Biomedical subjects

J Lipski

Publications and source records attributed to J Lipski.

At least 19 recordsLinked to original sources

PubMed2006

Differential expression of group I metabotropic glutamate receptors in human motoneurons at low and high risk of degeneration in amyotrophic lateral sclerosis.

Glutamate excitotoxicity has been suggested to play a role in amyotrophic lateral sclerosis, since overstimulation of post-synaptic glutamate receptors by accumulated extracellular glutamate leads to motoneuron cell death. It is however unclear as to why some groups of motoneurons degenerate in this disease while other groups remain relatively intact even during terminal stages of the disease. Our previous studies in the rat showed differential expression of group I metabotropic glutamate receptors in motoneurons at low and high risk of degeneration in amyotrophic lateral sclerosis. Here we have extended this study to normal human brains. In situ hybridization showed that transcripts of both metabotropic glutamate receptor (mGluR) 1 and mGluR5 were expressed in motoneurons in both the resistant motor nucleus III and the vulnerable motor nucleus XII. Immunolabeling of mGluR1alpha and mGluR5 was found in both motoneurons and glia-like cells in all the motor nuclei and the ventral horn of the cervical spinal cord studied. Quantitative analysis of optical density measurements showed higher levels of mGluR1alpha protein expression but lower levels of mGluR5 protein expression in the vulnerable motoneuron pool (VII, XII and spinal cord) than in the resistant motoneuron pool (III, IV and VI). This differential expression of group I metabotropic glutamate receptor proteins within vulnerable motoneuron pools may predispose these neurons to degeneration as seen in amyotrophic lateral sclerosis.

Central Nervous System↗

PubMed2002

Changes of the excitability of rat trigeminal root ganglion neurons evoked by alpha(2)-adrenoreceptors.

The aim of the study was to examine the effects of alpha(2)-adrenoreceptor agonists on the excitability of trigeminal root ganglion (TRG) neurons using the perforated patch-clamp technique, and to determine whether these neurons express mRNA for alpha(2)-adrenoreceptors. In current-clamp mode, the resting membrane potential was -57.4+/-1.2 mV (n=26). Most neurons (71%) were hyperpolarized by clonidine (5-50 microM) in a concentration-dependent manner. The response was associated with an increase of cell input resistance. In addition, clonidine reduced the repetitive firing evoked by depolarizing current pulses. An alpha(2)-adrenergic agonist, UK14,304, (10-20 microM) also hyperpolarized TRG neurons. The clonidine- and UK14,304-induced hyperpolarization was blocked by idazoxan (alpha(2)-adrenoreceptor antagonist). In voltage-clamp, clonidine (1-50 microM) reversibly reduced the hyperpolarization- and time-dependent cationic current. The effect was mimicked by UK14,304 (10-20 microM), and antagonized by idazoxan. Hyperpolarization-activated cationic current was blocked by extracellular Cs(+) (2 mM) or a specific blocker, ZD7288 (20 microM). Analysis of tail currents revealed that a reversal potential of the clonidine-sensitive component of hyperpolarization-activated cationic current was -46 mV. Single-cell reverse transcription-polymerase chain reaction analysis demonstrated the expression of mRNA for alpha(2A)- and alpha(2C)-adrenoreceptors. These results demonstrate that activation of alpha(2)-adrenoreceptors can hyperpolarize TRG neurons, and that the inhibitory effect is associated with inhibition of hyperpolarization-activated cationic current. Our results suggest that activation of alpha(2)-adrenoreceptors in the absence of nerve injury may have an inhibitory effect on nociceptive transmission in the trigeminal system at the level of both TRG neuronal cell bodies and primary afferent terminals.

Action Potentials↗

PubMed2001

Differential expression of catecholamine synthetic enzymes in the caudal ventral pons.

The analysis of colocalization of multiple catecholamine biosynthetic enzymes within the ventrolateral part of the medulla oblongata of the rat revealed distinct subpopulations of neurons within the C1 region (Phillips et al., J Comp Neurol 2001, 432:20-34). In extending this study to include the caudal pons, it was shown for the first time that the A5 cell group could be distinguished by the presence of immunoreactivity to tyrosine hydroxylase (TH), aromatic l-amino acid decarboxylase (AADC), and dopamine beta hydroxylase (DBH). A novel cell group was also identified. The cells within this new group were immunoreactive to DBH but not TH, AADC, or phenylethanolamine N-methyltransferase (PNMT) and will be referred to as the TH-, DBH+ cell group. The TH-, DBH+ neurons were not immunoreactive for either the dopamine or noradrenaline transporters, suggesting that these neurons do not take up these transmitters. A5 neurons were immunoreactive for the noradrenaline transporter but not the dopamine transporter (as previously shown). Retrograde tracing with cholera toxin B revealed that the TH-, DBH+ neurons do not project to the thoracic spinal cord or to the rostral ventrolateral medulla, but A5 neurons do. A calbindin immunoreactive cell group is located in a region overlapping TH-, DBH+ cell group. However, only a few neurons were immunoreactive for both markers. The physiological role of the TH-, DBH+ cell group remains to be determined.

Animals↗

PubMed2001

Differential expression of Group I metabotropic glutamate receptors in motoneurons at low and high risk for degeneration in ALS.

Glutamate excitotoxicity has been suggested to play a role in amyotrophic lateral sclerosis (ALS), yet it remains unclear why some groups of motoneurons (MNs) are more vulnerable to degeneration than others. Our aim was to compare, in normal adult rats, the expression of Group I metabotropic glutamate receptors (mGluR1 and mGluR5) in MNs normally affected in ALS (XII and spinal MNs) with those which are spared (III and IV MNs). RT-PCR analysis of tissue punches taken from III and XII motor nuclei revealed mRNA for both 'a' and 'b' splice variants of the mGluR1 and mGluR5 receptor subtypes, with expression of the 'a' variant dominant for both receptor subtypes in III and XII nuclei. Immunolabeling for mGluR1a protein was strong in vulnerable (XII and spinal) but negligible in the resistant (III and IV) MNs. Immunoreactivity for mGluR5 was not detected in the cell bodies or proximal dendrites of any MN pool examined. Greater expression of mGluR1a receptor protein within vulnerable MN pools may predispose these neurons to neurodegeneration as seen in ALS.

Alternative Splicing↗

PubMed2001

Modulation of ACh-induced currents in rat adrenal chromaffin cells by ligands of alpha2 adrenergic and imidazoline receptors.

The aim of this study was to investigate the expression of the alpha2-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 2A-, alpha2B- and alpha2C-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 microM) evoked inward currents with a peak amplitude of 117.8+/-9.3 pA (n = 45; Vhol = -60 mV). The currents were inhibited in a dose-dependent manner (0.5-50 microM) by clonidine, UK 14,304 and rilmenidine (agonists of alpha2/imidazoline receptors), as well as by SKF 86466 and efaroxan (antagonists). Adrenaline and noradrenaline (50-100 microM) had no significant effect. Thus, although the adrenal medulla expresses mRNA for the alpha2-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 alpha2 or imidazoline receptor interactions.

Acetylcholine↗

PubMed2001

Differential expression of catecholamine biosynthetic enzymes in the rat ventrolateral medulla.

Adrenergic (C1) neurons located in the rostral ventrolateral medulla are considered a key component in the control of arterial blood pressure. Classically, C1 cells have been identified by their immunoreactivity for the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH) and/or phenylethanolamine N-methyltransferase (PNMT). However, no studies have simultaneously demonstrated the expression of aromatic L-amino acid decarboxylase (AADC) and dopamine beta-hydroxylase (DBH) in these neurons. We examined the expression and colocalization of all four enzymes in the rat ventrolateral medulla using immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) analysis. Retrograde tracer injected into thoracic spinal segments T2-T4 was used to identify bulbospinal neurons. Using fluorescence and confocal microscopy, most cells of the C1 group were shown to be double or triple labeled with TH, DBH, and PNMT, whereas only 65-78% were immunoreactive for AADC. Cells that lacked detectable immunoreactivity for AADC were located in the rostral C1 region, and approximately 50% were spinally projecting. Some cells in this area lacked DBH immunoreactivity (6.5-8.3%) but were positive for TH and/or PNMT. Small numbers of cells were immunoreactive for only one of the four enzymes. Numerous fibres that were immunoreactive for DBH but not for TH or PNMT were noted in the rostral C1 region. Single-cell RT-PCR analysis conducted on spinally projecting C1 neurons indicated that only 76.5% of cells that contained mRNA for TH, DBH, and PNMT contained detectable message for AADC. These experiments suggest that a proportion of C1 cells may not express all of the enzymes necessary for adrenaline synthesis.

Animals↗

PubMed2001

GluR2 AMPA receptor subunit expression in motoneurons at low and high risk for degeneration in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder that results in selective degeneration of most, but not all, groups of motoneurons. The greater susceptibility of vulnerable motoneurons to glutamate excitotoxicity and neurodegeneration has been hypothesized to result from their lower expression of the GluR2 AMPA receptor subunit under control conditions, which renders these receptors permeable to calcium. To address the question of whether there is differential expression of the GluR2 subunit in motoneurons, we compared in normal adult rats expression of GluR2 mRNA and protein within two cranial motor nuclei that are either resistant (III; oculomotor nucleus) or vulnerable (XII; hypoglossal nucleus) to degeneration in ALS. RT-PCR analysis of tissue punched from III and XII motor nuclei detected mRNA for all AMPA subunits (GluR1-R4). In situ hybridization demonstrated no significant difference in GluR2 mRNA expression between III and XII nuclei. Immunohistochemical examination of GluR2 (and GluR4) protein levels demonstrated a similar pattern of the subunit expression in both motor nuclei. This equivalent expression of GluR2 mRNA and protein in motoneurons that differ in their vulnerability to degeneration in ALS suggests that reduced expression of GluR2 is not a factor predisposing motoneurons to degeneration.

Animals↗

PubMed2001

Differential expression of the noradrenaline transporter in adrenergic chromaffin cells, ganglion cells and nerve fibres of the rat adrenal medulla.

Expression of the noradrenaline transporter (NAT) was identified in various cell and fibre populations of the rat adrenal medulla, examined with immunohistochemistry and confocal microscopy. Immunoreactivity for the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH), aromatic-L-amino-acid decarboxylase (AADC) and dopamine beta-hydroxylase (DBH) was present in all chromaffin cells, while phenylethanolamine N-methyltransferase (PNMT) was used to determine adrenergic chromaffin cell groups. Labelling with NAT antibody was predominantly cytoplasmic and colocalised with PNMT immunoreactivity. Noradrenergic chromaffin cells were not NAT immunoreactive. Additionally, NAT antibody labelling demonstrated clusters of ganglion cells (presumably Type I) and nerve fibres. Expression of TH, AADC, DBH, PNMT and NAT mRNA was examined using reverse transcription-polymerase chain reaction (RT-PCR) from adrenal medulla punches and single chromaffin cells, and results were consistent with those obtained with immunocytochemistry. Chromaffin cells and fibres labelled with antibodies against growth associated protein-43 (GAP-43) were not NAT immunoreactive, while ganglion cells were doubled labelled with the two antibodies. The presence of NAT in adrenergic chromaffin cells, and its absence from noradrenergic cells, suggests that the adrenergic cell type is primarily responsible for uptake of catecholamines in the adrenal medulla.

Adrenal Medulla↗

PubMed2001

Dopaminergic substantia nigra neurons express functional nmda receptors in postnatal rats.

Activation of N-methyl-D-aspartate (NMDA) receptors in the Substantia Nigra zona compacta (SNc) may determine the degree of physiological apoptosis during the early postnatal period. However, the expression of these receptors during this stage of development is uncertain, as a recent study failed to detect responses to NMDA in unidentified SNc neurons isolated from 2-wk-old rats. Using conventional or perforated-patch whole cell recordings, we examined the presence of NMDA-evoked responses in SNc neurons acutely dissociated from P4 to P16 rats, applying strict criteria for identification of these neurons as nigrostriatal and dopaminergic. The SNc neurons were identified by retrograde labeling after striatal injection of Fluoro-Gold; the presence of I(h) current; and the inhibition of firing by dopamine (50 microM). NMDA (100 microM, V(hold) = -60 mV) evoked inward, APV-sensitive currents (56.4 +/- 8.6 pA) in all tested neurons (n = 29). Strong depolarizing responses were observed under current-clamp. These results indicate that NMDA receptors play a functional role in SNc neurons during the first two postnatal weeks.

2-Amino-5-phosphonovalerate↗

PubMed2000

Single-cell RT-PCR as a tool to study gene expression in central and peripheral autonomic neurones.

In studies of the central and peripheral autonomic nervous system, it has become increasingly important to be able to investigate mRNA expression patterns within specific neuronal populations. Traditionally, the identification of mRNA species in discrete populations of cells has relied upon in situ hybridization. An alternative, relatively simple procedure is 'multiplex' reverse transcription-polymerase chain reaction (RT-PCR), conducted on single neurons after their in vitro isolation. Multiplex single-cell RT-PCR can be used to examine the expression of multiple genes within individual cells, and can be combined with electrophysiological, pharmacological and anatomical (retrograde labelling) studies. This review focuses on a number of key aspects of this approach, methodology, and both the advantages and the limitations of the technique. We also provide specific examples of work performed in our laboratory, examining the expression of alpha 2-adrenergic receptors in catecholaminergic cells of the rat brainstem and adrenal medulla. The application of single-cell RT-PCR to future studies of the autonomic nervous system will hopefully provide information on how physiological and pathological conditions affect gene expression in autonomic neurones.

Animals↗

PubMed2000

c-Jun promotes neurite outgrowth and survival in PC12 cells.

We investigated the function of c-Jun in PC12 cells by transfecting them with a plasmid containing a c-Jun cDNA transcription cassette. Transfected cells expressed high levels of c-Jun mRNA and protein and demonstrated an increase in both AP-1 DNA binding and gene activation. The c-Jun over-expressing cells showed marked neurite outgrowth but no evidence of spontaneous cell death. In fact, c-Jun over-expressing cells were more resistant to okadaic acid-induced apoptosis. The process outgrowth was not indicative of a full neuronal differentiation response as the transfected PC12 cells did not display action potentials when examined with whole-cell patch-clamping. The phosphorylation of c-Jun on serine 73 appears to be important for this neurite sprouting effect as mutagenesis at this site reduced sprouting whereas a serine 63 mutant tended to increase sprouting. Thus, in PC12 cells c-Jun expression does not induce apoptosis, but rather functions as a neurite outgrowth and neuronal survival signal.

Action Potentials↗

PubMed2000

Calcium binding proteins in motoneurons at low and high risk for degeneration in ALS.

Recent reports challenge the hypothesis that expression of calcium binding proteins contributes to the greater resistance of some motoneurons to degeneration in amyotrophic lateral sclerosis (ALS). We therefore re-examined, using immunohistochemistry, the expression of calbindin, calretinin and parvalbumin in vulnerable (hypoglossal, XII; and cervical spinal) and resistant (oculomotor, III) motoneurons of adult rats. Calbindin immunoreactivity was lacking in motor nuclei but strong in the dorsal horn. Calretinin was expressed in spinal, but not III or XII, motoneurons. Parvalbumin immunoreactivity, tested with a polyclonal antibody, was intense in spinal and III, but not XII, motoneurons; however, no staining in the ventral horn was observed with a monoclonal antibody. Differential expression of calretinin and parvalbumin within vulnerable motoneurons suggests that immunoreactivity for these proteins is not a reliable marker for resistance to degeneration in ALS.

Amyotrophic Lateral Sclerosis↗

PubMed2000

An oral vaccine against NMDAR1 with efficacy in experimental stroke and epilepsy.

The brain is generally considered immunoprivileged, although increasing examples of immunological responses to brain antigens, neuronal expression of major histocompatibility class I genes, and neurological autoimmunity have been recognized. An adeno-associated virus (AAV) vaccine generated autoantibodies that targeted a specific brain protein, the NR1 subunit of the N-methyl-D-aspartate (NMDA) receptor. After peroral administration of the AAV vaccine, transgene expression persisted for at least 5 months and was associated with a robust humoral response in the absence of a significant cell-mediated response. This single-dose vaccine was associated with strong anti-epileptic and neuroprotective activity in rats for both a kainate-induced seizure model and also a middle cerebral artery occlusion stroke model at 1 to 5 months following vaccination. Thus, a vaccination strategy targeting brain proteins is feasible and may have therapeutic potential for neurological disorders.

Administration, Oral↗

PubMed1999

Neurotrophic requirements of rat embryonic catecholaminergic neurons from the rostral ventrolateral medulla.

The factors that regulate the ontogeny and differentiation of C1 adrenergic neurons located in the rostral ventrolateral medulla (RVLM) are completely unknown. In the present study, we have investigated the effects of a number of neurotrophic factors on the survival of E18-19 rat C1 adrenergic neurons in culture. Immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) were used to study the expression of tyrosine hydroxylase (TH), an enzyme present in all catecholaminergic neurons, and of phenylethanolamine N-methyltransferase (PNMT), the final enzyme in the synthesis of adrenalin, as markers for the C1 RVLM neurons. Our results show that GDNF, CNTF BDNF, NT-3 and NT-4/5 increase the number of TH-immunoreactive neurons surviving in vitro. The effects of NGF, TGFbeta and bFGF were not significant. The E18-19 C1 neurons appeared to loose their ability to express PNMT in culture as examined with immunocytochemistry and RT-PCR, and none of the tested neurotrophic factors was able to sustain or induce this expression. Our results indicate that the adrenergic phenotype of C1 neurons, or the survival of these neurons, is determined by environmental factors other than the neurotrophic factors examined in this study.

Animals↗

PubMed1999

Effects of cyanide and hypoxia on membrane currents in neurones acutely dissociated from the rostral ventrolateral medulla of the rat.

Previous reports suggested that some neurones located in the rostral ventrolateral medulla (RVL) can act as fast oxygen sensors which enhance the sympathetic activity and blood pressure independent of peripheral chemoreceptors. The aim of this study was to compare hypoxic responses of different subpopulations of RVL neurones to ascertain whether the hypoxic sensitivity is restricted to one group of these neurones. Whole-cell patch-clamp recordings were made from acutely dissociated neurones obtained from RVL of P13-P19 rats. Short-lasting hypoxia (1-2 min) was evoked by pressure injection of NaCN or lowering pO2. Cells projecting to the upper thoracic segments were retrogradely labelled with fluorescent beads. Catecholaminergic (CA) or non-catecholaminergic (non-CA) neurones were identified using single-cell reverse-transcription polymerase chain reaction (RT-PCR) or immunocytochemistry. Recordings were made from 38 neurones (26 spinally-projecting, 12 non-spinal) using Cs+/TEA or K+-containing pipettes. In most of the cells tested with slow depolarising ramp commands (78%; including spinally-projecting and non-spinal neurones, as well as CA and non-CA neurones), NaCN or hypoxia evoked a reversible increase of the sustained inward current. Extracellular application of 1 mM Co2+ or 25 nM TTX revealed three components of the hypoxia-sensitive inward current which resembled the persistent sodium (INaP), low threshold calcium (LVA Ca2+) and high threshold calcium (HVA Ca2+) currents. The NaCN or hypoxia induced increase of the current could also be observed during step commands. Recordings with K+-containing pipettes during similar depolarising ramps revealed, in addition, a reversible increase of IK in 78% of tested cells (in all four types of examined neurones). These results are consistent with the concepts that RVL neurones can act as a central oxygen sensor. However, in contrast to the previously published data demonstrating that in pentobarbital anaesthetised rats only the barosensitive and spinally projecting cells were affected by a short-lasting hypoxia, our findings obtained with dissociated RVL neurones indicate that sensitivity to hypoxia is widely distributed within this part of the medulla oblongata.

Animals↗

PubMed1999

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

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.

Animals↗

PubMed1999

Localization of the noradrenaline transporter in rat adrenal medulla and PC12 cells: evidence for its association with secretory granules in PC12 cells.

The noradrenaline transporter (NAT) is present in noradrenergic neurons and a few other specialized cells such as adrenal medullary chromaffin cells and the rat pheochromocytoma (PC12) cell line. We have raised antibodies to a 49-residue segment (NATM2) of the extracellular region (residues 184-232) of bovine NAT. Affinity-purified NATM2 antibodies specifically recognized an 80-kDa band in PC12 cell membranes by western blotting. Bands of a similar size were also detected in membranes from human neuroblastoma (SK-N-SH) cells expressing endogenous NAT and human embryonic kidney (HEK293) cells stably expressing bovine NAT. Immunocytochemistry of rat adrenal tissue showed that NAT staining was colocalized with tyrosine hydroxylase in medullary chromaffin cells. Most NAT immunoreactivity in rat adrenal chromaffin and PC12 cells was present in the cytoplasm and had a punctate appearance. Cell surface biotinylation experiments in PC12 cells confirmed that only a minor fraction of the NAT was present at the cell surface. Subcellular fractionation of PC12 cells showed that relatively little NAT colocalized with plasma membrane, synaptic-like microvesicles, recycling endosomes, or trans-Golgi vesicles. Most of the NAT was associated with [3H]noradrenaline-containing secretory granules. Following nerve growth factor treatment, NAT was localized to the growing tip of neurites. This distribution was similar to the secretory granule marker secretogranin I. We conclude that the majority of NAT is present intracellularly in secretory granules and suggest that NAT may undergo regulated trafficking in PC12 cells.

Adrenal Medulla↗

PubMed1998

Noradrenaline transporter expression in the pons and medulla oblongata of the rat: localisation to noradrenergic and some C1 adrenergic neurones.

Catecholaminergic neurotransmission is normally terminated by rapid re-uptake of the neurotransmitter by a high-affinity Na+/Cl--dependent plasma membrane transporter. Specific transporters have been cloned for both dopamine (DAT) and noradrenaline (NAT) in the rat. While DAT has been studied extensively, NAT expression has received less attention, particularly at the protein level. We used an antibody generated against a 49 residue segment of an extracellular loop region of NAT to study expression of the transporter protein throughout the rat pons and medulla oblongata. NAT was expressed in over 95% of noradrenergic neurones in the A1, A2/area postrema, A5, A6/locus subcoeruleus, and A7 noradrenergic groups. Approximately 10% of C1 adrenergic neurones located in the rostral ventrolateral medulla (RVL) also expressed NAT. Expression of NAT mRNA in bulbospinal C1 cells was confirmed using single-cell reverse transcription polymerase chain reaction (RT-PCR) of acutely isolated RVL neurones. Spinally projecting neurones were identified by retrograde labelling with rhodamine beads, and C1 neurones were identified by RT-PCR using primers specific for tyrosine hydroxylase (TH) or phenylethanolamine N-methyltransferase (PNMT) mRNAs. Thirteen percent of adrenergic bulbospinal neurones tested expressed NAT mRNA. C1 neurones are potentially important in cardiovascular control and blood pressure regulation, and the identification of NAT expression in a sub-population of these neurones provides further evidence for the heterogeneity of this neuronal population.

Animals↗