Cell Tissue Res (2015) 359:279–294 DOI 10.1007/s00441-014-1988-4

REVIEW

Immunocytochemical markers of neuronal maturation in human diagnostic neuropathology Harvey B. Sarnat

Received: 23 April 2014 / Accepted: 8 August 2014 / Published online: 17 September 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Histological descriptions of morphogenesis in human fetal brain and in malformations and tumours can now be supplemented by the timing and sequence of the maturation of individual neurons. In human neuropathology, this is principally achieved by immunocytochemical reactivities used as maturational markers of neuronal properties denoted by molecules and cell products. Cytological markers can appear early and then regress, often being replaced by more mature molecules, or might not exhibit the onset of immunoreactivity until a certain stage of neuronal differentiation is achieved, some early, others intermediate and some late during the maturational process. Inter-specific differences occur in some structures of the brain. The classification of markers of neuronal maturation can be based, in addition to those mentioned above, on several criteria: cytological localisation, water solubility, biochemical nature of the antigen, specificity and various technical factors. The most useful immunocytochemical markers of neuronal maturation in human neuropathology are NeuN, synaptophysin, calretinin and other calciumbinding molecules, various microtubule-associated proteins and chromogranins. Non-antibody histochemical stains that denote maturational processes include luxol fast blue for myelination, acridine orange fluorochrome for nucleic acids, mitochondrial respiratory chain enzymes and argentophilic impregnations. Neural crest derivatives of the peripheral nervous system, including chromaffin and neuroendocrine cells, have special features that are shared and others that differ

The author has no conflicts of interest or financial disclosures to declare. H. B. Sarnat (*) Departments of Paediatrics, Pathology and Laboratory Medicine (Neuropathology) and Clinical Neurosciences, University of Calgary Faculty of Medicine and Alberta Children’s Hospital Research Institute, 2888 Shaganappi Trail NW, Calgary, AB T3B 6A8, Canada e-mail: [email protected]

greatly between lineages. Other techniques used in human diagnostic neuropathology, particularly as applied to tumours, include chromosomal and genetic analyses, the mTOR signalling pathway, BRAF V600E and other tumour-suppressor gene products, transcription products of developmental genes and the proliferation index of the tumour cells and of mitotic neuroepithelial cells. Keywords Markers of neuronal maturation . Immunocytochemistry . Histochemistry . Calcium-binding proteins . Microtubule-associated proteins . Mitochondrial enzymes . Neural crest derivatives

Introduction The histopathological examination of brain tissue in humans and experimental animals often involves similar methods but differences also exist for several reasons. The primary goal in human diagnostic neuropathology is patient care. Animal studies, by contrast, are designed to improve our understanding of cellular development and function. Human neuropathologists rely heavily upon immunocytochemistry (ICC) to supplement histological observations with haematoxylineosin (H&E) or Nissl (cresylviolet) stains. Many other techniques further confirm diagnoses, particularly in neural tumours but nevertheless ICC retains its place in diagnostic neuropathology as the primary tool. Brain tumours In addition to the immunoreactivities of cellspecific markers to help distinguish neuronal and glial lineages of neoplastic cells, several other approaches are valuable as supplements to help confirm diagnosis. (1) Chromosomal and genetic analyses might be useful. For example, more than 25 % of oligodendrogliomas and mixed oligodendroglioma/ astrocytoma neoplasms exhibit a unique co-deletion of

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chromosomal loci at 1p and 19q; this is not only useful for primary diagnosis but also has a predictive value of the response to treatment and prognosis (Lin et al. 2014; Hewer et al. 2014; Rodríguez et al. 2014). Pre-operative neuroimaging by magnetic resonance imaging (MRI) does not identify this co-deletion demonstrated in individual tumour cells (Reyes-Botero et al. 2014), although magnetic resonance spectroscopy demonstrates the biochemical composition of tumours to some extent, for example, the relative ratios of choline, N-acetyl-aspartate and taurine. Neoplastic neuroepithelial cell derivatives often show aberrations in karyotype. (2) Germ-line polymorphism is associated with isocitric dehydrogenase (IDH1) enzymatic mutations, many of which are also 1p-19q co-deletions and an R132H protein of IDH1 can be demonstrated immunocytochemically in tissue sections (Rodríguez et al. 2014; Esmaeili et al. 2014). Distinct phospholipid metabolic profiles are seen in gliomas with defective IDH1, expressed as decreased phosphoethanolamine and increased glycerophosphocholine (Esmaeili et al. 2014). (3) Molecular testing, also modified for ICC, is available to demonstrate mutations in BRAF V600E, part of the RAS signalling pathway, in melanoma, in many diverse carcinomas and also in some brain tumours such as some gangliogliomas (Brown et al. 2014; Pearlstein et al. 2014; Fisher et al. 2014; Bledsoe et al. 2014; Dvorak et al. 2014). However, other gangliogliomas are BRAF-negative (Lummus et al. 2014). (4) In neural tumours that are germline or somatic mutations, particularly if BRAF V600E mutations are present, CD34, a marker of stem cells and endothelial cells, is over-expressed in cells and neuropil (Chappé et al. 2013; Prabowo et al. 2014; Aronica and Crino 2014). The tumour that most strongly shows CD34 is the ganglioglioma but, at times, dysembryoplastic neuroepithelial tumours and pseudoxanthomatous astrocytomas also exhibit this feature (Chappé et al. 2013). (5) Other oncogenes are also useful for the diagnosis of neoplasms involving the brain, such as the loss of function of p53, one of the earliest tumour-suppressor genes discovered (Mulligan and Mole 1993; Crawford et al. 2007). (6) Some developmental genes that are not oncogenes are useful in diagnosis of some brain neoplasms; Sonic hedgehog (SHH), Wingless (WNT) and NOTCH or their growth factor receptors in cerebellar medulloblastoma in children are useful not only diagnostically but also e.g., in targeting treatment and predicting prognosis (Mulligan and Mole 1993). (7) In addition to estimating the number of mitoses in a field, proliferating cell nuclear antigen (PCNA; Ki67; MIB-1) is useful for determining the proliferative index in tumours and also in sites of expected neuroepithelial proliferation, such as the ventricular zone of the fetal cerebral hemisphere in the first half of gestation and the outer molecular zone of the cerebellar cortex for several postnatal months. (8) In situ hybridisation and autoradiography techniques are not well suited to human neuropathology and are rarely

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used. (9) Some neuroanatomical techniques, such as postmortem implantation of DiI crystals, a fluorescent carbocyanin dye for axonal tracing (Honig and Hume 1989; Auclair et al. 1993), are occasionally applied to human brain for research (Hevner 2000) but are not used as diagnostic tools. (10) Traditional histochemical stains of frozen sections are still routinely performed for muscle biopsies but only occasionally for human brain tissue, except for frozen sections stained with H&E for intraoperative provisional diagnosis. Fetal brain and malformations In immature brain and in developmental malformations, ICC remains the principal technique for examination and for the determination of maturation by neuropathologists (Sarnat 2013). Nevertheless, the genetic basis underlying many malformations is known, particularly for neuroblast migratory disorders such as the lissencephalies and periventricular and subcortical laminar heterotopia and hence genetic studies can be performed but even some of these are now studied by ICC. Examples are the transcription products of the genes Doublecortin (DCX), Aristless (ARX), lissencephaly-1 (LIS1), L1-CAM and other microtubule-associated proteins and many other gene transcription products such as those of SHH. The genetic alterations associated with somatic mutations can also be tested: TSC1 and TSC2 for tuberous sclerosis complex, AKT3 for isolated hemimegalencephaly and AKT1 for hemimegalencephaly in Proteus syndrome, one of the epidermal naevus syndromes (Lindhurst et al. 2011; Poduri et al. 2012, 2013; Lee et al. 2012). Studies of the mTOR (mammalian target of rapomycin) signalling pathway and RAS pathway and also the demonstration of an abnormally phosphorylated and upregulated tau protein can be demonstrated to confirm the diagnosis (Crino 2005, 2011; Sarnat et al. 2012; Prabowo et al. 2013; Tsai et al. 2014; Wang et al. 2014). The ultrastructure of the neurons is abnormal (Sarnat et al. 2012). Differences in the performance of studies of neuropathy between humans and laboratory animals Amongst differences between human neuropathology and experimental animal neuropathology, residency training and experience are not the same as research training for a doctorate degree or postdoctoral fellowship in neuroscience. The resources available in neuropathology depend upon hospital laboratory budgets in most countries and on private insurance in the U.S. rather than on research grant support and, hence, costs must be factored into human pathology, although grant-funded research must also correspond to allocated funds. Requirements of university and government Ethics Committees in approving research projects differ substantially between the use of human tissues and those of laboratory animals and funding is also closely tied to this approval; even the application of diagnostic tests to

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human tissue not for research and the disposition of such tissues after the diagnostic report is complete must comply with constraints imposed by an Ethics Committee. Post-mortem protein degradation or autolysis is generally not a factor in animal studies but can be a major impediment to the interpretation and reliability of autopsy studies in human brain tissue, because of the inevitable delay in tissue fixation. However unpleasant, this is an unavoidable topic that must be addressed by all human neuropathologists who perform autopsies. Some cellular proteins are quite resistant to autolysis and ICC is reliable even after 5 days, e.g., vimentin and synaptophysin. Others are more fragile and undergo postmortem breakdown so that they are no longer reliably recognised by the antibody after the first 24 h, e.g., neuronal nuclear antigen (NeuN) and chromogranins (Sarnat 2013). Inter-specific differences in some brain structures Some structures are uniquely human without a counterpart in other species, most notably the speech regions of the left cerebral cortex. Other human structures are shared only with apes and not even with lower primates such as the macaque monkey. An example is the medullary arcuate nucleus, which surrounds the pyramidal tracts (Baizer 2014), the agenesis of which has been implicated as a cause of sudden infant death syndrome (Filiano and Kinney 1992; Matturi et al. 2000). The arcuate nuclei were regarded by Olszewski and Baxter in 1954 as merely a caudal extension of the pontine nuclei of the basis pontis, because of their continuity at the pontomedullary junction and their similar cytoarchitecture with Nissl stains (cresylviolet) but recent immunocytochemical data show differences in reactivity for calcium-binding water-soluble proteins, such as parvalbumin and calretinin (Baizer 2014; Baizer and Broussard 2010; H.B. Sarnat, unpublished personal observations). Another example is the immunoreactivity of the red nuclei of the midbrain to calbindin D28k, another calcium-binding protein; in the rodent, little or no difference has been seen between the parvocellular and magnocellular neurons (Wang et al. 1996) but, in the human, the distinction is sharp (Sarnat 2013; Wang et al. 1996; Ulfig and Chan 2002; Ulfig 2002). Dopaminergic neurons of the substantia nigra are highly variable in NeuN reactivity in rodents (Kumar and Buckmaster 2007; Cannon and Greenamyre 2009) but are consistently reactive in humans (Sarnat 2013).

Criteria of neuronal maturation Maturation of the undifferentiated neuroepithelial cell to neuroblast and then to neuron can be defined by several processes. The neuron is unique amongst cells, because it is both secretory and has an electrically polarised, excitable

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membrane; the axon might be just as important. Muscle cells have an excitable membrane but are non-secretory; many epithelial, endocrine and exocrine cells are secretory and their membranes might be weakly polarised and associated with adhesion molecules that ensure the separation of apical and baso-lateral membrane constituents to enable an epithelium to be maintained as adherent cells but the polarised membrane of such cells is not excitable and does not generate an action potential. These two criteria thus are primordial in defining the maturation of the neuron. However, pancreatic islet cells can also trigger action potentials in response to increasing serum glucose concentrations, causing the elevation of calcium influx and enhanced insulin granule exocytosis (Drews et al. 2010; Rorsman and Braun 2013; Fridyland et al. 2013). Genetic programming of neuronal differentiation probably begins even during the mitotic phase of the neuroepithelial cell. Microtubules form the strands of the mitotic spindle and are important in the post-mitotic cell, because they determine cellular polarity, growth, differentiation and migration. Microtubules and actin intermediate filaments accumulate first to form filopodia and then polymerisation of the actin causes an interaction between these cytoskeletal elements to induce the formation of neurites, both dendrites and axons, including collateral axonal branching (Sainath and Gallo 2014). The sequence of cellular migration and of the formation of cytoskeletal elements is of primordial importance in neuronal maturation and is associated with the progressive synthesis of specific proteins that can be immunocytochemically demonstrated in human neurons during maturation. Various gene regulatory programmes govern the expression of synaptic proteins in the neuronal and neuroendocrine branches of the sympatho-adrenal system; Dicer-1-dependent regulation facilitates the establishment of high neuronal mRNA levels for synaptic proteins and represses neurofilament messages in neuroendocrine cells so that neurites do not form (Stubbusch et al. 2013). The development of an electrically polarised plasma membrane and a resting membrane potential first requires the formation of the Na+/K+ energy pump to produce ion gradients between the intracellular and extracellular spaces. Closely associated are the formation of ion channels within the membrane and membrane receptors for chemical neurotransmitters, which further develop into specialised synaptic membranes. As the neuroblast matures, the sprouting of the axon and later of collateral axonal branches occurs during the course of migration. The growth of dendrites always follows axonal sprouting and generally does not take place until migration is complete and the cell is in its final position. Dendritic branching and spine formation follow the development of the primary dendritic trunk. Synaptogenesis is closely associated. The neurites also help to determine the mature cytomorphology of the neuron.

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Neurotransmitter biosynthesis marks the initiation of the secretory function of the neuron and is accompanied by a proliferation of mitochondria and increase in cytoplasmic ribosomes. Enzymes of biosynthesis and of degradation of the transmitter must also be produced by the cell. An example is acetylcholine (ACh) for which choline acetyltransferase (CAT) and acetylcholinesterase (AChE) are the principal enzymatic proteins. The development of neuron-specific proteins is not the same as that of transmitter biosynthesis but the various proteins of the nucleus and cytoplasm not only distinguish neurons but also the types of neuron. This feature of maturation provides a basis for the immunocytochemical tissue markers used to denote maturation in normal human fetal brain and in malformations.

Neural crest derivatives of the peripheral nervous system No neurons in the central nervous system (CNS) have been shown to be derived from the neural crest, although a few neural crest cells possibly do not migrate peripherally and remain expressing neural crest markers. All nerve cells of the peripheral nervous system, by contrast, are neural crest derivatives. These include: (1) sensory neurons of the dorsal root ganglia; (2) autonomic neurons of the sympathetic chain and parasympathetic neurons, such as those of the enteric plexi; (3) chromaffin cells, such as those of the adrenal medulla; (4) neuroendocrine cells, such as the pancreatic islets of Langerhans (some investigators would not classify islet cells as neuroendocrine, because they are endodermal derivatives but concepts of the traditional three embryonic germ layers are rapidly changing, in part because many of the same organiser genes are expressed in all three layers, although their functions might differ and neural crest cells are now recognised to be able to give origin to mature tissues of each germ layer; (Hall 2009); and (5) chemoreceptor glomus cells, such as those of the carotid body. Although chromaffin and neuroendocrine cells fulfil the basic criteria of neurons, because they are secretory and have electrically polarised membranes, they differ fundamentally from most neurons in that they do not form neurites and their secretory products (transmitters) are released into the blood stream rather than into synaptic clefts. Nevertheless, as with sympathetic chain neurons, chromaffin cells of the adrenal medulla receive afferent innervation from pre-ganglionic sympathetic neurons in the lumbar spinal cord, whose fibres reach the adrenal gland via the splanchnic nerve (Schober and Unsicker 2001). The early development of the progenitors of chromaffin cells and sympathetic neurons depends upon a common set of transcription factors with overlapping but quite distinct functions during development (Huber 2014). Proto-neural genes also direct the development of a wide range of endocrine cells in addition to chromaffin

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cells, including pancreatic islet cells (Arda et al. 2013), pituitary cells (Zhu et al. 2007) and diffuse neuroendocrine cells throughout the mucosa lining the lumen of the digestive and respiratory tracts (Ito et al. 2001). Many differences between neurons and chromaffin/neuroendocrine cells are so wide that some investigators do not even consider the latter to be neurons. A treatise by Huber, distinguishing neuronal and neuroendocrine lineage and differentiation, is included in this current special issue (Huber 2014). Sympathetic chain neurons and chromaffin cells such as those of the carotid body both require Hes1 expression for their differentiation, perhaps because both are of neural crest origin (Kameda et al. 2012). Nerve growth factor signalling is another important factor required not only for survival but also for the initial differentiation of nociceptor dorsal root ganglionic and sympathetic neurons (Ernsberger 2009). Cell surface receptors for the glial-derived neurotrophic factor ligands, namely neurturin and artemin, are expressed in subpopulations of sympathetic and dorsal root ganglionic neurons (Ernsberger 2008).

Classifying tissue markers of neuronal maturation Tissue markers applied to sections are distinguished from the more general term, biomarkers, which generally denote the quantitative measurement of a protein or gene product in a body fluid of a living subject, usually blood or cerebrospinal fluid (CSF). Examples of biomarkers useful in detecting neuronal death in humans, particularly in the CSF of infants and children, are S-100β protein, neuron-specific enolase (NSE) and neurofilament protein (NFP; Shahim et al. 2013). These biomarkers are now used to predict the outcome of encephalopathic neonates treated with hypothermia (Massaro et al. 2014). Because antibodies for ICC are available for all of these proteins, if the clinical biomarker results are available and particularly if elevated, the tissue marker can be applied for comparison in a brain resection or at autopsy, together with appropriate age-matched controls (Sarnat 2013). In considering the many immunocytochemical reactivities that can be applied as tissue markers of neuronal maturation, these specific immunoreactivities can be classified in several ways in modern neuropathology. Classification is important to demonstrate the relationships of one to another from various perspectives; these are identified by the subheadings below. Most immunoreactivities can be applied to standard formalinfixed paraffin-embedded sections, a great advantage in human neuropathology. Tables 1 and 2 summarise these markers by combining several perspectives of neuronal maturation and Table 3 deals with glial maturation. Many more criteria could be cited, for example, gene expression, transcription factors, RNA analysis, membrane receptors, ion channels and cytoskeletal development but these are not yet practical for measurements in human neuropathology at this time and remain as

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Table 1 Immunocytochemical markers of maturation of neurons and neuroendocrine cells Antibody against

Nature of protein

Water solubility

Timing in cellular maturation

Neuronal nuclear antigen (NeuN) Synaptophysin Chromogranins Calretinin

Nucleoprotein Glycoprotein of synaptic vesicle membrane Glycosylated calcium-binding Calcium-binding for GABAergic interneurons

No No Yes Yes

Late Late Late Early

Paravalbumin Calbindin D28k S-100α protein Enolasa especifica para neuronas Microtubule-associated protein 2 (MAP2) Microtubule-associated protein 1/5 (MAP1/5) Doublecortin microtubule-associated protein (DCX) Tau microtubule-associated protein Neurofilament protein (NFP; SMI32) Protein gene product 9.5 (PGP9.5)

Calcium-binding Calcium-binding Cytoplasmic calciumbinding neuronal protein Glycolytic enzyme Cytoskeletal Cytoskeletal Cytoskeletal Cytoskeletal Cytoskeletal Non-specific cytoplasmic

Yes Yes Yes Yes No No No No No Yes

Intermediate Intermediate Intermediate/late Intermediate Early Early Early Early Intermediate Intermediate

future goals for augmenting the existing immunocytochemical and histochemical criteria. Positive (progressive increase) and negative (progressive restriction) markers A positive marker is one that appears at a certain stage of cellular maturation. Positive markers represent the majority of immunoreactivities used for the purpose of determining cellular differentiation. A negative marker, by contrast, is one that is expressed in primitive, poorly differentiated cells and regresses or disappears with maturation, often being replaced by a positive marker. An example is vimentin and nestin intermediate filaments being replaced by NFPs in neurons and by glial fibrillary acidic protein (GFAP) in astrocytes; they might co-exist for a transitional period of overlap. Another example is non-neuronal enolase (NNE) expressed in early differentiating neuroblasts, but then replaced by NSE. Alternative terms for positive and negative markers are progressive increase and progressive restriction markers (Ernsberger 2009). Whereas positive and negative are rather bland and non-descriptive, they are understood by most human neuropathologists.

Time of onset of expression: early, intermediate, late Early neuronal markers are those expressed even before cellular migration is initiated or other signs of neuroblast maturation are evident. Examples include calretinin and most of the microtubule-associated proteins (MAP), including some that that are specifically defective in certain neuroblast migratory disorders, such as the DCX transcription product, which is a cause of subcortical laminar or “band” heterotopia in humans. Intermediate markers are those expressed during and/or after migration. Examples are protein gene product 9.5, NSE and NFP. Late markers occur during the final stages of neuronal maturation when the excitable plasma membrane is established with membrane ion channels and receptors and when neurotransmitter biosynthesis has been initiated. Examples are NeuN, synaptophysin and chromogranin-A (CgrA). Although CgrA is more commonly used as a neuroendocrine marker, it is also an important neuronal marker in the CNS, for example, in the hippocampus (Sarnat 2013). In general, late markers are expressed only after neuroblast migration is complete and the neuron is at its mature site, although some neurons mature and develop both axonal and

Table 2 Histochemical (non-immuno-) markers of neuronal maturation Marker

Subcellular product identified

Water solubility

Timing in cellular maturation

Luxol fast blue (LFB) Silver impregnations: Golgi, Bielschowsky Sevier-Munger, Bodian Mitochondrial respiratory chain oxidative enzymes: complexes I, II, IV Periodic acid-Schiff

Myelin Neurofilaments and other cytoskeletal elements

No No

Late Intermediate

Mitochondrial activity

Yes

Intermediate

Glycogen, glycoproteins

No

Intermediate (poor reliability)

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Table 3 Immunocytochemical markers of maturation of glial cells Antibody against

Nature of protein

Water solubility

Timing in cellular maturation

Glial fibrillary acidic protein (GFAP)

Mature astrocytic filaments; not oligo-dendrocytes, ependyma Transitory early filaments; also for regenerating or proliferating glia Transitory early filaments; also for regenerating or proliferating glia Cytoplasmic protein of astrocytes and oligodendrocytes Nucleoprotein of oligodendrocytes

No

Intermediate/late

No

Early/Intermediate

No

Early

Yes Yes

Intermediate Late

Vimentin Nestin S-100β protein Olig2

dendritic connections before or during migration, such as cerebellar granular cells and can express NeuN earlier than would be expected (Weyer and Schilling 2003; Sarnat 2013). Antibodies that identify the synaptic vesicle membranes, such as synaptophysin, are another example of late markers. Cytological localisation The subcellular localisation of expression is another criterion of classification: nuclear (e.g., NeuN); cytoplasmic (e.g., calretinin, CgrA, NSE) or at the surface of the plasma membrane (e.g., synapse-related soluble calcium-binding proteins); synaptic vesicle membranes (e.g., synaptophysin). Nuclear markers have an advantage over cytoplasmic markers in fetal brain, because nuclei are usually relatively large in relation to the sparse cytoplasm of immature neurons and hence reactivity is easy to detect in the nucleoplasm and is sometimes subtle and more difficult to discern in the thin cytoplasmic rim of the cell in cut sections. Other examples of nucleoprotein markers, apart from NeuN, include Olig2 for oligodendrocytes (Sakuma et al. 2014), Ki67 proliferating cell nuclear antigen and many antibodies against viruses such as Herpes. Water-soluble or insoluble Water-soluble molecules might be secretory but are not structural proteins (e.g., calbinden D28k; CgrA), whereas insoluble proteins might serve as structural elements of the cytoskeleton or membranes (e.g., NFP; MAP proteins; synaptophysin). Water-soluble proteins are distributed throughout the cytoplasm and often display the small ramifications of the dendritic tree (e.g., calbinden D28k in Purkinje neurons of the cerebellar cortex, reminiscent of Golgi impregnations; Katsetos et al. 1993) but different concentration gradients might occur between the soma and axon (e.g., Cgr). Molecular structure of the antigen, specificity and physiological factors The biochemical structure of the antigen against which immunocytochemical antibodies are applied and their physiological functions provide other potential bases of classification of markers. Examples are nucleoproteins; structural components of plasma or synaptic vesicle membranes;

calcium-binding molecules; secretory products; mitochondrial enzymes; nucleic acids; and glycogen. Various physiological conditions of the tissue, including its vascular perfusion, availability of oxygen and means of eliminating toxic degradation products, must be considered as factors that can alter the timing of expression of immunocytochemical markers. Tissue pH is another factor that might alter cellular maturation (Carlin 2013). The thickness of the section must be uniform when comparing different brains, because the intensity of reactivity might appear weaker in thin sections than in thicker ones. In our laboratory, we generally use paraffin sections at a thickness of 6 μm for human brain.

Usefulness of immunocytochemical markers in assesssing neuronal maturation The current standard of practice in human neuropathology is based on immunoreactivity. Except for the simplest of cases that can be diagnosed by H&E alone, various immunocytochemical markers are applied and are particularly useful in the diagnosis of tumours and neurodegenerative diseases and even to localise neural elements within non-neural tissues. More recently, immunocytochemical markers have been applied to developing fetal and neonatal brain and especially to malformations of the nervous system (Sarnat 2013). Whereas more refined methods of nucleic acid analysis and the identification of membrane receptors, ion channels and cytoskeletal elements provide useful supplementary data, these procedures are not generally available to the practicing neuropathologist for application to human tissues. Gene analysis is now applied routinely to some CNS tumours but neoplastic cells have a different profile from normal neurons and glial cells and this aspect of neoplasia is beyond the scope of the present treatise. Below are listed the most useful and frequently applied antibodies with a brief explanation of each. Neuronal nuclear antigen This 46–kDa to 48-kDa nucleoprotein is a late neuronal marker during fetal and early postnatal

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life (Sarnat 2013; Sarnat et al. 1998; Fig. 1). After the appearance of nuclear reactivity in fetal brain, a cytoplasmic epitope

becomes expressed after a few months into the post-natal period. NeuN is encoded by the Fox3 gene of the FOX family

Fig. 1 Neuronal nuclear antigen (NeuN) immunoreactivity in (a, b) cerebral cortex shows progressive neuronal maturation from deep to middle layers. a At 26 weeks gestation (26wk GA), nuclear reactivity is seen only in the deep layers of the cortex and in white matter neurons just beneath the cortical plate. b At 34 weeks gestation (36wk GA), most neurons of layer 2 are still not yet mature enough to exhibit NeuNreactive nuclei but most of those in all other layers are reactive. By term, neurons in all layers are reactive (not shown). Cajal-Retzius (CR) neurons (thin arrows) normally are non-reactive at all ages, whereas reactive nuclei in the molecular zone (thick arrows) identify over-migrated nonCR neurons. c The cytoplasmic epitope of NeuN appears during postnatal development, as seen in this frontal neocortex of a 3-year-old girl; the nuclear reactivity continues to be strongly expressed. Cytoplasmic NeuN extends into the proximal dendritic trunk, although not the axon,

thus showing the orientation of the neuron. d Cerebellar cortex of a 26week fetus. Nuclear NeuN labelling in half of the internal granule cells and in the deep lamina of the external granular layer. Pre-migratory granule cells have extended axons as longitudinal fibres in the molecular zone, forming synapses with Purkinje cell dendrites and hence are mature neurons before migration. Purkinje cells (arrows) are not labelled at any age. e Olfactory bulb of a 24-week human fetus. NeuN labelling of the more mature granule cells in the periphery of this layer but not as yet of the less mature neurons in the deep inner part of the granular layer. All granule cells are marked by NeuN by term (not shown). Mitral and tufted neurons are not recognised by the anti-NeuN antibody at this or any other gestational age or in the adult but smaller neurons in the mitral cell layer are reactive (a-d is reproduced with permission from Sarnat 2013; e is original)

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of regulatory splicing factors (Kim et al. 2009; Dent et al. 2010); properly, it is the Rbfox3 gene that encodes NeuN (Darnell 2013). The Fox3 gene more specificially encodes peroxisomal 3-oxoacyl-coenzyme-A thiolase in Saccharomyces cerevisiae, a yeast. NeuN is strongly reactive in most neurons but a group of neurons has been found for which NeuN never reacts at any gestational age, unrelated by site, shape, size, axonal and dendritic connections or type of neurotransmitter: olfactory bulb mitral and tufted neurons; retinal photoreceptor cells; cerebellar Purkinje cells; inferior olivary and dentate nuclear neurons; and sympathetic chain autonomic neurons (but parasympathetic ganglion cells are reactive). Non-reactivity in these neurons is probably attributable to the reciprocal activity of neuron-restrictive silencing factor (NRSF; REST), which inhibits NeuN expression in this mixed population of specific neurons (Schoenherr and Anderson 1995a, 1995b; Ernsberger 2012). The molecular basis, specificity and cross-reactivity of the NeuN epitope of the RbFox3 REST are discussed in depth by Maxeiner et al. (2014). REST also targets some but not all, of the genes encoding synaptic and ion channel proteins (Ernsberger 2012). In NeuN-expressing neurons, perinatal hypoxic/ ischaemic encephalopathy can cause a transitory loss of nuclear expression that could erroneously be interpreted as cellular death. As with all nucleoprotein immunomarkers, postmortem degradation is rapid and NeuN reactivity is not reliable if more than 24 h transpire between the time of death and fixation of the brain tissue at autopsy. NeuN is never expressed in glial cells or ependymal cells and is also nonreactive in the neurons of all invertebrates. Synaptophysin Synaptophysin is the most important of several glycolipid proteins (others are synaptobrevin and synaptotagmin) that form the structure of synaptic vesicle membranes in the axonal terminal, regardless of the identity of the neurotransmitter substance contained, the site of the synapse (axodendritic or axosomatic) or whether the synapse is functionally excitatory or inhibitory (Sarnat and Born 1999; Jahn et al. 1985; Wiedenmann and Francke 1985; Gincel and Shoshan-Barmatz 2003; Linstedt and Kelly 1991). The formation of synapses at the somatic plasma membrane of the neuron is seen as punctuate reactivity surrounding the neuronal soma; dendritic synapses show punctuate reactivity in the neuropil. Synaptophysin is, therefore, an excellent means of showing the temporal sequence of synapse formation in any part of the fetal CNS and hence is also a late marker of neuronal maturation. Synaptogenesis has been demonstrated by synaptophysin immunoreactivity in the human cerebral neocortex and hippocampus (Sarnat and Born 1999; Sarnat et al. 2010), corpus striatum and globus pallidus (Ulfig et al. 2000; Sarnat et al. 2013a), cerebellar system (Sarnat et al. 2013b, 2013c; Fig. 2) and other regions. An advantage of synaptophysin is that it is an extremely robust molecule that

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resists autolysis for as long as 5 days post-mortem (unlike NeuN, which degrades rapidly) rendering it quite reliable in autopsies of human fetuses and infants. In addition to denoting the formation of synapses, the contrast that the antibody against it provides with surrounding white matter enable it more clearly to define the shape or form of certain nuclei than histological stains (Fig. 2). The 38-kDa synaptophysin molecule is synthesized in the neuronal soma, in the perinuclear cytoplasm and uses axonal transport to reach the distal axonal terminals in which it is then arranged to form the synaptic vesicle. Even during axonal transport, the molecule is recognised by the synaptophysin antibody; hence, in immature neurons, immunoreactivity shows axonal reactivity, which disappears with further maturation. Nevertheless, the axons of some neurons continue to show a beaded reactivity along their length as late as maturity. An example of the latter is the subcortical white matter heterotopic neurons that are increased in number in zones of cortical dysplasia, contrasting with the long white matter axons in the centrum semiovale. These subcortical white matter neurons are often described as “isolated” following histological staining but synaptophysin immunoreactivity demonstrates that they are not isolated but rather occur in synaptic contact with each other and with neurons in the overlying cortex and are hence capable of contributing to epileptic circuitry (Sarnat et al. 2010). Synaptophysin is also seen in the perinuclear zones of large neurons, such as the primary sensory neurons of the dorsal root ganglia that lack synapses, demarcating the site of production before axonal transport. The synaptophysin molecule has been purified and characterised for its ion channel activity (Jahn et al. 1985). Endocytosis of synaptophysin requires the carboxy-terminus of the molecule (Wiedenmann and Francke 1985). Various gene regulatory programs govern the expression of synaptic proteins between sympathetic chain neurons and adrenal medullary chromaffin cells (Sarnat 2013). Calcium-binding proteins This family of proteins (calretinin, parvalbumin and calbindin D28k) that bind calcium ions serve as modulators of neurotransmission but are not themselves neurotransmitters (Baimbridge et al. 1992; Ulfig 2002). They are important because levels of intracellular Ca2+ ions are involved in regulating neurite outgrowth, growth cone motility in axonal pathfinding, cellular migration and expression of neurotransmitter receptors. Calcium-binding proteins are involved in neuroblast migration because calcium ions regulate cell movement (Frassoni et al. 1998). Calretinin and parvalbumin are also involved in neuroepithelial cell mitoses (Gotzos et al. 1992; Rasmussen and Means 1992). These calcium-binding proteins are all water-soluble and hence diffuse freely throughout the cytoplasm often enabling the visualisation of small dendritic branches. Calcium-binding proteins are markers of GABAergic neurons (Baimbridge

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Fig. 2 Synaptophysin immunoreactivity in transverse sections of the medulla oblongata and cerebellum in human fetuses of (a) 20 weeks and (b) 33 weeks gestation. The crenated shape of the dentate and inferior olivary nuclei are not yet evident at 20 weeks but synaptophysin reactivity indicates synapse formation even before the morphological form of these nuclei is mature. The reactivity is strongest in a medio-lateral gradient, being stronger in the more medial nuclei fastigius, globosus and

emboliformis than in the more lateral dentate nucleus at 20 weeks. In the inferior olives, reactivity is more intense in the periphery of the nuclei than in its core in the U-shaped nucleus at 20 weeks gestation but, by 33 weeks, it is uniform throughout. Progressive synaptogenesis is also evident in the cerebellar cortex and follows a precise histological sequence, not evident at these low magnifications. ×20. Reproduced with permission from Fridyland et al. (2013)

et al. 1992; Ulfig 2002). In the cerebral cortex, they identify the GABAergic inhibitory interneurons that arrive by tangential migration from the ganglionic eminence, as a specialised part of the periventricular neuroepithelium or “germinal matrix”. They are also good markers of Cajal-Retzius neurons in the molecular zone of the cortex, cells that are not identified by NeuN and are strongly reactive in most subplate neurons. The various calcium-binding molecules identify subclasses of neurons, the majority being reactive for calretinin (Sarnat 2013) but a minority reacting for parvalbumin are disinhibitory neurons that suppress inhibitory interneurons (Pi et al. 2013). In the cerebral cortex, calretinin and parvalbumin mark different inhibitory neurons; in the cerebellar cortex, Purkinje cells are well demonstrated by calbindin D28k but not by calretinin and a differential reactivity has also been seen amongst the other cerebellar cortical neurons (Katsetos et al. 1993; Yu et al. 1996; Yew et al. 1997). Various calcium-binding proteins also identify brainstem neurons of the vestibular nuclei and other tegmental structures (Baizer 2014). In the retina, calretinin is an excellent marker of ganglion cells and photoreceptor neurons. Calretinin is an early marker of neuronal differentiation and can be seen in pre-migratory cells of the ganglionic eminence (Sarnat 2013;

Ulfig 2001) but parvalbumin and calbindin D28k are intermediate markers in temporal expression. Chromogranins A–C This family of small, 68–kDa to 75kDa, glycosylated, calcium-binding, acidic proteins, formerly called secretogranins, has three forms, widely distributed in the CNS (Muñoz et al. 1990; Erickson et al. 1992) and a peripheral nervous system including chromaffin and other neuroendocrine cells such as insulin-secreting cells of the pancreatic islets of Langerhans (Reiffen and Gratzl 1986; Gratzl 1987; Lloyd et al. 1988). In human neuropathology, a section of pancreas with islet cells serves as a control for brain Cgr reactivity, as it also does with NSE and S-100β protein. CgrA is the most frequently seen form in the CNS and is developmentally a late marker. Although traditionally used more as neuroendocrine cell markers, the chromogranins have more recently been applied as neuronal markers within the brain, especially in the hippocampus. Ultrastructurally, Cgr localises to synaptic vesicles but because it is water-soluble, it cannot serve as a structural protein of the vesicular wall as does synaptophysin but rather is an associated secretory protein of the synapse. In the hippocampus, CgrA is strongly expressed in some but not the

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neuropil, of the CA2 sector of Ammon’s horn and is seen as beaded axons of CA2 neurons in CA3 without reactivity in the soma of CA3 neurons (Sarnat 2013); CA2 has a nonreciprocal projection to CA3. CgrA is the major protein component of chromaffin vesicles and large dense-core vesicles but is lacking in the small dense-core vesicles of sympathetic nerve terminals, although the degrading enzyme dopamine-βhydroxylase is present in both large and small dense-core vesicles (Huber 2014; Neuman et al. 1984). CgrA occurs in axons of all sizes but CgrB is limited to large axons. CgrB is most closely associated with cholinergic neurons, including spinal motor neurons; CgrA has been demonstrated in both cholinergic and adrenergic neurons (Banks and Helle 1967). CgrA also distinguishes monodendritic neurons of the cerebral cortex (Muñoz 1990) and inhibits dopamine release in the retina (Gibson and Muñoz 1993). Enolases These glycolytic enzymes are found in many cells and tissue of the body. They catalyze the inter-conversion between 2-phosphoglyceric acid and phyphoenoylpyruvate. In the CNS, non-neuronal enolase (NNE or γ-enolase) is expressed in mature glial cells and also in the early stages of cells differentiating in neuronal lineages but NNE is later replaced by NSE (or α-enolase) as the neuron matures (Ghandour et al. 1981; Royds et al. 1982; Marangos et al. 1980). The molecule is water-soluble and reactive in neurites and in the soma and so poor contrast often occurs between the neuron and surrounding neuropil. NSE is expressed in all neuronal types but not in the neuroepithelial cells of the germinal matrix. Chromaffin and neuroendocrine cells of the peripheral nervous system also express NSE. The development of NSE in the human striate cortex of the occipital lobe was demonstrated by Nishimura et al. (1985) and in the rat somatosensory cortex by Hamre et al. (1989). Microtubule-associated proteins This large family of cytoskeletal stiffening proteins changes in degree of phosphorylation with neuronal maturation. They are expressed even in the mitotic spindle and are involved in several early developmental processes of neuronal maturation, including cellular polarity, shape, motility, remodelling of dendritic spines and intracellular transport. Regulation of gene expression during early neuronal differentiation largely involves microtubules (Ernsberger 2012). The role of MAPs and other cytoskeletal elements in the formation of neurites is discussed by Saineth in this issue (Sainath and Gallo 2014). MAPs are expressed in both immature and mature neurons as an early marker but not in mature glial or ependymal cells. The most frequently used anti-MAP antibodies for human brain tissue are: MAP 1/5, which marks the soma and axon but not dendrites; MAP2, which marks the soma and dendritic trunk but not the axon; class III β-tubulin (TUJ1); transcription products of the genes

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DCX, ARX and LIS1, genes that, in mutated form, underlie neuroblast migratory disorders and the disruption in cortical plate organisation in humans and rodent models. Specific MAPs define neuronal types in the cerebellum (Katsetos et al. 1993). Not all MAPs have the same function, either during development or in adult life and they could be subclassified in this regard. Tau protein is a particularly important MAP. This normal protein, in abnormal phosphorylated form, is over-expressed in several adult neurodegenerative diseases associated with dementia, known as a grouped category of “tauopathies” (Mackenzie et al. 2012). In developing brain, upregulation of tau protein is seen in tuberous sclerosis complex, in hemimegalencephaly, in focal cortical dysplasia type 2 and in a paediatric brain tumour, the ganglioglioma, in which dysmorphic and megalocytic neurons are dysplasia and the astrocytic component of the lesion is neoplastic (Sarnat and Flores-Sarnat 2014; Brat et al. 2001). A disorder of microtubular function during early cellular differentiation could explain a common pathogenesis of these conditions. All infantile tauopathies are thus a tetrad of dysmorphic and megalocytic neurons, focal somatic mosaicism, activated mTOR and upregulated phosphorylated tau (Sarnat and Flores-Sarnat 2014). Neurofilament proteins This family of neuron-specific intermediate filament proteins (NFP in North America; SMI-32 in Europe) serves as cytoskeletal elements in the somatic and neuritic cytoplasm of both mature and immature neurons but not of glial cells. Three major subgroups of NFPs are distinguished by molecular weight: 68–70 kDa, 150–160 kDa and 200 kDa. All are non-specific for types of neurons but are not equally well expressed in the different neuronal classes. In rodent dorsal root ganglia, NF-200 is used for subtype classification. During development, NFPs replace the less mature intermediate filaments of vimentin and nestin. Dicerdependent mechanisms differ between neurons and neuroendocrine cells and this difference is expressed in the formation of many NFP in the former but not in the latter (Sarnat 2013). Dicer is essential to the survival of differentiating neural crest cells (Zehir et al. 2010). In dysmorphic neurons, as in tuberous sclerosis and hemimegalencephaly and in the neurodegenerative diseases during adult life, NFP might aggregate in the soma and appear as neurofibrillary tangles of adult neurodegenerative diseases, as demonstrated by argentophilic impregnations but they are simply aggregates of neurofilaments without the double helical ultrastructure. Protein gene product 9.5 This non-specific cytoplasmic intermediate marker of all types of neurons has been studied extensively in ganglion cells and interstitial cells of Cajal of the submucosal and myenteric plexi and particularly as a tumour-suppressor gene in the intestinal tract (Geramizadeh

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et al. 2009; Tokumaru et al. 2008) but it is also reactive in neurons of the CNS. Studies in developing human brain are still too few to conclude its reliability as a marker of neuronal maturation.

Histochemical markers of neuronal maturation Certain histochemical stains, not based upon immunoreactivity, can also serve as useful markers of maturation. Luxol fast blue for myelination Luxol fast blue (LFB) is one of the earliest histochemical stains to be widely used to demonstrate axonal myelination in the developing CNS, usually being counterstained by H&E or with the Periodic acid-Schiff reaction (PAS), a technique developed by Klüver and Barrera (1953). Its use in immature brain to show the sequence of myelination is well described (Yakovlev and Lecours 1967; Rorke and Riggs 1969; Gilles 1976). Although gallocyanin histochemistry, peroxidise-catalyzed gold chloride impregnations and immunoreactivity for myelin basic protein may be more precise, LFB remains the most widely used myelin stain in human neuropathology because of tradition, familiarity and low cost. In focal cortical dysplasia type 2, focal white matter hypomyelination, as seen with LFB, can be correlated with delayed oligodendrocyte maturation (Shepherd et al. 2013). LFB is also useful in demonstrating Schwann-cell-produced myelin in peripheral nerves, including intramuscular and cutaneous nerves. LFB-stained sections correlate well with the MRI demonstration of myelination in the living patient. Acridine orange fluorochrome for nucleic acids Aminoacridine compounds form highly fluorescent complexes with nucleic acids when viewed by fluorescence microscopy. Acridine orange (AO) is the most useful in the nervous system. Because of the different wavelengths of the absorption of ultraviolet light and the emission of visible light between AO-DNA and AO-RNA complexes, DNA is perceived as yellow-green and ribosomal RNA appears brilliant orange-red (Sarnat 1985). As a marker of maturation, the increase in cytoplasmic RNA in neurons denotes the onset of neurotransmitter synthesis (Sarnat 1989). AO preparations are temporary water mounts and the fluorescence rapidly quenches or fades with continued exposure to UV light after a few minutes. Documentation must therefore be photographic. The AO preparations can then be rinsed after the removal of the water-mounted coverslip and the same section can be restained with H&E, cresylviolet or any other histological stain as a permanent mount. AO is equally reliable for unfixed frozen sections and for formalin-fixed paraffin-embedded sections.

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Argentophilic impregnations Heavy metal (mainly silver) impregnations of neurons to demonstrate tiny details of their neurites and synaptic spines were developed by Camillo Golgi of Verona, Italy and enabled Santiago Ramón y Cajal of Madrid, Spain to describe the structure and development of the nervous system in a manner that is as valid today as when Golgi and Ramón y Cajal shared the Nobel Prize in Medicine in 1909. The first publication of the Golgi impregnation of the human brain was the description of the human olfactory bulb by Golgi (1875). Many contributions by Cajal were published in the late 19th and early 20th centuries (Ramón y Cajal 1881; Ramón y Cajal 1909-1911). Developmental studies with the Golgi technique continue to contribute new understanding, as exemplified by the work of Marín-Padilla (2011). Because these techniques are extremely labourious, expensive and somewhat capricious, they are not widely used in human diagnostic neuropathology. Simpler silver impregnation methods are used, however, such as the techniques of Bielschowsky, Sevier-Munger and Gallyas and the protargol method of Bodian. These can demonstrate growing axons in the CNS and peripheral nervous system as early as 10 weeks gestation, such as both the central and peripheral extensions of dorsal root ganglion cells (Sarnat 2013). Despite not providing the fine detail of Golgi silver stains, they do provide useful information about neurofibrillary aggregates or tangles in infantile tauopathies and in morphogenesis of developing neurites. In my personal experience, the Golgi-Cox method of mercury impregnations does not work as well in immature brain as in adult brain and I have long abandoned this technique for fetal and neonatal brains. Mitochondrial respiratory chain enzymes The number and concentration of mitochondria is a reflection of the metabolic activity of the neuron and increases with the onset of neurotransmitter synthesis and neurosecretion. Hence, the histochemical activity of respiratory chain enzymes might serve as a maturational marker in immature neurons. Mitochondria are also greatly increased in “hypermetabolic” neurons with a high rate of discharge at the epileptic focus (see below; Sarnat et al. 2011). The enzymes most applicable for this purpose are nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR; Respiratory Complex I), succinic dehydrogenase (SDH; Complex II) and cytochrome-c-oxidase (COX; Complex IV). The disadvantage is that they require freshly frozen sections; however, new immunocytochemical methods for demonstrating these enzymes in formalin-fixed paraffinembedded sections have recently been developed but are not as yet fully confirmed and validated by comparison with the traditional frozen section histochemistry of the same tissue samples. Biochemical quantitative assay of each of the enzymes is available, generally applied to muscle biopsies of patients suspected of having mitochondrial cytopathies but, for brain, this method is not as specific as histochemical

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sections, despite being quantitative rather than qualitative, because it requires a tissue homogenate. If only a few scattered neurons are hypermetabolic, they might be diluted and the abnormality not demonstrated, whereas histochemistry can show individual neurons. Periodic acid-Schiff reaction for glycogen Glycogen stores in neurons are highly variable and influenced by many factors, including nutritional status and metabolic activity that consumes glycogen. The turnover of glycogen is nine times greater in glial cells than in neurons (Pellerin et al. 2001; Hamprecht et al. 2005). Digestion of glycogen continues post-mortem, rendering autopsy tissue more difficult to interpret. Because of these variables, PAS staining is not a reliable marker of neuronal maturation (Sarnat 2013), despite being one of the oldest and most traditional of histochemical stains, applicable to both freshly frozen and paraffin sections. Nevertheless, these factors that render PAS unreliable as a maturational marker do not signify that the function of glycogen in cells of the nervous system is not important. For example, Schwann cell glycogen supports the function of myelinated axons in peripheral nerves during hypoglycaemia and is passed to axons in the form of lactate; unmyelinated axons not so protected are more vulnerable to dysfunction in hypoglycaemia (Brown et al. 2012).

Metablic tissue markers of epileptic foci In our laboratory, one of our projects is to identify tissue markers that identify epileptic foci in surgical resections of cerebral cortex and hippocampus in children with intractable epilepsy that resists pharmacological control. Surgical specimens submitted by the neurosurgeon are marked at the surface with the site of most epileptic activity being recorded intraoperatively by electrocorticography of the exposed brain in order to supplement pre-operative electroencephalographic (EEG) monitoring. During the preparation of sections, a cut is made at the site of the focus and at 1 cm intervals on either side of it, all being noted in labelled cassettes; a thin frozen section is also taken at the site of the epileptic focus. To date, we have identified two types of markers that are useful for this purpose. Mitochondrial respiratory chain enzymes The three histochemically reliable enzymes of the respiratory chain to be demonstrated in frozen sections have previously been noted (see above) as markers of neuronal maturation, with an increase in mitochondrial activity corresponding to the initiation of neurotransmitter synthesis. As a marker in epileptic foci, however, at any age from infancy to adolescence, scattered cortical and hippocampal neurons exhibit much more intense

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histochemical activity for all three of the enzymes than do the normal neurons and their neurites in the surrounding neuropil (Sarnat et al. 2011). Ultrastructural examination of the same region has disclosed scattered neurons with cytoplasm filled with excessive mitochondria, almost masking all other organelles. We interpret these findings as hypermetabolic epileptogenic neurons with a greater energy requirement than normal neurons because of their continuous plasma membrane depolarisations leading to paroxysmal discharges. Diminished mitochondrial respiratory chain activity, by contrast, might correlate with and indeed contribute to an early stage of neuronal degeneration (Pathak et al. 2013). α-B-crystallin This small heat-shock protein is similar, although with a slightly higher molecular weight, to heatshock protein-27 (HSP27). Both are upregulated in the CNS under certain adverse conditions, epileptic foci being one of several. Others include chronic hypoxic/ischaemic encephalopathy, exposure to x-irradiation or to anti-metabolic and immunosuppressive drugs used as chemotherapy in the treatment of brain tumours, recurrent hypoglycaemia, traumatic brain injury and some primary CNS tumours. Despite the presence of α-B-crystallin being a non-specific response to cellular injury, other conditions can be excluded by medical history and histopathology, so that it can be used as a specific marker for chronic epileptic activity (Sarnat and Flores-Sarnat 2009). The cytoplasmic expression of α-B-crystallin in formalin-fixed paraffin-embedded sections is most intense not only in the glial cells of the white matter just underlying the epileptic cortex but also in the cortical astrocytes and occasionally in the large pyramidal neurons, both in deep layers of the neocortex and in Ammon’s horn of the hippocampus. In about one third of cases, a gradient of diminishing reactivity is seen away from the epileptic focus, with the reactivity disappearing almost completely at 2.5–3.0 cm from the focus. In the remaining cases, reactivity remains more diffuse in all parts of the examined tissue. However, the size of the resected brain tissue specimen varies greatly between patients and this might be a factor in some cases.

Comments Studies of developing brain, whether in humans or in animals, traditionally are gross and microscopic/histological descriptions of morphogenesis defining the structural architecture of the CNS and the organisation of cellular elements. The critical study of the maturation of individual cells during development provides a new dimension of understanding of ontogenesis. Modern techniques of ICC and histochemistry can provide the tools needed to accomplish this goal. The establishment of criteria of normal development, including the critical aspect of

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timing, provides a basis for the diagnosis and interpretation of abnormal development at the cellular level by light microscopy. These methods also denote individual maturational processes, such as neurotransmitter biosynthesis, synaptogenesis and myelination. Certain cellular types can be identified with greater certainty than with histological stains alone. For example, calretinin in the developing cerebral cortical plate identifies the GABAergic inhibitory interneurons that arrive by tangential migration, distinguishing them from the glutaminergic neurons that arrive by radial migration. Ultrastructural study by transmission electron microscopy can confirm many of the findings obtained by light microscopy, such as dendritic spine maturation and synapse formation but is beyond the scope of this review. Various means of classification of the immunocytochemical and histochemical markers of neuroblast maturation are available, depending upon the needs of the investigator. Some can be combined with other markers in a classification scheme. The most useful criteria, at least in human developmental neuropathology, are positive markers that exhibit a specific timing of expression in the maturing neuroblast as early, intermediate or late expression. Subcellular localisation and the nature of the molecule are also important. Some markers, such as NeuN, are expressed in abnormally developing, dysmorphic or megalocytic (hypertrophic) neurons that occur in malformations such as tuberous sclerosis, hemimegalencephaly and focal cortical dysplasia type 2. Others, such as CgrA, are expressed only in normally developing neurons, even if displaced within an abnormal tissue architecture and are not reactive in dysplastic neurons. This provides another criterion of normal or abnormal cytological development, particularly if dysmorphic and normal neurons are both present in the same field to provide an internal control. The precisely time-linked process of ontogenesis can be either delayed or accelerated as precocious maturation. Delay not only accompanies many genetically determined diseases but can also be attributable to epigenetic factors such as chronic fetal ischaemia, hypothyroidism and the adverse effects of certain drugs and neurotoxins to which the pregnant mother and thus her fetus might be exposed. An example is fetal alcohol syndrome, which is associated not only with intrauterine growth retardation and dysmorphic facies but with developmental abnormalities of the cerebral cortex, delayed synaptogenesis and delayed myelination (Sarnat et al. 2014a). Precocious synaptogenesis in the cerebral cortex occurs in the structurally abnormal cortex of fetuses and infants with h o l o p r o s e n c e p h a l y, w h e t h e r a s s o c i a t e d w i t h a chromosomopathy or with normal karyotype (Sarnat and Flores-Sarnat 2013; Sarnat et al. 2014b). Precocious synapse formation in the cortex, out of synchrony with other maturational processes, is not advantageous but rather might facilitate the early establishment of epileptic circuitry resulting in severe infantile epilepsies in the post-natal period. Precocious synaptogenesis is also seen in the dysplastic retina of the

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medial cyclopean eye in holoprosencephaly (Sarnat et al. 2014b). Although this review is focused upon markers of neuronal maturation, the same principles can be applied to markers of glial, ependymal and choroid plexus epithelial cell maturation by using different protein and transcription product markers (Sarnat 1998a). Similar principles apply, for example, with regard to vimentin; this is a negative marker of ependymal cells, is strongly expressed during development but regresses with maturation (Sarnat 1998b), continues to be expressed in the ependyma lining the 3rd or lateral ventricles until the 3rd trimester in normal human brain and persists in dysplastic regions of the 4th ventricle and hydromyelic regions of spinal cord in Chiari II malformations (Sarnat 2004). The importance of glia in neural maturation is also increasingly being recognised but this topic cannot be addressed adequately in the present review of neuronal maturational markers.

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