The corpus striatum, being a part of the basal ganglia, consists of groups of neurons deeply embedded inside the telencephalon and links functionally with the cortex and other parts of the basal ganglion (e.g., substantia nigra and the subthalamic nucleus). This essential area comprises the caudate, putamen, and globus pallidus and has been long known to contain various neurotransmitters and their receptors. These include acetylcholine, dopamine, glutamine, and serotonin (5-hydroxytryptamine; 5-HT) 2A/2C receptors (Bubser et al., 2001; Porras et al., 2002; Bishop et al., 2003; Napier et al., 2008), NMDA and AMPA glutamate receptors (Pollack et al., 1999, 2005), and dopamine D1 and D2 receptors in rat (Pollack et al., 1999; Yamada et al., 2007). Nicotinic ACh receptors have been found in mouse striatum (Grady et al., 2007). In recent years, nNOS neurons have been discovered to be essential functional subunits in the striatum of animals (Aguilera et al., 2007; Domek-Lopacińska et al., 2008; Galati et al., 2008; Watanabe et al., 2008). Concomitantly, recent psychiatric studies in patients advocated the involvement of 5-HT and NO in psychiatric patients (e.g., 5-HT2A receptor binding decreased in human OCD and NOS striatal neurons decreased in schizophrenics) (Adams et al., 2005; Lauer et al., 2005). However, most of these studies, with the exception of the patient studies, were done on animals with results being extrapolated for humans. In addition, the majority of striatal studies were focused on adults, with few on development. Human development of the corpus striatum is still very much an uncharted territory. The objective of this study was therefore to examine the development of the corpus striatum in the human fetus and to search whether serotonin2A receptors and nNOS positive cells are present in development.
This study focussed on the development of the corpus striatum in the fetus, using silver impregnation and immunohistochemistry. For the latter, we looked for nNOS positive cells and 5-HT2A receptors positive cells in the corpus striatum during development. During the initial formation of the corpus striatum, there was migration cells of the ganglionic eminence toward the putamen by 15–17 weeks of gestation. Process formation in the neurons started by week 17 and became very complex before term (31/32 weeks of gestation). By 25–27 gestational weeks, the globus pallidus already had two parts and the corpus striatum was similar to the adult in configuration. The nNOS positive cells appeared early (21–23 weeks in gestation) while 5-HT2A receptors positive cells were not observed until 31/32 weeks gestation. The number of positive cells in both groups was relatively small. It is anticipated that further developmental changes would occur in the postnatal/neonatal phases. Anat Rec, 2012. © 2011 Wiley Periodicals, Inc.
MATERIALS AND METHODS
Twenty-two aborted and dead fetuses were used in the study, with the permission of both the hospital and the parents. They were of the following gestation weeks: 15 (n = 7) to 17 weeks (n = 1), 21 weeks (n = 5), 23 weeks (n = 3), 25 weeks (n = 6), 27 weeks (n = 2), 31 weeks (n = 4), 32 weeks (n = 2). The protocol of this study was approved by the institutional ethics committee at Hangzhou Red Cross Hospital. The calvaria of the fetuses were opened approximately 1 h after death and the brains were dissected out (Wai et al., 2008). The regions of the corpus striatum with the neighboring diencephalon were isolated. The isolated brain tissues were sectioned coronally and fixed immediately in 4% paraformaldehyde overnight at 4°C. The specimens were then dehydrated in graded ethanol, cleared in xylene, and embedded in paraffin wax simultaneously in an automatic processing machine (Pathcenter, Shandon). Coronal paraffin sections of 6 μm thickness were serially cut from each block anteroposteriorly. For every 10 sections, the first five consecutive sections were taken for immunohistochemistry of neuronal nitric oxide synthase (nNOS) and serotonin2A receptor (5-HT2A), and morphological studies (i.e., cresyl violet and Bielschowsky silver staining) (Fang, et al., 2005). All brain sections were processed simultaneously in the same bath of solutions in our study.
For immunostaining, all steps were carried out at room temperature (See Fang et al., 2011) unless otherwise stated. In brief, brain sections were first dewaxed, rehydrated, and permeabilized (antigen retrieval) with 1x phosphate buffered saline (PBS) supplemented with 0.1% Triton-X and 0.05% Tween 20 for 10 min, followed by three rinses in 1x PBS (5 min each). The endogenous peroxidase activity was blocked by 3% hydrogen peroxidase in absolute methanol for 45 min. After three rinses in 1x PBS, non-specific binding was suppressed by 1.5% normal blocking serum (prepared from the same animal species for the secondary antibody) for 30 min. Thereafter, the sections were either incubated with diluted primary antibodies against neuronal nitric oxide synthase (nNOS 1) (1:500; BD Biosciences, USA; N31020) and serotonin2A receptors (5-HT2A) (1:500; Abcam Inc., USA; ab16028) overnight at 4°C. The next day, after three rinses with 1x PBS, the sections were incubated with respective diluted biotinylated secondary antibodies (1:200; Zymed® Laboratories, Inc., San Francisco, CA) for 2 h, and rinsed again. Subsequently, the sections were incubated with diluted streptavidin-HRP (horseradish peroxidase) conjugated solution (1:200; Zymed® Laboratories, Inc., San Francisco, CA) for 2 h, and rinsed again. The immunohistological positive stainings were visualized by 0.05% 3,3′-diaminobenzidine tetrahydrochloride in 1 x PBS containing 0.01% hydrogen peroxide (H2O2). The above staining included negative controls with omission of the primary antibody, which did not show any immunoreaction. The sections were then dehydrated, cleared and mounted with Permount® (Fisher Scientific, Hampton, VA, USA).
Bielschowsky Sliver Staining
For Bielschowsky silver staining, brain sections were first rehydrated in graded ethanol and then immersed in 20% sliver nitrate solution for 2 h at 37°C. Subsequently, the sections were washed with reducer solution (5% formaldehyde and 20% absolute ethanol) for 4 min, followed by a 2-min wash in 10% ethanol. After that, the sections were incubated in ammonia sliver nitrate solution (10% silver nitrate; 49.5% ammonia in ethanol) for 2 min. After incubation, the sections were put in reducer solution for 5 min and rinsed again with distilled water. Fixation of the color was done by immersing the sections in 5% sodium thiosulfate for 2 min and then washing in distilled water before dehydration and mounting.
Quantitative analysis of the density of nNOS and 5-HT2A receptor positive cells was performed by randomly selecting 30 microscopic fields (total n = 30, size 1,500 μm2, magnification 200×) from sections of the corpus striatum of four arbitrarily assigned groups (group 1: 15–17 gestation weeks; group 2: 21–23 gestation weeks; group 3: 25–27 gestation weeks; group 4: 31–32 gestation weeks). From each specimen, three slides spaced 60 μm apart were taken and from each slide, 10 random fields were observed and computed for mean±SD.
Statistical analysis was performed by SPSS 16.0 software (Statistical Package for the Social Sciences). First, all data was analyzed by the Kolmogorov-Smirnov test for normal distribution and Levene's test of Equal Variances for variance homogeneity. A one-way ANOVA was used for comparison of three-group design (in case of nNOS), with post-hoc T-tests. A level of P < 0.05 was considered to be statistically significant. For the positive 5-HT2A cells, preliminary observation showed that they were only present in the corpus striatum at the late stage (31/32 weeks) of gestation. Furthermore, some differences between the caudate and lentiform nucleus were observed. In this case, the caudate and lentiform nucleus were separately considered as two groups. The data were analyzed by a two sample t-test.
Our study indicated that vigorous development was evident in the corpus striatum in our earliest specimen at 15–17 weeks gestation group, where strands of cells from the ganglionic eminence ran from the ventricular surface of the diencephalon toward the putamen of the lentiform nucleus (Fig. 1). Here, both the caudate and the lentiform nuclei were clearly present, and by 17 weeks of gestation the lentiform nucleus separated into a denser (putamen) and a lighter region (globus pallidus) (Fig. 2). Higher magnification using silver impregnation revealed that some cells (i.e., the majority of neurons by morphology) began to send out processes, especially those in the globus pallidus (Fig. 3), while most other cells in the caudate and putamen still lacked conspicuous processes (Fig. 4). By 21 weeks of gestation, short conspicuous processes were evident in the cells of the caudate and putamen (Fig. 5) and one could indeed identify both bipolar and multipolar neurons. By 27 gestational weeks, a more complicated branching of the processes (Fig. 6) in neurons was observed and spicules was also easily visualized (Fig. 7). The configuration of the lentiform nucleus was very much like those seen in adults, with a putamen and two parts of the globus pallidus, at the even earlier stage of 25–27 weeks of gestation, with the cells in the putamen also appearing to have a columnar arrangement (Fig. 8). Increase of fibers (processes of neurons and glia as well as afferent and efferent fibers) was also a feature from 27 to 31/32 gestation weeks (Fig. 9).
No nNOS positive cells could be identified in the corpus striatum from earliest specimen of 15–17 weeks gestation. At mid (21–23 weeks) gestation, within either the caudate or the lentiform nucleus, nNOS positive neurons were sparse. But at the 25–27 weeks, the density of nNOS-positive cells in the stratium increased significantly and was distributed homogeneously Fig. 12A. It then reduced markedly at 31–32 weeks. The nNOS positive neurons were of large and small sizes (Fig. 10).
In contrast, no 5-HT2A receptor positive cells could be detected in the corpus striatum at earlier (15–17 weeks) and mid (21–23 or 25–27 weeks) gestation. 5-HT2A receptor positive cells were seen only at the late stages before birth (31/32 weeks of gestation) and did not appear in large numbers. There were a slightly greater number of 5-HT2A receptor positive cells in the caudate (histogram B, p < 0.05) (Fig. 12B). 5-HT2A receptors positive cells were of a small size (Fig. 11).1-12
This study presented two de novo points: (1) the development of the corpus striatum in the human started before 20 weeks and became morphologically similar by architecture to those of the adult by 27 gestation weeks, with refinement in branching of processes and increase in the amount fibers until late gestation (31/32 weeks); (2) there were nNOS positive cells and cells with serotonin2A receptors, but the latter started late in development and were only few in number. It is likely that more positive cells of both nNOS and 5-HT2A receptor will develop after birth, a point important for future psychiatric research and behavior (Lauer et al., 2005; Fritzen et al., 2007). nNOS, apart from its involvement in psychiatric disease, may also play a role in cell death, as has been observed to occur in Alzheimer's disease (Yew et al., 1999). However, in our preliminary studies, we have colocalized nNOS positive cells in the fetal striatum using the TUNEL technique for DNA damage, and out of 300 randomly chosen cells positive with nNOS at stage 32 weeks of gestation, and TUNEL positivity was only present in 43 cells. Twelve types of cells with nNOS positivity were described by Johannes et al. (2003). In our study we reported large and small cells. The latter was akin to Johannes's category of large efferent NOS striatal neurons.
The striatum and the neocortex are the brain regions most known to be particularly vulnerable to acute injuries such as hypoxia or ischemia. The fluctuation in the number of nNOS positive cells in these areas during fetal development is interesting. At late (31–32 weeks) gestation, a transient increase in nNOS neurons within the striatum provides additional insight into the vulnerability of the striatum, and their possible involvement in neurodegenerative pathologies (Ezquer et al., 2006). To some extent, our data were consistent with developing patterns of nNOS neurons in the rat striatum. By embryonic day 18, the density of nNOS neurons begins to increase and peaks on postnatal day 5. It then decreases as the striatum grows. Such age-related changes for nNOS has also been documented for the hippocampi of postnatal mice (Hayakawa et al., 2008), in the cerebral cortices of rats of ages 3–14 months (Bustamante et al., 2008) and in the hippocampi and the cerebral cortices of rapid aging (SAMP8) mice (Colas et al., 2006; Han et al., 2010). The fluctuation might be tied with onset of normal/abnormal but specific behavior (Colas et al., 2006; Balda et al., 2008). It is highly likely that the numbers of nNOS and 5-HT2A receptor positive cells may further fluctuate perinatally and postnatally and that the postnatal developmental period is also a very critical period of transmitter development and interaction in the brains of children. Unfortunately specimens in this period are hard to obtain. In some cases, in the rabbit for example, the continuation of neurogenesis has been recorded in the caudates of adults (Luzzati et al., 2006). The further study of nNOS and 5-HT2A receptor positive cells during development is therefore warranted.
We thank Henry Davies for assistance in the final preparation of this manuscript.