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NeuN is not a reliable marker of dopamine neurons in rat substantia nigra
Neuroscience Letters 464 (2009) 14–17
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
NeuN is not a reliable marker of dopamine neurons in rat substantia nigra Jason R. Cannon, J. Timothy Greenamyre ∗ Pittsburgh Institute for Neurodegenerative Diseases, Department of Neurology, University of Pittsburgh, Pittsburgh, PA 15260, United States
a r t i c l e
i n f o
Article history: Received 3 June 2009 Received in revised form 30 July 2009 Accepted 6 August 2009 Keywords: NeuN Substantia nigra Dopamine neuron Parkinson’s disease
a b s t r a c t Quantification of neuronal cell number is a key endpoint in the characterization of neurodegenerative disease models and neuroprotective regimens. Immunohistochemistry for phenotypic markers, followed by unbiased stereology is often used to quantify the relevant neuronal population. To control for loss of phenotypic markers in the absence of cell death, or to determine if other types of neurons are lost, a general neuronal marker is often desired. Vertebrate neuron-specific nuclear protein (NeuN) is reportedly expressed in most mammalian neurons. In Parkinson’s disease models, NeuN has been widely used to determine if there is actual nigral dopamine neuron loss or simply loss of tyrosine hydroxylase expression, a prominent phenotypic marker. To date, the qualitative value of NeuN expression as such a marker in the substantia nigra has not been assessed. Midbrain tissue sections from control rats were stained for NeuN and tyrosine hydroxylase and assessed by light or confocal microscopy. Here we report that NeuN expression level in the rat substantia nigra was highly variable, with many faintly stained cells that would not be meet stereological scoring criteria. Additionally, dopamine neurons with little or no NeuN expression were readily identified. Subcellular compartmentalization of NeuN expression was also variable, with many cells dorsal and ventral to the nigra exhibiting expression in both the nucleus and cytoplasm. NeuN expression also appeared to be much higher in non-dopamine neurons within the ventral midbrain. This characterization of nigral NeuN expression suggests that it is not useful as a quantitative general neuronal marker in the substantia nigra. © 2009 Elsevier Ireland Ltd. All rights reserved.
Characterization of neuronal cell loss in the brain is a complex and tedious process. Unbiased stereological cell counting is now the accepted standard for quantitative assessment of neuronal cell number [16]. For a given treatment, it is often desirable to determine the specific types of neurons that are lost. In Parkinson’s disease, the loss of dopamine neurons in the substantia nigra is a key pathological feature and elicits motor symptoms [2,6]. Indeed, prominent neurotoxicant PD models including 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP), paraquat, and rotenone have all reported selective nigral dopamine neuron loss [3,5,8,12,14,15]. The claim of a ‘selective’ lesion typically needs to be supported by assessment of loss of other types of neurons or total neuronal loss. Additionally, it is possible that a treatment may result in loss of phenotypic marker expression, rather than actual neuronal cell loss. For example, a 6-hydroxydopamine regimen was found to elicit a 50% loss of fluorogold labeled neurons (retrograde neuronal tracer) and an 85% loss of tyrosine hydroxylase
∗ Corresponding author at: University of Pittsburgh, 3501 Fifth Avenue, Suite 7039, Pittsburgh, PA 15260, United States. Tel.: +1 412 648 9793; fax: +1 412 648 9766. E-mail address: [email protected] (J.T. Greenamyre). 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2009.08.023
positive neurons—evidence that loss of phenotype exceeded neuronal cell death [4]. While fluorogold is a useful marker for such a determination, it must be delivered by stereotaxic infusion. Vertebrate neuron-specific nuclear protein (NeuN) is reportedly expressed in the nuclei of the majority of nervous system neurons [9]. It is one of the most commonly used general immunohistochemical markers for neurons. NeuN immunohistochemistry in conjunction with specific phenotypic markers may be used to compare total neuronal loss with loss of a specific population of neurons. Indeed, NeuN immunohistochemistry has been widely used to test for loss of phenotypic marker expression and cell-type specificity of the lesions in PD models [1,10,18]. While NeuN is reportedly expressed in most neuronal populations, it is not expressed in cerebellar Purkinje cells, olfactory bulb mitral cells, and retinal photoreceptor cells [9]. To date, the utility of NeuN as a control for loss of expression of phenotypic markers or for overall neuronal loss in the midbrain has not been reported, although it is widely used for this purpose. Indeed, reports in other regions of the central nervous system and in disease models have questioned the utility of NeuN expression as a general neuronal marker. For example, after cerebral ischemia there is a loss of NeuN immunoreactivity, but healthy-appearing neurons were still evident with H&E staining [13]. Additionally spinal cord neurons
J.R. Cannon, J.T. Greenamyre / Neuroscience Letters 464 (2009) 14–17
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Fig. 1. Nigral NeuN immunohistochemistry. Representative low magnification image (A) indicates dramatic differences in staining intensity in different ventral midbrain neuronal populations. Red dashes indicate boundary of the substantia nigra. Bar = 100 m. High magnification image in the medial pars compacta region of the substantia nigra (B) shows variability in intensity and cellular localization. White arrows indicate faintly stained cells that would not meet threshold criteria for stereological counting. Localization of NeuN ranges from primarily nuclear to whole-cell immunoreactivity. Bar = 10 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
exhibit loss of expression during normal aging, while expression of other neuronal markers persists [11]. During routine analysis of midbrain sections stained for tyrosine hydroxylase and NeuN, we noticed several dopamine neurons with faint or no NeuN staining. The goal of the current study was to determine if NeuN could serve as a reliable marker for ventral midbrain neurons. Our data strongly cautions against the use of NeuN as such a marker. Adult male Lewis rats (n = 5) from multiple batches (∼3–5 months old) were used for all experiments (Hilltop Lab Animals Inc., Scottdale, PA, USA). The animals were maintained under standard conditions of 12-h light/dark cycles, 22 ± 1 ◦ C temperature-controlled room and 50–70% humidity and they were provided water and food ad libitum. All studies were approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh and in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. Three animals were deeply anesthetized with pentobarbital (50 mg/kg, i.p.) and perfusion-fixed, first with 50 mL ice-cold 0.1 M PBS, and then with 250–350 mL 4% paraformaldehyde. Brains were then post-fixed overnight and transferred to 30% sucrose, where they were stored until fully infiltrated and further processed. An additional two animals were euthanized by CO2 exposure, and brains were rapidly removed and bisected mid-sagittally. One hemisphere was post-fixed in 4% paraformaldehyde for 7 days and then placed in 30% sucrose for at least 3 days, until infiltration was complete. This fixation method is commonly used in our lab and others, to obtain many endpoints from the same tissue (the opposite hemisphere can be used for biochemical studies) and was employed here to determine if NeuN expression was influenced by fixation protocol. Brains were cut coronally on a frozen sliding microtome at 35 m and stored in cryoprotectant at −20 ◦ C until use. Sections for chromagen development were then removed from cryoprotectant, washed in PBS 6× for 5 min each then treated with 3% hydrogen peroxide for 10 min followed by three PBS washes and blocked for 1 h in 10% normal donkey serum, containing 0.3% Triton X100. The sections were incubated in a primary antibody for NeuN (1:1000, mouse anti-NeuN; Millipore #MAB377; Bilerica, MA, USA) for 72 h. The optimal antibody concentration was determined by testing a range of dilutions and choosing the concentration that produced the most intense staining in the absence of extracellular ‘background’ staining. The sections were then washed in PBS 3 × 5 min and incubated at room temperature for 1 h in biotinylated donkey anti-mouse secondary antibody (1:200, Jackson Immuno Research; West Grove, PA, USA). The sections were then washed
3× in PBS and incubated in Avidin–Biotin Complex solution (Vectastain, Vector Labs; Burligame, CA, USA). After washing in PBS, the sections were developed using DAB (Vector Labs). The tissue was mounted onto slides after staining, air dried, dehydrated using graded alcohols and Histoclear (National Diagnostics, Atlanta, GA, USA), followed by mounting with Histomount (National Diagnostics). For immunofluorescence, the staining was done as above except using primary antibodies for tyrosine hydroxylase (1:2000; rabbit anti-TH; AB152; Millipore) and NeuN, and one of the following fluorescent secondary antibodies was used: Cy3-conjugated anti-mouse (1:500; Jackson Immuno), Cy5-conjugated anti-rabbit (1:500; Jackson Immuno). Following secondary antibody incubation, the sections were treated with the nuclear dye Hoechst 33342 (Sigma–Aldrich, St. Louis, MO, USA) for 5 min. These sections were then washed and mounted using gelvatol mounting media. Images were captured on a light microscope (Olympus BX51) using the software DP controller (Olympus) or a confocal microscope (Olympus Fluoview FV1000), using the software FV10-ASW (Olympus). Lasers and detectors were optimized over several sections and the settings at each magnification were maintained throughout acquisition. Optimization for NeuN expression was conducted using several NeuN and tyrosine hydroxylase positive neurons, where the settings for NeuN expression were adjusted near saturation. Adjustments to intensity, brightness and contrast were consistently made to every image at a given magnification. Within the ventral midbrain NeuN expression level was highly variable. Low magnification images of NeuN immunohistochemistry of the substantia nigra clearly indicated a wide range in intensity of staining (Fig. 1A); many neurons were stained very faintly. Neuronal populations dorsal to the nigra had more intense staining than nigral neurons. Additionally, these extra-nigral neuronal populations showed both nuclear and cytoplasmic expression of NeuN. High magnification images show faintly stained NeuN cells that would not meet threshold criteria for stereological scoring (Fig. 1B). Expression within the nigra also ranges from purely nuclear to whole-cell immunoreactivity. These findings were observed in all animals examined, irrespective of fixation method. NeuN expression in dopamine neurons ranged from undetectable to intense (Fig. 2). Confocal microscopy of fluorescently labeled sections at low magnification showed that NeuN expression was highly variable in the medial tier of the pars compacta region of the substantia nigra (Fig. 2B). While nuclear staining (Fig. 2A) and dopamine neurons were clearly evident (Fig. 2C), NeuN expression levels varied widely between dopamine neurons (Fig. 2B). Non-
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J.R. Cannon, J.T. Greenamyre / Neuroscience Letters 464 (2009) 14–17
Fig. 2. Low magnification nigral NeuN confocal immunohistochemistry. The medial tier of the substantia nigra is stained for Hoechst 33342 (nuclear marker; blue; A), NeuN (green; B), and tyrosine hydroxylase (red; C). NeuN immunoreactivity is lower in the tyrosine hydroxylase positive neurons compared to neuronal populations dorsal or ventral to the pars compacta. Within the nigra, variability in NeuN immunoreactivity is also readily apparent. Bar = 100 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
dopamine neurons both dorsal and ventral to the nigra expressed NeuN at higher levels. Dopamine neurons that express little or no NeuN are readily apparent at high magnification (Fig. 3). Evidence of faint or undetectable NeuN expression in dopamine neurons was observed in all animals examined. NeuN is reportedly expressed in most vertebrate neuronal nuclei [9] and is often used as a general neuronal marker. In PD research it is often used in conjunction with phenotypic markers, such as tyrosine hydroxylase, to characterize dopamine neuron loss in the substantia nigra [1,10,18]. While NeuN has been widely utilized for this purpose, the utility of this marker has not been critically assessed. Here we report that NeuN expression magnitude varies dramatically from cell-to-cell within the substantia nigra, and dopamine neurons without detectable NeuN are readily identifiable. Additionally, localization ranges from primarily nuclear to both nuclear and cytoplasmic. Therefore, our data suggests that NeuN is not a reliable, quantitative marker for neurons of the substantia nigra.
Quantification of dopaminergic neuron number is one of the primary endpoints of PD model characterization; it is also critical for quantitative assessment of neuroprotection conferred by putative therapeutic regimens. However, this seemingly simple measurement may be confounded by issues related to cell-type specificity of the lesion (or lack thereof), or by loss of phenotype instead of cell loss. For example, it is possible that a given model may be associated with loss of both dopaminergic and non-dopaminergic neurons in the substantia nigra. Such a lesion would have less relevance to clinical PD, considering the rather specific loss of dopamine neurons that occurs in the human PD substantia nigra [6]. To address this possibility, the total number of nigral neurons – or another identified neuronal population – may be counted in addition to tyrosine hydroxylase positive neurons. Similarly, counts of a general neuronal marker can be used to determine whether loss of tyrosine hydroxylase staining represents loss of neurons or loss of phenotype. While these issues should ideally be addressed when characterizing a new model, or a ‘neuroprotective’ treatment, our
Fig. 3. High magnification nigral NeuN confocal immunohistochemistry. Sections were stained for stained for Hoechst 33342 (nuclear marker; blue; A, E), NeuN (green; B, F), and tyrosine hydroxylase (red; C, G). Examples of NeuN negative (D) and NeuN positive (H) dopamine neurons (tyrosine hydroxylase positive) from the dorsolateral substantia nigra are presented. NeuN immunoreactivity is typically lower in dopamine neurons compared to non-dopamine neurons (H). Bar = 10 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
J.R. Cannon, J.T. Greenamyre / Neuroscience Letters 464 (2009) 14–17
results suggest that NeuN expression is not suitable for this purpose. Our findings also show that nigral NeuN expression may be both nuclear and cytoplasmic. This makes interpretation of doublestained sections with two chromagens difficult to interpret. As a result, in stereological studies, counting both tyrosine hydroxylaseand NeuN-positive cells may be very difficult. Indeed these results are in agreement with a previous study, in which NeuN was found to be a phosphoprotein with both cytoplasmic and nuclear localization in the mouse cortex [7]. Moreover, although histological procedures, such as antigen retrieval, may improve staining quality/intensity [17], such methods may not be appropriate for stereology because they would likely affect tissue volume. Discrepancies in NeuN staining outside the substantia nigra have been reported previously. During normal aging in the rat, NeuN immunoreactivity is lost in spinal cord neurons, in the absence of cell loss [11]. Additionally, in mice under pathological conditions such as cerebral ischemia, NeuN immunoreactivity is lost, while apparently healthy H&E-stained neurons persist [13]. Therefore, NeuN expression may not be a reliable neuronal marker in aged animals or in certain disease models. Given that variability in NeuN expression has been reported in both rats and mice, this phenomenon is likely species independent. Our results further suggest that even in control animals significant variability exists in the substantia nigra. The percentage of dopamine neurons that are NeuN-negative was not quantified in this study. However, our finding of an easily observed absence of NeuN expression in dopamine neurons is sufficient to preclude its use as a quantitative marker for nigral neurons. In summary, we have shown that NeuN is not a reliable marker of neurons in the substantia nigra and that this marker should not be used to control for loss of phenotype or to assess total neuronal counts in the ventral midbrain. Acknowledgements This work was supported by a postdoctoral fellowship from the American Parkinson Disease Association (J.R.C.). References [1] Z.C. Baquet, P.C. Bickford, K.R. Jones, Brain-derived neurotrophic factor is required for the establishment of the proper number of dopaminergic neurons in the substantia nigra pars compacta, J. Neurosci. 25 (2005) 6251–6259.
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[2] H. Bernheimer, W. Birkmayer, O. Hornykiewicz, K. Jellinger, F. Seitelberger, Brain dopamine and the syndromes of Parkinson and Huntington. Clinical, morphological and neurochemical correlations, J. Neurol. Sci. 20 (1973) 415– 455. [3] R. Betarbet, T.B. Sherer, G. MacKenzie, M. Garcia-Osuna, A.V. Panov, J.T. Greenamyre, Chronic systemic pesticide exposure reproduces features of Parkinson’s disease, Nat. Neurosci. 3 (2000) 1301–1306. [4] K.E. Bowenkamp, D. David, P.L. Lapchak, M.A. Henry, A.C. Granholm, B.J. Hoffer, T.J. Mahalik, 6-Hydroxydopamine induces the loss of the dopaminergic phenotype in substantia nigra neurons of the rat. A possible mechanism for restoration of the nigrostriatal circuit mediated by glial cell line-derived neurotrophic factor, Exp. Brain Res. 111 (1996) 1–7. [5] J.R. Cannon, V. Tapias, H.M. Na, A.S. Honick, R.E. Drolet, J.T. Greenamyre, A highly reproducible rotenone model of Parkinson’s disease, Neurobiol. Dis. 34 (2009) 279–290. [6] L.S. Forno, Neuropathology of Parkinson’s disease, J. Neuropathol. Exp. Neurol. 55 (1996) 259–272. [7] D. Lind, S. Franken, J. Kappler, J. Jankowski, K. Schilling, Characterization of the neuronal marker NeuN as a multiply phosphorylated antigen with discrete subcellular localization, J. Neurosci. Res. 79 (2005) 295–302. [8] A.L. McCormack, M. Thiruchelvam, A.B. Manning-Bog, C. Thiffault, J.W. Langston, D.A. Cory-Slechta, D.A. Di Monte, Environmental risk factors and Parkinson’s disease: selective degeneration of nigral dopaminergic neurons caused by the herbicide paraquat, Neurobiol. Dis. 10 (2002) 119–127. [9] R.J. Mullen, C.R. Buck, A.M. Smith, NeuN, a neuronal specific nuclear protein in vertebrates, Development 116 (1992) 201–211. [10] L. Novikova, B.L. Garris, D.R. Garris, Y.S. Lau, Early signs of neuronal apoptosis in the substantia nigra pars compacta of the progressive neurodegenerative mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/probenecid model of Parkinson’s disease, Neuroscience 140 (2006) 67–76. [11] E.L. Portiansky, C.G. Barbeito, E.J. Gimeno, G.O. Zuccolilli, R.G. Goya, Loss of NeuN immunoreactivity in rat spinal cord neurons during aging, Exp. Neurol. 202 (2006) 519–521. [12] D.J. Sirinathsinghji, A. Kupsch, E. Mayer, M. Zivin, D. Pufal, W.H. Oertel, Cellular localization of tyrosine hydroxylase mRNA and cholecystokinin mRNAcontaining cells in the ventral mesencephalon of the common marmoset: effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, Brain Res. Mol. Brain Res. 12 (1992) 267–274. [13] I. Unal-Cevik, M. Kilinc, Y. Gursoy-Ozdemir, G. Gurer, T. Dalkara, Loss of NeuN immunoreactivity after cerebral ischemia does not indicate neuronal cell loss: a cautionary note, Brain Res. 1015 (2004) 169–174. [14] U. Ungerstedt, 6-Hydroxy-dopamine induced degeneration of central monoamine neurons, Eur. J. Pharmacol. 5 (1968) 107–110. [15] M. Varastet, D. Riche, M. Maziere, P. Hantraye, Chronic MPTP treatment reproduces in baboons the differential vulnerability of mesencephalic dopaminergic neurons observed in Parkinson’s disease, Neuroscience 63 (1994) 47– 56. [16] M.J. West, L. Slomianka, H.J. Gundersen, Unbiased stereological estimation of the total number of neurons in the subdivisions of the rat hippocampus using the optical fractionator, Anat. Rec. 231 (1991) 482–497. [17] H.K. Wolf, R. Buslei, R. Schmidt-Kastner, P.K. Schmidt-Kastner, T. Pietsch, O.D. Wiestler, I. Blumcke, NeuN: a useful neuronal marker for diagnostic histopathology, J. Histochem. Cytochem. 44 (1996) 1167–1171. [18] C. Zhu, P. Vourc’h, P.O. Fernagut, S.M. Fleming, S. Lacan, C.D. Dicarlo, R.L. Seaman, M.F. Chesselet, Variable effects of chronic subcutaneous administration of rotenone on striatal histology, J. Comp. Neurol. 478 (2004) 418–426.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
NeuN is not a reliable marker of dopamine neurons in rat substantia nigra Jason R. Cannon, J. Timothy Greenamyre ∗ Pittsburgh Institute for Neurodegenerative Diseases, Department of Neurology, University of Pittsburgh, Pittsburgh, PA 15260, United States
a r t i c l e
i n f o
Article history: Received 3 June 2009 Received in revised form 30 July 2009 Accepted 6 August 2009 Keywords: NeuN Substantia nigra Dopamine neuron Parkinson’s disease
a b s t r a c t Quantification of neuronal cell number is a key endpoint in the characterization of neurodegenerative disease models and neuroprotective regimens. Immunohistochemistry for phenotypic markers, followed by unbiased stereology is often used to quantify the relevant neuronal population. To control for loss of phenotypic markers in the absence of cell death, or to determine if other types of neurons are lost, a general neuronal marker is often desired. Vertebrate neuron-specific nuclear protein (NeuN) is reportedly expressed in most mammalian neurons. In Parkinson’s disease models, NeuN has been widely used to determine if there is actual nigral dopamine neuron loss or simply loss of tyrosine hydroxylase expression, a prominent phenotypic marker. To date, the qualitative value of NeuN expression as such a marker in the substantia nigra has not been assessed. Midbrain tissue sections from control rats were stained for NeuN and tyrosine hydroxylase and assessed by light or confocal microscopy. Here we report that NeuN expression level in the rat substantia nigra was highly variable, with many faintly stained cells that would not be meet stereological scoring criteria. Additionally, dopamine neurons with little or no NeuN expression were readily identified. Subcellular compartmentalization of NeuN expression was also variable, with many cells dorsal and ventral to the nigra exhibiting expression in both the nucleus and cytoplasm. NeuN expression also appeared to be much higher in non-dopamine neurons within the ventral midbrain. This characterization of nigral NeuN expression suggests that it is not useful as a quantitative general neuronal marker in the substantia nigra. © 2009 Elsevier Ireland Ltd. All rights reserved.
Characterization of neuronal cell loss in the brain is a complex and tedious process. Unbiased stereological cell counting is now the accepted standard for quantitative assessment of neuronal cell number [16]. For a given treatment, it is often desirable to determine the specific types of neurons that are lost. In Parkinson’s disease, the loss of dopamine neurons in the substantia nigra is a key pathological feature and elicits motor symptoms [2,6]. Indeed, prominent neurotoxicant PD models including 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP), paraquat, and rotenone have all reported selective nigral dopamine neuron loss [3,5,8,12,14,15]. The claim of a ‘selective’ lesion typically needs to be supported by assessment of loss of other types of neurons or total neuronal loss. Additionally, it is possible that a treatment may result in loss of phenotypic marker expression, rather than actual neuronal cell loss. For example, a 6-hydroxydopamine regimen was found to elicit a 50% loss of fluorogold labeled neurons (retrograde neuronal tracer) and an 85% loss of tyrosine hydroxylase
∗ Corresponding author at: University of Pittsburgh, 3501 Fifth Avenue, Suite 7039, Pittsburgh, PA 15260, United States. Tel.: +1 412 648 9793; fax: +1 412 648 9766. E-mail address: [email protected] (J.T. Greenamyre). 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2009.08.023
positive neurons—evidence that loss of phenotype exceeded neuronal cell death [4]. While fluorogold is a useful marker for such a determination, it must be delivered by stereotaxic infusion. Vertebrate neuron-specific nuclear protein (NeuN) is reportedly expressed in the nuclei of the majority of nervous system neurons [9]. It is one of the most commonly used general immunohistochemical markers for neurons. NeuN immunohistochemistry in conjunction with specific phenotypic markers may be used to compare total neuronal loss with loss of a specific population of neurons. Indeed, NeuN immunohistochemistry has been widely used to test for loss of phenotypic marker expression and cell-type specificity of the lesions in PD models [1,10,18]. While NeuN is reportedly expressed in most neuronal populations, it is not expressed in cerebellar Purkinje cells, olfactory bulb mitral cells, and retinal photoreceptor cells [9]. To date, the utility of NeuN as a control for loss of expression of phenotypic markers or for overall neuronal loss in the midbrain has not been reported, although it is widely used for this purpose. Indeed, reports in other regions of the central nervous system and in disease models have questioned the utility of NeuN expression as a general neuronal marker. For example, after cerebral ischemia there is a loss of NeuN immunoreactivity, but healthy-appearing neurons were still evident with H&E staining [13]. Additionally spinal cord neurons
J.R. Cannon, J.T. Greenamyre / Neuroscience Letters 464 (2009) 14–17
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Fig. 1. Nigral NeuN immunohistochemistry. Representative low magnification image (A) indicates dramatic differences in staining intensity in different ventral midbrain neuronal populations. Red dashes indicate boundary of the substantia nigra. Bar = 100 m. High magnification image in the medial pars compacta region of the substantia nigra (B) shows variability in intensity and cellular localization. White arrows indicate faintly stained cells that would not meet threshold criteria for stereological counting. Localization of NeuN ranges from primarily nuclear to whole-cell immunoreactivity. Bar = 10 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
exhibit loss of expression during normal aging, while expression of other neuronal markers persists [11]. During routine analysis of midbrain sections stained for tyrosine hydroxylase and NeuN, we noticed several dopamine neurons with faint or no NeuN staining. The goal of the current study was to determine if NeuN could serve as a reliable marker for ventral midbrain neurons. Our data strongly cautions against the use of NeuN as such a marker. Adult male Lewis rats (n = 5) from multiple batches (∼3–5 months old) were used for all experiments (Hilltop Lab Animals Inc., Scottdale, PA, USA). The animals were maintained under standard conditions of 12-h light/dark cycles, 22 ± 1 ◦ C temperature-controlled room and 50–70% humidity and they were provided water and food ad libitum. All studies were approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh and in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. Three animals were deeply anesthetized with pentobarbital (50 mg/kg, i.p.) and perfusion-fixed, first with 50 mL ice-cold 0.1 M PBS, and then with 250–350 mL 4% paraformaldehyde. Brains were then post-fixed overnight and transferred to 30% sucrose, where they were stored until fully infiltrated and further processed. An additional two animals were euthanized by CO2 exposure, and brains were rapidly removed and bisected mid-sagittally. One hemisphere was post-fixed in 4% paraformaldehyde for 7 days and then placed in 30% sucrose for at least 3 days, until infiltration was complete. This fixation method is commonly used in our lab and others, to obtain many endpoints from the same tissue (the opposite hemisphere can be used for biochemical studies) and was employed here to determine if NeuN expression was influenced by fixation protocol. Brains were cut coronally on a frozen sliding microtome at 35 m and stored in cryoprotectant at −20 ◦ C until use. Sections for chromagen development were then removed from cryoprotectant, washed in PBS 6× for 5 min each then treated with 3% hydrogen peroxide for 10 min followed by three PBS washes and blocked for 1 h in 10% normal donkey serum, containing 0.3% Triton X100. The sections were incubated in a primary antibody for NeuN (1:1000, mouse anti-NeuN; Millipore #MAB377; Bilerica, MA, USA) for 72 h. The optimal antibody concentration was determined by testing a range of dilutions and choosing the concentration that produced the most intense staining in the absence of extracellular ‘background’ staining. The sections were then washed in PBS 3 × 5 min and incubated at room temperature for 1 h in biotinylated donkey anti-mouse secondary antibody (1:200, Jackson Immuno Research; West Grove, PA, USA). The sections were then washed
3× in PBS and incubated in Avidin–Biotin Complex solution (Vectastain, Vector Labs; Burligame, CA, USA). After washing in PBS, the sections were developed using DAB (Vector Labs). The tissue was mounted onto slides after staining, air dried, dehydrated using graded alcohols and Histoclear (National Diagnostics, Atlanta, GA, USA), followed by mounting with Histomount (National Diagnostics). For immunofluorescence, the staining was done as above except using primary antibodies for tyrosine hydroxylase (1:2000; rabbit anti-TH; AB152; Millipore) and NeuN, and one of the following fluorescent secondary antibodies was used: Cy3-conjugated anti-mouse (1:500; Jackson Immuno), Cy5-conjugated anti-rabbit (1:500; Jackson Immuno). Following secondary antibody incubation, the sections were treated with the nuclear dye Hoechst 33342 (Sigma–Aldrich, St. Louis, MO, USA) for 5 min. These sections were then washed and mounted using gelvatol mounting media. Images were captured on a light microscope (Olympus BX51) using the software DP controller (Olympus) or a confocal microscope (Olympus Fluoview FV1000), using the software FV10-ASW (Olympus). Lasers and detectors were optimized over several sections and the settings at each magnification were maintained throughout acquisition. Optimization for NeuN expression was conducted using several NeuN and tyrosine hydroxylase positive neurons, where the settings for NeuN expression were adjusted near saturation. Adjustments to intensity, brightness and contrast were consistently made to every image at a given magnification. Within the ventral midbrain NeuN expression level was highly variable. Low magnification images of NeuN immunohistochemistry of the substantia nigra clearly indicated a wide range in intensity of staining (Fig. 1A); many neurons were stained very faintly. Neuronal populations dorsal to the nigra had more intense staining than nigral neurons. Additionally, these extra-nigral neuronal populations showed both nuclear and cytoplasmic expression of NeuN. High magnification images show faintly stained NeuN cells that would not meet threshold criteria for stereological scoring (Fig. 1B). Expression within the nigra also ranges from purely nuclear to whole-cell immunoreactivity. These findings were observed in all animals examined, irrespective of fixation method. NeuN expression in dopamine neurons ranged from undetectable to intense (Fig. 2). Confocal microscopy of fluorescently labeled sections at low magnification showed that NeuN expression was highly variable in the medial tier of the pars compacta region of the substantia nigra (Fig. 2B). While nuclear staining (Fig. 2A) and dopamine neurons were clearly evident (Fig. 2C), NeuN expression levels varied widely between dopamine neurons (Fig. 2B). Non-
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J.R. Cannon, J.T. Greenamyre / Neuroscience Letters 464 (2009) 14–17
Fig. 2. Low magnification nigral NeuN confocal immunohistochemistry. The medial tier of the substantia nigra is stained for Hoechst 33342 (nuclear marker; blue; A), NeuN (green; B), and tyrosine hydroxylase (red; C). NeuN immunoreactivity is lower in the tyrosine hydroxylase positive neurons compared to neuronal populations dorsal or ventral to the pars compacta. Within the nigra, variability in NeuN immunoreactivity is also readily apparent. Bar = 100 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
dopamine neurons both dorsal and ventral to the nigra expressed NeuN at higher levels. Dopamine neurons that express little or no NeuN are readily apparent at high magnification (Fig. 3). Evidence of faint or undetectable NeuN expression in dopamine neurons was observed in all animals examined. NeuN is reportedly expressed in most vertebrate neuronal nuclei [9] and is often used as a general neuronal marker. In PD research it is often used in conjunction with phenotypic markers, such as tyrosine hydroxylase, to characterize dopamine neuron loss in the substantia nigra [1,10,18]. While NeuN has been widely utilized for this purpose, the utility of this marker has not been critically assessed. Here we report that NeuN expression magnitude varies dramatically from cell-to-cell within the substantia nigra, and dopamine neurons without detectable NeuN are readily identifiable. Additionally, localization ranges from primarily nuclear to both nuclear and cytoplasmic. Therefore, our data suggests that NeuN is not a reliable, quantitative marker for neurons of the substantia nigra.
Quantification of dopaminergic neuron number is one of the primary endpoints of PD model characterization; it is also critical for quantitative assessment of neuroprotection conferred by putative therapeutic regimens. However, this seemingly simple measurement may be confounded by issues related to cell-type specificity of the lesion (or lack thereof), or by loss of phenotype instead of cell loss. For example, it is possible that a given model may be associated with loss of both dopaminergic and non-dopaminergic neurons in the substantia nigra. Such a lesion would have less relevance to clinical PD, considering the rather specific loss of dopamine neurons that occurs in the human PD substantia nigra [6]. To address this possibility, the total number of nigral neurons – or another identified neuronal population – may be counted in addition to tyrosine hydroxylase positive neurons. Similarly, counts of a general neuronal marker can be used to determine whether loss of tyrosine hydroxylase staining represents loss of neurons or loss of phenotype. While these issues should ideally be addressed when characterizing a new model, or a ‘neuroprotective’ treatment, our
Fig. 3. High magnification nigral NeuN confocal immunohistochemistry. Sections were stained for stained for Hoechst 33342 (nuclear marker; blue; A, E), NeuN (green; B, F), and tyrosine hydroxylase (red; C, G). Examples of NeuN negative (D) and NeuN positive (H) dopamine neurons (tyrosine hydroxylase positive) from the dorsolateral substantia nigra are presented. NeuN immunoreactivity is typically lower in dopamine neurons compared to non-dopamine neurons (H). Bar = 10 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
J.R. Cannon, J.T. Greenamyre / Neuroscience Letters 464 (2009) 14–17
results suggest that NeuN expression is not suitable for this purpose. Our findings also show that nigral NeuN expression may be both nuclear and cytoplasmic. This makes interpretation of doublestained sections with two chromagens difficult to interpret. As a result, in stereological studies, counting both tyrosine hydroxylaseand NeuN-positive cells may be very difficult. Indeed these results are in agreement with a previous study, in which NeuN was found to be a phosphoprotein with both cytoplasmic and nuclear localization in the mouse cortex [7]. Moreover, although histological procedures, such as antigen retrieval, may improve staining quality/intensity [17], such methods may not be appropriate for stereology because they would likely affect tissue volume. Discrepancies in NeuN staining outside the substantia nigra have been reported previously. During normal aging in the rat, NeuN immunoreactivity is lost in spinal cord neurons, in the absence of cell loss [11]. Additionally, in mice under pathological conditions such as cerebral ischemia, NeuN immunoreactivity is lost, while apparently healthy H&E-stained neurons persist [13]. Therefore, NeuN expression may not be a reliable neuronal marker in aged animals or in certain disease models. Given that variability in NeuN expression has been reported in both rats and mice, this phenomenon is likely species independent. Our results further suggest that even in control animals significant variability exists in the substantia nigra. The percentage of dopamine neurons that are NeuN-negative was not quantified in this study. However, our finding of an easily observed absence of NeuN expression in dopamine neurons is sufficient to preclude its use as a quantitative marker for nigral neurons. In summary, we have shown that NeuN is not a reliable marker of neurons in the substantia nigra and that this marker should not be used to control for loss of phenotype or to assess total neuronal counts in the ventral midbrain. Acknowledgements This work was supported by a postdoctoral fellowship from the American Parkinson Disease Association (J.R.C.). References [1] Z.C. Baquet, P.C. Bickford, K.R. Jones, Brain-derived neurotrophic factor is required for the establishment of the proper number of dopaminergic neurons in the substantia nigra pars compacta, J. Neurosci. 25 (2005) 6251–6259.
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