A histochemical and immunohistochemical analysis of the subependymal layer in the normal and Huntington's disease brain

doi:10.1016/j.jchemneu.2005.05.001

Journal of Chemical Neuroanatomy

Volume 30, Issue 1, July 2005, Pages 55-66

https://doi.org/10.1016/j.jchemneu.2005.05.001 Get rights and content

Abstract

Previous studies in the rodent brain have characterised the cell types present in the subependymal layer, however the general organisation and cellular morphology of the adult human subependymal layer has not been demonstrated previously. In this study, we have demonstrated that the normal human brain subependymal layer contains three morphologically distinct types of cells, A, B and C type cells. The type A cells resembling migrating neuroblasts were located in the superficial part of the subependymal layer, type B cells resembling glial cells were evenly distributed throughout the subependymal layer and caudate nucleus, and type C cells that resembled progenitor cells were located in the deeper regions of the subependymal layer close to the caudate nucleus. We also examined the subependymal layer in the Huntington's disease brain to determine whether neurodegenerative pathology of the caudate nucleus (adjacent to the subependymal layer) altered the cellular composition of the subependymal layer. In the Huntington's disease subependymal layer there was a significant increase in the thickness of the subependymal layer compared with the normal subependymal layer (p < 0.01) and there was a 2.8-fold increase in the number of cells present in the Huntington's disease subependymal layer compared with the normal subependymal layer but the density of cells remained unchanged. As the grade of Huntington's disease increased, so did the overall number of cells in the subependymal layer. An increase in the number of type B cells was responsible for most of the increase demonstrated, however there was also an increase in the numbers of type A and C cells. To further characterise the human normal and Huntington's disease subependymal layer we used immunohistochemistry and antibodies against a range of projection neuron markers, interneuron markers, glial cell markers and GABA_A receptor subunits. The results demonstrated the presence of increased numbers of neuropeptide Y positive cells in the Huntington's disease subependymal layer compared with the normal subependymal layer, suggesting that neuropeptide Y neurons may play a role in progenitor cell proliferation. Also there was an increased level of the developmentally active GABA_A receptor subunit γ2 that indicates that the adult subependymal layer still retains the ability to proliferate. Taken together our results give a detailed description of the adult human subependymal layer and also demonstrate the plasticity of the human subependymal layer in response to Huntington's disease.

Introduction

In the developing brain the subependymal layer (SEL) is the birthplace of neurons and the time course of the development of neurons from stem/progenitor cells in the SEL has been well documented (Altman and Das, 1966, Altman, 1969, Smart, 1973, Smart, 1976). In the adult rodent brain, cells in the SEL are still mitotically active and retain a neurogenic potential (Tattersfield et al., 2004), however, the thickness of the SEL is reduced postnatally as the brain matures and the SEL becomes considerably less active with developmental maturity (Privat and Leblond, 1972, Smart, 1972, Smart, 1973, Smart, 1976, Garcia-Verdugo et al., 2002).

We have previously demonstrated that the adult human SEL contains progenitor cells that proliferate and form new neurons in response to Huntington's disease (HD) (Connor et al., 2001, Curtis et al., 2003a, Curtis et al., 2003b). However, the SEL in the adult human brain has not been previously described in terms of its detailed morphological and histochemical staining characteristics neither has it been described in the HD brain where the underlying caudate nucleus undergoes extensive degeneration (Ferrante et al., 1991, Hedreen and Folstein, 1995, Aylward et al., 1996, Vonsattel and DiFiglia, 1998). Doetsch et al. (1997) described the anatomy of the mouse SEL in terms of the distribution, morphology and staining characteristics of five main cell types (types A–E). Type A cells were described as migrating neuroblasts, believed to migrate within the rostral migratory stream to the olfactory bulb where they formed replacement interneurons; type B cells were glial cells that support the migration and differentiation of the type A cells and type C cells were putative precursor cells capable of differentiation down a number of different cell lineages. Type D cells were rare tanycytes and Type E cells were ciliated ependymal cells that line the lateral ventricle (Doetsch et al., 1997). In our study, the same criteria have been adopted for classifying the various cell types in the SEL of the human brain. We undertook this study to examine the morphology of mature cell types (neurons, glia, microglia) in the human SEL. In order to be comprehensive in our examination of the SEL we have immunostained the SEL for the presence of neurons that contain the neurotransmitter γ-amino butyric acid (GABA), as evidenced by calbindin, as well as their cotransmission factors enkephalin (ENK) and substance P (SP) (DiFiglia and Christakos, 1989, Waldvogel et al., 1991, Holt et al., 1997, Hontanilla et al., 1998). We also immunostained for the four different types of striatal interneurons containing parvalbumin, calretinin, choline-acetyl transferase (ChAT) and neuropeptide Y (NPY) (Kawaguchi et al., 1995). In this study, astrocytes were evidenced by glial fibrillary acidic protein (GFAP), immature astrocytes by vimentin and microglial cells by ferritin antibodies (Kaneko et al., 1989, Connor et al., 1994). Finally, we studied the SEL for the most common GABA_A receptor subunits in the basal ganglia, the α1, β2, 3 and γ2 subunits (Olsen and Tobin, 1990, Seeburg et al., 1990, Waldvogel et al., 1990, Wisden and Seeburg, 1992, Veenman et al., 1994).

Thus, we have investigated the human normal and HD SEL anatomy and morphology with special reference to the cellular composition (types A–E), mature cell types present in the striatum and SEL, cell numbers present and SEL thickness. The plasticity evident in the SEL during development suggests there may be significant alterations in the composition of the SEL in response to HD.

Section snippets

Human tissue collection

For this study, the basal ganglia from post-mortem human brains were obtained from the Neurological Foundation Human Brain Bank (Department of Anatomy with Radiology, the University of Auckland). The University of Auckland Human Subjects Ethics Committee approved the protocols used in these studies. Normal brains (detailed in Table 1) were received from cases with no history of neurological disease and on pathological examination showed no pathological abnormalities. HD brains (detailed in

The anatomy of the SEL in the normal and Huntington's disease brain

In the normal adult human brain the ependymal region was comprised of two layers, the ependymal layer (EPL) and the subependymal layer (SEL). The EPL was a single cell layer of darkly haemotoxylin and eosin stained, tightly packed ependymal cells forming a barrier between the cerebrospinal fluid of the lateral ventricle and the underlying brain tissue (Fig. 1). Immediately beneath the EPL was the SEL, a heterogeneous layer bound superficially by the EPL and by a deep myelin layer adjacent to

Discussion

The results from this study have demonstrated that the human SEL is a heterogeneous region mostly comprised of type A, B and C cells. This is in agreement with the results from studies of the mouse SEL performed by Doetsch et al. (1997). In HD, the SEL was on average 2.8 times thicker than that of normal brains. Also, there were 2.8 times as many cells in the HD brain SEL compared with normal brains. The major increase in cell numbers was predominantly a result of increased numbers of type B

Acknowledgements

We thank the Biomedical Imaging Unit in the Department of Anatomy with Radiology at the University of Auckland for their assistance in image production. This research was supported by the Health Research Council of New Zealand Programme Grant and by the Neurological Foundation of New Zealand. M.A. Curtis was funded by a Neurological Foundation of New Zealand, Miller Postgraduate Scholarship.

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A histochemical and immunohistochemical analysis of the subependymal layer in the normal and Huntington's disease brain

Abstract

Introduction

Section snippets

Human tissue collection

The anatomy of the SEL in the normal and Huntington's disease brain

Discussion

Acknowledgements

Brain Res. Brain Res. Rev.

J. Chem. Neuroanat.

Cell

Brain Res.

Neurosci. Lett.

Brain Res. Bull.

Neuroscience

Brain Res. Bull.

Trends Neurosci.

Brain Res.

Neuron

Brain Res.

Neuroscience

Brain Res.

Brain Res.

Neuroscience

Curr. Opin. Neurobiol.

Migration and differentiation of neural precursor cells can be directed by microglia

Proc. Natl. Acad. Sci. U.S.A.

Autoradiographic and histological studies of postnatal neurogenesis. IV. Cell proliferation and migration in the anterior forebrain, with special reference to persisting neurogenesis in the olfactory bulb

J. Comp. Neurol.

Autoradiographic and histological studies of postnatal neurogenesis. I. A longitudinal investigation of the kinetics, migration and transformation of cells incorporating tritiated thymidine in neonate rats, with special reference to postnatal neurogenesis in some brain regions

J. Comp. Neurol.

Basal ganglia volume and proximity to onset in presymptomatic Huntington disease

Arch. Neurol.

Endothelial nitric oxide synthase regulates brain-derived neurotrophic factor expression and neurogenesis after stroke in mice

J. Neurosci.

Stem cells and neurodegenerative diseases

N Z Med. J.

Isoforms of ferritin have a specific cellular distribution in the brain

J. Neurosci. Res.

Neurogenesis in the diseased adult human brain: new therapeutic strategies for neurodegenerative diseases

Cellcycle

Increased cell proliferation and neurogenesis in the adult human Huntington's disease brain

Proc. Natl. Acad. Sci. U.S.A.

Ultrastructural localisation of immunoreactive calbindin-D28k in the rat and monkey basal ganglia, including subcellular distribution with colloidal gold labeling

J. Comp. Neurol.