Lindsay M. Parker

Differential Regulation of NADPH Oxidase in Sympathetic and Sensory Ganglia in Deoxycorticosterone Acetate–Salt Hypertension

We demonstrated recently that superoxide anion levels are elevated in prevertebral sympathetic ganglia of deoxycorticosterone acetate–salt hypertensive rats and that this superoxide anion is generated by reduced nicotinamide-adenine dinucleotide phosphate oxidase. In this study we compared the reduced nicotinamide-adenine dinucleotide phosphate oxidase enzyme system of dorsal root ganglion (DRG) and sympathetic celiac ganglion (CG) and its regulation in hypertension. The reduced nicotinamide-adenine dinucleotide phosphate oxidase activity of ganglion extracts was measured using fluorescence spectrometry of dihydroethidine; the activity in hypertensive dorsal root ganglion was 34% lower than in normotensive DRG. In contrast, activity was 79% higher in hypertensive CG than normotensive CG. mRNA for the oxidase subunits NOX1, NOX2, NOX4, p47phox, and p22phox were present in both CG and DRG; mRNA for NOX4 was significantly higher in CG than in DRG. The levels of mRNA and protein expression of the membrane-bound catalytic subunit p22phox and of the regulatory subunits p47phox and Rac-1 were measured in CG and DRG in normotensive and hypertensive rats. p22phox mRNA and protein expression was greater in CG of hypertensive rats but not in DRG. Compared with normotensive controls, p47phox mRNA and protein, as well as Rac-1 protein, were significantly decreased in hypertensive DRG but not in CG. Immunohistochemical staining of p47phox showed translocation from cytoplasm to membrane in hypertensive CG but not in hypertensive DRG. This suggests that reduced nicotinamide-adenine dinucleotide phosphate oxidase activation in sympathetic neurons and sensory neurons is regulated in opposite directions in hypertension. This differential regulation may contribute to unbalanced vasomotor control and enhanced vasoconstriction in the splanchnic circulation.

Chronic Methamphetamine Self-Administration Dysregulates Oxytocin Plasma Levels and Oxytocin Receptor Fibre Density in the Nucleus Accumbens Core and Subthalamic Nucleus of the Rat.

The neuropeptide oxytocin attenuates reward and abuse for the psychostimulant methamphetamine (METH). Recent findings have implicated the nucleus accumbens (NAc) core and subthalamic nucleus (STh) in oxytocin modulation of acute METH reward and relapse to METH‐seeking behaviour. Surprisingly, the oxytocin receptor (OTR) is only modestly involved in both regions in oxytocin attenuation of METH‐primed reinstatement. Coupled with the limited investigation of the role of the OTR in psychostimulant‐induced behaviours, we primarily investigated whether there are cellular changes to the OTR in the NAc core and STh, as well as changes to oxytocin plasma levels, after chronic METH i.v. self‐administration (IVSA) and after extinction of drug‐taking. An additional aim was to examine whether changes to central corticotrophin‐releasing factor (CRF) and plasma corticosterone levels were also apparent because of the interaction of oxytocin with stress‐regulatory mechanisms. Male Sprague–Dawley rats were trained to lever press for i.v. METH (0.1 mg/kg/infusion) under a fixed‐ratio 1 schedule or received yoked saline infusions during 2‐h sessions for 20 days. An additional cohort of rats underwent behavioural extinction for 15 days after METH IVSA. Subsequent to the last day of IVSA or extinction, blood plasma was collected for enzyme immunoassay, and immunofluorescence was conducted on NAc core and STh coronal sections. Rats that self‐administered METH had higher oxytocin plasma levels, and decreased OTR‐immunoreactive (‐IR) fibres in the NAc core than yoked controls. In animals that self‐administered METH and underwent extinction, oxytocin plasma levels remained elevated, OTR‐IR fibre density increased in the STh, and a trend towards normalisation of OTR‐IR fibre density was evident in the NAc core. CRF‐IR fibre density in both brain regions and corticosterone plasma levels did not change across treatment groups. These findings demonstrate that oxytocin systems, both centrally within the NAc core and STh, as well as peripherally through plasma measures, are dysregulated after METH abuse.

Neuropeptide coding of sympathetic preganglionic neurons; focus on adrenally projecting populations

Chemical coding of sympathetic preganglionic neurons (SPN) suggests that the chemical content of subpopulations of SPN can define their function. Since neuropeptides, once synthesized are transported to the axon terminal, most demonstrated chemical coding has been identified using immunoreactive terminals at the target organ. Here, we use a different approach to identify and quantify the subpopulations of SPN that contain the mRNA for pituitary adenylate cyclase activating polypeptide (PACAP) or enkephalin. Using double-labeled immunohistochemistry combined with in situ hybridization (ISH) we firstly identified the distribution of these mRNAs in the spinal cord and determined quantitatively, in Sprague-Dawley rats, that many SPN at the T4-T10 spinal level contain preproPACAP (PPP+, 80 ± 3%, n=3), whereas a very small percentage contain preproenkephalin (PPE+, 4 ± 2%, n=4). A similar neurochemical distribution was found at C8-T3 spinal level. These data suggest that PACAP potentially regulates a large number of functions dictated by SPN whereas enkephalins are involved in few functions. We extended the study to explore those SPN that control adrenal chromaffin cells. We found 97 ± 5% of adrenally projecting SPN (AP-SPN) to be PPP+ (n=4) with only 47 ± 3% that were PPE+ (n=5). These data indicate that adrenally projecting PACAPergic SPN regulate both adrenal adrenaline (Ad) and noradrenaline (NAd) release whereas the enkephalinergic SPN subpopulation must control a (sub) population of chromaffin cells - most likely those that release Ad. The sensory innervation of the adrenal gland was also determined. Of the few adrenally projecting dorsal root ganglia (AP-DRG) observed, 74 ± 12% were PPP+ (n=3), whereas 1 ± 1% were PPE+ (n=3). Therefore, if sensory neurons release peptides to the adrenal medulla, PACAP is most likely involved. Together, these data provide a neurochemical basis for differential control of sympathetic outflow particularly that to the adrenal medulla.

Cardiac norepinephrine transporter protein expression is inversely correlated to chamber norepinephrine content.

The cardiac neuronal norepinephrine (NE) transporter (NET) in sympathetic neurons is responsible for uptake of released NE from the neuroeffector junction. The purpose of this study was to assess the chamber distribution of cardiac NET protein measured using [(3)H]nisoxetine binding in rat heart membranes and to correlate NE content to NET amount. In whole mounts of atria, NET was colocalized in nerve fibers with tyrosine hydroxylase (TH) immunoreactivity. NE content expressed as micrograms NE per gram tissue was lowest in the ventricles; however, NET binding was significantly higher in the left ventricle than the right ventricle and atria (P < 0.05), resulting in a significant negative correlation (r(2) = 0.922; P < 0.05) of NET to NE content. The neurotoxin 6-hydroxydopamine, an NET substrate, reduced NE content more in the ventricles than the atria, demonstrating functional significance of high ventricular NET binding. In summary, there is a ventricular predominance of NET binding that corresponds to a high NE reuptake capacity in the ventricles, yet negatively correlates to tissue NE content.

Neurochemical codes of sympathetic preganglionic neurons activated by glucoprivation

Glucoprivation or hypoglycemia induces a range of counterregulatory responses, including glucose mobilization, reduced glucose utilization, and de novo glucose synthesis. These responses are mediated in part by the sympathetic nervous system. The aim of this study was to determine the chemical codes of sympathetic preganglionic neurons (SPN) activated by glucoprivation, induced by 2‐deoxy‐D‐glucose (2DG). SPN controlling the adrenal glands and celiac ganglia, which ultimately can innervate the liver and pancreas, were targeted together with the superior cervical ganglia (control). 23.9% ± 1.3% of SPN in the T4–T11 region contained c‐Fos immunoreactivity following 2DG; 70.3% ± 1.8% of SPN innervating the adrenal glands and 37.4% ± 3% of SPN innervating celiac ganglia were activated. 14.8% ± 3.5% of SPN (C8–T3) innervating superior cervical ganglia were activated. In the C8–T3 region 55% ± 10% of SPN activated contained PPCART, with only 12% ± 3% expressing PPE mRNA, whereas, in the T4–T11 region, 78% ± 4% contained PPE, with only 6.0% ± 0.6% expressing PPCART mRNA. Thus CART is not involved in glucose mobilization. Two chemically distinct populations of SPN (PPE+ 57.4% ± 5%, PPE− ∼40%) were identified to regulate adrenaline release in response to glucoprivation. Multiple chemically distinct SPN populations innervating a specific target could suggest their graded recruitment. The two distinct populations of SPN (PPE+ 67.6% ± 9%, PPE− ∼30%) projecting to celiac ganglia activated by glucoprivation could direct pancreatic and hepatic or other counterregulatory responses. Nearly all SPN that expressed PPE mRNA and projected to the adrenal glands or celiac ganglia were activated, suggesting a role for the inhibitory peptide enkephalin in responses evoked by glucoprivation. J. Comp. Neurol. 521:2703–2718, 2013.

Proteome Analysis of Ground State Pluripotency.

The differentiation potential of pluripotent embryonic stem cells (ESCs) can be manipulated via serum and medium conditions for direct cellular development or to maintain a naïve ground state. The self-renewal state of ESCs can thus be induced by adding inhibitors of mitogen activated protein kinase (MAPK) and glycogen synthase kinase-3 (Gsk3), known as 2 inhibitors (2i) treatment. We have used a shotgun proteomics approach to investigate differences in protein expressions between 2i- and serum-grown mESCs. The results indicated that 164 proteins were significantly upregulated and 107 proteins downregulated in 2i-grown cells compared to serum. Protein pathways in 2i-grown cells with the highest enrichment were associated with glycolysis and gluconeogenesis. Protein pathways related to organ development were downregulated in 2i-grown cells. In serum-grown ESCs, protein pathways involved in integrin and focal adhesion, and signaling proteins involved in the actin cytoskeleton regulation were enriched. We observed a number of nuclear proteins which were mostly involved in self-renewal maintenance and were expressed at higher levels in 2i compared to serum - Dnmt1, Map2k1, Parp1, Xpo4, Eif3g, Smarca4/Brg1 and Smarcc1/Baf155. Collectively, the results provided an insight into the key protein pathways used by ESCs in the ground state or metastable conditions through 2i or serum culture medium, respectively.

DDX3Y, a male-specific region of y chromosome gene, may modulate neuronal differentiation

Although it is apparent that chromosome complement mediates sexually dimorphic expression patterns of some proteins that lead to functional differences, there has been insufficient evidence following the manipulation of the male-specific region of the Y chromosome (MSY) gene expression during neural development. In this study, we profiled the expression of 23 MSY genes and 15 of their X-linked homologues during neural cell differentiation of NTERA-2 human embryonal carcinoma cell line (NT2) cells in three different developmental stages using qRT-PCR, Western blotting, and immunofluorescence. The expression level of 12 Y-linked genes significantly increased over neural differentiation, including RBMY1, EIF1AY, DDX3Y, HSFY1, BPY2, PCDH11Y, UTY, RPS4Y1, USP9Y, SRY, PRY, and ZFY. We showed that siRNA-mediated knockdown of DDX3Y, a DEAD box RNA helicase enzyme, in neural progenitor cells impaired cell cycle progression and increased apoptosis, consequently interrupting differentiation. Label-free quantitative shotgun proteomics based on a spectral counting approach was then used to characterize the proteomic profile of the cells after DDX3Y knockdown. Among 917 reproducibly identified proteins detected, 71 proteins were differentially expressed following DDX3Y siRNA treatment compared with mock treated cells. Functional grouping indicated that these proteins were involved in cell cycle, RNA splicing, and apoptosis, among other biological functions. Our results suggest that MSY genes may play an important role in neural differentiation and demonstrate that DDX3Y could play a multifunctional role in neural cell development, probably in a sexually dimorphic manner.

GABAergic mRNA expression is upregulated in the prefrontal cortex of rats sensitized to methamphetamine.

Inhibitory gamma-aminobutyric acid (GABA)-mediated neurotransmission plays an important role in the regulation of the prefrontal cortex (PFC), with increasing evidence suggesting that dysfunctional GABAergic processing of the PFC may underlie certain deficits reported across psychotic disorders. Methamphetamine (METH) is a psychostimulant that induces chronic psychosis in a subset of users, with repeat administration producing a progressively increased vulnerability to psychotic relapse following subsequent drug administration (sensitization). The aim here was to investigate changes to GABAergic mRNA expression in the PFC of rats sensitized to METH using quantitative polymerase chain reaction (qPCR). Male Sprague-Dawley rats (n=12) underwent repeated methamphetamine (intraperitoneal (i.p.) or saline injections for 7 days. Following 14 days of withdrawal, rats were challenged with acute methamphetamine (1mg/kg i.p.) and RNA was isolated from the PFC to compare the relative mRNA expression of a range of GABA enzymes, transporters and receptors subunits. METH challenge resulted in a significant sensitized behavioral (locomotor) response in METH pre-treated animals compared with saline pre-treated controls. The mRNAs of transporters (GAT1 and GAT3), ionotropic GABAA receptor subunits (α3 and β1), together with the metabotropic GABAB1 receptor, were upregulated in the PFC of sensitized rats compared with saline controls. These findings indicate that GABAergic mRNA expression is significantly altered at the pre and postsynaptic level following sensitization to METH, with sensitization resulting in the transcriptional upregulation of several inhibitory genes. These changes likely have significant consequences on GABA-mediated neurotransmission in the PFC and may underlie certain symptoms conserved across psychotic disorders, such as executive dysfunction.

Distribution and localisation of Gα proteins in the rostral ventrolateral medulla of normotensive and hypertensive rats: focus on catecholaminergic neurons.

About 860 G-protein-coupled receptors (GPCRs) mediate their actions via heterotrimeric G-proteins. Their activation releases Gα from Gβλ subunits. The type of Gα subunit dictates the major signalling proteins involved: adenylyl cyclase, PLC and rhoGEF. The rostral ventrolateral medulla (RVLM), containing the rostral C1 (rC1) cell group, sets and maintains the tonic and reflex control of blood pressure and a plethora of inputs converge onto these neurons. We determined the relative abundance of 10 Gα subunit mRNAs, representing the four major families, within the RVLM, using quantitative RT-PCR. In situ hybridisation (ISH) combined with immunohistochemistry (IHC) was used to quantify and compare this expression in rC1 with that in the A1 and A5 cell groups. The relative abundance of Gα subunit mRNAs and a comparison of gene expression levels were quantitatively determined in normotensive and hypertensive rat strains. All 10 Gα mRNAs were detected in the RVLM of Sprague-Dawley (SD) rats with relative abundance such that Gαs>Gαi2>Gαo>Gαq>GαL>Gα11>Gαi3>Gαi1>Gα12>Gα13. The high abundance of Gα mRNAs signalling via adenylyl cyclase indicates the importance of associated GPCRs. Within the rC1 and A1 groups similar differential Gα mRNA expression profiles were seen with Gαs being found in all rC1 cells, Gα11 absent and Gαi3 rarely expressed. Thus functionally distinct subgroups exist within the rC1 and A1 cell groups as differing distributions of Gα subunits must reflect the array of GPCRs that influence their activity. In contrast, all A5 cells expressed all Gα mRNAs suggesting a functionally homogeneous group. When the 10 Gα mRNAs of the RVLM in spontaneously hypertensive rats (SHR) were compared quantitatively to Wistar-Kyoto (WKY), only Gαs and Gα12 were significantly elevated. However when the expression in normotensive SD and WKY was compared with SHR no significant differences were evident. These findings demonstrate a range of GPCR signalling capabilities in brainstem neurons important for homeostasis and suggest a prominent role for signalling via adenylyl cyclase.

Neurochemistry of neurons in the ventrolateral medulla activated by hypotension: are the same neurons activated by glucoprivation?

Previous studies have demonstrated that a range of stimuli activate neurons, including catecholaminergic neurons, in the ventrolateral medulla. Not all catecholaminergic neurons are activated and other neurochemical content is largely unknown hence whether stimulus specific populations exist is unclear. Here we determine the neurochemistry (using in situ hybridization) of catecholaminergic and noncatecholaminergic neurons which express c‐Fos immunoreactivity throughout the rostrocaudal extent of the ventrolateral medulla, in Sprague Dawley rats treated with hydralazine or saline. Distinct neuronal populations containing PPCART, PPPACAP, and PPNPY mRNAs, which were largely catecholaminergic, were activated by hydralazine but not saline. Both catecholaminergic and noncatecholaminergic neurons containing preprotachykinin and prepro‐enkephalin (PPE) mRNAs were also activated, with the noncatecholaminergic population located in the rostral C1 region. Few GlyT2 neurons were activated. A subset of these data was then used to compare the neuronal populations activated by 2‐deoxyglucose evoked glucoprivation (Brain Structure and Function (2015) 220:117). Hydralazine activated more neurons than 2‐deoxyglucose but similar numbers of catecholaminergic neurons. Commonly activated populations expressing PPNPY and PPE mRNAs were defined. These likely include PPNPY expressing catecholaminergic neurons projecting to vasopressinergic and corticotrophin releasing factor neurons in the paraventricular nucleus, which when activated result in elevated plasma vasopressin and corticosterone. Stimulus specific neurons included noncatecholaminergic neurons and a few PPE positive catecholaminergic neuron but neurochemical codes were largely unidentified. Reasons for the lack of identification of stimulus specific neurons, readily detectable using electrophysiology in anaesthetized preparations and for which neural circuits can be defined, are discussed.