Detection of mRNA species in bulbospinal neurons isolated from the rostral ventrolateral medulla using single-cell RT–PCR

doi:10.1016/S1385-299X(99)00042-2

Brain Research Protocols

Volume 4, Issue 3, December 1999, Pages 367-377

https://doi.org/10.1016/S1385-299X(99)00042-2 Get rights and content

Abstract

The rostral ventrolateral medulla (RVL) contains neurons which are critically involved in the tonic and reflex control of blood pressure. Some of these neurons project to the intermediolateral cell column of the thoracolumbar spinal cord and excite preganglionic sympathetic neurons. In order to gain a better understanding of the properties of the RVL neurons at the cellular and molecular level, a protocol was developed utilizing acute dissociation and the reverse transcription–polymerase chain reaction (RT–PCR) to study the expression of several genes in single RVL neurons. Neurons were dissociated from the RVL region of young rats, and classified as spinally projecting or non-spinal by the presence or absence of retrogradely transported fluorescent beads injected into the upper thoracic segments of the spinal cord. Individual neurons were collected by aspiration into a glass micropipette and analysed by RT–PCR. The presence of either glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or neuron-specific enolase (NSE) mRNA was used as the criterion for selecting cells for further analysis. A subpopulation (50%) of spinally projecting, GAPDH- or NSE-positive neurons expressed mRNA for tyrosine hydroxylase (TH) or phenylethanolamine N-methyltransferase (PNMT), indicative of catecholaminergic or C1 adrenergic neurons, respectively. Some bulbospinal RVL neurons, including those that were TH- or PNMT-positive, were also found to express mRNA for the mineralocorticoid receptor (MR), the glucocorticoid receptor (GR), noradrenaline transporter (NET), and neuronal glutamate transporter (EAAC1). The glial glutamate transporter (GLT), glycine transporter (GLYT2), glutamic acid decarboxylase (GAD67) and gamma-amino butyric acid (GABA) transporter (GAT-1) were not expressed. The single-cell RT–PCR protocol is a powerful, yet simple and relatively rapid method for analysis of mRNA expression in a defined neuronal population. It can be combined with whole-cell patch-clamp recording prior to RT–PCR analysis, allowing linkage of the molecular analysis of mRNA expression to the electrophysiological and pharmacological properties of single neurons. The method is very sensitive, enabling mRNA transcripts in low abundance to be detected, and its application in our recent studies provided novel information about neurons involved in blood-pressure regulation at the molecular and cellular level.
Theme E: Endocrine and autonomic regulation
Topic 76: Cardiovascular regulation — central

Section snippets

Type of research

Identification of genes expressed in single neurons acutely dissociated from the rostral ventrolateral medulla of young rats using reverse transcription–polymerase chain reaction (RT–PCR) analysis of mRNA 5, 19, 23. The mRNA species investigated in the present study included those coding for two catecholamine synthesizing enzymes, two receptors, and several neurotransmitter transporters. The technique can be used to establish expression of any gene for which cDNA or genomic sequence is known.

Time required

The whole protocol can be completed within two working days, or within a 24-h period if necessary. The specific breakdown of the time required is as follows:

•
Cell dissociation: 2–3 h.
•
Electrophysiological recording: variable (optional).
•
Cell collection: 0.5–1 h depending on the number of cells taken.
•
Reverse transcription (RT) of RNA: 1.5 h.
•
PCR amplification of genes of interest: 8–10 h, depending on the number of amplification cycles required for each of the two rounds of amplification, and the

Animals

Wistar rat pups (7–19 days old) were obtained from the Animal Facility in the Faculty of Medicine and Health Science, University of Auckland. They were housed with their mothers until required for surgery/experimental use. Approval was obtained for all animal experimentation from the University of Auckland Animal Ethics Committee.

Special equipment

•
Vibratome (Series 1000, TPI, St. Louis, MO, USA).
•
Incubation chamber for tissue dissociation (similar to that described in Ref. [20]) and a magnetic stirrer.
•
Inverted

Retrograde labelling of RVL bulbospinal neurons

⋅ Anaesthetize Wistar rat pups (P7–P19) with halothane, and under aseptic conditions surgically expose the dorsal surface of the upper thoracic spinal cord segments.

⋅ Inject rhodamine-labelled latex microspheres (Lumafluor) unilaterally or bilaterally into the T2–T4 segments using either a Hamilton syringe (30 gauge needle) or pressure injection through a glass micropipette (tip diameter of about 40 μm). Make one or two 0.5-μl deposits per side.

⋅ Close the wound with acrylic glue, and allow the

Results

Each dissociation of the RVL region typically resulted in tens of healthy looking neuron-like cells (Fig. 1AFig. 2A). Approximately 5% of the neurons were identified as spinally projecting by the presence of rhodamine-labelled microspheres in the cell body and proximal dendrites (Fig. 1B). The cell bodies had various shapes, including oval, fusiform, multipolar and triangular, with 2–6 processes (average 3.5). The average soma size (n=23; mean±S.E.M.) was 24.5±1.2 μm (major axis) by 13.4±0.4 μm

General

We describe here a protocol for acute dissociation of neurons from the medulla oblongata of young rats, which allows functional and molecular characterization of these neurons under conditions where all cell-to-cell interactions are eliminated. The neurons were dissociated from a small, well-defined medullary region, and identified as spinally projecting or non-spinal by the presence of fluorescent rhodamine microbeads injected 3–6 days earlier into the animal's thoracic spinal cord. The cell

Retrograde labelling of bulbospinal RVL neurons

•
Anaesthetize Wistar rat pups (P7–P19) with halothane.
•
Expose upper thoracic spinal cord.
•
Inject rhodamine-labelled latex microspheres into the T2–T4 spinal segments.
•
Close wound, allow pups to recover.

Cell dissociation

•
Three to six days after retrograde labelling, anaesthetize rat pups by CO₂ inhalation, decapitate, remove brain rapidly and immerse it in ice-cold aCSF.
•
Cut transverse 350 μm sections on a Vibratome.
•
Select a slice containing the RVL area and enzymatically digest using papain.
•
Dissect RVL region out of

Essential references

Refs. 5, 9, 20, 21, 23, 33.

Acknowledgements

This study was supported by the Health Research Council of New Zealand and the Lottery Health Board. The authors are grateful to Dr. C. Jiang (Georgia State University) and Ms. M.S. Lim (Australian National University) for their helpful advice on neuron dissociation techniques, and to Dr. L. Kubin (University of Pennsylvania) for comments on the manuscript.

References (33)

J.L Arriza et al.
The neuronal mineralocorticoid receptor as a mediator of glucocorticoid response
Neuron
(1988)
A.M Comer et al.
Noradrenaline transporter expression in the pons and medulla oblongata of the rat: localisation to noradrenergic and some C1 adrenergic neurones
Mol. Brain Res.
(1998)
K Fuxe et al.
Immunocytochemical studies on the localization of glucocorticoid receptor immunoreactive nerve cells in the lower brain stem and spinal cord of the male rat using a monoclonal antibody against rat liver glucocorticoid receptor
Neurosci. Lett.
(1985)
G.M Halliday et al.
Four groups of tyrosine hydroxylase-immunoreactive neurons in the ventrolateral medulla of rats, guinea-pigs and cats identified on the basis of chemistry, topography and morphology
Neuroscience
(1991)
J.R Haselton et al.
Electrophysiological characterization of putative C1 adrenergic neurons in the rat
Neuroscience
(1989)
A Hayar et al.
GABA-induced responses in electrophysiologically characterized neurons within the rat rostro-ventrolateral medulla in vitro
Brain Res.
(1996)
F.F Johansen et al.
Single cell RT–PCR proceeds without the risk of genomic DNA amplification
Neurochem. Int.
(1995)
I.M Kangrga et al.
Whole-cell recordings from visualized C1 adrenergic bulbospinal neurons: ionic mechanisms underlying vasomotor tone
Brain Res.
(1995)
Y Kawai et al.
Effects of cyanide and hypoxia on neurones acutely dissociated from the rostral ventrolateral medulla of the rat
Brain Res.
(1999)
A.R Kay et al.
Isolation of neurons suitable for patch-clamping from adult mammalian central nervous systems
J. Neurosci. Methods
(1986)

S.A Mackler et al.

Stimulus-induced coordinate changes in mRNA abundance in single postsynaptic hippocampal CA1 neurons

Neuron

(1992)

J.D Rothstein et al.

Localization of neuronal and glial glutamate transporters

Neuron

(1994)

D.A Ruggiero et al.

Adrenergic and non-adrenergic spinal projections of a cardiovascular-active pressor area of medulla oblongata: quantitative topographic analysis

Brain Res.

(1994)

R Ahima et al.

Type I corticosteroid receptor-like immunoreactivity in the rat CNS: distribution and regulation by corticosteroids

J. Comp. Neurol.

(1991)

M Aronsson et al.

Localization of glucocorticoid receptor mRNA in the male rat brain by in situ hybridization

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

(1988)

A Cintra et al.

Glial and neuronal glucocorticoid receptor immunoreactive cell populations in developing, adult, and aging brain

Ann. N. Y. Acad. Sci.

(1994)

Cited by (27)

Gene Expression: Analysis and Quantitation
2013, Animal Biotechnology: Models in Discovery and Translation
Detection of thromboxane A<inf>2</inf> receptor mRNA in rabbit nodose ganglion neurons
2005, Neuroscience Letters
Thromboxane A₂ (TXA₂) is an arachidonic acid metabolite that is released during tissue trauma and elicits platelet aggregation and vascular smooth muscle contraction. Previous research has shown that TXA₂ stimulates pulmonary and cardiac vagal afferent neurons. Therefore, we hypothesized that the presence of the TXA₂ receptor (TP) in vagal neurons would allow for stimulation or modulation of these neurons by TXA₂. To test this hypothesis, single cell RT-PCR was employed using neurons obtained from primary cell cultures of nodose ganglia excised from adult rabbits. Since the sequence for the rabbit TP gene was unknown, a portion of the rabbit TP cDNA was first amplified, cloned, and sequenced. Primer sets for TP were then designed based on this sequence and used in conjunction with a neuronal marker, medium weight neurofilament (NFM), in multiplex RT-PCR reactions. Ninety-three cells were isolated from culture and RT-PCR was carried out on individual cells. Using an aliquot from the initial RT-PCR reaction, a second round of PCR was then employed in which the NFM and TP primer sets were split up into separate reactions. Twenty-three of the 82 cells that were positive for NFM were also positive for TP. Therefore, we conclude that the presence of TP mRNA in a subset of cultured nodose ganglion neurons allows for the possibility that TXA₂ may directly stimulate or modulate vagal afferent neurons.
Single-cell RT-PCR gene expression profiling of acutely dissociated and immunocytochemically identified central neurons
2004, Journal of Neuroscience Methods
Identification of neurons for single-cell mRNA profiling is difficult when cells of interest are located in heterogeneous brain regions. We developed a protocol in which acutely dissociated neurons are immunocytochemically labeled prior to single-cell reverse transcription-polymerase chain reaction (RT-PCR). We tested the protocol on hypothalamic melanin-concentrating hormone (MCH) and prepro-orexin (PPO) neurons, which are similarly distributed but functionally different. Cells dissociated from the perifornical region of the posterior hypothalamus of juvenile or adult rats were incubated with anti-MCH or anti-PPO primary antibodies, followed by washout and incubation with fluorescein-tagged secondary antibodies. Individual labeled cells were subjected to RT-PCR with primers for PPO and MCH. MCH mRNA was detected in 26 out of the 38 successfully reverse-transcribed cells identified as MCH-containing, and 28 cells out of the 42 identified as PPO-containing expressed PPO mRNA. No cell expressed both mRNAs. Most MCH neurons tested (five out of six) expressed the adrenergic α_2A receptor mRNA, whereas it was absent from all seven PPO neurons tested. Neither PPO (n=11) nor MCH (n=6) cells expressed the type 2 orexin receptor mRNA. Thus, the method allows, with at least 66% confidence, immunocytochemical cell identification prior to mRNA studies of single neurons located in heterogeneous brain regions.
Cytokine suppression of dopamine-β-hydroxylase by extracellular signal-regulated kinase-dependent and -independent pathways
2003, Journal of Biological Chemistry
Citation Excerpt :
Each 20-μl reaction included 2.0 mmMgCl2 for GAPDH or 3 mm MgCl2 for DBH, 0.5 μm of each primer, and 2 μl of DNA Master. The intron-spanning rat GAPDH primers generate a 238-bp fragment (27): 5′-CCTGCACCACCAACTGCTTAGC and 3′-GCCAGTGAGCTTCCCGTTCAGC. The mouse DBH primers generate a 211-bp fragment: 5′-AAGGTGGTTACTGTGCTCGC and 3′-CACACATCTCCTCCAAGATTCC.
Cholinergic differentiation factors (CDFs) suppress noradrenergic properties and induce cholinergic properties in sympathetic neurons. The CDFs leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) bind to a LIFR·gp130 receptor complex to activate Jak/signal transducers and activators of transcription and Ras/mitogen-activated protein kinases signaling pathways. Little is known about how these differentiation factors suppress noradrenergic properties. We used sympathetic neurons and SK-N-BE(2)M17 neuroblastoma cells to investigate CDF down-regulation of the norepinephrine synthetic enzyme dopamine-β-hydroxylase (DBH). LIF and CNTF activated extracellular signal-regulated kinases (ERKs) 1 and 2 but not p38 or Jun N-terminal kinases in both cell types. Preventing ERK activation with PD98059 blocked CNTF suppression of DBH protein in sympathetic neurons but did not prevent the loss of DBH mRNA. CNTF decreased transcription of a DBH promoter-luciferase reporter construct in SK-N-BE(2)M17 cells, and this was also ERK-independent. Cytokine inhibition of DBH promoter activity did not require a silencer element but was prevented by overexpression of the transcriptional activator Phox2a. Inhibiting ERK activation increased basal DBH transcription in SK-N-BE(2)M17 cells, and DBH mRNA in sympathetic neurons. Transfection of Phox2a into PD98059-treated M17 cells resulted in a synergistic increase in DBH promoter activity compared with Phox2a or PD98059 alone. These data suggest that CDFs down-regulate DBH protein via an ERK-dependent pathway but inhibit DBH gene expression through an ERK-independent pathway. They further suggest that ERK activity inhibits basal DBH gene expression.
Tyrosine hydroxylase gene expression in ventrolateral medulla oblongata of WKY and SHR: A quantitative real-time polymerase chain reaction study
2002, Autonomic Neuroscience: Basic and Clinical
The aims of this study were, first, to determine quantitatively the levels of tyrosine hydroxylase (TH) gene expression in both peripheral and central sites related to blood pressure regulation, and to compare the level of expression in Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). Second, to see if any relationship exists between TH gene expression and systolic arterial blood pressure. Total RNA was isolated from adrenal glands and from tissue punches taken from the C1 and A1 cell groups in the rostral and caudal ventrolateral medulla oblongata of the brainstem, respectively. Total RNA was reverse-transcribed into cDNA followed by quantitative fluorescence detection polymerase chain reaction for TH cDNA. The levels of TH gene expression measured as a percentage of the house-keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in SHR, were significantly higher (∼2.5-fold) compared to WKY in all sites examined (P<0.01). There was a positive and significant relationship between systolic blood pressure and TH gene expression in the C1 area of the brainstem in both WKY (n=5, P<0.05) and SHR (n=6, P<0.05). Taken together, these results suggest that elevated gene expression of the TH gene is associated with the phenotypic characteristic of SHR.
Modulation of ACh-induced currents in rat adrenal chromaffin cells by ligands of α<inf>2</inf> adrenergic and imidazoline receptors
2001, Autonomic Neuroscience: Basic and Clinical
The aim of this study was to investigate the expression of the α₂-adrenergic receptors in the adrenal medulla, and to examine the mechanism by which clonidine and related drugs inhibit acetylcholine (ACh)-induced whole-cell currents in adrenal chromaffin cells. Reverse transcription-polymerase chain reaction (RT-PCR) performed on punches of rat adrenal medulla demonstrated expression of mRNA for the α_2A-, α_2B- and α_2C-adrenergic receptors. Similar experiments conducted with tissue punches obtained from the adrenal cortex did not reveal expression of these receptor subtypes. Whole-cell currents were recorded in isolated chromaffin cells using the perforated-patch configuration. ACh (50 μM) evoked inward currents with a peak amplitude of 117.8±9.3 pA (n=45; V_hol=−60 mV). The currents were inhibited in a dose-dependent manner (0.5–50 μM) by clonidine, UK 14,304 and rilmenidine (agonists of α₂/imidazoline receptors), as well as by SKF 86466 and efaroxan (antagonists). Adrenaline and noradrenaline (50–100 μM) had no significant effect. Thus, although the adrenal medulla expresses mRNA for the α₂-adrenergic receptors, the lack of agonist–antagonist specificity observed in our whole-cell recordings (in the absence of intracellular dialysis) provides additional evidence against the possibility that these inhibitory effects are mediated by classical α₂ or imidazoline receptor interactions.

View all citing articles on Scopus

¹: Present address: Department of Histology and Embryology, West China University of Medical Sciences, Chengdu, China.

²: Present address: Department of Anatomy and Neurobiology, Wakayama Medical College, 27 Kyubancho, Wakayama 640, Japan.

View full text

ProtocolDetection of mRNA species in bulbospinal neurons isolated from the rostral ventrolateral medulla using single-cell RT–PCR

Abstract

Section snippets

Type of research

Time required

Animals

Special equipment

Retrograde labelling of RVL bulbospinal neurons

Results

General

Retrograde labelling of bulbospinal RVL neurons

Cell dissociation

Essential references

Acknowledgements

Neuron

Mol. Brain Res.

Neurosci. Lett.

Neuroscience

Neuroscience

Brain Res.

Neurochem. Int.

Brain Res.

Brain Res.

J. Neurosci. Methods

Neuron

Neuron

Brain Res.

Type I corticosteroid receptor-like immunoreactivity in the rat CNS: distribution and regulation by corticosteroids

J. Comp. Neurol.

Localization of glucocorticoid receptor mRNA in the male rat brain by in situ hybridization

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

Glial and neuronal glucocorticoid receptor immunoreactive cell populations in developing, adult, and aging brain

Ann. N. Y. Acad. Sci.

Protocol
Detection of mRNA species in bulbospinal neurons isolated from the rostral ventrolateral medulla using single-cell RT–PCR