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NTRK2 expression levels are reduced in laser captured pyramidal neurons from the anterior cingulate cortex in males with autism spectrum disorder.

Chandley MJ, Crawford JD, Szebeni A, Szebeni K, Ordway GA - Mol Autism (2015)

Bottom Line: The level of NTRK2 expression was robustly and significantly lower in pyramidal neurons from ASD donors as compared to typically developing donors.Levels of expression of GRIN1, GRM8, SLC1A1, and GRIP1 were modestly lower in pyramidal neurons from ASD donors, but statistical significance for these latter genes did not survive correction for multiple comparisons.No significant expression differences of any genes were found in astrocytes laser captured from the same neocortical area.

View Article: PubMed Central - PubMed

Affiliation: Department of Health Sciences, College of Public Health, East Tennessee State University, P.O. Box 70673, Johnson City, TN 37614 USA.

ABSTRACT

Background: The anterior cingulate cortex (ACC) is a brain area involved in modulating behavior associated with social interaction, disruption of which is a core feature of autism spectrum disorder (ASD). Functional brain imaging studies demonstrate abnormalities of the ACC in ASD as compared to typically developing control patients. However, little is known regarding the cellular basis of these functional deficits in ASD. Pyramidal neurons in the ACC are excitatory glutamatergic neurons and key cellular mediators of the neural output of the ACC. This study was designed to investigate the potential role of ACC pyramidal neurons in ASD brain pathology.

Methods: Postmortem ACC tissue from carefully matched ASD and typically developing control donors was obtained from two national brain collections. Pyramidal neurons and surrounding astrocytes were separately collected from layer III of the ACC by laser capture microdissection. Isolated RNA was subjected to reverse transcription and endpoint PCR to determine gene expression levels for 16 synaptic genes relevant to glutamatergic neurotransmission. Cells were also collected from the prefrontal cortex (Brodmann area 10) to examine those genes demonstrating differences in expression in the ACC comparing typically developing and ASD donors.

Results: The level of NTRK2 expression was robustly and significantly lower in pyramidal neurons from ASD donors as compared to typically developing donors. Levels of expression of GRIN1, GRM8, SLC1A1, and GRIP1 were modestly lower in pyramidal neurons from ASD donors, but statistical significance for these latter genes did not survive correction for multiple comparisons. No significant expression differences of any genes were found in astrocytes laser captured from the same neocortical area. In addition, expression levels of NTRK2 and other synaptic genes were normal in pyramidal neurons laser captured from the prefrontal cortex.

Conclusions: These studies demonstrate a unique pathology of neocortical pyramidal neurons of the ACC in ASD. NTRK2 encodes the tropomyosin receptor kinase B (TrkB), transmission through which neurotrophic factors modify differentiation, plasticity, and synaptic transmission. Reduced pyramidal neuron NTRK2 expression in the ACC could thereby contribute to abnormal neuronal activity and disrupt social behavior mediated by this brain region.

No MeSH data available.


Related in: MedlinePlus

Laser capture microdissection of pyramidal neurons and astrocytes. Shown in (A) is the absence of laser captured nissl-stained pyramidal neurons from cortical layer 3 in BA24 gray matter tissue following capture (20× magnification). (B) shows those cells adhered to the polymer cap (20× magnification). (C) and (D) illustrate the before (C) and after (D) capture images for nissl-stained BA24 pyramidal neurons (40× magnification). (E) and (F) show the same laser capture process for GFAP immunostained astrocytes in BA24 gray matter (60× magnification).
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Fig1: Laser capture microdissection of pyramidal neurons and astrocytes. Shown in (A) is the absence of laser captured nissl-stained pyramidal neurons from cortical layer 3 in BA24 gray matter tissue following capture (20× magnification). (B) shows those cells adhered to the polymer cap (20× magnification). (C) and (D) illustrate the before (C) and after (D) capture images for nissl-stained BA24 pyramidal neurons (40× magnification). (E) and (F) show the same laser capture process for GFAP immunostained astrocytes in BA24 gray matter (60× magnification).

Mentions: Neurons were visualized by staining frozen tissue sections with the Histogene staining kit (Life Technologies; Grand Island, NY, USA) according to the manufacturer’s instructions (Figure 1C). In short, the protocol used a cresyl violet stain on an ethanol-fixed slide followed by a series of dehydration steps. Stained slides were placed in a vacuum chamber until ready for LCM. Astrocytes were identified using a modified glial fibrillary acidic protein (GFAP) rapid immunohistochemistry protocol as previously described (Figure 1E) [36,37]. Briefly, tissue-mounted slides were fixed in acetone (5 min), blocked in horse serum (10 min), incubated with GFAP antibody (10 min; ThermoFisher; Pittsburgh, PA, USA), anti-mouse antibody (5 min; Vectastain; Burlingame, CA, USA), and avidin-biotinylated horseradish peroxidase (5 min; Vectastain; Burlingame, CA, USA). GFAP immunoreactivity was visualized by exposure to diaminobenzidine (DAB) (Sigma; St. Louis, MO, USA) with ammonium nickel sulfate (ANS) (Sigma; St. Louis, MO, USA) for 5 min, then exposed to DAB/ANS with 0.1% H2O2 for an additional 5 min. Afterwards, the slide was ethanol dehydrated then placed in xylene for 5 min. The slide was kept in a vacuum chamber for 10 min prior to LCM.Figure 1


NTRK2 expression levels are reduced in laser captured pyramidal neurons from the anterior cingulate cortex in males with autism spectrum disorder.

Chandley MJ, Crawford JD, Szebeni A, Szebeni K, Ordway GA - Mol Autism (2015)

Laser capture microdissection of pyramidal neurons and astrocytes. Shown in (A) is the absence of laser captured nissl-stained pyramidal neurons from cortical layer 3 in BA24 gray matter tissue following capture (20× magnification). (B) shows those cells adhered to the polymer cap (20× magnification). (C) and (D) illustrate the before (C) and after (D) capture images for nissl-stained BA24 pyramidal neurons (40× magnification). (E) and (F) show the same laser capture process for GFAP immunostained astrocytes in BA24 gray matter (60× magnification).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4440594&req=5

Fig1: Laser capture microdissection of pyramidal neurons and astrocytes. Shown in (A) is the absence of laser captured nissl-stained pyramidal neurons from cortical layer 3 in BA24 gray matter tissue following capture (20× magnification). (B) shows those cells adhered to the polymer cap (20× magnification). (C) and (D) illustrate the before (C) and after (D) capture images for nissl-stained BA24 pyramidal neurons (40× magnification). (E) and (F) show the same laser capture process for GFAP immunostained astrocytes in BA24 gray matter (60× magnification).
Mentions: Neurons were visualized by staining frozen tissue sections with the Histogene staining kit (Life Technologies; Grand Island, NY, USA) according to the manufacturer’s instructions (Figure 1C). In short, the protocol used a cresyl violet stain on an ethanol-fixed slide followed by a series of dehydration steps. Stained slides were placed in a vacuum chamber until ready for LCM. Astrocytes were identified using a modified glial fibrillary acidic protein (GFAP) rapid immunohistochemistry protocol as previously described (Figure 1E) [36,37]. Briefly, tissue-mounted slides were fixed in acetone (5 min), blocked in horse serum (10 min), incubated with GFAP antibody (10 min; ThermoFisher; Pittsburgh, PA, USA), anti-mouse antibody (5 min; Vectastain; Burlingame, CA, USA), and avidin-biotinylated horseradish peroxidase (5 min; Vectastain; Burlingame, CA, USA). GFAP immunoreactivity was visualized by exposure to diaminobenzidine (DAB) (Sigma; St. Louis, MO, USA) with ammonium nickel sulfate (ANS) (Sigma; St. Louis, MO, USA) for 5 min, then exposed to DAB/ANS with 0.1% H2O2 for an additional 5 min. Afterwards, the slide was ethanol dehydrated then placed in xylene for 5 min. The slide was kept in a vacuum chamber for 10 min prior to LCM.Figure 1

Bottom Line: The level of NTRK2 expression was robustly and significantly lower in pyramidal neurons from ASD donors as compared to typically developing donors.Levels of expression of GRIN1, GRM8, SLC1A1, and GRIP1 were modestly lower in pyramidal neurons from ASD donors, but statistical significance for these latter genes did not survive correction for multiple comparisons.No significant expression differences of any genes were found in astrocytes laser captured from the same neocortical area.

View Article: PubMed Central - PubMed

Affiliation: Department of Health Sciences, College of Public Health, East Tennessee State University, P.O. Box 70673, Johnson City, TN 37614 USA.

ABSTRACT

Background: The anterior cingulate cortex (ACC) is a brain area involved in modulating behavior associated with social interaction, disruption of which is a core feature of autism spectrum disorder (ASD). Functional brain imaging studies demonstrate abnormalities of the ACC in ASD as compared to typically developing control patients. However, little is known regarding the cellular basis of these functional deficits in ASD. Pyramidal neurons in the ACC are excitatory glutamatergic neurons and key cellular mediators of the neural output of the ACC. This study was designed to investigate the potential role of ACC pyramidal neurons in ASD brain pathology.

Methods: Postmortem ACC tissue from carefully matched ASD and typically developing control donors was obtained from two national brain collections. Pyramidal neurons and surrounding astrocytes were separately collected from layer III of the ACC by laser capture microdissection. Isolated RNA was subjected to reverse transcription and endpoint PCR to determine gene expression levels for 16 synaptic genes relevant to glutamatergic neurotransmission. Cells were also collected from the prefrontal cortex (Brodmann area 10) to examine those genes demonstrating differences in expression in the ACC comparing typically developing and ASD donors.

Results: The level of NTRK2 expression was robustly and significantly lower in pyramidal neurons from ASD donors as compared to typically developing donors. Levels of expression of GRIN1, GRM8, SLC1A1, and GRIP1 were modestly lower in pyramidal neurons from ASD donors, but statistical significance for these latter genes did not survive correction for multiple comparisons. No significant expression differences of any genes were found in astrocytes laser captured from the same neocortical area. In addition, expression levels of NTRK2 and other synaptic genes were normal in pyramidal neurons laser captured from the prefrontal cortex.

Conclusions: These studies demonstrate a unique pathology of neocortical pyramidal neurons of the ACC in ASD. NTRK2 encodes the tropomyosin receptor kinase B (TrkB), transmission through which neurotrophic factors modify differentiation, plasticity, and synaptic transmission. Reduced pyramidal neuron NTRK2 expression in the ACC could thereby contribute to abnormal neuronal activity and disrupt social behavior mediated by this brain region.

No MeSH data available.


Related in: MedlinePlus