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Visualization of Sensory Neurons and Their Projections in an Upper Motor Neuron Reporter Line.

Genç B, Lagrimas AK, Kuru P, Hess R, Tu MW, Menichella DM, Miller RJ, Paller AS, Özdinler PH - PLoS ONE (2015)

Bottom Line: Our findings corroborate previous studies, and suggest that more than five months is required for significant skin denervation.Crossing UCHL1-eGFP with hSOD1G93A mice generated hSOD1G93A-UeGFP reporter line of amyotrophic lateral sclerosis, and revealed sensory nervous system defects, especially towards disease end-stage.Our studies not only emphasize the complexity of the disease in ALS, but also reveal that UCHL1-eGFP reporter line would be a valuable tool to visualize and study various aspects of sensory nervous system development and degeneration in the context of numerous diseases.

View Article: PubMed Central - PubMed

Affiliation: Davee Department of Neurology and Clinical Neurological Sciences, Northwestern University, Feinberg School of Medicine, Chicago, IL, United States of America.

ABSTRACT
Visualization of peripheral nervous system axons and cell bodies is important to understand their development, target recognition, and integration into complex circuitries. Numerous studies have used protein gene product (PGP) 9.5 [a.k.a. ubiquitin carboxy-terminal hydrolase L1 (UCHL1)] expression as a marker to label sensory neurons and their axons. Enhanced green fluorescent protein (eGFP) expression, under the control of UCHL1 promoter, is stable and long lasting in the UCHL1-eGFP reporter line. In addition to the genetic labeling of corticospinal motor neurons in the motor cortex and degeneration-resistant spinal motor neurons in the spinal cord, here we report that neurons of the peripheral nervous system are also fluorescently labeled in the UCHL1-eGFP reporter line. eGFP expression is turned on at embryonic ages and lasts through adulthood, allowing detailed studies of cell bodies, axons and target innervation patterns of all sensory neurons in vivo. In addition, visualization of both the sensory and the motor neurons in the same animal offers many advantages. In this report, we used UCHL1-eGFP reporter line in two different disease paradigms: diabetes and motor neuron disease. eGFP expression in sensory axons helped determine changes in epidermal nerve fiber density in a high-fat diet induced diabetes model. Our findings corroborate previous studies, and suggest that more than five months is required for significant skin denervation. Crossing UCHL1-eGFP with hSOD1G93A mice generated hSOD1G93A-UeGFP reporter line of amyotrophic lateral sclerosis, and revealed sensory nervous system defects, especially towards disease end-stage. Our studies not only emphasize the complexity of the disease in ALS, but also reveal that UCHL1-eGFP reporter line would be a valuable tool to visualize and study various aspects of sensory nervous system development and degeneration in the context of numerous diseases.

No MeSH data available.


Related in: MedlinePlus

All DRG neurons are eGFP+ and nociceptive neurons display bright GFP.(A-B) eGFP is expressed in all DRG neurons in UCHL1-eGFP reporter mice (A) and is completely absent in the wild type (WT) mice (B). (C) Pie-chart graph showing distribution of eGFP+ neurons. (D) eGFP co-localizes with UCHL1 in P30 DRG neurons. (E) Co-localization of bright eGFP+ with Parvalbumin (PV), a marker for proprioceptive neurons. (F) Bar graph representation of average percentage of bright and dim eGFP+ DRG neurons among PV+ neurons. (G) Co-localization of bright eGFP+ with Isolectin IB4, a marker for nociceptive neurons. (H) Bar graph representation of average percentage of bright and dim eGFP+ DRG neurons among IB4+ neurons. (I) Co-localization of bright eGFP+ with calcitonin gene related peptide (CGRP), a marker for nociceptive neurons. (J) Bar graph representation of average percentage of bright and dim eGFP+ DRG neurons among CGRP+ neurons. Bar graphs represent mean ± SEM. Student’s t-test, * P < 0.05, ** P < 0.01, *** P < 0.001. Scale bars A-B,D,E,G,I 100 μm.
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pone.0132815.g002: All DRG neurons are eGFP+ and nociceptive neurons display bright GFP.(A-B) eGFP is expressed in all DRG neurons in UCHL1-eGFP reporter mice (A) and is completely absent in the wild type (WT) mice (B). (C) Pie-chart graph showing distribution of eGFP+ neurons. (D) eGFP co-localizes with UCHL1 in P30 DRG neurons. (E) Co-localization of bright eGFP+ with Parvalbumin (PV), a marker for proprioceptive neurons. (F) Bar graph representation of average percentage of bright and dim eGFP+ DRG neurons among PV+ neurons. (G) Co-localization of bright eGFP+ with Isolectin IB4, a marker for nociceptive neurons. (H) Bar graph representation of average percentage of bright and dim eGFP+ DRG neurons among IB4+ neurons. (I) Co-localization of bright eGFP+ with calcitonin gene related peptide (CGRP), a marker for nociceptive neurons. (J) Bar graph representation of average percentage of bright and dim eGFP+ DRG neurons among CGRP+ neurons. Bar graphs represent mean ± SEM. Student’s t-test, * P < 0.05, ** P < 0.01, *** P < 0.001. Scale bars A-B,D,E,G,I 100 μm.

Mentions: eGFP expression is present in all neurons (100%; n = 3924 neurons) in the P30 lumbar DRG of the UCHL1-eGFP reporter line (Fig 2A), and not detected in the control WT littermates (Fig 2B). Close inspection of the DRG reveals that sensory neurons vary both in size and also in the intensity of eGFP and UCHL1 expression. Even though UCHL1 is used as a marker for all sensory neurons, similar to eGFP expression it displayed varied levels of intensity. Quantitative analysis of eGFP expression showed that even though all DRG neurons express eGFP, about 1/4th of them are relatively brighter (Fig 2C; 27 ± 1% bright, 73 ± 1% dim; P < 0.0001). To investigate the possibility that these very bright GFP+ neurons represent a specific subpopulation of sensory neurons, we performed co-labeling studies using molecular markers for different classes, such as isolectin B4 (IB4), calcitonin gene related peptide (CGRP), and parvalbumin (PV), which are markers for peptidergic [22,23] and non-peptidergic [24] nociceptive, and proprioceptive [25,26] neurons, respectively (Fig 2E and 2G, and 2I). 64 ± 3% of bright eGFP+ neurons expressed CGRP (n = 2397 neurons; P = 0.002202173), 44 ± 3% co-localized with IB4 (n = 1687 neurons), and only 3% were PV+ (n = 5279 neurons; P < 0.0001), suggesting that the very bright neurons were mainly nociceptive, but not propriceptive. However, all DRG neurons expressed eGFP, albeit at different levels. Out of all the PV+ cells 13% were bright and 87% of dim (Fig 2F; n = 1419 neurons; P < 0.0001), but there was not a single PV+ neurons that did not express eGFP. Likewise, out of all the IB4+ neurons, 60 ± 4% were bright (Fig 2H; n = 1188 neurons; P = 0.0246), and out of all CGRP+ neurons 25 ± 1% were bright (Fig 2J; n = 1539 neurons, P < 0.0001). These quantitative analyses revealed that all DRG neurons were genetically labeled with eGFP expression in the UCHL1-eGFP mice, and mainly the nociceptive neurons displayed very bright eGFP expression.


Visualization of Sensory Neurons and Their Projections in an Upper Motor Neuron Reporter Line.

Genç B, Lagrimas AK, Kuru P, Hess R, Tu MW, Menichella DM, Miller RJ, Paller AS, Özdinler PH - PLoS ONE (2015)

All DRG neurons are eGFP+ and nociceptive neurons display bright GFP.(A-B) eGFP is expressed in all DRG neurons in UCHL1-eGFP reporter mice (A) and is completely absent in the wild type (WT) mice (B). (C) Pie-chart graph showing distribution of eGFP+ neurons. (D) eGFP co-localizes with UCHL1 in P30 DRG neurons. (E) Co-localization of bright eGFP+ with Parvalbumin (PV), a marker for proprioceptive neurons. (F) Bar graph representation of average percentage of bright and dim eGFP+ DRG neurons among PV+ neurons. (G) Co-localization of bright eGFP+ with Isolectin IB4, a marker for nociceptive neurons. (H) Bar graph representation of average percentage of bright and dim eGFP+ DRG neurons among IB4+ neurons. (I) Co-localization of bright eGFP+ with calcitonin gene related peptide (CGRP), a marker for nociceptive neurons. (J) Bar graph representation of average percentage of bright and dim eGFP+ DRG neurons among CGRP+ neurons. Bar graphs represent mean ± SEM. Student’s t-test, * P < 0.05, ** P < 0.01, *** P < 0.001. Scale bars A-B,D,E,G,I 100 μm.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4519325&req=5

pone.0132815.g002: All DRG neurons are eGFP+ and nociceptive neurons display bright GFP.(A-B) eGFP is expressed in all DRG neurons in UCHL1-eGFP reporter mice (A) and is completely absent in the wild type (WT) mice (B). (C) Pie-chart graph showing distribution of eGFP+ neurons. (D) eGFP co-localizes with UCHL1 in P30 DRG neurons. (E) Co-localization of bright eGFP+ with Parvalbumin (PV), a marker for proprioceptive neurons. (F) Bar graph representation of average percentage of bright and dim eGFP+ DRG neurons among PV+ neurons. (G) Co-localization of bright eGFP+ with Isolectin IB4, a marker for nociceptive neurons. (H) Bar graph representation of average percentage of bright and dim eGFP+ DRG neurons among IB4+ neurons. (I) Co-localization of bright eGFP+ with calcitonin gene related peptide (CGRP), a marker for nociceptive neurons. (J) Bar graph representation of average percentage of bright and dim eGFP+ DRG neurons among CGRP+ neurons. Bar graphs represent mean ± SEM. Student’s t-test, * P < 0.05, ** P < 0.01, *** P < 0.001. Scale bars A-B,D,E,G,I 100 μm.
Mentions: eGFP expression is present in all neurons (100%; n = 3924 neurons) in the P30 lumbar DRG of the UCHL1-eGFP reporter line (Fig 2A), and not detected in the control WT littermates (Fig 2B). Close inspection of the DRG reveals that sensory neurons vary both in size and also in the intensity of eGFP and UCHL1 expression. Even though UCHL1 is used as a marker for all sensory neurons, similar to eGFP expression it displayed varied levels of intensity. Quantitative analysis of eGFP expression showed that even though all DRG neurons express eGFP, about 1/4th of them are relatively brighter (Fig 2C; 27 ± 1% bright, 73 ± 1% dim; P < 0.0001). To investigate the possibility that these very bright GFP+ neurons represent a specific subpopulation of sensory neurons, we performed co-labeling studies using molecular markers for different classes, such as isolectin B4 (IB4), calcitonin gene related peptide (CGRP), and parvalbumin (PV), which are markers for peptidergic [22,23] and non-peptidergic [24] nociceptive, and proprioceptive [25,26] neurons, respectively (Fig 2E and 2G, and 2I). 64 ± 3% of bright eGFP+ neurons expressed CGRP (n = 2397 neurons; P = 0.002202173), 44 ± 3% co-localized with IB4 (n = 1687 neurons), and only 3% were PV+ (n = 5279 neurons; P < 0.0001), suggesting that the very bright neurons were mainly nociceptive, but not propriceptive. However, all DRG neurons expressed eGFP, albeit at different levels. Out of all the PV+ cells 13% were bright and 87% of dim (Fig 2F; n = 1419 neurons; P < 0.0001), but there was not a single PV+ neurons that did not express eGFP. Likewise, out of all the IB4+ neurons, 60 ± 4% were bright (Fig 2H; n = 1188 neurons; P = 0.0246), and out of all CGRP+ neurons 25 ± 1% were bright (Fig 2J; n = 1539 neurons, P < 0.0001). These quantitative analyses revealed that all DRG neurons were genetically labeled with eGFP expression in the UCHL1-eGFP mice, and mainly the nociceptive neurons displayed very bright eGFP expression.

Bottom Line: Our findings corroborate previous studies, and suggest that more than five months is required for significant skin denervation.Crossing UCHL1-eGFP with hSOD1G93A mice generated hSOD1G93A-UeGFP reporter line of amyotrophic lateral sclerosis, and revealed sensory nervous system defects, especially towards disease end-stage.Our studies not only emphasize the complexity of the disease in ALS, but also reveal that UCHL1-eGFP reporter line would be a valuable tool to visualize and study various aspects of sensory nervous system development and degeneration in the context of numerous diseases.

View Article: PubMed Central - PubMed

Affiliation: Davee Department of Neurology and Clinical Neurological Sciences, Northwestern University, Feinberg School of Medicine, Chicago, IL, United States of America.

ABSTRACT
Visualization of peripheral nervous system axons and cell bodies is important to understand their development, target recognition, and integration into complex circuitries. Numerous studies have used protein gene product (PGP) 9.5 [a.k.a. ubiquitin carboxy-terminal hydrolase L1 (UCHL1)] expression as a marker to label sensory neurons and their axons. Enhanced green fluorescent protein (eGFP) expression, under the control of UCHL1 promoter, is stable and long lasting in the UCHL1-eGFP reporter line. In addition to the genetic labeling of corticospinal motor neurons in the motor cortex and degeneration-resistant spinal motor neurons in the spinal cord, here we report that neurons of the peripheral nervous system are also fluorescently labeled in the UCHL1-eGFP reporter line. eGFP expression is turned on at embryonic ages and lasts through adulthood, allowing detailed studies of cell bodies, axons and target innervation patterns of all sensory neurons in vivo. In addition, visualization of both the sensory and the motor neurons in the same animal offers many advantages. In this report, we used UCHL1-eGFP reporter line in two different disease paradigms: diabetes and motor neuron disease. eGFP expression in sensory axons helped determine changes in epidermal nerve fiber density in a high-fat diet induced diabetes model. Our findings corroborate previous studies, and suggest that more than five months is required for significant skin denervation. Crossing UCHL1-eGFP with hSOD1G93A mice generated hSOD1G93A-UeGFP reporter line of amyotrophic lateral sclerosis, and revealed sensory nervous system defects, especially towards disease end-stage. Our studies not only emphasize the complexity of the disease in ALS, but also reveal that UCHL1-eGFP reporter line would be a valuable tool to visualize and study various aspects of sensory nervous system development and degeneration in the context of numerous diseases.

No MeSH data available.


Related in: MedlinePlus