervation in the middle layers may ref lect changes in the DA

modulation of other afferents to the middle layers, such as

excitatory corticocortical connections (Insausti et al., 1987b). In

addition, the ERC receives inputs from multiple thalamic nuclei,

including centralis, paraventricularis, reuniens, lateral dorsalis

and pulvinaris (Insausti et al., 1987a). Most of these thalamic

nuclei are considered diffuse-projection nuclei, associated with

cortical terminations in layer I. Axons from reuniens, however,

have been shown to terminate across cortical layers I–IV in the

ERC (Ohtake and Yamada, 1989), and axons from pulvinaris

terminate in the middle layers in other cortical regions (Levitt et

al., 1995; Romanski et al., 1997). These findings suggest that

afferents from these thalamic nuclei may be candidates for

modulation by DA in layer III, and therefore inf luenced by the

marked developmental changes in DA innervation of this layer.

The mechanisms responsible for the laminar specificity of

these developmental changes remain unclear. One possibility is

that the branching patterns of individual axons may be

differentially inf luenced locally by neurotrophins or other

factors in each layer. Alternatively, the DA innervation of the

middle layers may derive from a different source than the

superficial and deep layers. For example, in the rat the DA

innervation of cortical layers I–III originates in cell groups

located in lateral A10/A9 in the ventral mesencephalon, whereas

the DA projection to the deep layers arises from medial A10

(Berger et al., 1991). Although comparable data are lacking

in the nonhuman primate, such an arrangement for the DA

innervation of the monkey entorhinal cortex would permit

regulation of axon branching at the level of the DA cell body,

perhaps ref lecting different inputs to the mesencephalic cell

groups.

The timecourse of developmental changes in DA innervation

may also be associated with regionally specific functional

consequences. Developmental changes in the DA innervation of

the prefrontal cortex are protracted and continue through

adolescence. Interestingly, measures of the functional maturity

of prefrontal cortex indicate that these regions do not reach their

full capacity until this same age (Alexander and Goldman, 1978;

Figure 8. Percentage change in the mean total axon length (A) and number ofvaricosities (B) for the five oldest groups of animals relative to animals <1 monthof age. Data shown are from deep layer III, the laminar location where the greatestdevelopmental change occurred in both ER (solid bars) and area 9 (open bars). Notethat the temporal pattern of change is different for the two areas, with peak values forboth axon length and number of varicosities occurring earlier in ER.

Cerebral Cortex Jul/Aug 1998, V 8 N 5 425

Diamond, 1990). The timecourse of the changes in DA inner-

vation of the prefrontal cortex thus parallels the developmental

changes in functional capacity of this region. It is possible that

the temporal pattern of changes in the DA innervation of

the ERC similarly parallels the functional maturation of the

ERC. Although the available data are limited, Bachevalier and

Beauregard (1993) have suggested that the contribution of

medial temporal lobe structures to some memory functions

in monkeys may reach adult levels by 3–6 months of age.

Consequently, the timecourse of developmental changes in the

DA innervation of the ERC identified in this study, with a peak

density of innervation occurring in the middle of the first year of

life and adult patterns established by 1 year of age, is consistent

with the idea that DA inputs play an important role in the

functional maturation of the ERC.

The regional differences in the timecourse of DA development

suggest that consequences of decreased DA innervation in the

ERC (Lewis and Akil, 1997) and prefrontal cortex (Akil and

Lewis, 1996; Okuba et al., 1997) in subjects with schizophrenia

may become manifest at different times. In the ERC, decreased

DA innervation may be related to the appearance of early,

prodromal cognitive deficits (Jones et al., 1993, 1994) due to

compromised medial temporal lobe function. In contrast,

abnormal DA innervation in the prefrontal cortex may be linked

to the emergence of the clinical symptoms of the disorder during

late adolescence (Waddington, 1993). However, it is not clear

when during development the DA innervation of these cortical

regions becomes abnormal in schizophrenics. This last point is

critical to understanding the consequences of decreased DA

innervation, since DA may have distinctly different roles in the

adult cortex and developing cortex.

In addition to modulating excitatory inputs in the adult

cortex, DA may have additional effects unique to development.

For example, DA may serve as a trophic factor. This idea has

been suggested by Berger et al. (1993), based upon the

observation that DA axons first arrive in the ERC early in

prenatal development, at embryonic day 56 in a 165 day

gestation. Neurogenesis in this region takes place between

embryonic days 38 and 70–75 (Rakic and Nowakowski, 1981),

and the arrival of DA afferents during this time raises the

possibility that DA may play a role in neuronal migration or

differentiation. Consistent with this idea, Lankford et al. (1988)

have shown that DA can inf luence growth cone motility, at least

in cultured neurons. Later in development, DA has been shown

to affect the morphology of cortical cells. For example, in rats

with neonatal lesions of the ventral tegmental area, the total

length of basilar dendritic arbors of pyramidal cells in the

prefrontal cortex is reduced by 30% (Kalsbeek et al., 1989).

These observations suggest a role for DA in development beyond

modulation of excitatory synaptic transmission.

In summary, the DA innervation of the primate ERC changes

substantially during postnatal development. The laminar

specificity of these changes is identical to that in frontal regions

of neocortex, but the timecourse is quite different and may be

related to the functional maturation of the regions. Together,

these findings suggest an important role for DA in shaping

cortical circuitry, and the current study has identified the period

during which the impact of DA on the circuitry of the primate

ERC may be particularly critical.

NotesThe authors thank Mary Brady and Richard Whitehead for excellent

technical assistance, Dr Allan Sampson and Claudia Matus for statistical

consultation, and Dr Susan Sesack for helpful comments in the

preparation of this manuscript. This work was supported by USPHS

grants MH43784, MH45156 and NS31937, NIMH Independent Scientist

Award MH00519 and the American Parkinson’s Disease Association.

Address correspondence to David A. Lewis, MD, Western Psychiatric

Institute and Clinic, University of Pittsburgh, Biomedical Science Tower

W1651, 3811 O’Hara Street, Pittsburgh, PA 15213, USA. Email: lewis@

cortex.psychiatry.pitt.edu.

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